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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">04260</article-id><article-id pub-id-type="doi">10.7554/eLife.04260</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Microbiology and infectious disease</subject></subj-group><subj-group subj-group-type="heading"><subject>Neuroscience</subject></subj-group></article-categories><title-group><article-title>The sheddase ADAM10 is a potent modulator of prion disease</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-17378"><name><surname>Altmeppen</surname><given-names>Hermann C</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-8"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17379"><name><surname>Prox</surname><given-names>Johannes</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="other" rid="par-1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17380"><name><surname>Krasemann</surname><given-names>Susanne</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17381"><name><surname>Puig</surname><given-names>Berta</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17382"><name><surname>Kruszewski</surname><given-names>Katharina</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17383"><name><surname>Dohler</surname><given-names>Frank</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17384"><name><surname>Bernreuther</surname><given-names>Christian</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con7"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17385"><name><surname>Hoxha</surname><given-names>Ana</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="con8"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17386"><name><surname>Linsenmeier</surname><given-names>Luise</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con9"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17387"><name><surname>Sikorska</surname><given-names>Beata</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con10"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17388"><name><surname>Liberski</surname><given-names>Pawel P</given-names></name><xref ref-type="aff" rid="aff4">4</xref><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con11"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17389"><name><surname>Bartsch</surname><given-names>Udo</given-names></name><xref ref-type="aff" rid="aff3">3</xref><xref ref-type="fn" rid="con12"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17390" equal-contrib="yes"><name><surname>Saftig</surname><given-names>Paul</given-names></name><xref ref-type="aff" rid="aff2">2</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-5"/><xref ref-type="other" rid="par-6"/><xref ref-type="fn" rid="con13"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-17220" corresp="yes" equal-contrib="yes"><name><surname>Glatzel</surname><given-names>Markus</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-7720-8817</contrib-id><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="fn" rid="equal-contrib">†</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con14"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><label>1</label><institution content-type="dept">Institute of Neuropathology</institution>, <institution>University Medical Center Hamburg-Eppendorf</institution>, <addr-line><named-content content-type="city">Hamburg</named-content></addr-line>, <country>Germany</country></aff><aff id="aff2"><label>2</label><institution content-type="dept">Institute of Biochemistry</institution>, <institution>Christian Albrechts University</institution>, <addr-line><named-content content-type="city">Kiel</named-content></addr-line>, <country>Germany</country></aff><aff id="aff3"><label>3</label><institution content-type="dept">Department of Ophthalmology</institution>, <institution>University Medical Center Hamburg-Eppendorf</institution>, <addr-line><named-content content-type="city">Hamburg</named-content></addr-line>, <country>Germany</country></aff><aff id="aff4"><label>4</label><institution content-type="dept">Department of Molecular Pathology and Neuropathology</institution>, <institution>Medical University Lodz</institution>, <addr-line><named-content content-type="city">Lodz</named-content></addr-line>, <country>Poland</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>De Strooper</surname><given-names>Bart</given-names></name><role>Reviewing editor</role><aff><institution>VIB Center for the Biology of Disease, KU Leuven</institution>, <country>Belgium</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>m.glatzel@uke.de</email></corresp><fn fn-type="con" id="equal-contrib"><label>†</label><p>These authors contributed equally to this work</p></fn></author-notes><pub-date publication-format="electronic" date-type="pub"><day>05</day><month>02</month><year>2015</year></pub-date><pub-date pub-type="collection"><year>2015</year></pub-date><volume>4</volume><elocation-id>e04260</elocation-id><history><date date-type="received"><day>06</day><month>08</month><year>2014</year></date><date date-type="accepted"><day>04</day><month>02</month><year>2015</year></date></history><permissions><copyright-statement>© 2015, Altmeppen et al</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>Altmeppen et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife04260.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.04260.001</object-id><p>The prion protein (PrP<sup>C</sup>) is highly expressed in the nervous system and critically involved in prion diseases where it misfolds into pathogenic PrP<sup>Sc</sup>. Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer's disease. PrP<sup>C</sup> is shed at the plasma membrane by the metalloprotease ADAM10, yet the impact of this on prion disease remains enigmatic. Employing conditional knockout mice, we show that depletion of ADAM10 in forebrain neurons leads to posttranslational increase of PrP<sup>C</sup> levels. Upon prion infection of these mice, clinical, biochemical, and morphological data reveal that lack of ADAM10 significantly reduces incubation times and increases PrP<sup>Sc</sup> formation. In contrast, spatiotemporal analysis indicates that absence of shedding impairs spread of prion pathology. Our data support a dual role for ADAM10-mediated shedding and highlight the role of proteolytic processing in prion disease.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.001">http://dx.doi.org/10.7554/eLife.04260.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.04260.002</object-id><title>eLife digest</title><p>Prion proteins are anchored to the surface of brain cells called neurons. Normally, prion proteins are folded into a specific three-dimensional shape that enables them to carry out their normal roles in the brain. However, they can be misfolded into a different shape known as PrP<sup>Sc</sup>, which can cause Creutzfeldt-Jakob disease and other serious conditions that affect brain function and ultimately lead to death.</p><p>The PrP<sup>Sc</sup> proteins can force normal prion proteins to change into the PrP<sup>Sc</sup> form, so that over time this form accumulates in the brain. They are essential components of infectious particles termed ‘prions’ and this is why prion diseases are infectious: if prions from one individual enter the brain of another individual they can cause disease in the recipient. The UK outbreak of variant Creutzfeldt-Jakob disease in humans in the 1990s is thought to be due to the consumption of meat from cattle with a prion disease known as mad cow disease.</p><p>An enzyme called ADAM10 can cut normal prion proteins from the surface of neurons. However, it is not clear whether ADAM10 can also target the PrP<sup>Sc</sup> proteins and what impact this may have on the development of prion diseases.</p><p>Here, Altmeppen et al. studied mutant mice that were missing ADAM10 in neurons in the front portion of their brain. These mice had a higher number of normal prion proteins on the surface of their neurons than normal mice did. When mice missing ADAM10 were infected with prions, more PrP<sup>Sc</sup> accumulated in their brain and disease symptoms developed sooner than when normal mice were infected. This supports the view that mice with higher numbers of prion proteins are more vulnerable to prion disease. However, disease symptoms did not spread as quickly to other parts of the brain in the mice missing ADAM10. This suggests that by releasing prion proteins from the surface of neurons, ADAM10 helps PrP<sup>Sc</sup> proteins to spread around the brain.</p><p>Recently, it has been suggested that prion proteins may also play a role in Alzheimer's disease and other neurodegenerative conditions. Therefore, Altmeppen et al.'s findings may help to develop new therapies for other forms of dementia. The next challenge is to understand the precise details of how ADAM10 works.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.002">http://dx.doi.org/10.7554/eLife.04260.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>ADAM10</kwd><kwd>neurodegeneration</kwd><kwd>prion disease</kwd><kwd>proteolytic processing</kwd><kwd>shedding</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>SFB877</award-id><principal-award-recipient><name><surname>Altmeppen</surname><given-names>Hermann C</given-names></name><name><surname>Prox</surname><given-names>Johannes</given-names></name><name><surname>Linsenmeier</surname><given-names>Luise</given-names></name><name><surname>Saftig</surname><given-names>Paul</given-names></name><name><surname>Glatzel</surname><given-names>Markus</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001664</institution-id><institution>Leibniz Association</institution></institution-wrap></funding-source><award-id>Leibniz Graduate School</award-id><principal-award-recipient><name><surname>Altmeppen</surname><given-names>Hermann C</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100004431</institution-id><institution>European Commission Directorate-General for Research and Innovation</institution></institution-wrap></funding-source><award-id>REGPOT-2012-2013-1,7FP</award-id><principal-award-recipient><name><surname>Sikorska</surname><given-names>Beata</given-names></name><name><surname>Liberski</surname><given-names>Pawel P</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Werner Otto Stiftung</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Altmeppen</surname><given-names>Hermann C</given-names></name><name><surname>Linsenmeier</surname><given-names>Luise</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution>Hans & Ilse Breuer Stiftung</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Saftig</surname><given-names>Paul</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>GRK1459</award-id><principal-award-recipient><name><surname>Saftig</surname><given-names>Paul</given-names></name><name><surname>Glatzel</surname><given-names>Markus</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001659</institution-id><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>FOR885 (GL 589/5)</award-id><principal-award-recipient><name><surname>Glatzel</surname><given-names>Markus</given-names></name></principal-award-recipient></award-group><award-group id="par-8"><funding-source><institution-wrap><institution content-type="university">University of Hamburg</institution></institution-wrap></funding-source><award-id>Young investigator research grant of the Medical Faculty</award-id><principal-award-recipient><name><surname>Altmeppen</surname><given-names>Hermann C</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2.0</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A lack of ADAM10-mediated shedding increases prion protein levels at the plasma membrane and promotes the generation of pathological prion proteins, which accelerates prion disease in mice.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="s1"><title>Introduction</title><p>The cellular prion protein (PrP<sup>C</sup>) is a glycosylphosphatidylinositol (GPI)-anchored lipid raft constituent highly expressed in neurons of the central nervous system (CNS). Misfolding of PrP<sup>C</sup> into its pathogenic isoform, PrP<sup>Sc</sup>, occurs by a self-perpetuating process of templated conformational conversion and causes transmissible and invariably fatal prion diseases (<xref ref-type="bibr" rid="bib84">Prusiner, 1982</xref>). In these diseases, PrP<sup>C</sup> not only represents the substrate for conversion but also a prerequisite of prion-associated neurotoxicity (<xref ref-type="bibr" rid="bib19">Büeler et al., 1993</xref>; <xref ref-type="bibr" rid="bib17">Brandner et al., 1996</xref>; <xref ref-type="bibr" rid="bib65">Mallucci et al., 2003</xref>). In contrast to PrP<sup>C</sup>, PrP<sup>Sc</sup> is prone to aggregation and is characterized by a high β-sheet content and partial resistance to digestion with proteinase K (PK). Moreover, PrP<sup>Sc</sup> constitutes an essential component of infectious prion particles or ‘prions’ (<xref ref-type="bibr" rid="bib84">Prusiner, 1982</xref>; <xref ref-type="bibr" rid="bib92">Silveira et al., 2005</xref>).</p><p>Recently, a central role in more common proteinopathies, such as Alzheimer's disease (AD), has been attributed to PrP<sup>C</sup> where it was shown to act as a neuronal receptor for neurotoxic amyloid-β (Aβ) oligomers (<xref ref-type="bibr" rid="bib55">Lauren et al., 2009</xref>; <xref ref-type="bibr" rid="bib30">Dohler et al., 2014</xref>) and a variety of other disease-associated β-sheet rich protein assemblies including PrP<sup>Sc</sup> (<xref ref-type="bibr" rid="bib85">Resenberger et al., 2011</xref>). Despite some degree of controversy regarding this receptor function and its relevance in AD (<xref ref-type="bibr" rid="bib9">Balducci et al., 2010</xref>; <xref ref-type="bibr" rid="bib14">Benilova and De Strooper, 2010</xref>; <xref ref-type="bibr" rid="bib22">Calella et al., 2010</xref>; <xref ref-type="bibr" rid="bib48">Kessels et al., 2010</xref>), binding of pathogenic oligomers to the flexible N-terminus of PrP<sup>C</sup> is thought to initiate a cascade of events that mediates their neurotoxicity and results in synaptic degeneration and neuronal loss (<xref ref-type="bibr" rid="bib85">Resenberger et al., 2011</xref>; <xref ref-type="bibr" rid="bib53">Larson et al., 2012</xref>; <xref ref-type="bibr" rid="bib99">Um et al., 2012</xref>).</p><p>PrP<sup>C</sup> is subject to two conserved proteolytic cleavage events under physiological conditions, termed α-cleavage and shedding (<xref ref-type="bibr" rid="bib16">Borchelt et al., 1993</xref>; <xref ref-type="bibr" rid="bib42">Harris et al., 1993</xref>; <xref ref-type="bibr" rid="bib26">Chen et al., 1995</xref>; <xref ref-type="bibr" rid="bib66">Mange et al., 2004</xref>). These cleavages likely influence physiological functions of PrP<sup>C</sup> (<xref ref-type="bibr" rid="bib2">Aguzzi et al., 2008</xref>; <xref ref-type="bibr" rid="bib63">Linden et al., 2008</xref>) and, importantly, its role in neurodegenerative diseases (reviewed in <xref ref-type="bibr" rid="bib6">Altmeppen et al., 2013</xref>). While α-cleavage, occurring in the middle of the PrP<sup>C</sup> sequence and producing a soluble N1 and a membrane-attached C1 fragment, confers neuroprotection with regard to prion diseases (<xref ref-type="bibr" rid="bib59">Lewis et al., 2009</xref>; <xref ref-type="bibr" rid="bib103">Westergard et al., 2011</xref>; <xref ref-type="bibr" rid="bib97">Turnbaugh et al., 2012</xref>; <xref ref-type="bibr" rid="bib23">Campbell et al., 2013</xref>) and Aβ-associated neurotoxicity (<xref ref-type="bibr" rid="bib40">Guillot-Sestier et al., 2009</xref>; <xref ref-type="bibr" rid="bib85">Resenberger et al., 2011</xref>; <xref ref-type="bibr" rid="bib13">Beland et al., 2012</xref>; <xref ref-type="bibr" rid="bib41">Guillot-Sestier et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Fluharty et al., 2013</xref>), the role of an extreme C-terminal cleavage in close proximity to the plasma membrane, termed shedding, remains enigmatic. On the one hand, by reducing the substrate for prion conversion and the receptor for neurotoxicity, this proteolytic release of almost full length PrP<sup>C</sup> from the plasma membrane could be protective with regard to prion diseases (<xref ref-type="bibr" rid="bib67">Marella et al., 2002</xref>; <xref ref-type="bibr" rid="bib44">Heiseke et al., 2008</xref>; <xref ref-type="bibr" rid="bib8">Altmeppen et al., 2012</xref>). Moreover, recombinant or transgenically expressed anchorless PrP<sup>C</sup> mimicking shed PrP<sup>C</sup> has been shown to antagonize both PrP<sup>Sc</sup> propagation and Aβ neurotoxicity, respectively, thus indicating a protective activity by blocking toxic conformers and preventing their access to the cell (<xref ref-type="bibr" rid="bib70">Meier et al., 2003</xref>; <xref ref-type="bibr" rid="bib22">Calella et al., 2010</xref>; <xref ref-type="bibr" rid="bib72">Nieznanski et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Fluharty et al., 2013</xref>; <xref ref-type="bibr" rid="bib106">Yuan et al., 2013</xref>). On the other hand, shedding could accelerate an ongoing prion disease by increasing production and subsequent spread of anchorless prions within the CNS. In fact, PrP<sup>Sc</sup> can be shed from the cellular surface in vitro (<xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>) and anchorless PrP<sup>C</sup> can, in principle, be converted to PrP<sup>Sc</sup>, resulting in an altered type of prion disease in transgenic mice (<xref ref-type="bibr" rid="bib28">Chesebro et al., 2005</xref>, <xref ref-type="bibr" rid="bib27">2010</xref>). High expression of anchorless PrP<sup>C</sup> leads to formation of prions and a late onset neurological disease (<xref ref-type="bibr" rid="bib94">Stöhr et al., 2011</xref>).</p><p>While the in vivo identity of the protease(s) responsible for the α-cleavage of PrP<sup>C</sup> remains a matter of controversy (<xref ref-type="bibr" rid="bib100">Vincent et al., 2001</xref>; <xref ref-type="bibr" rid="bib98">Tveit et al., 2005</xref>; <xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>; <xref ref-type="bibr" rid="bib73">Oliveira-Martins et al., 2010</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>, <xref ref-type="bibr" rid="bib8">2012</xref>; <xref ref-type="bibr" rid="bib13">Beland et al., 2012</xref>; <xref ref-type="bibr" rid="bib60">Liang et al., 2012</xref>; <xref ref-type="bibr" rid="bib104">Wik et al., 2012</xref>; <xref ref-type="bibr" rid="bib68">Mays et al., 2014</xref>; <xref ref-type="bibr" rid="bib69">McDonald et al., 2014</xref>), we recently showed that <underline>a</underline> <underline>d</underline>isintegrin <underline>a</underline>nd <underline>m</underline>etalloproteinase ADAM10 is the relevant sheddase of PrP<sup>C</sup> in vivo (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>), as previously suggested (<xref ref-type="bibr" rid="bib76">Parkin et al., 2004</xref>; <xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>) and subsequently confirmed (<xref ref-type="bibr" rid="bib104">Wik et al., 2012</xref>; <xref ref-type="bibr" rid="bib74">Ostapchenko et al., 2013</xref>; <xref ref-type="bibr" rid="bib69">McDonald et al., 2014</xref>) by in vitro experiments of others. Using conditional <sup>Nestin</sup>ADAM10 knockout mice (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>), we recently showed that depletion of the protease in neural precursors abolishes shedding and leads to posttranslational accumulation of PrP<sup>C</sup> in the early secretory pathway indicative of a regulatory role of ADAM10 in PrP<sup>C</sup> membrane homeostasis (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). However, due to the perinatal lethality of these mice, the influence of ADAM10 on the course of prion disease remained unsolved. In this study we used novel viable ADAM10 conditional knockout (ADAM10 cKO or A10 cKO) mice with specific postnatal deletion of the protease in forebrain neurons (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). We show that lack of ADAM10 leads to (i) elevated (membrane) levels of PrP<sup>C</sup>, (ii) drastically shortened incubation times of prion disease, (iii) increased prion conversion, and (iv) upregulation of calpain levels. In contrast, spread of prion-associated pathology within the brain was reduced. It is intriguing that the absence of a protease involved in the constitutive processing of PrP<sup>C</sup> significantly impacts the course of prion disease, thus enforcing the relevance of proteolytic processing events in neurodegeneration.</p></sec><sec sec-type="results" id="s2"><title>Results</title><sec id="s2-1"><title>Increased (membrane) levels and lack of shedding of PrP<sup>C</sup> in different cellular models of ADAM10 depletion</title><p>Maintenance of plasma membrane levels of PrP<sup>C</sup> by ADAM10-mediated shedding may represent a conserved mechanism in different cellular lineages. To further investigate this we generated neural stem cells (NSCs) from embryonic day (E) 14 wild-type and A10 cKO mice with inactivation of the <italic>Adam10</italic> gene in neuroectodermal progenitor cells (<sup>Nestin</sup>A10 KO mice) (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>). As shown in <xref ref-type="fig" rid="fig1">Figure 1A</xref>, surface biotinylation experiments on neuronally differentiated NSCs revealed that membrane levels of PrP<sup>C</sup> were increased 1.56-fold (±0.12; SEM) in the absence of ADAM10 (n = 9 independent samples) compared with wild-type controls (set to 1 ± 0.13; n = 9). Moreover, genetic reintroduction of <italic>Adam10</italic> into NSC cultures of <sup>Nestin</sup>A10 KO mice was sufficient to reduce membrane levels of PrP<sup>C</sup> (0.95 ± 0.11; n = 8) and thus to restore physiological wild-type conditions. Nucleofection of <sup>Nestin</sup>A10 KO cells with a vector lacking the <italic>Adam10</italic> cDNA did not show any effect on PrP<sup>C</sup> membrane levels (1.55 ± 0.18; n = 5). Indirect immunofluorescence analyses of non-permeabilized neuronally differentiated NSCs confirmed the biochemical results by showing increased intensity of PrP<sup>C</sup> surface immunostaining in <sup>Nestin</sup>A10 KO cells and <sup>Nestin</sup>A10 KO cells nucleofected with a control vector compared with wild-type control cells, and a similar intensity of PrP<sup>C</sup> surface immunostaining in A10-nucleofected <sup>Nestin</sup>A10 KO and control cells (<xref ref-type="fig" rid="fig1">Figure 1B</xref>).<fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.003</object-id><label>Figure 1.</label><caption><title>Characterization of PrP<sup>C</sup> levels in different cellular models of ADAM10 deficiency.</title><p>(<bold>A</bold>) Representative Western blots showing membrane levels of PrP<sup>C</sup> as revealed by surface biotinylation (I; on the left) and total PrP<sup>C</sup> levels in lysates (I; on the right) as well as ADAM10 surface expression (II) of neuronally differentiated neural stem cells (NSCs) from <sup>Nestin</sup>A10 KO and littermate control mice and after genetic reintroduction of <italic>Adam10</italic> (A10 KO + ADAM10) or nucleofection with control vector (A10 KO + Vector) into NSCs of <sup>Nestin</sup>A10 KO mice. Flotillin served as loading control. (III) Quantification of densitometric analysis of PrP<sup>C</sup> membrane levels of experimental groups mentioned above (n = 9 independent samples for controls [set to 1]; n = 9 for <sup>Nestin</sup>A10 KO; n = 8 for <sup>Nestin</sup>A10 KO + ADAM10; n = 5 for <sup>Nestin</sup>A10 KO + Vector; significance: **p = 0.0054; <sup>##</sup>p = 0.0014; *p = 0.0336 ; <sup>#</sup>p = 0.0212). Error bars indicate SEM. (<bold>B</bold>) Representative immunofluorescent PrP<sup>C</sup> (green) surface staining of neuronally differentiated NSCs derived from <sup>Nestin</sup>A10 KO (without [second row] or with genetic reintroduction of ADAM10 [third row] or vector only [fourth row]) and littermate control mice (first row), respectively. Tubulin (red) was stained after permeabilization of cells to confirm neuronal differentiation of NSCs. DAPI (blue) marks nuclei. (<bold>C</bold>) Representative immunostaining of PrP<sup>C</sup> (green) and ADAM10 (red) in permeabilized (upper two rows) and non-permeabilized (lower three rows) murine embryonic fibroblasts (MEFs) derived from mice with a complete knockout of ADAM10 (ADAM10 KO) or wild-type mice (control). Higher resolution of white boxes is shown in the bottom row and reveals colocalization of PrP<sup>C</sup> and ADAM10 at the plasma membrane of wild-type control MEFs. Scale bars in <bold>B</bold> and <bold>C</bold> represent 10 µm. (<bold>D</bold>) Western blot analysis of cell-associated PrP<sup>C</sup> levels in ADAM10 knockout (A10 KO) and wild-type (wt) MEF lysates (left part: actin served as loading control). Levels of shed PrP<sup>C</sup> were assessed in cell culture media supernatants of ADAM10 knockout and wild-type MEFs by filter column concentration (conc. media) and immunoprecipitation (IP) with a PrP<sup>C</sup>-specific antibody respectively (right part). (<bold>E</bold>) Levels of cell-associated (neuronal lysates) and shed PrP<sup>C</sup> (IP of media supernatants) in primary neuronal cultures of prion protein knockout (<italic>Prnp</italic><sup>0/0</sup>), wild-type (wt; C57BL/6), prion protein overexpressing (<italic>tg</italic>a<italic>20</italic>), and <sup>Nestin</sup>A10 KO mice at embryonic day 14. IgG-HC and IgG-LC mark signals for heavy and light chain of the capturing antibody POM2.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.003">http://dx.doi.org/10.7554/eLife.04260.003</ext-link></p></caption><graphic xlink:href="elife04260f001"/></fig></p><p>In addition, we analyzed murine embryonic fibroblasts (MEFs) derived from mice with a complete knockout of ADAM10 (<xref ref-type="bibr" rid="bib43">Hartmann et al., 2002</xref>) with regard to PrP<sup>C</sup> levels (<xref ref-type="fig" rid="fig1">Figure 1C and D</xref>). As expected, we found increased total PrP<sup>C</sup> levels in ADAM10 knockout MEFs by (i) immunofluorescence analysis of permeabilized cells (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, upper part) and (ii) Western blot assessment of MEF lysates (<xref ref-type="fig" rid="fig1">Figure 1D</xref>, left part). In non-permeabilized wild-type MEFs, colocalization could be observed between the protease ADAM10 and its substrate PrP<sup>C</sup> at the plasma membrane (<xref ref-type="fig" rid="fig1">Figure 1C</xref>, bottom row). Next, we directly investigated the shedding of PrP<sup>C</sup> in ADAM10 knockout and wild-type MEFs by biochemical analysis of culture supernatants. Results obtained with concentrated media and with immunoprecipitation of shed PrP<sup>C</sup> from media showed that shedding is impaired in ADAM10 knockout MEFs compared with wild-type MEFs (<xref ref-type="fig" rid="fig1">Figure 1D</xref>, right part).</p><p>Finally, we assessed PrP<sup>C</sup> levels and the shedding of PrP<sup>C</sup> in primary neurons of <sup>Nestin</sup>A10 KO and wild-type control mice as well as in mice deficient for PrP<sup>C</sup> (<italic>Prnp</italic><sup>0/0</sup>) and in PrP<sup>C</sup> overexpressing <italic>Tg(Prnp)a20</italic> mice (known and hereafter referred to as <italic>tg</italic>a<italic>20</italic>) (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). Confirming our previous study (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>), shedding of PrP<sup>C</sup> was absent in <sup>Nestin</sup>A10 KO neurons while <italic>tg</italic>a<italic>20</italic> neurons showed increased levels of shed PrP<sup>C</sup> compared with wild-type controls.</p><p>Taken together, data obtained from different murine cellular models having a deletion of <italic>Adam10</italic> confirmed the role of this protease as the functionally relevant sheddase of PrP<sup>C</sup> and, thus, as a regulator of PrP<sup>C</sup> membrane homeostasis.</p></sec><sec id="s2-2"><title>Lack of ADAM10 in neurons of the forebrain results in increased levels of PrP<sup>C</sup></title><p>Using conditional <sup>Nestin</sup>A10 knockout mice, we previously showed that lack of ADAM10-mediated shedding leads to increased neuronal levels of PrP<sup>C</sup> (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). However, due to the perinatal lethality of these mice we were unable to investigate the impact of ADAM10 deficiency on the course of prion disease. Therefore, new conditional <sup>Camk2a</sup>ADAM10 knockout mice (ADAM10 cKO or A10 cKO) lacking Adam10 in neurons of the forebrain were produced and characterized (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). These mice were viable and used for prion inoculations performed in this study. First, we analyzed PrP<sup>C</sup> levels in A10 cKO and littermate controls at postnatal day (P) 19. Reduction of ADAM10 expression was accompanied by increased PrP<sup>C</sup> amounts as revealed by Western blot analysis of cortical homogenates (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Residual ADAM10 most likely resulted from glial cells not depleted of ADAM10. In contrast to the cortex, differences in ADAM10 expression and PrP<sup>C</sup> levels between A10 cKO and littermate controls were not seen in the cerebellum, a brain region not affected by our knockout strategy (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>) (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Immunohistochemical analysis in P19 mouse brains confirmed the Western blot data by showing increased immunostaining for PrP<sup>C</sup> in A10 cKO mice in several regions of the forebrain, as exemplified by the hippocampus and cortex, whereas no such increase was observed in the cerebellum (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). In addition, a coronal brain section showing costaining of PrP<sup>C</sup> with the neuronal marker NeuN correlates with the <italic>Camk2a</italic> driven ADAM10 knockout strategy (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>) by showing increased PrP<sup>C</sup> expression in hippocampal and cortical areas as well as in the striatum (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Increased PrP<sup>C</sup> levels in A10 cKO mice did not result from transcriptional upregulation as there were no significant differences in PrP<sup>C</sup> mRNA levels in the forebrain at P19 as assessed by quantitative RT-PCR (mean ± SD: controls: 5.96 ± 0.86; A10 cKO: 6.32 ± 1.04; p = 0.23; n = 5) (<xref ref-type="fig" rid="fig2">Figure 2C</xref>). We also analyzed PrP<sup>C</sup> levels in 35-week-old A10 cKO and littermate control mice. In contrast to newborn A10 cKO mice (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>), adult A10 cKO mice did not show differences regarding weight and body size (<xref ref-type="fig" rid="fig2">Figure 2D</xref> and <xref ref-type="other" rid="video1">Video 1</xref>). Again, as shown at P19, adult A10 cKO mice also showed increased levels of PrP<sup>C</sup> in the cortex and hippocampus compared with their littermate controls, as assessed by immunohistochemistry at 35 weeks of age (<xref ref-type="fig" rid="fig2">Figure 2E</xref>). Taking these observations together, A10 cKO mice are viable and show a temporally unrestricted posttranslational increase of PrP<sup>C</sup> in neurons.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.004</object-id><label>Figure 2.</label><caption><title>Characterization of PrP<sup>C</sup> expression in juvenile and adult <italic>Camk2a</italic>-Cre A10 cKO mice and littermate controls.</title><p>(<bold>A</bold>) Western blot analysis of mature ADAM10 (mADAM10) and PrP<sup>C</sup> expression in different brain homogenates from the cortex (left) and cerebellum (right) of A10 cKO and control mice at postnatal day (P) 19. Actin served as a loading control. (<bold>B</bold>) Immunohistochemical detection of PrP<sup>C</sup> in forebrain of P19 mice of both genotypes. Overview (top) and magnifications showing cortex (Cx) and hippocampal CA2 and CA3 regions. Overview and details of cerebellum (Cb) are shown below. Scale bars represent 100 µm (insets for Cb: 50 µm). (<bold>C</bold>) Quantitative RT-PCR analysis of PrP<sup>C</sup> mRNA levels in A10 cKO mice and controls at P19 (n = 5 for each genotype). GAPDH served as a control for normalization. Error bars indicate SD. (<bold>D</bold>) Adult age-matched A10 cKO and wild-type littermates had a comparable body size at 35 weeks of age. (<bold>E</bold>) Representative immunohistochemical staining of PrP<sup>C</sup> in cortex (Cx) and hippocampal CA1 region of adult (35 weeks) A10 cO and control mice. Prion protein knockout (<italic>Prnp</italic><sup>0/0</sup>) and overexpressing mice (<italic>tg</italic>a<italic>20</italic>) served as negative and positive controls, respectively (scale bar: 100 µm).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.004">http://dx.doi.org/10.7554/eLife.04260.004</ext-link></p></caption><graphic xlink:href="elife04260f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.005</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Regional distribution of increased PrP<sup>C</sup> levels in a coronal brain section of an ADAM10 cKO mouse compared with a wild-type littermate control.</title><p>Brain sections derived from postnatal day 19 mice of both genotypes co-stained for PrP<sup>C</sup> (brownish signal) and neuronal marker NeuN (red signal). Scale bar represents 200 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.005">http://dx.doi.org/10.7554/eLife.04260.005</ext-link></p></caption><graphic xlink:href="elife04260fs001"/></fig></fig-group><media id="video1" content-type="glencoe play-in-place height-250 width-310" xlink:href="elife04260v001.MOV" mimetype="video" mime-subtype="MOV"><object-id pub-id-type="doi">10.7554/eLife.04260.006</object-id><label>Video 1.</label><caption><title>Clinical presentation of a prion-infected ADAM10 cKO and littermate control mouse at 95 days post inoculation (dpi).</title><p>Terminally diseased ADAM10 cKO mouse (cage on the left) directly prior to termination of the experiment showing lack of nest-building behaviour and late hypoactivity whereas a wild-type littermate (cage on the right) matched for dpi presents with a regular nest and normal activity.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.006">http://dx.doi.org/10.7554/eLife.04260.006</ext-link></p></caption></media></p><p>Based on previous reports (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>) and on our findings presented here (<xref ref-type="fig" rid="fig2">Figure 2</xref>), a qualitative representation showing the regional distribution of the neuronal <italic>Camk2a</italic>-Cre expression (and thus of the ADAM10 knockout and increased PrP<sup>C</sup> expression) is provided in <xref ref-type="fig" rid="fig3">Figure 3A</xref>. In addition, this scheme depicts information on our experimental design described in detail below, including the site of prion inoculation and brain regions chosen for immunohistochemical or biochemical analyses.<fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.007</object-id><label>Figure 3.</label><caption><title>Overview of the experimental design and summary of most important findings.</title><p>(<bold>A</bold>) Scheme of a mouse brain combining a qualitative representation of the <italic>Camk2a</italic> driven ADAM10 knockout strategy and information on the sampling of specimen. The site of intracerebral inoculation of mice with RML prions is indicated by the red encircled dot. Samples of frontal brain (dotted box) were taken for biochemical analysis and determination of infectivity titers (bioassay). The rest of the brain was formalin-fixed and embedded in paraffin. Coronal sections were prepared from different layers (dashed lines) with varying distance to the site of prion inoculation (as indicated by blue arrows) and assessed by immunohistochemical analysis. (<bold>B</bold>) Qualitative comparison of mouse genotypes (A10 cKO, controls, <italic>tg</italic>a<italic>20</italic>) with regard to PrP<sup>C</sup> or PrP<sup>Sc</sup> levels, prion-associated neuropathology (including spongiosis, astrocytosis, and microglia activation) and prion infectivity titers according to brain region and time point. Reference to corresponding figures showing original data is provided. Cb = Cerebellum; Cx = Cortex; Hc = Hippocampus; Stri = Striatum; Tha = Thalamus; n.a. = not assessed; Qualities: +++ = high/strong; ++ = medium/moderate; + = low/basal; (+) = very low/weak; o = none.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.007">http://dx.doi.org/10.7554/eLife.04260.007</ext-link></p></caption><graphic xlink:href="elife04260f003"/></fig></p></sec><sec id="s2-3"><title>Drastically reduced incubation time to terminal prion disease in A10 cKO mice</title><p>Next, we studied how the absence of the PrP<sup>C</sup> sheddase impacts on the course of prion disease. For better orientation, <xref ref-type="fig" rid="fig3">Figure 3B</xref> summarizes the most important findings presented in the following paragraphs including comparisons of PrP<sup>Sc</sup> levels, prion-associated neuropathology, and infectivity titers for the different genotypes, brain regions, and time points investigated in this study. Moreover, this overview provides reference to the corresponding figures showing the original data described below.</p><p>We inoculated A10 cKO (n = 7) and littermate control mice (n = 8) at 6 weeks of age intracerebrally with the Rocky Mountain laboratory (RML) strain of prions. Mock (CD1)-inoculated A10 cKO mice (n = 5) or wild-type littermates (n = 11) and prion inoculated, prion hypersensitive, PrP<sup>C</sup> overexpressing <italic>tg</italic>a<italic>20</italic> mice (n = 8) served as negative and positive controls, respectively. Interestingly, we found significantly reduced incubation times until onset of terminal prion disease in the A10 cKO mice (103 ± 7 days post inoculation (dpi); SD) compared with littermate controls (146 ± 2 dpi) (<xref ref-type="fig" rid="fig4">Figure 4</xref> and <xref ref-type="table" rid="tbl1">Table 1</xref>). Clinical manifestations (starting with a neglect of nest formation and fur cleaning, followed by presentation of a stiff tail and gait abnormalities, and terminating with kyphosis, weight loss, and hypoactivity) and disease duration (35 ± 4 days for A10 cKO mice and 40 ± 2.4 days for controls) were comparable between A10 cKO and littermate control mice (<xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="table" rid="tbl1">Table 1</xref>, <xref ref-type="other" rid="video1">Video 1</xref>). In line with other studies (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>; <xref ref-type="bibr" rid="bib87">Sandberg et al., 2011</xref>), <italic>tg</italic>a<italic>20</italic> mice showed fastest disease development and had to be sacrificed between 59 and 65 dpi (<xref ref-type="fig" rid="fig3">Figure 3</xref>) whereas mock-infected A10 cKO and control mice were taken out of the experiment at 200 dpi without any signs of prion disease (<xref ref-type="fig" rid="fig4">Figure 4</xref> and <xref ref-type="table" rid="tbl1">Table 1</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.008</object-id><label>Figure 4.</label><caption><title>Survival curves of mice upon intracerebral inoculation with RML prions.</title><p>Age-matched juvenile (6-week-old) A10 cKO mice (n = 7), littermate control mice (n = 8), and <italic>tg</italic>a<italic>20</italic> mice (n = 8) were intracerebrally inoculated with RML prions (+RML) and time until development of terminal prion disease was measured as days post inoculation (dpi; ***p = 7.6 × 10<sup>−7</sup>). As a negative control (mock), 5 A10 cKO mice and 11 littermate controls were intracerebrally inoculated with brain homogenate of CD1 mice and sacrificed without clinical signs at 200 dpi.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.008">http://dx.doi.org/10.7554/eLife.04260.008</ext-link></p></caption><graphic xlink:href="elife04260f004"/></fig><table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.009</object-id><label>Table 1.</label><caption><p>Clinical presentation of A10 cKO, littermate control and <italic>tg</italic>a<italic>20</italic> mice inoculated with RML prions</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.009">http://dx.doi.org/10.7554/eLife.04260.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th/><th colspan="3">RML prions</th><th colspan="2">CD1 mock</th></tr><tr><th>Clinical manifestations</th><th>A10 cKO (n = 7)</th><th>Controls (n = 8)</th><th><italic>tg</italic>a20 (n = 8)</th><th>A10 cKO (n = 5)</th><th>Controls (n = 11)</th></tr></thead><tbody><tr><td>First clinical signs (dpi)</td><td>68 (±3.5)</td><td>106 (±2.5)</td><td>n.a.</td><td>None</td><td>None</td></tr><tr><td>Duration of clinical signs (d)</td><td>35 (±3.8)</td><td>40 (±2.4)</td><td>n.a.</td><td>-</td><td>-</td></tr><tr><td>Time to terminal disease (dpi)</td><td>103 (±6.6)</td><td>146 (±2.1)</td><td>62 (±2.8)</td><td>*</td><td>*</td></tr><tr><td>Progression of signs</td><td>Steady</td><td>Steady</td><td>Rapid</td><td>–</td><td>–</td></tr><tr><td>Quitting of nest-building</td><td>All</td><td>All</td><td>All</td><td>–</td><td>–</td></tr><tr><td>Ungroomed coat</td><td>Most (6/7)</td><td>Most (6/8)</td><td>Rare (2/8)</td><td>–</td><td>–</td></tr><tr><td>Stiff tail</td><td>All</td><td>Most (7/8)</td><td>Most (5/8)</td><td>–</td><td>–</td></tr><tr><td>Gait disturbance</td><td>All</td><td>All</td><td>All</td><td>–</td><td>–</td></tr><tr><td>Hind leg paresis</td><td>Rare (2/7)</td><td>Rare (3/8)</td><td>Most (7/8)</td><td>–</td><td>–</td></tr><tr><td>Kyphosis</td><td>Most (6/7)</td><td>Most (6/8)</td><td>Rare (3/8)</td><td>–</td><td>–</td></tr><tr><td>Weight loss</td><td>All</td><td>All</td><td>Most (6/8)</td><td>–</td><td>–</td></tr><tr><td>Late hypoactivity</td><td>All</td><td>All</td><td>Most (5/8)</td><td>–</td><td>–</td></tr></tbody></table><table-wrap-foot><fn><p>Negative controls included A10 cKO and littermate controls inoculated with CD1 brain homogenates (mock). Asterisks indicate that CD1 mock-inoculated animals were sacrificed at 200 days post inoculation (dpi) without any clinical signs.</p></fn></table-wrap-foot></table-wrap></p><p>In summary, depletion of the PrP<sup>C</sup> sheddase ADAM10 in neurons of the forebrain led to a significant reduction in incubation times of more than 40 days until onset of terminal prion disease without affecting overall clinical presentation.</p></sec><sec id="s2-4"><title>Lack of ADAM10 does not influence PrP<sup>Sc</sup> loads and neuropathological features of prion disease at terminal disease state</title><p>We performed biochemical and neuropathological analyses of terminally prion-diseased A10 cKO and control mice in order to elucidate the reasons for reduced incubation times in the A10 cKO mice. In terminally diseased mice, comparable levels of PrP<sup>Sc</sup> were found in frontal brain homogenates of both genotypes as assessed by Western blot analysis after digestion of samples with PK (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). As expected, no PrP<sup>Sc</sup> was found in mock-inoculated controls of each genotype (<xref ref-type="fig" rid="fig5">Figure 5A</xref> on the right). Since alterations in incubation times may indicate generation of different prion strains (<xref ref-type="bibr" rid="bib11">Barron et al., 2003</xref>) with altered PrP<sup>Sc</sup> glycosylation pattern and altered sizes of core fragments of PrP<sup>Sc</sup>, we investigated whether lack of ADAM10-mediated shedding resulted in an altered PrP<sup>Sc</sup> banding pattern. To this end, we measured the relative proportion of di-, mono-, and unglycosylated forms of PrP<sup>Sc</sup> and sizes of PrP<sup>Sc</sup> core fragments. No differences in the PrP<sup>Sc</sup> glycopattern (<xref ref-type="fig" rid="fig5">Figure 5B</xref>) or sizes of PrP<sup>Sc</sup> core fragments (<xref ref-type="fig" rid="fig5">Figure 5A</xref>) between terminally diseased A10 cKO and control mice were detected, which argues against the generation of a modified prion strain.<fig-group><fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.010</object-id><label>Figure 5.</label><caption><title>Analysis of terminally prion-diseased A10 cKO mice and littermate controls.</title><p>(<bold>A</bold>) Detection of PrP<sup>Sc</sup> by Western blot analysis of proteinase K (PK)-digested (PK+) forebrain homogenates of prion-infected (RML+; blot on the left) and mock-inoculated (RML−; blot on the right) A10 cKO mice and controls at terminal stage of disease. Samples from three (RML+) or two (RML−) representative animals per genotype are shown. Digestion controls included prion-diseased/undigested (RML+/PK−), prion-negative/PK-digested (RML−/PK+), or prion-diseased/digested (RML+/PK+) brain homogenates. (<bold>B</bold>) Comparison of proportions of di-, mono, and unglycosylated PrP<sup>Sc</sup> forms between A10 cKO (n = 4) and control mice (n = 4; error bars indicate SEM). (<bold>C</bold>) Representative histological analysis in the forebrain of terminally prion-diseased (RML terminal) and healthy mock-inoculated (CD1 mock) A10 cKO and control mice including H&E staining and immunostaining of PrP<sup>Sc</sup>, GFAP (for detection of astrocytes), and Iba-1 (for detection of microglia). Scale bar in overviews: 200 µm; scale bar for inlays showing magnifications of representative areas: 100 µm. (<bold>D</bold>) Electron microscopy photographs showing intraneuronal vacuoles (asterisk in (a) and (b)) containing membranous structures (small arrowheads), enlarged and densely packed multivesicular bodies (arrows in (b)) as well as autophagic membranes (arrow in (c)) and autolysosomes that were found in terminally prion-infected forebrains of both genotypes (exemplified here for an A10 cKO brain). Scale bars represent 500 nm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.010">http://dx.doi.org/10.7554/eLife.04260.010</ext-link></p></caption><graphic xlink:href="elife04260f005"/></fig><fig id="fig5s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.011</object-id><label>Figure 5—figure supplement 1.</label><caption><title>Neuropathological features in the thalamus of ADAM10 cKO and control mice.</title><p>Representative histological analysis in the thalamic brain region of terminally prion-diseased ADAM10 cKO and control mice including H&E staining and immunohistochemical detection of PrP<sup>Sc</sup>, GFAP (for detection of astrocytes), and Iba-1 (for detection of microglia). Scale bar: 100 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.011">http://dx.doi.org/10.7554/eLife.04260.011</ext-link></p></caption><graphic xlink:href="elife04260fs002"/></fig><fig id="fig5s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.012</object-id><label>Figure 5—figure supplement 2.</label><caption><title>Electron microscopic analysis.</title><p>Overviews showing vacuolization (asterisks) in forebrain samples of ADAM10 cKO (<bold>A</bold>) and control mice (<bold>B</bold>) at a terminal stage of prion disease. High abundance of clusters containing tubulovesicular structures (TVS; arrows in (<bold>A</bold>) and (<bold>C</bold>)) was only found in terminal ADAM10 cKO brain (5 out of 13 square grids showed clusters of TVS whereas only 1 out of 15 square grids presented with TVS in terminal wild-type control mice). Scale bars represent 500 nm (<bold>A</bold>, <bold>B</bold>) or 200 nm (<bold>C</bold>). Inset in (<bold>C</bold>) shows magnification of TVS.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.012">http://dx.doi.org/10.7554/eLife.04260.012</ext-link></p></caption><graphic xlink:href="elife04260fs003"/></fig></fig-group></p><p>Prion infection caused spongiform lesions, massive astrocytosis, and microgliosis in both genotypes (<xref ref-type="fig" rid="fig5">Figure 5C</xref> on the left). Similar levels of PrP<sup>Sc</sup> immunopositivity were found at this terminal stage of prion disease (<xref ref-type="fig" rid="fig5">Figure 5C</xref>), thus confirming our biochemical data (<xref ref-type="fig" rid="fig5">Figure 5A</xref>). Comparable morphological findings were observed in the thalamic brain region (<xref ref-type="fig" rid="fig5s1">Figure 5—figure supplement 1</xref>).</p><p>Electron microscopic analysis revealed the presence of typical spongiform changes, dystrophic neurites, and abundant autophagy including enlarged multivesicular bodies and autolysosomes in both genotypes (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). Remarkably, prion-diseased A10 cKO mice presented with an extraordinarily high abundance of tubulovesicular structures (TVS) (<xref ref-type="fig" rid="fig5s2">Figure 5—figure supplement 2</xref>) (<xref ref-type="bibr" rid="bib45">Jeffrey and Fraser, 2000</xref>; <xref ref-type="bibr" rid="bib61">Liberski et al., 2010</xref>). These are spherical structures of 25–37 nm diameter that are specific for prion diseases though devoid of PrP (<xref ref-type="bibr" rid="bib45">Jeffrey and Fraser, 2000</xref>; <xref ref-type="bibr" rid="bib62">Liberski et al., 2008</xref>, <xref ref-type="bibr" rid="bib61">2010</xref>). Thus, our model may allow purification of these structures and could contribute to unraveling the nature and relevance of TVS in prion diseases.</p><p>Taken together, these data indicate that, at terminal prion disease, the lack of PrP<sup>C</sup> shedding does not influence local PrP<sup>Sc</sup> distribution while it does affect the appearance of TVS.</p></sec><sec id="s2-5"><title>Enhanced PrP<sup>Sc</sup> formation and calpain levels in A10 cKO mice</title><p>To elucidate the order of events underlying changed disease kinetics, we measured PrP<sup>Sc</sup> production in A10 cKO mice and littermate controls. Since in A10 cKO mice levels of PrP<sup>C</sup> are posttranslationally increased, we also investigated this issue in <italic>tg</italic>a<italic>20</italic> mice with genetically increased <italic>Prnp</italic> expression. To this aim, we performed Western blot analysis of total PrP (i.e., PrP<sup>C</sup> plus PrP<sup>Sc</sup>) and PK-resistant PrP<sup>Sc</sup> in A10 cKO and littermate control mice matched for days post prion inoculation (95 dpi) as well as in terminally prion-diseased <italic>tg</italic>a<italic>20</italic> mice (65 dpi). Relative levels of total PrP were highest in <italic>tg</italic>a<italic>20</italic> mice (5.0 ± 0.3; SEM), with increased levels in A10 cKO mice (2.8 ± 0.4) compared with controls (set to 1 ± 0.1) (A10 cKO vs control: **p = 0.0022; <italic>tg</italic>a<italic>20</italic> vs control: ***p = 0.0005; A10 cKO vs <italic>tg</italic>a<italic>20</italic>: **p = 0.0015) (<xref ref-type="fig" rid="fig6">Figure 6A</xref>). Surprisingly, relative levels of PrP<sup>Sc</sup> were significantly elevated in A10 cKO mice (3.9 ± 0.8), with moderate levels in littermate controls (set to 1 ± 0.2) and reduced levels in <italic>tg</italic>a<italic>20</italic> mice (0.6 ± 0.3) (A10 cKO vs control: *p = 0.049; A10 cKO vs <italic>tg</italic>a<italic>20</italic>: *p = 0.032). There were no atypical PrP patterns as assessed by PK digestion at 4°C (‘cold PK’) or with lower dilutions of PK (<xref ref-type="fig" rid="fig6s1">Figure 6—figure supplement 1</xref>). In summary, A10 cKO mice showed significantly increased PrP<sup>Sc</sup> formation compared with littermate controls and <italic>tg</italic>a<italic>20</italic> mice, which cannot solely be explained by increased neuronal PrP<sup>C</sup> levels.<fig-group><fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.013</object-id><label>Figure 6.</label><caption><title>PrP<sup>Sc</sup> formation, neuropathology, toxic signaling, and calpain levels at a matched time point.</title><p>(<bold>A</bold>) Assessment of total PrP (no proteinase K (PK)) and PrP<sup>Sc</sup> amounts (+PK; blot is shown with short and longer exposition) by parallel replica Western blot analysis in forebrain homogenates of age-matched A10 cKO mice and littermate controls (both at 95 days post inoculation (dpi); n = 3 for each genotype) as well as terminally diseased <italic>tg</italic>a<italic>20</italic> mice (at 65 dpi; n = 3). Actin was detected in the undigested homogenates (no PK) and served as loading control. Densitometric quantification of relative protein amounts from two technical replicates is shown on the right. (<bold>B</bold>) Morphological analysis of neuropathological lesions in forebrains (showing hippocampal and cortical brain regions) of A10 cKO, littermate controls, and <italic>tg</italic>a<italic>20</italic> mice at the aforementioned time points (scale bars: 200 µm in overviews and 100 µm in insets and for PrP<sup>Sc</sup>). (<bold>C</bold>) Biochemical assessment of candidate toxic signaling pathways showing protein levels of total Fyn (t-Fyn), phosphorylated (Tyr416) Src (p-Src) as well as total (t-Erk1/2) and phosporylated (Thr202/Tyr204) Erk1/2 (p-Erk1/2). Actin served as a loading control and for normalization (# and § indicate use of the same actins as corresponding signaling proteins were detected on the same Western blot). Quantitative densitometric analysis of relative p-Src/t-Fyn (left) and p-Erk/t-Erk ratio (right) (n.s. = not significant). (<bold>D</bold>) Representative Western blot analysis (left) and quantification of three technical replicates (right) of calpain levels in aforementioned samples. Levels of ADAM10 are shown in (<bold>C</bold>) and (<bold>D</bold>) to confirm the ADAM10 status. Error bars indicate SEM; *p <0.05; **p <0.01, ***p <0.001 (p values of Student's t-test are given in the main text).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.013">http://dx.doi.org/10.7554/eLife.04260.013</ext-link></p></caption><graphic xlink:href="elife04260f006"/></fig><fig id="fig6s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.014</object-id><label>Figure 6—figure supplement 1.</label><caption><title>‘Cold proteinase K (PK)’ and partial PK digestion of forebrain samples from ADAM10 cKO, control and <italic>tg</italic>a<italic>20</italic> mice at 95 days post inoculation (dpi).</title><p>(<bold>A</bold>) Western blot analysis showing ‘cold PK’ digestions of forebrain homogenates from two animals per genotype with 200 µg/ml PK at 4°C for 1 hr compared with classical digestion with 20 µg/ml PK at 37°C. No atypical digestion pattern is detected. Antibody POM1 was used for detection. (<bold>B</bold>) Representative Western blot analysis of frontal brain homogenates incubated with decreasing amounts of PK or without PK. Blot was first incubated with 1E4 antibody and then re-probed with POM1. Apart from differences in total PrP and PrP<sup>Sc</sup> amounts between the genotypes described in the main <xref ref-type="fig" rid="fig6">Figure 6</xref>, neither atypical bands nor low abundance or small PrP digestion fragments (∼10 kDa) are detected.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.014">http://dx.doi.org/10.7554/eLife.04260.014</ext-link></p></caption><graphic xlink:href="elife04260fs004"/></fig><fig id="fig6s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.015</object-id><label>Figure 6—figure supplement 2.</label><caption><title>Calpain levels and calpain substrates in forebrain homogenates of ADAM10 cKO and control mice.</title><p>(<bold>A</bold>) Representative Western blot analysis of calpain expression in non-prion infected ADAM10 cKO and control mice. In addition, premature (pADAM10), mature ADAM10 (mADAM10) and PrP<sup>C</sup> were detected. Quantification of calpain levels by densitometric analysis of ADAM10 cKO (n = 4) and control mice (n = 5) is shown below. Actin served as loading control. (<bold>B</bold>) Western blot analysis of spectrin (FL = full length) and spectrin breakdown products (SBDP) in prion-infected ADAM10 cKO and control mice at 95 days post inoculation (dpi; n = 3 per genotype). FL spectrin was reduced in ADAM10 cKO mice whereas calpain-dependent SBDP (marked by asterisk at 150/145 kDa) were not increased. (<bold>C</bold>) Western blot analysis of p35 and p25 levels in prion-infected samples mentioned in (<bold>B</bold>). A reduction in p35 levels was found in two out of three ADAM10 cKO mice. A band corresponding to p25 was not detectable. # indicates that blots used for calpain detection (<xref ref-type="fig" rid="fig6">Figure 6D</xref>) were re-probed with an antibody against p35/p25 and thus the same actin signals were used as loading controls.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.015">http://dx.doi.org/10.7554/eLife.04260.015</ext-link></p></caption><graphic xlink:href="elife04260fs005"/></fig></fig-group></p><p>Neuropathological changes in the forebrain, including spongiosis, astrocytosis and microgliosis, were more prominent in A10 cKO mice (<xref ref-type="fig" rid="fig6">Figure 6B</xref>). Remarkably, severe differences could be observed for PrP<sup>Sc</sup> immunopositivity, with A10 cKO mice showing prominent PrP<sup>Sc</sup> deposits in cortical and hippocampal regions while controls and <italic>tg</italic>a<italic>20</italic> mice showed only moderate levels, thus confirming the biochemical data (<xref ref-type="fig" rid="fig6">Figure 6A</xref>).</p><p>Next, we addressed the question whether elevated (membrane) levels of PrP<sup>C</sup> in A10 cKO and <italic>tg</italic>a<italic>20</italic> mice caused an increase in PrP<sup>C</sup>-mediated neurotoxic signaling events. Therefore, we biochemically assessed two candidate signaling pathways, via the Src kinase Fyn and the MAP kinases Erk1/2, that have previously been reported to be activated upon binding of toxic oligomers to PrP<sup>C</sup> (<xref ref-type="bibr" rid="bib71">Mouillet-Richard et al., 2000</xref>; <xref ref-type="bibr" rid="bib88">Schneider et al., 2003</xref>; <xref ref-type="bibr" rid="bib53">Larson et al., 2012</xref>; <xref ref-type="bibr" rid="bib99">Um et al., 2012</xref>; <xref ref-type="bibr" rid="bib80">Pradines et al., 2013</xref>). However, we did not observe any significant differences in the activation state of these kinases in forebrain homogenates between <italic>tg</italic>a<italic>20</italic>, A10 cKO, and littermate control mice at the chosen time point (<xref ref-type="fig" rid="fig6">Figure 6C</xref>).</p><p>A10 cKO mice showed significantly increased PrP<sup>Sc</sup> production (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>) compared with controls (at 95 dpi) and <italic>tg</italic>a<italic>20</italic> mice (at 65 dpi). Since PrP<sup>Sc</sup>-associated neurotoxicity and cell death have been linked to formation of membrane pores by binding and clustering of PrP<sup>Sc</sup> to PrP<sup>C</sup> at the plasma membrane resulting in calpain upregulation (<xref ref-type="bibr" rid="bib32">Falsig et al., 2012</xref>; <xref ref-type="bibr" rid="bib93">Sonati et al., 2013</xref>), we investigated this in our mice. Specifically, we biochemically analyzed calpain expression in the frontal brain of all experimental groups at the aforementioned time points. Interestingly, we found significant upregulation of calpain expression in A10 cKO mice (1.78 ± 0.24; SEM) compared with littermate controls (set to 1 ± 0.07; *p = 0.034) and <italic>tg</italic>a<italic>20</italic> mice (0.93 ± 0.14; *p = 0.022) (<xref ref-type="fig" rid="fig6">Figure 6D</xref>), thus correlating with PrP<sup>Sc</sup> levels (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>). This seemed to be specific for prion disease as no differences were detected in non-prion infected A10 cKO mice (mean: 0.90 ± 0.26 SEM; n = 4) when compared with age-matched wild-type controls (set to 1 ± 0.19; n = 5; p = 0.7) (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2A</xref>). In prion disease we observed a reduction of the described calpain substrates spectrin (<xref ref-type="bibr" rid="bib32">Falsig et al., 2012</xref>) and neuron-specific activator p35 (<xref ref-type="bibr" rid="bib58">Lee et al., 2000</xref>), suggesting that increased expression in A10 cKO mice correlated with the activity of calpain (<xref ref-type="fig" rid="fig6s2">Figure 6—figure supplement 2B,C</xref>). However, it should be noted that we failed to detect differences in specific cleavage products of spectrin and p35.</p><p>Our data suggest that lack of ADAM10-mediated PrP<sup>C</sup> shedding leads to increased production of PrP<sup>Sc</sup> and upregulation of calpain expression, whereas activation of proposed toxic PrP<sup>C</sup>-dependent signaling pathways was not observed.</p></sec><sec id="s2-6"><title>Similar titers of prion infectivity in brain samples of A10 cKO and control mice at terminal and preclinical phases of prion disease</title><p>We next investigated whether reduced incubation times and increased PrP<sup>Sc</sup> conversion rates in A10 cKO mice were accompanied by elevated prion infectivity titers and therefore performed bioassays (<xref ref-type="fig" rid="fig7">Figure 7</xref>). We intracerebrally inoculated forebrain homogenates of A10 cKO and littermate control mice at terminal (∼103 dpi for A10 cKO and ∼146 dpi for controls; see <xref ref-type="table" rid="tbl1">Table 1</xref>) and preclinical (60 dpi, 35 dpi) prion disease into <italic>tg</italic>a<italic>20</italic> (n = 4 for each sample) reporter mice. In addition, we assessed the 95 dpi time point (n = 6 <italic>tg</italic>a<italic>20</italic> mice per sample) at which A10 cKO mice were clinically affected while controls were still asymptomatic and where we observed significant differences in PrP<sup>Sc</sup> loads (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>). Titers of infectious prions increased with progression of disease, showed only some inter-individual variation, and reached a plateau phase towards the terminal stage of disease. However, they were independent of ADAM10 expression since we did not find differences between the two genotypes. At early time points (35 dpi) prion titers were low (mean: A10 cKO: 3.9 ± 0.2 logLD50; control: 4.0 ± 0.2), intermediate titers were seen at 60 dpi (A10 cKO: 5.0 ± 0.5; control: 4.9 ± 0.8), and highest titers were observed in terminally ill mice (A10 cKO: 5.8 ± 0.2; control: 6.0 ± 0.2) (<xref ref-type="fig" rid="fig7">Figure 7</xref>). Interestingly, significantly increased PrP<sup>Sc</sup> amounts in A10 cKO mice at 95 dpi (<xref ref-type="fig" rid="fig6">Figure 6A,B</xref>) did not result in elevated prion infectivity titers (A10 cKO: 5.74 ± 0.05; control: 5.66 ± 0.08; p = 0.2).<fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.016</object-id><label>Figure 7.</label><caption><title>Titers of prion infectivity in terminally diseased and preclinical A10 cKO mice and controls.</title><p>Prion titers in forebrain homogenates of A10 cKO mice and littermate controls at terminal or matched (preclinical) time points (95 days post inoculation [dpi], 60 dpi, and 35 dpi) after prion inoculation as assessed by bioassays in <italic>tg</italic>a<italic>20</italic> reporter mice. Each dot indicates the titer (shown as logLD50) assessed in a single reporter mice. Bars indicate mean values from six (for the 95 dpi time point) or four (all other time points) <italic>tg</italic>a<italic>20</italic> mice. Error bars indicate SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.016">http://dx.doi.org/10.7554/eLife.04260.016</ext-link></p></caption><graphic xlink:href="elife04260f007"/></fig></p></sec><sec id="s2-7"><title>ADAM10-mediated shedding might contribute to spreading of prion pathology</title><p>Since PrP<sup>Sc</sup> is shed from the plasma membrane in vitro (<xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>) and anchorless PrP<sup>C</sup> can convert into PrP<sup>Sc</sup> (<xref ref-type="bibr" rid="bib28">Chesebro et al., 2005</xref>, <xref ref-type="bibr" rid="bib27">2010</xref>; <xref ref-type="bibr" rid="bib94">Stöhr et al., 2011</xref>), we were interested to investigate if lack of PrP shedding affects the spread of prion pathology within the brain. Therefore, we performed a morphology-based spatiotemporal analysis in prion-infected A10 cKO and littermate control mice.</p><p>We assessed a brain region in direct proximity to the site of prion administration (striatum) and brain regions (cerebellum and brain stem) distant from the inoculation site and not directly affected by the depletion of ADAM10 (<xref ref-type="fig" rid="fig2">Figure 2</xref>; see <xref ref-type="fig" rid="fig3">Figure 3</xref> for overview) (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). Whereas for striatum we observed neuropathological changes typically found in terminal prion disease irrespective of the ADAM10 status, we found an apparently reduced degree of spongiosis in the cerebellum and brain stem of A10 cKO mice compared with terminally diseased littermate controls (<xref ref-type="fig" rid="fig8">Figure 8A</xref> and <xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>). In line with this, other prion-associated pathological hallmarks, including PrP<sup>Sc</sup> immunopositivity, astrocytosis involving Bergmann glia, and microglia activation, were observed in the cerebellum of controls, but were virtually lacking in this brain region in A10 cKO mice (<xref ref-type="fig" rid="fig8">Figure 8A</xref>).<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.017</object-id><label>Figure 8.</label><caption><title>Spatiotemporal analysis of prion-associated pathology.</title><p>(<bold>A</bold>) Comparison of neuropathological features including PrP<sup>Sc</sup> deposition, spongiotic changes (presented in H&E stainings), astrocyte (GFAP) and microglia (Iba-1) activation in striatum (as a site in close proximity to prion inoculation) and cerebellum (resembling a brain area distant to prion inoculation) in A10 cKO and littermate control mice at a terminal stage of prion disease (i.e., ∼103 days post inoculation [dpi] for A10 cKO and ∼146 dpi for controls). While similar pathological alterations were observed in the striatum of both genotypes, prion-associated lesions in the cerebellum were almost absent in A10 cKO mice. (<bold>B</bold>) At a matched time point (95 dpi for A10 cKO and littermate controls; 65 dpi [i.e., terminal disease] for <italic>tg</italic>a<italic>20</italic>), prion-related pathology was found in the striatum of all genotypes analyzed. As described earlier for the cortex and hippocampus (<xref ref-type="fig" rid="fig6">Figure 6</xref>), differences in PrP<sup>Sc</sup> amounts could also be observed in the striatum. In the cerebellum of both time-matched A10 cKO mice and littermate controls, prion-associated lesions were largely absent whereas <italic>tg</italic>a<italic>20</italic> mice showed all relevant neuropathological features already at 65 dpi. Representative pictures for at least three animals per genotype and time point are shown. Scale bars represent 200 µm (overviews) or 100 µm (insets).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.017">http://dx.doi.org/10.7554/eLife.04260.017</ext-link></p></caption><graphic xlink:href="elife04260f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.04260.018</object-id><label>Figure 8—figure supplement 1.</label><caption><title>Spongiosis in brain stem and cerebellum of ADAM10 cKO and control mice.</title><p>Representative histological analysis of spongiotic vacuolization as revealed by H&E staining of the cerebellum and brain stem of terminally prion-diseased control and ADAM10 cKO mice. Upper row shows overview and lower two rows show higher magnifications of cerebellum and brain stem (scale bars represent 100 µm).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.018">http://dx.doi.org/10.7554/eLife.04260.018</ext-link></p></caption><graphic xlink:href="elife04260fs006"/></fig></fig-group></p><p>Reduced disease duration in A10 cKO mice leading to diminished dissemination of prion-associated pathology may represent a possible explanation for these findings. In order to control for this, we assessed neuropathological alterations at a matched time point (95 dpi) in both A10 cKO mice and littermate controls. Additionally, we included terminally prion-diseased <italic>tg</italic>a<italic>20</italic> mice (65 dpi). In the striatum, typical neuropathological changes were seen in all groups of mice. Similar to hippocampus and cortex (<xref ref-type="fig" rid="fig6">Figure 6B</xref>), increased PrP<sup>Sc</sup> immunopositivity and astrogliosis were seen in A10 cKO mice (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). In line with our biochemical findings (<xref ref-type="fig" rid="fig6">Figure 6A</xref>), <italic>tg</italic>a<italic>20</italic> mice showed lowest PrP<sup>Sc</sup> amounts in the forebrain. Interestingly, in the cerebellum and brain stem, relevant prion-typical neuropathological changes were observed in <italic>tg</italic>a<italic>20</italic> mice at 65 dpi but were almost absent in both A10 cKO and littermate controls at 95 dpi (<xref ref-type="fig" rid="fig8">Figure 8B</xref>). This argues against a temporal cause for the observed differences and rather supports an accelerating role of shedding in the spread of prion disease within the CNS. Since <italic>tg</italic>a<italic>20</italic> mice show enhanced rates of PrP<sup>C</sup> shedding (<xref ref-type="fig" rid="fig1">Figure 1E</xref>) (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>), our spatiotemporal data may indicate a correlation between PrP<sup>C</sup> shedding and the efficiency of prion spread within the brain, with <italic>tg</italic>a<italic>20</italic> mice having the highest efficiency whereas A10 cKO mice show the least efficient dissemination of prion pathology (see model in <xref ref-type="fig" rid="fig9">Figure 9</xref>).<fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.04260.019</object-id><label>Figure 9.</label><caption><title>Model of the dual role of ADAM10-mediated shedding in prion disease.</title><p>ADAM10 regulates PrP<sup>C</sup> levels at the plasma membrane and releases almost full length PrP into the extracellular space. Thereby it affects (i) neurotoxicity, (ii) PrP<sup>Sc</sup> formation, and (iii) spreading of prion pathology. (i) Lack of ADAM10 (as assessed here by use of A10 cKO mice) results in elevated PrP<sup>C</sup> membrane levels. Membrane levels of PrP<sup>C</sup> (as a receptor) likely determine PrP<sup>Sc</sup>-associated neurotoxicity (as indicated by sizes of thunderbolts and skulls) and thereby incubation times with shortest survival in <italic>tg</italic>a<italic>20</italic> mice and reduced incubation times in A10 cKO mice compared with wild-type littermates with longest survival (order reflected by grey triangles on the left). (ii) Shed PrP, which is most efficiently produced in <italic>tg</italic>a<italic>20</italic> and absent in A10 cKO mice, might block formation of PrP<sup>Sc</sup>. This is reflected by the different PrP<sup>Sc</sup> amounts found in our different experimental groups (A10 cKO > wild-type littermates > <italic>tg</italic>a<italic>20</italic>). The combination of increased PrP<sup>C</sup> membrane levels and PrP<sup>Sc</sup> formation in A10 cKO mice might favor increased production of membrane pores (as indicated in the middle row on the left) and neurotoxic Ca<sup>2+</sup> influx with possible (‘?’) involvement of calpain. (iii) Finally, spread of prion-associated pathology within the brain also seems to be affected by the levels of ADAM10 expression since <italic>tg</italic>a<italic>20</italic> mice showed enhanced whereas A10 cKO mice showed reduced dissemination of neuropathological features (as indicated by size of arrowheads on the right). Key references supporting this model are given in the text. Based on this model, stimulation of ADAM10 might therefore offer a treatment option. With regard to incubation times, protective effects by reducing local membrane bound PrP<sup>C</sup> amounts and by producing a protective soluble fragment able to block PrP<sup>Sc</sup> formation seem to predominate the disadvantage of increased spread by production of anchorless prions.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.04260.019">http://dx.doi.org/10.7554/eLife.04260.019</ext-link></p></caption><graphic xlink:href="elife04260f009"/></fig></p></sec></sec><sec sec-type="discussion" id="s3"><title>Discussion</title><p>Proteolytic processing of the prion protein is critically involved in controlling its physiological functions. For α-cleavage, a protective role for both prion and AD is postulated and attributed to destruction of the neurotoxic domain and production of the neuroprotective N1 fragment respectively (<xref ref-type="bibr" rid="bib40">Guillot-Sestier et al., 2009</xref>; <xref ref-type="bibr" rid="bib85">Resenberger et al., 2011</xref>; <xref ref-type="bibr" rid="bib103">Westergard et al., 2011</xref>; <xref ref-type="bibr" rid="bib13">Beland et al., 2012</xref>; <xref ref-type="bibr" rid="bib41">Guillot-Sestier et al., 2012</xref>; <xref ref-type="bibr" rid="bib34">Fluharty et al., 2013</xref>). Moreover, the C1 fragment of PrP<sup>C</sup> is involved in myelination (<xref ref-type="bibr" rid="bib18">Bremer et al., 2010</xref>). The protective role of α-cleavage is also supported by the fact that α-cleavage-resistant PrP<sup>C</sup> deletion mutants are intrinsically neurotoxic upon expression in mice (<xref ref-type="bibr" rid="bib91">Shmerling et al., 1998</xref>; <xref ref-type="bibr" rid="bib12">Baumann et al., 2007</xref>) (reviewed in <xref ref-type="bibr" rid="bib107">Yusa et al., 2012</xref>). In contrast, our knowledge of the consequences of PrP<sup>C</sup> shedding, though being evolutionary conserved and constitutively occurring under physiological conditions, is limited. In a previous study we have shown that ADAM10 represents the principal PrP<sup>C</sup> sheddase in vivo with lack of ADAM10 resulting in a posttranslational increase of PrP<sup>C</sup> levels (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). This pointed to a key role for this protease in the regulation of PrP<sup>C</sup> levels at the neuronal surface, yet its impact on prion diseases remained unknown. We recently generated A10 cKO mice, a viable model with selective postnatal ablation of ADAM10 in neurons of the forebrain (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). In the present study we demonstrate that prion-inoculated A10 cKO mice show considerably reduced incubation times and increased conversion of PrP<sup>C</sup> to PrP<sup>Sc</sup>. In contrast, spread of prion pathology within the brain is reduced, thus indicating a dual role for ADAM10 in the pathophysiology of prion disease (<xref ref-type="fig" rid="fig9">Figure 9</xref>).</p><p>ADAM10 acts as a sheddase for a number of neuronally expressed proteins with important functions in the development and homeostasis of the brain (<xref ref-type="bibr" rid="bib105">Yang et al., 2006</xref>; <xref ref-type="bibr" rid="bib101">Weber and Saftig, 2012</xref>). For PrP<sup>C</sup> it is now widely accepted that ADAM10 is the PrP<sup>C</sup> sheddase (<xref ref-type="bibr" rid="bib76">Parkin et al., 2004</xref>; <xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>; <xref ref-type="bibr" rid="bib104">Wik et al., 2012</xref>; <xref ref-type="bibr" rid="bib74">Ostapchenko et al., 2013</xref>; <xref ref-type="bibr" rid="bib69">McDonald et al., 2014</xref>). Here we extend our previous findings on increased levels of PrP<sup>C</sup> in the absence of ADAM10 (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>) in different cellular models and observe colocalization between PrP<sup>C</sup> and ADAM10 at the plasma membrane, which is regarded as the primary site of ADAM10-mediated shedding events (<xref ref-type="bibr" rid="bib52">Lammich et al., 1999</xref>; <xref ref-type="bibr" rid="bib25">Chen et al., 2014</xref>). In addition to accumulation of PrP<sup>C</sup> in the early secretory pathway (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>), we here show that lack of ADAM10 also results in elevated membrane levels of PrP<sup>C</sup>. This supports a model where ADAM10-mediated shedding controls PrP<sup>C</sup> membrane homeostasis (<xref ref-type="bibr" rid="bib6">Altmeppen et al., 2013</xref>).</p><p>However, cell culture models as well as mice with pan-neuronal depletion of ADAM10 did not allow investigation of the role of PrP shedding in prion diseases (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). Therefore, we used A10 cKO mice (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>) in which we observed increased PrP<sup>C</sup> levels that were not caused by elevated PrP<sup>C</sup> mRNA levels.</p><p>Onset of terminal prion disease in mice upon intracerebral inoculation with defined prion strains occurs after defined incubation times. Once clinical symptoms appear, individual mice reliably succumb to disease within days. In contrast to peripheral prion inoculation where the integrity of the lymphoreticular or autonomous nervous system plays a decisive role in neuroinvasion of prions, incubation time after intracerebral inoculation is mainly influenced by PrP<sup>C</sup> levels (<xref ref-type="bibr" rid="bib64">Mabbott et al., 2000</xref>; <xref ref-type="bibr" rid="bib36">Glatzel et al., 2001</xref>; <xref ref-type="bibr" rid="bib82">Prinz et al., 2003</xref>). This is nicely exemplified by <italic>tg</italic>a<italic>20</italic> mice constitutively overexpressing PrP<sup>C</sup> (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>; <xref ref-type="bibr" rid="bib87">Sandberg et al., 2011</xref>). Our intracerebrally inoculated A10 cKO mice developed prion disease >40 days earlier than controls, corresponding to a shortening of incubation times of ∼30%. Such dramatic reductions upon intracerebral inoculation with high-dose RML prions have so far only been achieved when PrP<sup>C</sup> is massively overexpressed (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>). Less dramatic reductions were, for instance, described in mice lacking superoxide dismutase 1 (<xref ref-type="bibr" rid="bib4">Akhtar et al., 2013</xref>) or overexpressing the heat shock protein Hspa13 (<xref ref-type="bibr" rid="bib39">Grizenkova et al., 2012</xref>). The only other study investigating the role of ADAM10 in prion disease showed that transgenic overexpression of bovine ADAM10 in mice led to prolongation of prion disease (<xref ref-type="bibr" rid="bib31">Endres et al., 2009</xref>), a finding which is complementary to our data on the effect of ADAM10 deficiency. However, these authors linked this effect to transcriptional downregulation of PrP<sup>C</sup> and not to proteolytic processing events. In contrast, data from the present and previous studies show that transcriptional regulation of PrP<sup>C</sup> is not affected by ADAM10 whereas proteolytic processing clearly is (<xref ref-type="bibr" rid="bib76">Parkin et al., 2004</xref>; <xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>; <xref ref-type="bibr" rid="bib104">Wik et al., 2012</xref>; <xref ref-type="bibr" rid="bib74">Ostapchenko et al., 2013</xref>; <xref ref-type="bibr" rid="bib69">McDonald et al., 2014</xref>). We considered the possibility that non-PrP dependent effects resulting from ADAM10 deficiency may have contributed to disease acceleration. Indeed, ADAM10 is involved in a number of developmental and immunological processes and A10 cKO mice show learning deficits (<xref ref-type="bibr" rid="bib101">Weber and Saftig, 2012</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). It is virtually impossible to dissect out the individual contribution of the multitude of ADAM10 targets to the pathophysiology of prion disease. For instance, microglia contribute to prion pathophysiology and express CD40L, a known ADAM10 substrate. Prion-inoculated CD40L-deficient mice show decreased incubation times (<xref ref-type="bibr" rid="bib21">Burwinkel et al., 2004</xref>). As CD40L is not expressed on neurons and its receptor (CD40) is not processed by ADAM10, we do not expect that this dyad is altered by our neuron-specific knockout strategy (<xref ref-type="bibr" rid="bib89">Schönbeck and Libby, 2001</xref>; <xref ref-type="bibr" rid="bib21">Burwinkel et al., 2004</xref>). Another microglia-related mechanism relevant to prion disease and influenced by ADAM10 is the Cx3cl1/Cx3cr1 signaling complex. The receptor (Cx3cr1) is expressed on microglia whereas its ligand Cx3cl1 (Fractalkine) is an ADAM10 substrate expressed on neurons. Cx3cl1-deficient mice inoculated with prions show slightly decreased incubation times to disease (<xref ref-type="bibr" rid="bib38">Grizenkova et al., 2014</xref>). Therefore, we do not assume that these events explain the strong effect observed in our study. Most other immune system-mediated effects on prion pathogenesis are only relevant for peripheral prion inoculation and neuroinvasion and do not play major roles in intracerebral prion administration (<xref ref-type="bibr" rid="bib3">Aguzzi et al., 2003</xref>). Furthermore, A10 cKO mice do not show any gross morphological abnormalities in the CNS, have a normal life span once they have survived a critical weaning period, and mock inoculated mice behave normally (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). Finally, we consider that increased PrP<sup>Sc</sup> levels found in A10 cKO mice strongly argue in favor of PrP-dependent effects of ADAM10 depletion.</p><p>Analysis of A10 cKO mice and littermate controls at 95 dpi as well as <italic>tg</italic>a<italic>20</italic> mice at 65 dpi (which represents terminal disease for these mice) revealed a significant difference in PrP<sup>Sc</sup> formation between A10 cKO and the other two experimental groups. This cannot be explained by the increased presence of PrP<sup>C</sup>, since PrP<sup>C</sup> overexpressing <italic>tg</italic>a<italic>20</italic> mice showed low levels of PrP<sup>Sc</sup> even at the terminal stage of the disease. How can this be explained? The molecular basis underlying neurodegeneration in prion diseases is under debate and different mechanisms of neurotoxicity have been proposed. In the receptor model, PrP<sup>C</sup> binds oligomeric β-sheet rich protein species, such as PrP<sup>Sc</sup>, and mediates neurotoxic signaling (<xref ref-type="bibr" rid="bib71">Mouillet-Richard et al., 2000</xref>; <xref ref-type="bibr" rid="bib55">Lauren et al., 2009</xref>; <xref ref-type="bibr" rid="bib85">Resenberger et al., 2011</xref>; <xref ref-type="bibr" rid="bib53">Larson et al., 2012</xref>; <xref ref-type="bibr" rid="bib99">Um et al., 2012</xref>). In the pore formation model, PrP<sup>Sc</sup> oligomers, either directly or upon binding to PrP<sup>C</sup>, form a membrane channel leading to exaggerated Ca<sup>2+</sup> influx and calpain activation (<xref ref-type="bibr" rid="bib32">Falsig et al., 2012</xref>; <xref ref-type="bibr" rid="bib93">Sonati et al., 2013</xref>). In our study we found evidence for the link between excessive generation of PrP<sup>Sc</sup> and increased calpain levels in A10 cKO mice, whereas basal (in littermate controls) and low (in <italic>tg</italic>a<italic>20</italic> mice) PrP<sup>Sc</sup> levels were not associated with calpain upregulation.</p><p><italic>Tg</italic>a<italic>20</italic> mice are hypersensitive to prion disease yet do not show significant levels of PrP<sup>Sc</sup> even though PrP<sup>C</sup> is strongly overexpressed. In a previous study we have shown and re-evaluated here that ADAM10-mediated shedding of PrP<sup>C</sup> in neurons of <italic>tg</italic>a<italic>20</italic> mice is increased approximately threefold (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). Of note, soluble GPI-anchorless forms of PrP<sup>C</sup>, which may be regarded as correlates of shed PrP<sup>C</sup>, and experimentally-induced release of PrP<sup>C</sup> in vitro impair PrP<sup>Sc</sup> formation (<xref ref-type="bibr" rid="bib67">Marella et al., 2002</xref>; <xref ref-type="bibr" rid="bib70">Meier et al., 2003</xref>; <xref ref-type="bibr" rid="bib49">Kim et al., 2009</xref>; <xref ref-type="bibr" rid="bib106">Yuan et al., 2013</xref>). Thus, high levels of shed PrP<sup>C</sup> in <italic>tg</italic>a<italic>20</italic> mice may impair PrP<sup>Sc</sup> formation whereas lack of shed PrP<sup>C</sup> in A10 cKO mice may favor PrP<sup>Sc</sup> formation (<xref ref-type="fig" rid="fig9">Figure 9</xref>). In line with this concept, PrP<sup>Sc</sup> production was decreased in mice moderately overexpressing ADAM10 (<xref ref-type="bibr" rid="bib31">Endres et al., 2009</xref>). In both <italic>tg</italic>a<italic>20</italic> and A10 cKO mice, increased levels of PrP<sup>C</sup> at the plasma membrane should facilitate PrP<sup>C</sup>-mediated neurotoxicity and thereby determine incubation times. Interestingly, these data provide a potential explanation for the <italic>tg</italic>a<italic>20</italic> paradox of low PrP<sup>Sc</sup> formation despite high PrP<sup>C</sup> levels and short incubation times (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>).</p><p>A10 cKO mice and controls showed identical prion titers. On the other hand levels of PrP<sup>Sc</sup> were elevated and no atypical protease-sensitive forms of PrP<sup>Sc</sup> were found. It is becoming increasingly evident that titers of prion infectivity, PrP<sup>Sc</sup>, and the presence of potentially neurotoxic PrP conformers are not congruent (<xref ref-type="bibr" rid="bib54">Lasmezas et al., 1997</xref>; <xref ref-type="bibr" rid="bib10">Barron et al., 2007</xref>; <xref ref-type="bibr" rid="bib78">Piccardo et al., 2007</xref>; <xref ref-type="bibr" rid="bib51">Krasemann et al., 2013</xref>). In fact, the exact composition of infectious ‘prions’ (i.e., the entity determined by bioassay) is not fully understood and it may be that ADAM10-mediated shedding affects formation of PrP<sup>Sc</sup> while it does not influence production of ‘prions’.</p><p>We did not observe induction of proposed neurotoxic signaling pathways (<xref ref-type="bibr" rid="bib71">Mouillet-Richard et al., 2000</xref>; <xref ref-type="bibr" rid="bib88">Schneider et al., 2003</xref>; <xref ref-type="bibr" rid="bib53">Larson et al., 2012</xref>; <xref ref-type="bibr" rid="bib99">Um et al., 2012</xref>; <xref ref-type="bibr" rid="bib80">Pradines et al., 2013</xref>). It may be that these pathways occur spatially and temporally restricted and future studies will have to address this question using refined methods of analysis. Alternatively, other yet to be discovered pathways may be involved. In A10 cKO mice, the combination of elevated PrP<sup>C</sup> membrane levels and increased PrP<sup>Sc</sup> amounts may favor increased pore formation and Ca<sup>2+</sup> influx as indicated by elevated calpain levels. However, since we did not detect unequivocal evidence for calpain activation, further studies are required to substantiate this model.</p><p>In view of the fact that prion-infected mice expressing anchorless PrP<sup>C</sup> show PrP<sup>Sc</sup> formation and deposition in ectopic sites (<xref ref-type="bibr" rid="bib57">Lee et al., 2011</xref>) and prion propagation in the CNS (<xref ref-type="bibr" rid="bib28">Chesebro et al., 2005</xref>), we investigated whether shedding contributes to the spread of prion pathology. In fact, A10 cKO mice show prion-related pathology and PrP<sup>Sc</sup> deposition at the site of (striatum) and adjacent to prion administration (hippocampus, frontal cortex, thalamus), whereas brain regions distant from the inoculation site (cerebellum, brain stem) were unaffected, even at terminal stages of disease. In contrast, in <italic>tg</italic>a<italic>20</italic> mice, dissemination of prion-related pathology occurred efficiently at a much earlier time point. Non-PrP-dependent effects resulting from ADAM10 deficiency, such as altered microglial activity or reduced processing of proinflammatory molecules, may have contributed to impaired spreading (<xref ref-type="bibr" rid="bib102">Weber et al., 2013</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). However, the influence of the immune system, including microglial activation on intracerebral spread of prions, is limited, therefore a correlation between PrP shedding and the efficiency of prion spread is possible (<xref ref-type="bibr" rid="bib35">Glatzel et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Grizenkova et al., 2014</xref>). In line with our findings, it has been shown that, upon prion infection of mice, heterozygous expression of normal and anchorless PrP results in earlier death compared with wild-type mice (<xref ref-type="bibr" rid="bib28">Chesebro et al., 2005</xref>). Thus, shedding of either PrP<sup>C</sup> (followed by cell-free conversion [<xref ref-type="bibr" rid="bib50">Kocisko et al., 1994</xref>]) or PrP<sup>Sc</sup>, both of which have been shown to occur (<xref ref-type="bibr" rid="bib96">Taylor et al., 2009</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>), should be considered as an alternative mechanism of prion spread (<xref ref-type="fig" rid="fig9">Figure 9</xref>) aside from direct transsynaptic cell-to-cell transfer (<xref ref-type="bibr" rid="bib36">Glatzel et al., 2001</xref>; <xref ref-type="bibr" rid="bib90">Shearin and Bessen, 2014</xref>), exosomal (<xref ref-type="bibr" rid="bib5">Alais et al., 2008</xref>) and viral transfer (<xref ref-type="bibr" rid="bib56">Leblanc et al., 2006</xref>), or tunneling nanotubes (<xref ref-type="bibr" rid="bib37">Gousset et al., 2009</xref>). It remains to be investigated whether ADAM10-mediated shedding is also involved in the release of bona fide prions into body fluids such as cerebrospinal fluid, blood, or nasal secretions, which may potentially increase the risk of transmission (<xref ref-type="bibr" rid="bib95">Tagliavini et al., 1992</xref>; <xref ref-type="bibr" rid="bib77">Perini et al., 1996</xref>; <xref ref-type="bibr" rid="bib75">Parizek et al., 2001</xref>; <xref ref-type="bibr" rid="bib15">Bessen et al., 2010</xref>).</p><p>In conclusion, our data indicate that proteolytic processing, as described here for the shedding of the prion protein, represents a master switch in the pathophysiology of prion diseases by drastically affecting PrP<sup>Sc</sup> formation and incubation times. Interestingly, protective effects of ADAM10 might be hijacked in prion diseases since a reduction of the protease in prion disease has recently been reported in vitro and in vivo (<xref ref-type="bibr" rid="bib25">Chen et al., 2014</xref>). By contrast, shedding also seems to affect the spread of prion pathology within the CNS. Understanding this dual role of ADAM10 in prion disease brings together current concepts of prion biology and might reveal a mechanistic insight into important pathophysiological processes. Apart from prion disease, in other neurodegenerative proteinopathies, where PrP<sup>C</sup>-PrP<sup>Sc</sup> conversion does not play a role, ADAM10-mediated shedding of PrP<sup>C</sup> might solely be protective due to reduction of the receptor as well as the production of a soluble blocker of toxic oligomers. In view of a potential role of PrP<sup>C</sup> in AD, further research on proteolysis as a regulatory event in prion biology will likely impact on more common neurodegenerative conditions.</p></sec><sec sec-type="materials|methods" id="s4"><title>Materials and methods</title><sec id="s4-1"><title>Description of mice and ethics statement</title><p>In this study we took advantage of the recently generated <italic>Camk2a</italic>-Cre <italic>Adam10</italic> conditional knockout (A10 cKO) mouse model with a depletion of <italic>Adam10</italic> in neurons of the forebrain. Generation as well as a detailed description of these mice can be found elsewhere (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). Assessment of floxed alleles (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>) and the <italic>Camk2a</italic>-Cre status (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>) in transgenic mice was performed by PCR. Our breeding strategy of crossing <italic>Adam10</italic><sup>F/F</sup> with <italic>Camk2a</italic>-Cre:<italic>Adam10</italic><sup>F/+</sup> yielded approximately 15% viable Cre-positive <italic>Adam10</italic><sup>F/F</sup> offsprings (A10 cKO) (<xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). Cre-negative <italic>Adam10</italic><sup>F/F</sup> or <italic>Adam10</italic><sup>F/+</sup> littermates were used as wild-type controls. In addition, we used prion protein overexpressing <italic>Tg(Prnp)a20</italic> mice (herein referred to as <italic>tg</italic>a<italic>20</italic>) (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>) as controls for our biochemical and immunohistochemical analysis and as reporter mice in our bioassays for determination of prion infectivity titers as described below. Finally, brain sections of prion protein knockout (<italic>Prnp</italic><sup>0/0</sup>) mice (<xref ref-type="bibr" rid="bib20">Büeler et al., 1992</xref>) were used as an internal negative control for our immunohistochemical staining procedure.</p><p>Our study was carried out in accordance with the principles of laboratory animal care (NIH publication No. 86-23, revised 1985) as well as the recommendations in the Guide for the Care and Use of Laboratory Animals of the German Animal Welfare Act on protection of animals. The protocol was approved by the Committee on Ethics of the <italic>Freie und Hansestadt Hamburg—Amt für Gesundheit und Verbraucherschutz</italic> (permit number 48/09, 81/07 and 84/13).</p></sec><sec id="s4-2"><title>Prion inoculations</title><p>Inoculations of mice were performed under deep ketamine and xylazine hydrochloride anesthesia. All efforts were made to minimize suffering of the animals including careful observation and special treatment (i.e., administration of wet food and incubation on a warming plate) of mice immediately after inoculations until recovery. In brief, 6−9-week-old A10 cKO mice (n = 12), littermate controls (n = 17), and <italic>tg</italic>a<italic>20</italic> mice (n = 11) were inoculated with 30 µl of 1% homogenate of Rocky Mountain Laboratory (RML) prions (RML 5.0 inoculum, corresponding to 3 × 10<sup>5</sup> LD50) into the forebrain. Mice were checked on a weekly basis and observation was intensified to a two-day schedule following the appearance of first clinical signs. To assess disease characteristics in preclinical mice, A10 cKO, wild-type littermates, and <italic>tg</italic>a<italic>20</italic> mice (n = 3 for each genotype) were sacrificed at 35 dpi. Moreover, two A10 cKO mice and two wild-type control mice were taken at 60 days for determination of preclinical prion titers. For matched comparison with terminal A10 cKO mice, preclinical littermate controls (n = 4) were sacrificed at day 95 after inoculation. All other mice (A10 cKO: n = 7; controls: n = 8; <italic>tg</italic>a<italic>20</italic>: n = 8) were sacrificed and analyzed when they reached terminal prion disease (<xref ref-type="table" rid="tbl1">Table 1</xref>). Additionally, we performed mock inoculations with 30 µl of 1% brain homogenate from uninfected CD-1 mice into age-matched A10 cKO (n = 5) and littermate controls (n = 11). These animals were sacrificed at 200 dpi lacking any signs of prion disease (see <xref ref-type="table" rid="tbl1">Table 1</xref>).</p><p>For the assessment of titers of prion infectivity in our preclinical and terminally diseased A10 cKO and control mice, we performed bioassays in <italic>tg</italic>a<italic>20</italic> reporter mice. To this end, 20 µl of a 1% homogenate of frontal brain derived from an animal to be investigated were intracerebrally inoculated into four or six <italic>tg</italic>a<italic>20</italic> mice. Prion titers (<italic>y</italic>; shown as logLD50) in the original sample were then calculated according to the equation <italic>y</italic> = 11.45 − 0.088 × <italic>x</italic>, with <italic>x</italic> being the time to terminal disease of <italic>tg</italic>a<italic>20</italic> reporter mice (in dpi).</p></sec><sec id="s4-3"><title>Primary neurons, cell culture, and analysis of media supernatants</title><p>Generation of primary neurons of E14 embryos of <sup>Nestin</sup>A10 KO, wild-type C57BL/6, <italic>Prnp</italic><sup>0/0</sup> and <italic>tg</italic>a<italic>20</italic> mice as well as immunoprecipitation of shed PrP<sup>C</sup> from conditioned media was described (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>).</p><p>MEFs of A10 KO and control mice were initially described in (<xref ref-type="bibr" rid="bib43">Hartmann et al., 2002</xref>) and maintained under standard cell culture conditions. Conditioned media were collected after 18–24 hr and prepared for immunoprecipitation of PrP<sup>C</sup> (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). Alternatively, media supernatants were concentrated 10× using ultrafiltration spin columns (Vivaspin 500, Sartorius Stedim Biotech, Goettingen, Germany).</p></sec><sec id="s4-4"><title>Western blot analysis</title><p>Samples of frontal brain were processed as 10% (wt/vol) homogenates in RIPA buffer (50 mM Tris–HCl pH 8, 150 mM NaCl, 1% NP40, 0.5% Na-Deoxycholat, 0.1% SDS) supplemented with Complete Mini protease inhibitor cocktail (PI; Roche Diagnostics, Mannheim, Germany). Samples were smashed 30× on ice using a Dounce homogenizer and subsequently incubated on ice for 15 min before resuspending by 15× pipetting up and down. Homogenates were centrifuged at 12,000×<italic>g</italic> for 6 min at 4°C and total protein content in supernatants was determined by colorimetric analysis (QuickStart Bradford 1× Dye, Biorad, Hercules, CA) following the manufacturer's instructions. Samples were then normalized to yield equal protein amounts and boiled in 4× loading buffer (250 mM Tris–HCl, 8% SDS, 40% glycerol, 20% β-mercaptoethanol, 0.008% Bromophenol Blue, pH 6.8) for 6 min at 96°C. Gel electrophoresis was performed using 20–50 µg of total protein per lane and 8%, 10%, or 12% SDS-PAGE gels. Proteins were subsequently wet-blotted onto nitrocellulose membranes (Biorad) and membranes were blocked for 1 hr using 5% non-fat dry milk in TBS-T. Immunoblot analysis was performed using the following primary antibodies: rabbit anti-ADAM10 (1:1000; polyclonal antiserum, B42.1), mouse monoclonal antibodies against PrP<sup>C</sup> (POM1 [for most of the experiments] or POM2 [for detection of shed PrP (<xref ref-type="fig" rid="fig1">Figure 1E</xref>)] (<xref ref-type="bibr" rid="bib79">Polymenidou et al., 2008</xref>), 1:2500; A Aguzzi, Zurich, Switzerland), mouse monoclonal 1E4 (1:500; Sanquin, Amsterdam, The Netherlands), rabbit against p35/p25 (1:1000; mAB #2680; Cell Signaling), mouse Anti-Spectrin (1:500; MAB1622; Millipore, Bedford, MA), rabbit polyclonal antibody against calpain (H-240, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA), and mouse monoclonal antibody against β-actin (1:2000; Millipore) or rabbit anti-Actin (1:2000; A5060; Sigma). Incubation with primary antibodies (diluted in 5% non-fat dry milk) was carried out overnight at 4°C on a shaking platform. Membranes were then washed 3× for 5 min with TBS-T, incubated for 45 min at room temperature with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies (Promega, Madison, WI) diluted in 5% non-fat dry milk in TBS-T, and washed 6× for 5 min with TBS-T. The signal was detected after incubation of membranes with Pierce ECL or SuperSignal West Femto substrate (Thermo Scientific, Waltham, MA) using a CD camera imaging system (Biorad). If necessary, quantification of signal strengths was performed using QuantityOne software (Biorad) to measure the ratio of protein bands (glycotyping of PrP<sup>C</sup>) or to determine relative protein expression against β-actin.</p><p>For assessment of PrP<sup>Sc</sup> levels and for use in inoculation experiments (Bioassay), 20% (wt/vol) homogenates of frontal brain were prepared in sterile phosphate buffer saline (PBS) without protease inhibitors. Again, samples were smashed 30× on ice using a Dounce homogenizer and subsequently spun down at 1000 rpm for 2 min. The resulting supernatant was either further diluted in PBS to yield a 1% homogenate used for inoculation of <italic>tg</italic>a<italic>20</italic> reporter mice (see above) or 2–5 µl were digested with 20 µg/ml PK (Roche) in a total volume of 22 µl RIPA buffer for 1 hr at 37°C to assess the PrP<sup>Sc</sup> content. For detection of atypical prion fragments, selected samples were digested with 1 or 10 µg/ml or without PK for 1 hr at 37°C. ‘Cold PK’ treatment was performed with 200 µg/ml PK for 1 hr at 4°C. Digestion was stopped by adding 10× loading buffer and boiling for 6 min at 96°C. Subsequent SDS-PAGE and Western blot analysis was performed as described above with the exception that commercial Mini-PROTEAN TGX Any kD gels (Biorad) were used.</p></sec><sec id="s4-5"><title>Histological and immunohistochemical analysis</title><p>Morphological analysis was performed as described previously (<xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>; <xref ref-type="bibr" rid="bib83">Prox et al., 2013</xref>). In brief, brains were dissected and fixed by immersion in 4% buffered formalin overnight. In the case of prion- or mock-inoculated animals, samples were inactivated for 1 hr in 98–100% formic acid before export from the facility. These samples were then incubated overnight with an excess of 4% buffered formalin. Samples were dehydrated and embedded in low melting point paraffin following standard laboratory procedures. Sections of 4 μm were prepared and either stained with hematoxylin and eosin (HE) following standard laboratory procedures or submitted to immunostaining following standard immunohistochemistry procedures using the Ventana Benchmark XT machine (Ventana, Tucson, AZ). Briefly, deparaffinated sections were boiled for 30–60 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. All solutions were from Ventana. Sections were incubated with primary antibody in 5% goat serum (Dianova, Hamburg, Germany), 45% Tris buffered saline (TBS) pH 7.6, 0.1% Triton X-100 in antibody diluent solution (Zytomed, Berlin, Germany) for 1 hr. The following primary antibodies were used: monoclonal POM1 (<xref ref-type="bibr" rid="bib79">Polymenidou et al., 2008</xref>) (1:100; Prof Dr Aguzzi, Zürich, Switzerland), SAF84 antibody against PrP<sup>C</sup> and PrP<sup>Sc</sup> (1:100; Cayman Chemicals, Ann Arbor, MI), anti-GFAP (1:200; M0761, DAKO, Hamburg, Germany), anti-Iba-1 (1:500; 019-19741, Wako Chemicals, Neuss, Germany), anti-NeuN (1:50; MAB377, Millipore). Detection was with anti-rabbit or anti-goat histofine Simple Stain MAX PO Universal immunoperoxidase polymer or Mouse Stain Kit (for detection of mouse antibodies on mouse sections). All secondary antibody polymers were purchased from Nichirei Biosciences (Tokyo, Japan). Detection of antibodies was performed with Ultra View Universal DAB Detection Kit or Ultra View Universal Alkaline Phosphatase Red Detection Kit from Ventana according to standard settings of the machine. Experimental groups were stained in one run, thereby providing identical conditions. The counterstaining was also performed by the machine according to common protocols. Additional negative controls included sections treated with secondary antibody only. For PrP<sup>Sc</sup> detection, mounted tissue sections (4 µm) were pretreated with 98% formic acid for 5 min. Further processing was performed on an automated staining machine (Benchmark XT, Ventana). Briefly, sections were pretreated with 1.1 mM sodium citrate buffer (2.1 mM Tris–HCl, 1.3 mM EDTA, pH 7.8) at 95°C for 30 min, digested with low concentration of PK for 16 min, incubated in Superblock for 10 min and then incubated with the PrP-specific antibody SAF 84 (see above), followed by secondary antibody treatment and detection.</p></sec><sec id="s4-6"><title>Electron microscopy</title><p>Small pieces of forebrain (2–3 mm<sup>3</sup>) of terminally prion-diseased A10 cKO mice and littermate controls were fixed in glutaraldehyde, postfixed in osmium tetroxide for 1−2 hr, dehydrated through a series of graded ethanols and propylene oxide, and embedded in Epon. Semi-thin sections were stained with toluidine blue. Ultrathin sections were stained with lead citrate and uranyl acetate, and specimens were examined using a JEM 100 C transmission electron microscope (JEOL, Tokyo, Japan). For the assessment of TVS, sample grids were divided into grid squares of equal size and the number of grid squares presenting TVS clusters was determined as published previously (<xref ref-type="bibr" rid="bib32">Falsig et al., 2012</xref>).</p></sec><sec id="s4-7"><title>Isolation, cultivation, modification, and in vitro differentiation of NSCs</title><p>Adherently growing NSC cultures were established from the ganglionic eminence of 14-day-old wild-type and <sup>Nestin</sup>A10 KO mice as described elsewhere (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). In brief, we first established neurosphere cultures according to standard protocols (<xref ref-type="bibr" rid="bib1">Ader et al., 2001</xref>; <xref ref-type="bibr" rid="bib81">Pressmar et al., 2001</xref>). After two passages, neurospheres were enzymatically dissociated using Accutase (PAA Laboratories, Pasching, Austria) and cells were further cultivated in tissue culture flasks coated with 0.1% Matrigel (BD Biosciences, Franklin Lakes, NJ) in DMEM/F12 (Life Technologies, Carlsbad, CA) supplemented with 2 mM glutamine, 5 mM HEPES, 3 mM sodium bicarbonate, 0.3% glucose (all from Sigma–Aldrich, St Louis, MO; in the following termed NS medium) and 10 ng/ml epidermal growth factor (EGF), 10 ng/ml fibroblast growth factor-2 (FGF-2) (both from Tebu-Bio, Le-Perray-en-Yvelines, France), 1% N2 and 1% B27 (both from Life Technologies) to establish adherently growing NSC cultures (<xref ref-type="bibr" rid="bib29">Conti et al., 2005</xref>; <xref ref-type="bibr" rid="bib47">Jung et al., 2013</xref>). To express ADAM10 in <sup>Nestin</sup>A10 cKO NSCs, the mouse <italic>Adam10</italic> cDNA was cloned into pcDNA3.1/Zeo(−) (Life Technologies), and the linearized plasmid was used to transfect <sup>Nestin</sup>A10 KO NSC using the Nucleofector technology (Lonza, Basel, Switzerland) as described previously (<xref ref-type="bibr" rid="bib86">Richard et al., 2005</xref>; <xref ref-type="bibr" rid="bib7">Altmeppen et al., 2011</xref>). As control, <sup>Nestin</sup>A10 KO NSCs were nucleofected with pcDNA3.1/Zeo(−) lacking the <italic>Adam10</italic> cDNA. To select for positive cells, cells were further cultivated in NS medium supplemented with 10 ng/ml EGF, 10 ng/ml FGF-2, 1% N2, 1% B27, and 200 µg/ml zeocin.</p><p>To induce neuronal differentiation of wild-type, <sup>Nestin</sup>A10 KO and A10-nucleofected <sup>Nestin</sup>A10 KO NSCs, cells were plated onto coverslips coated with 1% Matrigel and maintained for 4 days in NS medium supplemented with 5 ng/ml FGF-2, 1% N2, and 2% B27. Subsequently, cells were cultivated for another 4 days in a 1:1 mixture of NS medium and Neurobasal medium (Life Technologies) containing 0.25% N2 and 2% B27.</p></sec><sec id="s4-8"><title>Surface biotinylation assay in neuronally differentiated NSCs</title><p>After neuronal differentiation of NSCs in six-well plates, cells were washed 2× with cold PBS and incubated for 30 min with 0.5 mg EZ-Link Sulfo-NHS-SS-Biotin (Thermo Scientific) in PBS at 4°C under gentle agitation on a rocking platform. Cells were then washed 3× for 5 min at 4°C with 0.1% BSA in PBS and lysed with 500 µl RIPA buffer as described above. After centrifugation, supernatants were diluted 1:1 with Triton dilution buffer (100 mM TEA, 100 mM NaCl, 5 mM EDTA, 0.02% NaN<sub>3</sub>, 2.5% Triton X-100, pH 8.6, +PI) and incubated for 1 hr with 200 µl pre-washed NeutrAvidin agarose beads (Thermo Scientific) at room temperature on a rotary wheel. Centrifugation was performed at 1000×<italic>g</italic> for 1 min and supernatant was taken as ‘lysate control’. Beads were washed 3× with wash buffer (20 mM TEA, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.2% SDS, 0.02% NaN<sub>3</sub>, pH 8.6, +PI) and spun down. Two more washing steps were performed with final wash buffer (20 mM TEA, 150 mM NaCl, 5 mM EDTA, pH 8.6, +PI) before adding 50 µl of 4× loading buffer including DTT and boiling for 6 min at 96°C to release biotinylated proteins from the beads. Supernatants were taken and loaded onto gels for SDS-PAGE as described above. ‘Lysate controls’ and biotinylated samples were detected via immunblotting using POM1 antibody. As reference and specificity control for surface biotinylated samples, membrane marker Flotillin-1 (murine purified anti-Flot-1; 1:1.000; BD Bioscience) was used.</p></sec><sec id="s4-9"><title>Immunofluorescence analysis</title><p>For surface staining of PrP<sup>C</sup> in neuronally differentiated NSCs, live cells were incubated with primary antibody against PrP<sup>C</sup> (POM1) for 1 hr at 4°C. Cells were then fixed in 4% paraformaldehyde in PBS (pH 7.4), blocked in PBS containing 0.1% bovine serum albumin and 0.3% Triton X-100 (both from Sigma), and incubated with polyclonal rabbit anti-β-tubulin III antibodies (Sigma) overnight at room temperature to identify nerve cells. Primary anti-PrP<sup>C</sup> and anti-β-tubulin III antibodies were detected with anti-mouse Cy2- and anti-rabbit Cy3-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA), respectively, and cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma).</p><p>For MEF cells, surface staining of PrP<sup>C</sup> and ADAM10 was achieved by incubating live cells for 1 hr at 4°C with primary antibodies POM1 and monoclonal rat anti-mouse ADAM10 ectodomain antibody (1:100, R&D Systems, Minneapolis, MN), respectively. These antibodies were also applied after permeabilization of cells with 0.2% Triton X-100 in PBS. Goat anti-mouse IgG Alexa Fluor 488 and goat anti-rat IgG Alexa Fluor 568 (both from Life Technologies) were used as secondary antibodies prior to mounting the coverslips with DAPI Fluoromount-G (SouthernBiotech, Birmingham, AL) onto object slides.</p></sec><sec id="s4-10"><title>Quantitative RT-PCR</title><p>Cortical regions from A10 cKO mice (n = 5) and littermate controls (n = 5) were prepared and total RNA was isolated using the NucleoSpin RNAII kit (Macherey Nagel, Dueren, Germany) according to the manufacturer's instructions. 2 µg of DNAse-treated RNA was used for cDNA synthesis using the RevertAid cDNA Synthesis Kit (Thermo Fisher Scientific). Gene expression analysis for mouse PrP<sup>C</sup> and GAPDH were performed using the mouse Universal ProbeLibrary System (Roche Applied Science) for qRT-PCR experiments (PrP<sup>C</sup>: 5′-GCCGACATCAGTCCACATAG, 5′-GGAGAGCCAAGCAGACTATCA, Probe: #71) on a LightCycler480 (Roche Diagnostics). PrP<sup>C</sup> gene expression levels were depicted as percentage of GAPDH expression using the ΔCT method for calculation. PCR efficiency of each assay was determined by serial dilutions of standards and these values were used for calculation.</p></sec><sec id="s4-11"><title>Statistical analysis</title><p>Statistical comparison of incubation times, Western blot quantifications, and qRT-PCR results between experimental groups was performed using Student's <italic>t</italic>-test for two-group comparisons with consideration of statistical significance at p values <0.05(*), <0.01(**), and <0.001 (***).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Dr Melanie Neumann and Kristin Hartmann from the Mouse Pathology Facility, University Medical Center Hamburg-Eppendorf (UKE), for their valuable support in the immunohistochemical stainings. Moreover, we thank Dr Bernd Zobiak and Dr Virgilio Failla from the UKE Microscopy and Imaging Facility (UMIF) for technical support. We thank Prof Dr Adriano Aguzzi (Zurich, Switzerland) for providing us with the POM antibodies.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>HCA, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>JP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>SK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con4"><p>BP, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con5"><p>KK, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con6"><p>FD, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con7"><p>CB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con8"><p>AH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con9"><p>LL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con10"><p>BS, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con11"><p>PPL, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con12"><p>UB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con13"><p>PS, Conception and design, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con14"><p>MG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group><fn-group content-type="ethics-information"><title>Ethics</title><fn fn-type="other"><p>Animal experimentation: Our study was carried out in accordance with the principles of laboratory animal care (NIH publication No. 86-23, revised 1985) as well as the recommendations in the Guide for the Care and Use of Laboratory Animals of the German Animal Welfare Act on protection of animals. The protocol was approved by the Committee on the Ethics of the Freie und Hansestadt Hamburg—Amt für Gesundheit und Verbraucherschutz (permit number 48/09, 81/07 and 84/13). Every effort was made to minimize suffering.</p></fn></fn-group></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ader</surname><given-names>M</given-names></name><name><surname>Schachner</surname><given-names>M</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name></person-group><year>2001</year><article-title>Transplantation of neural precursor cells into the dysmyelinated CNS of mutant mice deficient in the myelin-associated glycoprotein and Fyn tyrosine kinase</article-title><source>The European Journal of Neuroscience</source><volume>14</volume><fpage>561</fpage><lpage>566</lpage><pub-id pub-id-type="doi">10.1046/j.0953-816x.2001.01673.x</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Baumann</surname><given-names>F</given-names></name><name><surname>Bremer</surname><given-names>J</given-names></name></person-group><year>2008</year><article-title>The prion's elusive reason for being</article-title><source>Annual Review of Neuroscience</source><volume>31</volume><fpage>439</fpage><lpage>477</lpage><pub-id pub-id-type="doi">10.1146/annurev.neuro.31.060407.125620</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Heppner</surname><given-names>FL</given-names></name><name><surname>Heikenwalder</surname><given-names>M</given-names></name><name><surname>Prinz</surname><given-names>M</given-names></name><name><surname>Mertz</surname><given-names>K</given-names></name><name><surname>Seeger</surname><given-names>H</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2003</year><article-title>Immune system and peripheral nerves in propagation of prions to CNS</article-title><source>British Medical Bulletin</source><volume>66</volume><fpage>141</fpage><lpage>159</lpage><pub-id pub-id-type="doi">10.1093/bmb/66.1.141</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Akhtar</surname><given-names>S</given-names></name><name><surname>Grizenkova</surname><given-names>J</given-names></name><name><surname>Wenborn</surname><given-names>A</given-names></name><name><surname>Hummerich</surname><given-names>H</given-names></name><name><surname>Fernandez de Marco</surname><given-names>M</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name><name><surname>Lloyd</surname><given-names>SE</given-names></name></person-group><year>2013</year><article-title>Sod1 deficiency reduces incubation time in mouse models of prion disease</article-title><source>PLOS ONE</source><volume>8</volume><fpage>e54454</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0054454</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alais</surname><given-names>S</given-names></name><name><surname>Simoes</surname><given-names>S</given-names></name><name><surname>Baas</surname><given-names>D</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name><name><surname>Raposo</surname><given-names>G</given-names></name><name><surname>Darlix</surname><given-names>JL</given-names></name><name><surname>Leblanc</surname><given-names>P</given-names></name></person-group><year>2008</year><article-title>Mouse neuroblastoma cells release prion infectivity associated with exosomal vesicles</article-title><source>Biology of the Cell</source><volume>100</volume><fpage>603</fpage><lpage>615</lpage><pub-id pub-id-type="doi">10.1042/BC20080025</pub-id></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altmeppen</surname><given-names>HC</given-names></name><name><surname>Prox</surname><given-names>J</given-names></name><name><surname>Puig</surname><given-names>B</given-names></name><name><surname>Dohler</surname><given-names>F</given-names></name><name><surname>Falker</surname><given-names>C</given-names></name><name><surname>Krasemann</surname><given-names>S</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2013</year><article-title>Roles of endoproteolytic alpha-cleavage and shedding of the prion protein in neurodegeneration</article-title><source>The FEBS Journal</source><volume>280</volume><fpage>4338</fpage><lpage>4347</lpage><pub-id pub-id-type="doi">10.1111/febs.12196</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altmeppen</surname><given-names>HC</given-names></name><name><surname>Prox</surname><given-names>J</given-names></name><name><surname>Puig</surname><given-names>B</given-names></name><name><surname>Kluth</surname><given-names>MA</given-names></name><name><surname>Bernreuther</surname><given-names>C</given-names></name><name><surname>Thurm</surname><given-names>D</given-names></name><name><surname>Jorissen</surname><given-names>E</given-names></name><name><surname>Petrowitz</surname><given-names>B</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name><name><surname>De Strooper</surname><given-names>B</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2011</year><article-title>Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo</article-title><source>Molecular Neurodegeneration</source><volume>6</volume><fpage>36</fpage><pub-id pub-id-type="doi">10.1186/1750–1326-6–36</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Altmeppen</surname><given-names>HC</given-names></name><name><surname>Puig</surname><given-names>B</given-names></name><name><surname>Dohler</surname><given-names>F</given-names></name><name><surname>Thurm</surname><given-names>DK</given-names></name><name><surname>Falker</surname><given-names>C</given-names></name><name><surname>Krasemann</surname><given-names>S</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2012</year><article-title>Proteolytic processing of the prion protein in health and disease</article-title><source>American Journal of Neurodegenerative Disease</source><volume>1</volume><fpage>15</fpage><lpage>31</lpage></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Balducci</surname><given-names>C</given-names></name><name><surname>Beeg</surname><given-names>M</given-names></name><name><surname>Stravalaci</surname><given-names>M</given-names></name><name><surname>Bastone</surname><given-names>A</given-names></name><name><surname>Sclip</surname><given-names>A</given-names></name><name><surname>Biasini</surname><given-names>E</given-names></name><name><surname>Tapella</surname><given-names>L</given-names></name><name><surname>Colombo</surname><given-names>L</given-names></name><name><surname>Manzoni</surname><given-names>C</given-names></name><name><surname>Borsello</surname><given-names>T</given-names></name><name><surname>Chiesa</surname><given-names>R</given-names></name><name><surname>Gobbi</surname><given-names>M</given-names></name><name><surname>Salmona</surname><given-names>M</given-names></name><name><surname>Forloni</surname><given-names>G</given-names></name></person-group><year>2010</year><article-title>Synthetic amyloid-beta oligomers impair long-term memory independently of cellular prion protein</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>107</volume><fpage>2295</fpage><lpage>2300</lpage><pub-id pub-id-type="doi">10.1073/pnas.0911829107</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Barron</surname><given-names>RM</given-names></name><name><surname>Campbell</surname><given-names>SL</given-names></name><name><surname>King</surname><given-names>D</given-names></name><name><surname>Bellon</surname><given-names>A</given-names></name><name><surname>Chapman</surname><given-names>KE</given-names></name><name><surname>Williamson</surname><given-names>RA</given-names></name><name><surname>Manson</surname><given-names>JC</given-names></name></person-group><year>2007</year><article-title>High titers of transmissible spongiform encephalopathy infectivity associated with extremely low levels of PrPSc in vivo</article-title><source>The Journal of Biological Chemistry</source><volume>282</volume><fpage>35878</fpage><lpage>35886</lpage><pub-id pub-id-type="doi">10.1074/jbc.M704329200</pub-id></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Barron</surname><given-names>RM</given-names></name><name><surname>Thomson</surname><given-names>V</given-names></name><name><surname>King</surname><given-names>D</given-names></name><name><surname>Shaw</surname><given-names>J</given-names></name><name><surname>Melton</surname><given-names>DW</given-names></name><name><surname>Manson</surname><given-names>JC</given-names></name></person-group><year>2003</year><article-title>Transmission of murine scrapie to P101L transgenic mice</article-title><source>The Journal of General Virology</source><volume>84</volume><fpage>3165</fpage><lpage>3172</lpage><pub-id pub-id-type="doi">10.1099/vir.0.19147-0</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Baumann</surname><given-names>F</given-names></name><name><surname>Tolnay</surname><given-names>M</given-names></name><name><surname>Brabeck</surname><given-names>C</given-names></name><name><surname>Pahnke</surname><given-names>J</given-names></name><name><surname>Kloz</surname><given-names>U</given-names></name><name><surname>Niemann</surname><given-names>HH</given-names></name><name><surname>Heikenwalder</surname><given-names>M</given-names></name><name><surname>Rülicke</surname><given-names>T</given-names></name><name><surname>Bürkle</surname><given-names>A</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2007</year><article-title>Lethal recessive myelin toxicity of prion protein lacking its central domain</article-title><source>The EMBO Journal</source><volume>26</volume><fpage>538</fpage><lpage>547</lpage><pub-id pub-id-type="doi">10.1038/sj.emboj.7601510</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Béland</surname><given-names>M</given-names></name><name><surname>Motard</surname><given-names>J</given-names></name><name><surname>Barbarin</surname><given-names>A</given-names></name><name><surname>Roucou</surname><given-names>X</given-names></name></person-group><year>2012</year><article-title>PrP(C) homodimerization stimulates the production of PrPC cleaved fragments PrPN1 and PrPC1</article-title><source>The Journal of Neuroscience</source><volume>32</volume><fpage>13255</fpage><lpage>13263</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.2236-12.2012</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Benilova</surname><given-names>I</given-names></name><name><surname>De Strooper</surname><given-names>B</given-names></name></person-group><year>2010</year><article-title>Prion protein in Alzheimer's pathogenesis: a hot and controversial issue</article-title><source>EMBO Molecular Medicine</source><volume>2</volume><fpage>289</fpage><lpage>290</lpage><pub-id pub-id-type="doi">10.1002/emmm.201000088</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bessen</surname><given-names>RA</given-names></name><name><surname>Shearin</surname><given-names>H</given-names></name><name><surname>Martinka</surname><given-names>S</given-names></name><name><surname>Boharski</surname><given-names>R</given-names></name><name><surname>Lowe</surname><given-names>D</given-names></name><name><surname>Wilham</surname><given-names>JM</given-names></name><name><surname>Caughey</surname><given-names>B</given-names></name><name><surname>Wiley</surname><given-names>JA</given-names></name></person-group><year>2010</year><article-title>Prion shedding from olfactory neurons into nasal secretions</article-title><source>PLOS Pathogens</source><volume>6</volume><fpage>e1000837</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000837</pub-id></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Borchelt</surname><given-names>DR</given-names></name><name><surname>Rogers</surname><given-names>M</given-names></name><name><surname>Stahl</surname><given-names>N</given-names></name><name><surname>Telling</surname><given-names>G</given-names></name><name><surname>Prusiner</surname><given-names>SB</given-names></name></person-group><year>1993</year><article-title>Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor</article-title><source>Glycobiology</source><volume>3</volume><fpage>319</fpage><lpage>329</lpage><pub-id pub-id-type="doi">10.1093/glycob/3.4.319</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Isenmann</surname><given-names>S</given-names></name><name><surname>Raeber</surname><given-names>A</given-names></name><name><surname>Fischer</surname><given-names>M</given-names></name><name><surname>Sailer</surname><given-names>A</given-names></name><name><surname>Kobayashi</surname><given-names>Y</given-names></name><name><surname>Marino</surname><given-names>S</given-names></name><name><surname>Weissmann</surname><given-names>C</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>1996</year><article-title>Normal host prion protein necessary for scrapie-induced neurotoxicity</article-title><source>Nature</source><volume>379</volume><fpage>339</fpage><lpage>343</lpage><pub-id pub-id-type="doi">10.1038/379339a0</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bremer</surname><given-names>J</given-names></name><name><surname>Baumann</surname><given-names>F</given-names></name><name><surname>Tiberi</surname><given-names>C</given-names></name><name><surname>Wessig</surname><given-names>C</given-names></name><name><surname>Fischer</surname><given-names>H</given-names></name><name><surname>Schwarz</surname><given-names>P</given-names></name><name><surname>Steele</surname><given-names>AD</given-names></name><name><surname>Toyka</surname><given-names>KV</given-names></name><name><surname>Nave</surname><given-names>KA</given-names></name><name><surname>Weis</surname><given-names>J</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2010</year><article-title>Axonal prion protein is required for peripheral myelin maintenance</article-title><source>Nature Neuroscience</source><volume>13</volume><fpage>310</fpage><lpage>318</lpage><pub-id pub-id-type="doi">10.1038/nn.2483</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Büeler</surname><given-names>HR</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Sailer</surname><given-names>A</given-names></name><name><surname>Greiner</surname><given-names>RA</given-names></name><name><surname>Autenried</surname><given-names>P</given-names></name><name><surname>Aguet</surname><given-names>M</given-names></name><name><surname>Weissmann</surname><given-names>C</given-names></name></person-group><year>1993</year><article-title>Mice devoid of PrP are resistant to scrapie</article-title><source>Cell</source><volume>73</volume><fpage>1339</fpage><lpage>1347</lpage><pub-id pub-id-type="doi">10.1016/0092-8674(93)90360-3</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Büeler</surname><given-names>HR</given-names></name><name><surname>Fischer</surname><given-names>M</given-names></name><name><surname>Lang</surname><given-names>Y</given-names></name><name><surname>Bluethmann</surname><given-names>H</given-names></name><name><surname>Lipp</surname><given-names>HP</given-names></name><name><surname>DeArmond</surname><given-names>SJ</given-names></name><name><surname>Prusiner</surname><given-names>SB</given-names></name><name><surname>Aguet</surname><given-names>M</given-names></name><name><surname>Weissmann</surname><given-names>C</given-names></name></person-group><year>1992</year><article-title>Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein</article-title><source>Nature</source><volume>356</volume><fpage>577</fpage><lpage>582</lpage><pub-id pub-id-type="doi">10.1038/356577a0</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Burwinkel</surname><given-names>M</given-names></name><name><surname>Schwarz</surname><given-names>A</given-names></name><name><surname>Riemer</surname><given-names>C</given-names></name><name><surname>Schultz</surname><given-names>J</given-names></name><name><surname>Van Landeghem</surname><given-names>F</given-names></name><name><surname>Baier</surname><given-names>M</given-names></name></person-group><year>2004</year><article-title>Rapid disease development in scrapie-infected mice deficient for CD40 ligand</article-title><source>EMBO Reports</source><volume>5</volume><fpage>527</fpage><lpage>531</lpage><pub-id pub-id-type="doi">10.1038/sj.embor.7400125</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Calella</surname><given-names>AM</given-names></name><name><surname>Farinelli</surname><given-names>M</given-names></name><name><surname>Nuvolone</surname><given-names>M</given-names></name><name><surname>Mirante</surname><given-names>O</given-names></name><name><surname>Moos</surname><given-names>R</given-names></name><name><surname>Falsig</surname><given-names>J</given-names></name><name><surname>Mansuy</surname><given-names>IM</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2010</year><article-title>Prion protein and Abeta-related synaptic toxicity impairment</article-title><source>EMBO Molecular Medicine</source><volume>2</volume><fpage>306</fpage><lpage>314</lpage><pub-id pub-id-type="doi">10.1002/emmm.201000082</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Campbell</surname><given-names>L</given-names></name><name><surname>Gill</surname><given-names>AC</given-names></name><name><surname>McGovern</surname><given-names>G</given-names></name><name><surname>Jalland</surname><given-names>CM</given-names></name><name><surname>Hopkins</surname><given-names>J</given-names></name><name><surname>Tranulis</surname><given-names>MA</given-names></name><name><surname>Hunter</surname><given-names>N</given-names></name><name><surname>Goldmann</surname><given-names>W</given-names></name></person-group><year>2013</year><article-title>The PrP(C) C1 fragment derived from the ovine A136R154R171PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP(C) protein in vitro</article-title><source>Biochimica et Biophysica Acta</source><volume>1832</volume><fpage>826</fpage><lpage>836</lpage><pub-id pub-id-type="doi">10.1016/j.bbadis.2013.02.020</pub-id></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Casanova</surname><given-names>E</given-names></name><name><surname>Fehsenfeld</surname><given-names>S</given-names></name><name><surname>Mantamadiotis</surname><given-names>T</given-names></name><name><surname>Lemberger</surname><given-names>T</given-names></name><name><surname>Greiner</surname><given-names>E</given-names></name><name><surname>Stewart</surname><given-names>AF</given-names></name><name><surname>Schutz</surname><given-names>G</given-names></name></person-group><year>2001</year><article-title>A CamKIIalpha iCre BAC allows brain-specific gene inactivation</article-title><source>Genesis</source><volume>31</volume><fpage>37</fpage><lpage>42</lpage><pub-id pub-id-type="doi">10.1002/gene.1078</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>C</given-names></name><name><surname>Lv</surname><given-names>Y</given-names></name><name><surname>Zhang</surname><given-names>BY</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>Shi</surname><given-names>Q</given-names></name><name><surname>Wang</surname><given-names>J</given-names></name><name><surname>Tian</surname><given-names>C</given-names></name><name><surname>Gao</surname><given-names>C</given-names></name><name><surname>Xiao</surname><given-names>K</given-names></name><name><surname>Ren</surname><given-names>K</given-names></name><name><surname>Zhou</surname><given-names>W</given-names></name><name><surname>Dong</surname><given-names>XP</given-names></name></person-group><year>2014</year><article-title>Apparent reduction of ADAM10 in scrapie-infected cultured cells and in the brains of scrapie-infected rodents</article-title><source>Molecular Neurobiology</source><volume>50</volume><fpage>875</fpage><lpage>887</lpage><pub-id pub-id-type="doi">10.1007/s12035-014-8708-7</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chen</surname><given-names>SG</given-names></name><name><surname>Teplow</surname><given-names>DB</given-names></name><name><surname>Parchi</surname><given-names>P</given-names></name><name><surname>Teller</surname><given-names>JK</given-names></name><name><surname>Gambetti</surname><given-names>P</given-names></name><name><surname>Autilio-Gambetti</surname><given-names>L</given-names></name></person-group><year>1995</year><article-title>Truncated forms of the human prion protein in normal brain and in prion diseases</article-title><source>The Journal of Biological Chemistry</source><volume>270</volume><fpage>19173</fpage><lpage>19180</lpage><pub-id pub-id-type="doi">10.1074/jbc.270.32.19173</pub-id></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chesebro</surname><given-names>B</given-names></name><name><surname>Race</surname><given-names>B</given-names></name><name><surname>Meade-White</surname><given-names>K</given-names></name><name><surname>Lacasse</surname><given-names>R</given-names></name><name><surname>Race</surname><given-names>R</given-names></name><name><surname>Klingeborn</surname><given-names>M</given-names></name><name><surname>Striebel</surname><given-names>J</given-names></name><name><surname>Dorward</surname><given-names>D</given-names></name><name><surname>McGovern</surname><given-names>G</given-names></name><name><surname>Jeffrey</surname><given-names>M</given-names></name></person-group><year>2010</year><article-title>Fatal transmissible amyloid encephalopathy: a new type of prion disease associated with lack of prion protein membrane anchoring</article-title><source>PLOS Pathogens</source><volume>6</volume><fpage>e1000800</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000800</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chesebro</surname><given-names>B</given-names></name><name><surname>Trifilo</surname><given-names>M</given-names></name><name><surname>Race</surname><given-names>R</given-names></name><name><surname>Meade-White</surname><given-names>K</given-names></name><name><surname>Teng</surname><given-names>C</given-names></name><name><surname>LaCasse</surname><given-names>R</given-names></name><name><surname>Raymond</surname><given-names>L</given-names></name><name><surname>Favara</surname><given-names>C</given-names></name><name><surname>Baron</surname><given-names>G</given-names></name><name><surname>Priola</surname><given-names>S</given-names></name><name><surname>Caughey</surname><given-names>B</given-names></name><name><surname>Masliah</surname><given-names>E</given-names></name><name><surname>Oldstone</surname><given-names>M</given-names></name></person-group><year>2005</year><article-title>Anchorless prion protein results in infectious amyloid disease without clinical scrapie</article-title><source>Science</source><volume>308</volume><fpage>1435</fpage><lpage>1439</lpage><pub-id pub-id-type="doi">10.1126/science.1110837</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Conti</surname><given-names>L</given-names></name><name><surname>Pollard</surname><given-names>SM</given-names></name><name><surname>Gorba</surname><given-names>T</given-names></name><name><surname>Reitano</surname><given-names>E</given-names></name><name><surname>Toselli</surname><given-names>M</given-names></name><name><surname>Biella</surname><given-names>G</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Sanzone</surname><given-names>S</given-names></name><name><surname>Ying</surname><given-names>QL</given-names></name><name><surname>Cattaneo</surname><given-names>E</given-names></name><name><surname>Smith</surname><given-names>A</given-names></name></person-group><year>2005</year><article-title>Niche-independent symmetrical self-renewal of a mammalian tissue stem cell</article-title><source>PLOS Biology</source><volume>3</volume><fpage>e283</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.0030283</pub-id></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Dohler</surname><given-names>F</given-names></name><name><surname>Sepulveda-Falla</surname><given-names>D</given-names></name><name><surname>Krasemann</surname><given-names>S</given-names></name><name><surname>Altmeppen</surname><given-names>H</given-names></name><name><surname>Schluter</surname><given-names>H</given-names></name><name><surname>Hildebrand</surname><given-names>D</given-names></name><name><surname>Zerr</surname><given-names>I</given-names></name><name><surname>Matschke</surname><given-names>J</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2014</year><article-title>High molecular mass assemblies of amyloid-beta oligomers bind prion protein in patients with Alzheimer's disease</article-title><source>Brain</source><volume>137</volume><fpage>873</fpage><lpage>886</lpage><pub-id pub-id-type="doi">10.1093/brain/awt375</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Endres</surname><given-names>K</given-names></name><name><surname>Mitteregger</surname><given-names>G</given-names></name><name><surname>Kojro</surname><given-names>E</given-names></name><name><surname>Kretzschmar</surname><given-names>H</given-names></name><name><surname>Fahrenholz</surname><given-names>F</given-names></name></person-group><year>2009</year><article-title>Influence of ADAM10 on prion protein processing and scrapie infectiosity in vivo</article-title><source>Neurobiology of Disease</source><volume>36</volume><fpage>233</fpage><lpage>241</lpage><pub-id pub-id-type="doi">10.1016/j.nbd.2009.07.015</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Falsig</surname><given-names>J</given-names></name><name><surname>Sonati</surname><given-names>T</given-names></name><name><surname>Herrmann</surname><given-names>US</given-names></name><name><surname>Saban</surname><given-names>D</given-names></name><name><surname>Li</surname><given-names>B</given-names></name><name><surname>Arroyo</surname><given-names>K</given-names></name><name><surname>Ballmer</surname><given-names>B</given-names></name><name><surname>Liberski</surname><given-names>PP</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2012</year><article-title>Prion pathogenesis is faithfully reproduced in cerebellar organotypic slice cultures</article-title><source>PLOS Pathogens</source><volume>8</volume><fpage>e1002985</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1002985</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fischer</surname><given-names>M</given-names></name><name><surname>Rülicke</surname><given-names>T</given-names></name><name><surname>Raeber</surname><given-names>A</given-names></name><name><surname>Sailer</surname><given-names>A</given-names></name><name><surname>Moser</surname><given-names>M</given-names></name><name><surname>Oesch</surname><given-names>B</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Weissmann</surname><given-names>C</given-names></name></person-group><year>1996</year><article-title>Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie</article-title><source>The EMBO Journal</source><volume>15</volume><fpage>1255</fpage><lpage>1264</lpage></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fluharty</surname><given-names>BR</given-names></name><name><surname>Biasini</surname><given-names>E</given-names></name><name><surname>Stravalaci</surname><given-names>M</given-names></name><name><surname>Sclip</surname><given-names>A</given-names></name><name><surname>Diomede</surname><given-names>L</given-names></name><name><surname>Balducci</surname><given-names>C</given-names></name><name><surname>La Vitola</surname><given-names>P</given-names></name><name><surname>Messa</surname><given-names>M</given-names></name><name><surname>Colombo</surname><given-names>L</given-names></name><name><surname>Forloni</surname><given-names>G</given-names></name><name><surname>Borsello</surname><given-names>T</given-names></name><name><surname>Gobbi</surname><given-names>M</given-names></name><name><surname>Harris</surname><given-names>DA</given-names></name></person-group><year>2013</year><article-title>An N-terminal fragment of the prion protein binds to amyloid-beta oligomers and inhibits their neurotoxicity in vivo</article-title><source>The Journal of Biological Chemistry</source><volume>288</volume><fpage>7857</fpage><lpage>7866</lpage><pub-id pub-id-type="doi">10.1074/jbc.M112.423954</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Glatzel</surname><given-names>M</given-names></name><name><surname>Giger</surname><given-names>O</given-names></name><name><surname>Seeger</surname><given-names>H</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2004</year><article-title>Variant Creutzfeldt-Jakob disease: between lymphoid organs and brain</article-title><source>Trends in Microbiology</source><volume>12</volume><fpage>51</fpage><lpage>53</lpage><pub-id pub-id-type="doi">10.1016/j.tim.2003.12.001</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Glatzel</surname><given-names>M</given-names></name><name><surname>Heppner</surname><given-names>FL</given-names></name><name><surname>Albers</surname><given-names>KM</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2001</year><article-title>Sympathetic innervation of lymphoreticular organs is rate limiting for prion neuroinvasion</article-title><source>Neuron</source><volume>31</volume><fpage>25</fpage><lpage>34</lpage><pub-id pub-id-type="doi">10.1016/S0896-6273(01)00331-2</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gousset</surname><given-names>K</given-names></name><name><surname>Schiff</surname><given-names>E</given-names></name><name><surname>Langevin</surname><given-names>C</given-names></name><name><surname>Marijanovic</surname><given-names>Z</given-names></name><name><surname>Caputo</surname><given-names>A</given-names></name><name><surname>Browman</surname><given-names>DT</given-names></name><name><surname>Chenouard</surname><given-names>N</given-names></name><name><surname>de Chaumont</surname><given-names>F</given-names></name><name><surname>Martino</surname><given-names>A</given-names></name><name><surname>Enninga</surname><given-names>J</given-names></name><name><surname>Olivo-Marin</surname><given-names>JC</given-names></name><name><surname>Männel</surname><given-names>D</given-names></name><name><surname>Zurzolo</surname><given-names>C</given-names></name></person-group><year>2009</year><article-title>Prions hijack tunnelling nanotubes for intercellular spread</article-title><source>Nature Cell Biology</source><volume>11</volume><fpage>328</fpage><lpage>336</lpage><pub-id pub-id-type="doi">10.1038/ncb1841</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Grizenkova</surname><given-names>J</given-names></name><name><surname>Akhtar</surname><given-names>S</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name><name><surname>Lloyd</surname><given-names>SE</given-names></name></person-group><year>2014</year><article-title>Microglial Cx3cr1 knockout reduces prion disease incubation time in mice</article-title><source>BMC Neuroscience</source><volume>15</volume><fpage>44</fpage><pub-id pub-id-type="doi">10.1186/1471–2202-15–44</pub-id></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Grizenkova</surname><given-names>J</given-names></name><name><surname>Akhtar</surname><given-names>S</given-names></name><name><surname>Hummerich</surname><given-names>H</given-names></name><name><surname>Tomlinson</surname><given-names>A</given-names></name><name><surname>Asante</surname><given-names>EA</given-names></name><name><surname>Wenborn</surname><given-names>A</given-names></name><name><surname>Fizet</surname><given-names>J</given-names></name><name><surname>Poulter</surname><given-names>M</given-names></name><name><surname>Wiseman</surname><given-names>FK</given-names></name><name><surname>Fisher</surname><given-names>EM</given-names></name><name><surname>Tybulewicz</surname><given-names>VL</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name><name><surname>Lloyd</surname><given-names>SE</given-names></name></person-group><year>2012</year><article-title>Overexpression of the Hspa13 (Stch) gene reduces prion disease incubation time in mice</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>109</volume><fpage>13722</fpage><lpage>13727</lpage><pub-id pub-id-type="doi">10.1073/pnas.1208917109</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guillot-Sestier</surname><given-names>MV</given-names></name><name><surname>Sunyach</surname><given-names>C</given-names></name><name><surname>Druon</surname><given-names>C</given-names></name><name><surname>Scarzello</surname><given-names>S</given-names></name><name><surname>Checler</surname><given-names>F</given-names></name></person-group><year>2009</year><article-title>The alpha-secretase-derived N-terminal product of cellular prion, N1, displays neuroprotective function in vitro and in vivo</article-title><source>The Journal of Biological Chemistry</source><volume>284</volume><fpage>35973</fpage><lpage>35986</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.051086</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Guillot-Sestier</surname><given-names>MV</given-names></name><name><surname>Sunyach</surname><given-names>C</given-names></name><name><surname>Ferreira</surname><given-names>ST</given-names></name><name><surname>Marzolo</surname><given-names>MP</given-names></name><name><surname>Bauer</surname><given-names>C</given-names></name><name><surname>Thevenet</surname><given-names>A</given-names></name><name><surname>Checler</surname><given-names>F</given-names></name></person-group><year>2012</year><article-title>Alpha-Secretase-derived fragment of cellular prion, N1, protects against monomeric and oligomeric amyloid beta (Abeta)-associated cell death</article-title><source>The Journal of Biological Chemistry</source><volume>287</volume><fpage>5021</fpage><lpage>5032</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.323626</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Harris</surname><given-names>DA</given-names></name><name><surname>Huber</surname><given-names>MT</given-names></name><name><surname>van Dijken</surname><given-names>P</given-names></name><name><surname>Shyng</surname><given-names>SL</given-names></name><name><surname>Chait</surname><given-names>BT</given-names></name><name><surname>Wang</surname><given-names>R</given-names></name></person-group><year>1993</year><article-title>Processing of a cellular prion protein: identification of N- and C-terminal cleavage sites</article-title><source>Biochemistry</source><volume>32</volume><fpage>1009</fpage><lpage>1016</lpage><pub-id pub-id-type="doi">10.1021/bi00055a003</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hartmann</surname><given-names>D</given-names></name><name><surname>de Strooper</surname><given-names>B</given-names></name><name><surname>Serneels</surname><given-names>L</given-names></name><name><surname>Craessaerts</surname><given-names>K</given-names></name><name><surname>Herreman</surname><given-names>A</given-names></name><name><surname>Annaert</surname><given-names>W</given-names></name><name><surname>Umans</surname><given-names>L</given-names></name><name><surname>Lubke</surname><given-names>T</given-names></name><name><surname>Lena Illert</surname><given-names>A</given-names></name><name><surname>von Figura</surname><given-names>K</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name></person-group><year>2002</year><article-title>The disintegrin/metalloprotease ADAM 10 is essential for Notch signalling but not for alpha-secretase activity in fibroblasts</article-title><source>Human Molecular Genetics</source><volume>11</volume><fpage>2615</fpage><lpage>2624</lpage><pub-id pub-id-type="doi">10.1093/hmg/11.21.2615</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Heiseke</surname><given-names>A</given-names></name><name><surname>Schöbel</surname><given-names>S</given-names></name><name><surname>Lichtenthaler</surname><given-names>SF</given-names></name><name><surname>Vorberg</surname><given-names>I</given-names></name><name><surname>Groschup</surname><given-names>MH</given-names></name><name><surname>Kretzschmar</surname><given-names>H</given-names></name><name><surname>Schätzl</surname><given-names>HM</given-names></name><name><surname>Nunziante</surname><given-names>M</given-names></name></person-group><year>2008</year><article-title>The novel sorting nexin SNX33 interferes with cellular PrP formation by modulation of PrP shedding</article-title><source>Traffic</source><volume>9</volume><fpage>1116</fpage><lpage>1129</lpage><pub-id pub-id-type="doi">10.1111/j.1600-0854.2008.00750.x</pub-id></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jeffrey</surname><given-names>M</given-names></name><name><surname>Fraser</surname><given-names>JR</given-names></name></person-group><year>2000</year><article-title>Tubulovesicular particles occur early in the incubation period of murine scrapie</article-title><source>Acta Neuropathologica</source><volume>99</volume><fpage>525</fpage><lpage>528</lpage><pub-id pub-id-type="doi">10.1007/s004010051155</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jorissen</surname><given-names>E</given-names></name><name><surname>Prox</surname><given-names>J</given-names></name><name><surname>Bernreuther</surname><given-names>C</given-names></name><name><surname>Weber</surname><given-names>S</given-names></name><name><surname>Schwanbeck</surname><given-names>R</given-names></name><name><surname>Serneels</surname><given-names>L</given-names></name><name><surname>Snellinx</surname><given-names>A</given-names></name><name><surname>Craessaerts</surname><given-names>K</given-names></name><name><surname>Thathiah</surname><given-names>A</given-names></name><name><surname>Tesseur</surname><given-names>I</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name><name><surname>Weskamp</surname><given-names>G</given-names></name><name><surname>Blobel</surname><given-names>CP</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name><name><surname>De Strooper</surname><given-names>B</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name></person-group><year>2010</year><article-title>The disintegrin/metalloproteinase ADAM10 is essential for the establishment of the brain cortex</article-title><source>The Journal of Neuroscience</source><volume>30</volume><fpage>4833</fpage><lpage>4844</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.5221-09.2010</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jung</surname><given-names>G</given-names></name><name><surname>Sun</surname><given-names>J</given-names></name><name><surname>Petrowitz</surname><given-names>B</given-names></name><name><surname>Riecken</surname><given-names>K</given-names></name><name><surname>Kruszewski</surname><given-names>K</given-names></name><name><surname>Jankowiak</surname><given-names>W</given-names></name><name><surname>Kunst</surname><given-names>F</given-names></name><name><surname>Skevas</surname><given-names>C</given-names></name><name><surname>Richard</surname><given-names>G</given-names></name><name><surname>Fehse</surname><given-names>B</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name></person-group><year>2013</year><article-title>Genetically modified neural stem cells for a local and sustained delivery of neuroprotective factors to the dystrophic mouse retina</article-title><source>Stem Cells Translational Medicine</source><volume>2</volume><fpage>1001</fpage><lpage>1010</lpage><pub-id pub-id-type="doi">10.5966/sctm.2013-0013</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kessels</surname><given-names>HW</given-names></name><name><surname>Nguyen</surname><given-names>LN</given-names></name><name><surname>Nabavi</surname><given-names>S</given-names></name><name><surname>Malinow</surname><given-names>R</given-names></name></person-group><year>2010</year><article-title>The prion protein as a receptor for amyloid-beta</article-title><source>Nature</source><volume>466</volume><fpage>E3</fpage><lpage>E4</lpage><pub-id pub-id-type="doi">10.1038/nature09217</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kim</surname><given-names>JI</given-names></name><name><surname>Surewicz</surname><given-names>K</given-names></name><name><surname>Gambetti</surname><given-names>P</given-names></name><name><surname>Surewicz</surname><given-names>WK</given-names></name></person-group><year>2009</year><article-title>The role of glycophosphatidylinositol anchor in the amplification of the scrapie isoform of prion protein in vitro</article-title><source>FEBS Letters</source><volume>583</volume><fpage>3671</fpage><lpage>3675</lpage><pub-id pub-id-type="doi">10.1016/j.febslet.2009.10.049</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kocisko</surname><given-names>DA</given-names></name><name><surname>Come</surname><given-names>JH</given-names></name><name><surname>Priola</surname><given-names>SA</given-names></name><name><surname>Chesebro</surname><given-names>B</given-names></name><name><surname>Raymond</surname><given-names>GJ</given-names></name><name><surname>Lansbury</surname><given-names>PT</given-names></name><name><surname>Caughey</surname><given-names>B</given-names></name></person-group><year>1994</year><article-title>Cell-free formation of protease-resistant prion protein</article-title><source>Nature</source><volume>370</volume><fpage>471</fpage><lpage>474</lpage><pub-id pub-id-type="doi">10.1038/370471a0</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Krasemann</surname><given-names>S</given-names></name><name><surname>Neumann</surname><given-names>M</given-names></name><name><surname>Szalay</surname><given-names>B</given-names></name><name><surname>Stocking</surname><given-names>C</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name></person-group><year>2013</year><article-title>Protease-sensitive prion species in neoplastic spleens of prion-infected mice with uncoupling of PrP(Sc) and prion infectivity</article-title><source>The Journal of General Virology</source><volume>94</volume><fpage>453</fpage><lpage>463</lpage><pub-id pub-id-type="doi">10.1099/vir.0.045922-0</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lammich</surname><given-names>S</given-names></name><name><surname>Kojro</surname><given-names>E</given-names></name><name><surname>Postina</surname><given-names>R</given-names></name><name><surname>Gilbert</surname><given-names>S</given-names></name><name><surname>Pfeiffer</surname><given-names>R</given-names></name><name><surname>Jasionowski</surname><given-names>M</given-names></name><name><surname>Haass</surname><given-names>C</given-names></name><name><surname>Fahrenholz</surname><given-names>F</given-names></name></person-group><year>1999</year><article-title>Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>96</volume><fpage>3922</fpage><lpage>3927</lpage><pub-id pub-id-type="doi">10.1073/pnas.96.7.3922</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Larson</surname><given-names>M</given-names></name><name><surname>Sherman</surname><given-names>MA</given-names></name><name><surname>Amar</surname><given-names>F</given-names></name><name><surname>Nuvolone</surname><given-names>M</given-names></name><name><surname>Schneider</surname><given-names>JA</given-names></name><name><surname>Bennett</surname><given-names>DA</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Lesne</surname><given-names>SE</given-names></name></person-group><year>2012</year><article-title>The complex PrP(c)-Fyn couples human oligomeric Abeta with pathological tau changes in Alzheimer's disease</article-title><source>The Journal of Neuroscience</source><volume>32</volume><fpage>16857</fpage><lpage>16871a</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.1858-12.2012</pub-id></element-citation></ref><ref id="bib54"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lasmezas</surname><given-names>CI</given-names></name><name><surname>Deslys</surname><given-names>JP</given-names></name><name><surname>Robain</surname><given-names>O</given-names></name><name><surname>Jaegly</surname><given-names>A</given-names></name><name><surname>Beringue</surname><given-names>V</given-names></name><name><surname>Peyrin</surname><given-names>JM</given-names></name><name><surname>Fournier</surname><given-names>JG</given-names></name><name><surname>Hauw</surname><given-names>JJ</given-names></name><name><surname>Rossier</surname><given-names>J</given-names></name><name><surname>Dormont</surname><given-names>D</given-names></name></person-group><year>1997</year><article-title>Transmission of the BSE agent to mice in the absence of detectable abnormal prion protein</article-title><source>Science</source><volume>275</volume><fpage>402</fpage><lpage>405</lpage><pub-id pub-id-type="doi">10.1126/science.275.5298.402</pub-id></element-citation></ref><ref id="bib55"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lauren</surname><given-names>J</given-names></name><name><surname>Gimbel</surname><given-names>DA</given-names></name><name><surname>Nygaard</surname><given-names>HB</given-names></name><name><surname>Gilbert</surname><given-names>JW</given-names></name><name><surname>Strittmatter</surname><given-names>SM</given-names></name></person-group><year>2009</year><article-title>Cellular prion protein mediates impairment of synaptic plasticity by amyloid-beta oligomers</article-title><source>Nature</source><volume>457</volume><fpage>1128</fpage><lpage>1132</lpage><pub-id pub-id-type="doi">10.1038/nature07761</pub-id></element-citation></ref><ref id="bib56"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Leblanc</surname><given-names>P</given-names></name><name><surname>Alais</surname><given-names>S</given-names></name><name><surname>Porto-Carreiro</surname><given-names>I</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name><name><surname>Grassi</surname><given-names>J</given-names></name><name><surname>Raposo</surname><given-names>G</given-names></name><name><surname>Darlix</surname><given-names>JL</given-names></name></person-group><year>2006</year><article-title>Retrovirus infection strongly enhances scrapie infectivity release in cell culture</article-title><source>The EMBO Journal</source><volume>25</volume><fpage>2674</fpage><lpage>2685</lpage><pub-id pub-id-type="doi">10.1038/sj.emboj.7601162</pub-id></element-citation></ref><ref id="bib57"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>AM</given-names></name><name><surname>Paulsson</surname><given-names>JF</given-names></name><name><surname>Cruite</surname><given-names>J</given-names></name><name><surname>Andaya</surname><given-names>AA</given-names></name><name><surname>Trifilo</surname><given-names>MJ</given-names></name><name><surname>Oldstone</surname><given-names>MB</given-names></name></person-group><year>2011</year><article-title>Extraneural manifestations of prion infection in GPI-anchorless transgenic mice</article-title><source>Virology</source><volume>411</volume><fpage>1</fpage><lpage>8</lpage><pub-id pub-id-type="doi">10.1016/j.virol.2010.12.012</pub-id></element-citation></ref><ref id="bib58"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lee</surname><given-names>MS</given-names></name><name><surname>Kwon</surname><given-names>YT</given-names></name><name><surname>Li</surname><given-names>M</given-names></name><name><surname>Peng</surname><given-names>J</given-names></name><name><surname>Friedlander</surname><given-names>RM</given-names></name><name><surname>Tsai</surname><given-names>LH</given-names></name></person-group><year>2000</year><article-title>Neurotoxicity induces cleavage of p35 to p25 by calpain</article-title><source>Nature</source><volume>405</volume><fpage>360</fpage><lpage>364</lpage><pub-id pub-id-type="doi">10.1038/35012636</pub-id></element-citation></ref><ref id="bib59"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lewis</surname><given-names>V</given-names></name><name><surname>Hill</surname><given-names>AF</given-names></name><name><surname>Haigh</surname><given-names>CL</given-names></name><name><surname>Klug</surname><given-names>GM</given-names></name><name><surname>Masters</surname><given-names>CL</given-names></name><name><surname>Lawson</surname><given-names>VA</given-names></name><name><surname>Collins</surname><given-names>SJ</given-names></name></person-group><year>2009</year><article-title>Increased proportions of C1 truncated prion protein protect against cellular M1000 prion infection</article-title><source>Journal of Neuropathology and Experimental Neurology</source><volume>68</volume><fpage>1125</fpage><lpage>1135</lpage><pub-id pub-id-type="doi">10.1097/NEN.0b013e3181b96981</pub-id></element-citation></ref><ref id="bib60"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liang</surname><given-names>J</given-names></name><name><surname>Wang</surname><given-names>W</given-names></name><name><surname>Sorensen</surname><given-names>D</given-names></name><name><surname>Medina</surname><given-names>S</given-names></name><name><surname>Ilchenko</surname><given-names>S</given-names></name><name><surname>Kiselar</surname><given-names>J</given-names></name><name><surname>Surewicz</surname><given-names>WK</given-names></name><name><surname>Booth</surname><given-names>SA</given-names></name><name><surname>Kong</surname><given-names>Q</given-names></name></person-group><year>2012</year><article-title>Cellular prion protein regulates its own alpha-cleavage through ADAM8 in skeletal muscle</article-title><source>The Journal of Biological Chemistry</source><volume>287</volume><fpage>16510</fpage><lpage>16520</lpage><pub-id pub-id-type="doi">10.1074/jbc.M112.360891</pub-id></element-citation></ref><ref id="bib61"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liberski</surname><given-names>PP</given-names></name><name><surname>Sikorska</surname><given-names>B</given-names></name><name><surname>Hauw</surname><given-names>JJ</given-names></name><name><surname>Kopp</surname><given-names>N</given-names></name><name><surname>Streichenberger</surname><given-names>N</given-names></name><name><surname>Giraud</surname><given-names>P</given-names></name><name><surname>Boellaard</surname><given-names>J</given-names></name><name><surname>Budka</surname><given-names>H</given-names></name><name><surname>Kovacs</surname><given-names>GG</given-names></name><name><surname>Ironside</surname><given-names>J</given-names></name><name><surname>Brown</surname><given-names>P</given-names></name></person-group><year>2010</year><article-title>Ultrastructural characteristics (or evaluation) of Creutzfeldt-Jakob disease and other human transmissible spongiform encephalopathies or prion diseases</article-title><source>Ultrastructural Pathology</source><volume>34</volume><fpage>351</fpage><lpage>361</lpage><pub-id pub-id-type="doi">10.3109/01913123.2010.491175</pub-id></element-citation></ref><ref id="bib62"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Liberski</surname><given-names>PP</given-names></name><name><surname>Sikorska</surname><given-names>B</given-names></name><name><surname>Hauw</surname><given-names>JJ</given-names></name><name><surname>Kopp</surname><given-names>N</given-names></name><name><surname>Streichenberger</surname><given-names>N</given-names></name><name><surname>Giraud</surname><given-names>P</given-names></name><name><surname>Budka</surname><given-names>H</given-names></name><name><surname>Boellaard</surname><given-names>JW</given-names></name><name><surname>Brown</surname><given-names>P</given-names></name></person-group><year>2008</year><article-title>Tubulovesicular structures are a consistent (and unexplained) finding in the brains of humans with prion diseases</article-title><source>Virus Research</source><volume>132</volume><fpage>226</fpage><lpage>228</lpage><pub-id pub-id-type="doi">10.1016/j.virusres.2007.11.008</pub-id></element-citation></ref><ref id="bib63"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Linden</surname><given-names>R</given-names></name><name><surname>Martins</surname><given-names>VR</given-names></name><name><surname>Prado</surname><given-names>MA</given-names></name><name><surname>Cammarota</surname><given-names>M</given-names></name><name><surname>Izquierdo</surname><given-names>I</given-names></name><name><surname>Brentani</surname><given-names>RR</given-names></name></person-group><year>2008</year><article-title>Physiology of the prion protein</article-title><source>Physiological Reviews</source><volume>88</volume><fpage>673</fpage><lpage>728</lpage><pub-id pub-id-type="doi">10.1152/physrev.00007.2007</pub-id></element-citation></ref><ref id="bib64"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mabbott</surname><given-names>NA</given-names></name><name><surname>Mackay</surname><given-names>F</given-names></name><name><surname>Minns</surname><given-names>F</given-names></name><name><surname>Bruce</surname><given-names>ME</given-names></name></person-group><year>2000</year><article-title>Temporary inactivation of follicular dendritic cells delays neuroinvasion of scrapie [letter]</article-title><source>Nature Medicine</source><volume>6</volume><fpage>719</fpage><lpage>720</lpage><pub-id pub-id-type="doi">10.1038/77401</pub-id></element-citation></ref><ref id="bib65"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mallucci</surname><given-names>G</given-names></name><name><surname>Dickinson</surname><given-names>A</given-names></name><name><surname>Linehan</surname><given-names>J</given-names></name><name><surname>Klohn</surname><given-names>PC</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name></person-group><year>2003</year><article-title>Depleting neuronal PrP in prion infection prevents disease and reverses spongiosis</article-title><source>Science</source><volume>302</volume><fpage>871</fpage><lpage>874</lpage><pub-id pub-id-type="doi">10.1126/science.1090187</pub-id></element-citation></ref><ref id="bib66"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mange</surname><given-names>A</given-names></name><name><surname>Beranger</surname><given-names>F</given-names></name><name><surname>Peoc'h</surname><given-names>K</given-names></name><name><surname>Onodera</surname><given-names>T</given-names></name><name><surname>Frobert</surname><given-names>Y</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name></person-group><year>2004</year><article-title>Alpha- and beta- cleavages of the amino-terminus of the cellular prion protein</article-title><source>Biology of the Cell</source><volume>96</volume><fpage>125</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1016/j.biolcel.2003.11.007</pub-id></element-citation></ref><ref id="bib67"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Marella</surname><given-names>M</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name><name><surname>Grassi</surname><given-names>J</given-names></name><name><surname>Chabry</surname><given-names>J</given-names></name></person-group><year>2002</year><article-title>Filipin prevents pathological prion protein accumulation by reducing endocytosis and inducing cellular PrP release</article-title><source>The Journal of Biological Chemistry</source><volume>277</volume><fpage>25457</fpage><lpage>25464</lpage><pub-id pub-id-type="doi">10.1074/jbc.M203248200</pub-id></element-citation></ref><ref id="bib68"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mays</surname><given-names>CE</given-names></name><name><surname>Coomaraswamy</surname><given-names>J</given-names></name><name><surname>Watts</surname><given-names>JC</given-names></name><name><surname>Yang</surname><given-names>J</given-names></name><name><surname>Ko</surname><given-names>KW</given-names></name><name><surname>Strome</surname><given-names>B</given-names></name><name><surname>Mercer</surname><given-names>RC</given-names></name><name><surname>Wohlgemuth</surname><given-names>SL</given-names></name><name><surname>Schmitt-Ulms</surname><given-names>G</given-names></name><name><surname>Westaway</surname><given-names>D</given-names></name></person-group><year>2014</year><article-title>Endoproteolytic processing of the mammalian prion glycoprotein family</article-title><source>The FEBS Journal</source><volume>281</volume><fpage>862</fpage><lpage>876</lpage><pub-id pub-id-type="doi">10.1111/febs.12654</pub-id></element-citation></ref><ref id="bib69"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McDonald</surname><given-names>AJ</given-names></name><name><surname>Dibble</surname><given-names>JP</given-names></name><name><surname>Evans</surname><given-names>EG</given-names></name><name><surname>Millhauser</surname><given-names>GL</given-names></name></person-group><year>2014</year><article-title>A new paradigm for enzymatic control of alpha-cleavage and beta-cleavage of the prion protein</article-title><source>The Journal of Biological Chemistry</source><volume>289</volume><fpage>803</fpage><lpage>813</lpage><pub-id pub-id-type="doi">10.1074/jbc.M113.502351</pub-id></element-citation></ref><ref id="bib70"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Meier</surname><given-names>P</given-names></name><name><surname>Genoud</surname><given-names>N</given-names></name><name><surname>Prinz</surname><given-names>M</given-names></name><name><surname>Maissen</surname><given-names>M</given-names></name><name><surname>Rulicke</surname><given-names>T</given-names></name><name><surname>Zurbriggen</surname><given-names>A</given-names></name><name><surname>Raeber</surname><given-names>AJ</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2003</year><article-title>Soluble dimeric prion protein binds PrP(Sc) in vivo and antagonizes prion disease</article-title><source>Cell</source><volume>113</volume><fpage>49</fpage><lpage>60</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(03)00201-0</pub-id></element-citation></ref><ref id="bib71"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mouillet-Richard</surname><given-names>S</given-names></name><name><surname>Ermonval</surname><given-names>M</given-names></name><name><surname>Chebassier</surname><given-names>C</given-names></name><name><surname>Laplanche</surname><given-names>JL</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name><name><surname>Launay</surname><given-names>JM</given-names></name><name><surname>Kellermann</surname><given-names>O</given-names></name></person-group><year>2000</year><article-title>Signal transduction through prion protein</article-title><source>Science</source><volume>289</volume><fpage>1925</fpage><lpage>1928</lpage><pub-id pub-id-type="doi">10.1126/science.289.5486.1925</pub-id></element-citation></ref><ref id="bib72"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nieznanski</surname><given-names>K</given-names></name><name><surname>Choi</surname><given-names>JK</given-names></name><name><surname>Chen</surname><given-names>S</given-names></name><name><surname>Surewicz</surname><given-names>K</given-names></name><name><surname>Surewicz</surname><given-names>WK</given-names></name></person-group><year>2012</year><article-title>Soluble prion protein inhibits amyloid-beta (Abeta) Fibrillization and toxicity</article-title><source>The Journal of Biological Chemistry</source><volume>287</volume><fpage>33104</fpage><lpage>33108</lpage><pub-id pub-id-type="doi">10.1074/jbc.C112.400614</pub-id></element-citation></ref><ref id="bib73"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Oliveira-Martins</surname><given-names>JB</given-names></name><name><surname>Yusa</surname><given-names>S</given-names></name><name><surname>Calella</surname><given-names>AM</given-names></name><name><surname>Bridel</surname><given-names>C</given-names></name><name><surname>Baumann</surname><given-names>F</given-names></name><name><surname>Dametto</surname><given-names>P</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2010</year><article-title>Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations</article-title><source>PLOS ONE</source><volume>5</volume><fpage>e9107</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0009107</pub-id></element-citation></ref><ref id="bib74"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ostapchenko</surname><given-names>VG</given-names></name><name><surname>Beraldo</surname><given-names>FH</given-names></name><name><surname>Guimaraes</surname><given-names>AL</given-names></name><name><surname>Mishra</surname><given-names>S</given-names></name><name><surname>Guzman</surname><given-names>M</given-names></name><name><surname>Fan</surname><given-names>J</given-names></name><name><surname>Martins</surname><given-names>VR</given-names></name><name><surname>Prado</surname><given-names>VF</given-names></name><name><surname>Prado</surname><given-names>MA</given-names></name></person-group><year>2013</year><article-title>Increased prion protein processing and expression of metabotropic glutamate receptor 1 in a mouse model of Alzheimer's disease</article-title><source>Journal of Neurochemistry</source><volume>127</volume><fpage>415</fpage><lpage>425</lpage><pub-id pub-id-type="doi">10.1111/jnc.12296</pub-id></element-citation></ref><ref id="bib75"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Parizek</surname><given-names>P</given-names></name><name><surname>Roeckl</surname><given-names>C</given-names></name><name><surname>Weber</surname><given-names>J</given-names></name><name><surname>Flechsig</surname><given-names>E</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Raeber</surname><given-names>AJ</given-names></name></person-group><year>2001</year><article-title>Similar turnover and shedding of the cellular prion protein in primary lymphoid and neuronal cells</article-title><source>The Journal of Biological Chemistry</source><volume>276</volume><fpage>44627</fpage><lpage>44632</lpage><pub-id pub-id-type="doi">10.1074/jbc.M107458200</pub-id></element-citation></ref><ref id="bib76"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Parkin</surname><given-names>ET</given-names></name><name><surname>Watt</surname><given-names>NT</given-names></name><name><surname>Turner</surname><given-names>AJ</given-names></name><name><surname>Hooper</surname><given-names>NM</given-names></name></person-group><year>2004</year><article-title>Dual mechanisms for shedding of the cellular prion protein</article-title><source>The Journal of Biological Chemistry</source><volume>279</volume><fpage>11170</fpage><lpage>11178</lpage><pub-id pub-id-type="doi">10.1074/jbc.M312105200</pub-id></element-citation></ref><ref id="bib77"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Perini</surname><given-names>F</given-names></name><name><surname>Vidal</surname><given-names>R</given-names></name><name><surname>Ghetti</surname><given-names>B</given-names></name><name><surname>Tagliavini</surname><given-names>F</given-names></name><name><surname>Frangione</surname><given-names>B</given-names></name><name><surname>Prelli</surname><given-names>F</given-names></name></person-group><year>1996</year><article-title>PrP27-30 is a normal soluble prion protein fragment released by human platelets</article-title><source>Biochemical and Biophysical Research Communications</source><volume>223</volume><fpage>572</fpage><lpage>577</lpage><pub-id pub-id-type="doi">10.1006/bbrc.1996.0936</pub-id></element-citation></ref><ref id="bib78"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Piccardo</surname><given-names>P</given-names></name><name><surname>Manson</surname><given-names>JC</given-names></name><name><surname>King</surname><given-names>D</given-names></name><name><surname>Ghetti</surname><given-names>B</given-names></name><name><surname>Barron</surname><given-names>RM</given-names></name></person-group><year>2007</year><article-title>Accumulation of prion protein in the brain that is not associated with transmissible disease</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>104</volume><fpage>4712</fpage><lpage>4717</lpage><pub-id pub-id-type="doi">10.1073/pnas.0609241104</pub-id></element-citation></ref><ref id="bib79"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Polymenidou</surname><given-names>M</given-names></name><name><surname>Moos</surname><given-names>R</given-names></name><name><surname>Scott</surname><given-names>M</given-names></name><name><surname>Sigurdson</surname><given-names>C</given-names></name><name><surname>Shi</surname><given-names>YZ</given-names></name><name><surname>Yajima</surname><given-names>B</given-names></name><name><surname>Hafner-Bratkovic</surname><given-names>I</given-names></name><name><surname>Jerala</surname><given-names>R</given-names></name><name><surname>Hornemann</surname><given-names>S</given-names></name><name><surname>Wuthrich</surname><given-names>K</given-names></name><name><surname>Bellon</surname><given-names>A</given-names></name><name><surname>Vey</surname><given-names>M</given-names></name><name><surname>Garen</surname><given-names>G</given-names></name><name><surname>James</surname><given-names>MN</given-names></name><name><surname>Kav</surname><given-names>N</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2008</year><article-title>The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes</article-title><source>PLOS ONE</source><volume>3</volume><fpage>e3872</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0003872</pub-id></element-citation></ref><ref id="bib80"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pradines</surname><given-names>E</given-names></name><name><surname>Hernandez-Rapp</surname><given-names>J</given-names></name><name><surname>Villa-Diaz</surname><given-names>A</given-names></name><name><surname>Dakowski</surname><given-names>C</given-names></name><name><surname>Ardila-Osorio</surname><given-names>H</given-names></name><name><surname>Haik</surname><given-names>S</given-names></name><name><surname>Schneider</surname><given-names>B</given-names></name><name><surname>Launay</surname><given-names>JM</given-names></name><name><surname>Kellermann</surname><given-names>O</given-names></name><name><surname>Torres</surname><given-names>JM</given-names></name><name><surname>Mouillet-Richard</surname><given-names>S</given-names></name></person-group><year>2013</year><article-title>Pathogenic prions deviate PrP(C) signaling in neuronal cells and impair A-beta clearance</article-title><source>Cell Death & Disease</source><volume>4</volume><fpage>e456</fpage><pub-id pub-id-type="doi">10.1038/cddis.2012.195</pub-id></element-citation></ref><ref id="bib81"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pressmar</surname><given-names>S</given-names></name><name><surname>Ader</surname><given-names>M</given-names></name><name><surname>Richard</surname><given-names>G</given-names></name><name><surname>Schachner</surname><given-names>M</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name></person-group><year>2001</year><article-title>The fate of heterotopically grafted neural precursor cells in the normal and dystrophic adult mouse retina</article-title><source>Investigative Ophthalmology & Visual Science</source><volume>42</volume><fpage>3311</fpage><lpage>3319</lpage></element-citation></ref><ref id="bib82"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Prinz</surname><given-names>M</given-names></name><name><surname>Heikenwalder</surname><given-names>M</given-names></name><name><surname>Junt</surname><given-names>T</given-names></name><name><surname>Schwarz</surname><given-names>P</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name><name><surname>Heppner</surname><given-names>FL</given-names></name><name><surname>Fu</surname><given-names>YX</given-names></name><name><surname>Lipp</surname><given-names>M</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2003</year><article-title>Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion</article-title><source>Nature</source><volume>425</volume><fpage>957</fpage><lpage>962</lpage><pub-id pub-id-type="doi">10.1038/nature02072</pub-id></element-citation></ref><ref id="bib83"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Prox</surname><given-names>J</given-names></name><name><surname>Bernreuther</surname><given-names>C</given-names></name><name><surname>Altmeppen</surname><given-names>H</given-names></name><name><surname>Grendel</surname><given-names>J</given-names></name><name><surname>Glatzel</surname><given-names>M</given-names></name><name><surname>D'Hooge</surname><given-names>R</given-names></name><name><surname>Stroobants</surname><given-names>S</given-names></name><name><surname>Ahmed</surname><given-names>T</given-names></name><name><surname>Balschun</surname><given-names>D</given-names></name><name><surname>Willem</surname><given-names>M</given-names></name><name><surname>Lammich</surname><given-names>S</given-names></name><name><surname>Isbrandt</surname><given-names>D</given-names></name><name><surname>Schweizer</surname><given-names>M</given-names></name><name><surname>Horré</surname><given-names>K</given-names></name><name><surname>De Strooper</surname><given-names>B</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Postnatal disruption of the Disintegrin/Metalloproteinase ADAM10 in brain causes epileptic seizures, learning deficits, altered spine morphology, and defective synaptic functions</article-title><source>The Journal of Neuroscience</source><volume>33</volume><fpage>12915</fpage><lpage>12928</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.5910-12.2013</pub-id></element-citation></ref><ref id="bib84"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Prusiner</surname><given-names>SB</given-names></name></person-group><year>1982</year><article-title>Novel proteinaceous infectious particles cause scrapie</article-title><source>Science</source><volume>216</volume><fpage>136</fpage><lpage>144</lpage><pub-id pub-id-type="doi">10.1126/science.6801762</pub-id></element-citation></ref><ref id="bib85"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Resenberger</surname><given-names>UK</given-names></name><name><surname>Harmeier</surname><given-names>A</given-names></name><name><surname>Woerner</surname><given-names>AC</given-names></name><name><surname>Goodman</surname><given-names>JL</given-names></name><name><surname>Muller</surname><given-names>V</given-names></name><name><surname>Krishnan</surname><given-names>R</given-names></name><name><surname>Vabulas</surname><given-names>RM</given-names></name><name><surname>Kretzschmar</surname><given-names>HA</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name><name><surname>Hartl</surname><given-names>FU</given-names></name><name><surname>Multhaup</surname><given-names>G</given-names></name><name><surname>Winklhofer</surname><given-names>KF</given-names></name><name><surname>Tatzelt</surname><given-names>J</given-names></name></person-group><year>2011</year><article-title>The cellular prion protein mediates neurotoxic signalling of beta-sheet-rich conformers independent of prion replication</article-title><source>The EMBO Journal</source><volume>30</volume><fpage>2057</fpage><lpage>2070</lpage><pub-id pub-id-type="doi">10.1038/emboj.2011.86</pub-id></element-citation></ref><ref id="bib86"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Richard</surname><given-names>I</given-names></name><name><surname>Ader</surname><given-names>M</given-names></name><name><surname>Sytnyk</surname><given-names>V</given-names></name><name><surname>Dityatev</surname><given-names>A</given-names></name><name><surname>Richard</surname><given-names>G</given-names></name><name><surname>Schachner</surname><given-names>M</given-names></name><name><surname>Bartsch</surname><given-names>U</given-names></name></person-group><year>2005</year><article-title>Electroporation-based gene transfer for efficient transfection of neural precursor cells</article-title><source>Brain Research Molecular Brain Research</source><volume>138</volume><fpage>182</fpage><lpage>190</lpage><pub-id pub-id-type="doi">10.1016/j.molbrainres.2005.04.010</pub-id></element-citation></ref><ref id="bib87"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sandberg</surname><given-names>MK</given-names></name><name><surname>Al-Doujaily</surname><given-names>H</given-names></name><name><surname>Sharps</surname><given-names>B</given-names></name><name><surname>Clarke</surname><given-names>AR</given-names></name><name><surname>Collinge</surname><given-names>J</given-names></name></person-group><year>2011</year><article-title>Prion propagation and toxicity in vivo occur in two distinct mechanistic phases</article-title><source>Nature</source><volume>470</volume><fpage>540</fpage><lpage>542</lpage><pub-id pub-id-type="doi">10.1038/nature09768</pub-id></element-citation></ref><ref id="bib88"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schneider</surname><given-names>B</given-names></name><name><surname>Mutel</surname><given-names>V</given-names></name><name><surname>Pietri</surname><given-names>M</given-names></name><name><surname>Ermonval</surname><given-names>M</given-names></name><name><surname>Mouillet-Richard</surname><given-names>S</given-names></name><name><surname>Kellermann</surname><given-names>O</given-names></name></person-group><year>2003</year><article-title>NADPH oxidase and extracellular regulated kinases 1/2 are targets of prion protein signaling in neuronal and nonneuronal cells</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>100</volume><fpage>13326</fpage><lpage>13331</lpage><pub-id pub-id-type="doi">10.1073/pnas.2235648100</pub-id></element-citation></ref><ref id="bib89"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Schönbeck</surname><given-names>U</given-names></name><name><surname>Libby</surname><given-names>P</given-names></name></person-group><year>2001</year><article-title>The CD40/CD154 receptor/ligand dyad</article-title><source>Cellular and Molecular Life Sciences</source><volume>58</volume><fpage>4</fpage><lpage>43</lpage><pub-id pub-id-type="doi">10.1007/PL00000776</pub-id></element-citation></ref><ref id="bib90"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shearin</surname><given-names>H</given-names></name><name><surname>Bessen</surname><given-names>RA</given-names></name></person-group><year>2014</year><article-title>Axonal and transynaptic spread of prions</article-title><source>Journal of Virology</source><volume>88</volume><fpage>8640</fpage><lpage>8655</lpage><pub-id pub-id-type="doi">10.1128/JVI.00378-14</pub-id></element-citation></ref><ref id="bib91"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shmerling</surname><given-names>D</given-names></name><name><surname>Hegyi</surname><given-names>I</given-names></name><name><surname>Fischer</surname><given-names>M</given-names></name><name><surname>Blattler</surname><given-names>T</given-names></name><name><surname>Brandner</surname><given-names>S</given-names></name><name><surname>Gotz</surname><given-names>J</given-names></name><name><surname>Rulicke</surname><given-names>T</given-names></name><name><surname>Flechsig</surname><given-names>E</given-names></name><name><surname>Cozzio</surname><given-names>A</given-names></name><name><surname>von Mering</surname><given-names>C</given-names></name><name><surname>Hangartner</surname><given-names>C</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name><name><surname>Weissmann</surname><given-names>C</given-names></name></person-group><year>1998</year><article-title>Expression of amino-terminally truncated PrP in the mouse leading to ataxia and specific cerebellar lesions</article-title><source>Cell</source><volume>93</volume><fpage>203</fpage><lpage>214</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)81572-X</pub-id></element-citation></ref><ref id="bib92"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Silveira</surname><given-names>JR</given-names></name><name><surname>Raymond</surname><given-names>GJ</given-names></name><name><surname>Hughson</surname><given-names>AG</given-names></name><name><surname>Race</surname><given-names>RE</given-names></name><name><surname>Sim</surname><given-names>VL</given-names></name><name><surname>Hayes</surname><given-names>SF</given-names></name><name><surname>Caughey</surname><given-names>B</given-names></name></person-group><year>2005</year><article-title>The most infectious prion protein particles</article-title><source>Nature</source><volume>437</volume><fpage>257</fpage><lpage>261</lpage><pub-id pub-id-type="doi">10.1038/nature03989</pub-id></element-citation></ref><ref id="bib93"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sonati</surname><given-names>T</given-names></name><name><surname>Reimann</surname><given-names>RR</given-names></name><name><surname>Falsig</surname><given-names>J</given-names></name><name><surname>Baral</surname><given-names>PK</given-names></name><name><surname>O'Connor</surname><given-names>T</given-names></name><name><surname>Hornemann</surname><given-names>S</given-names></name><name><surname>Yaganoglu</surname><given-names>S</given-names></name><name><surname>Li</surname><given-names>B</given-names></name><name><surname>Herrmann</surname><given-names>US</given-names></name><name><surname>Wieland</surname><given-names>B</given-names></name><name><surname>Swayampakula</surname><given-names>M</given-names></name><name><surname>Rahman</surname><given-names>MH</given-names></name><name><surname>Das</surname><given-names>D</given-names></name><name><surname>Kav</surname><given-names>N</given-names></name><name><surname>Riek</surname><given-names>R</given-names></name><name><surname>Liberski</surname><given-names>PP</given-names></name><name><surname>James</surname><given-names>MN</given-names></name><name><surname>Aguzzi</surname><given-names>A</given-names></name></person-group><year>2013</year><article-title>The toxicity of antiprion antibodies is mediated by the flexible tail of the prion protein</article-title><source>Nature</source><volume>501</volume><fpage>102</fpage><lpage>106</lpage><pub-id pub-id-type="doi">10.1038/nature12402</pub-id></element-citation></ref><ref id="bib94"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stöhr</surname><given-names>J</given-names></name><name><surname>Watts</surname><given-names>JC</given-names></name><name><surname>Legname</surname><given-names>G</given-names></name><name><surname>Oehler</surname><given-names>A</given-names></name><name><surname>Lemus</surname><given-names>A</given-names></name><name><surname>Nguyen</surname><given-names>HO</given-names></name><name><surname>Sussman</surname><given-names>J</given-names></name><name><surname>Wille</surname><given-names>H</given-names></name><name><surname>DeArmond</surname><given-names>SJ</given-names></name><name><surname>Prusiner</surname><given-names>SB</given-names></name><name><surname>Giles</surname><given-names>K</given-names></name></person-group><year>2011</year><article-title>Spontaneous generation of anchorless prions in transgenic mice</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>108</volume><fpage>21223</fpage><lpage>21228</lpage><pub-id pub-id-type="doi">10.1073/pnas.1117827108</pub-id></element-citation></ref><ref id="bib95"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tagliavini</surname><given-names>F</given-names></name><name><surname>Prelli</surname><given-names>F</given-names></name><name><surname>Porro</surname><given-names>M</given-names></name><name><surname>Salmona</surname><given-names>M</given-names></name><name><surname>Bugiani</surname><given-names>O</given-names></name><name><surname>Frangione</surname><given-names>B</given-names></name></person-group><year>1992</year><article-title>A soluble form of prion protein in human cerebrospinal fluid: implications for prion-related encephalopathies</article-title><source>Biochemical and Biophysical Research Communications</source><volume>184</volume><fpage>1398</fpage><lpage>1404</lpage><pub-id pub-id-type="doi">10.1016/S0006-291X(05)80038-5</pub-id></element-citation></ref><ref id="bib96"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taylor</surname><given-names>DR</given-names></name><name><surname>Parkin</surname><given-names>ET</given-names></name><name><surname>Cocklin</surname><given-names>SL</given-names></name><name><surname>Ault</surname><given-names>JR</given-names></name><name><surname>Ashcroft</surname><given-names>AE</given-names></name><name><surname>Turner</surname><given-names>AJ</given-names></name><name><surname>Hooper</surname><given-names>NM</given-names></name></person-group><year>2009</year><article-title>Role of ADAMs in the ectodomain shedding and conformational conversion of the prion protein</article-title><source>The Journal of Biological Chemistry</source><volume>284</volume><fpage>22590</fpage><lpage>22600</lpage><pub-id pub-id-type="doi">10.1074/jbc.M109.032599</pub-id></element-citation></ref><ref id="bib97"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Turnbaugh</surname><given-names>JA</given-names></name><name><surname>Unterberger</surname><given-names>U</given-names></name><name><surname>Saa</surname><given-names>P</given-names></name><name><surname>Massignan</surname><given-names>T</given-names></name><name><surname>Fluharty</surname><given-names>BR</given-names></name><name><surname>Bowman</surname><given-names>FP</given-names></name><name><surname>Miller</surname><given-names>MB</given-names></name><name><surname>Supattapone</surname><given-names>S</given-names></name><name><surname>Biasini</surname><given-names>E</given-names></name><name><surname>Harris</surname><given-names>DA</given-names></name></person-group><year>2012</year><article-title>The N-terminal, polybasic region of PrP(C) dictates the efficiency of prion propagation by binding to PrP(Sc)</article-title><source>The Journal of Neuroscience</source><volume>32</volume><fpage>8817</fpage><lpage>8830</lpage><pub-id pub-id-type="doi">10.1523/JNEUROSCI.1103-12.2012</pub-id></element-citation></ref><ref id="bib98"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tveit</surname><given-names>H</given-names></name><name><surname>Lund</surname><given-names>C</given-names></name><name><surname>Olsen</surname><given-names>CM</given-names></name><name><surname>Ersdal</surname><given-names>C</given-names></name><name><surname>Prydz</surname><given-names>K</given-names></name><name><surname>Harbitz</surname><given-names>I</given-names></name><name><surname>Tranulis</surname><given-names>MA</given-names></name></person-group><year>2005</year><article-title>Proteolytic processing of the ovine prion protein in cell cultures</article-title><source>Biochemical and Biophysical Research Communications</source><volume>337</volume><fpage>232</fpage><lpage>240</lpage><pub-id pub-id-type="doi">10.1016/j.bbrc.2005.09.031</pub-id></element-citation></ref><ref id="bib99"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Um</surname><given-names>JW</given-names></name><name><surname>Nygaard</surname><given-names>HB</given-names></name><name><surname>Heiss</surname><given-names>JK</given-names></name><name><surname>Kostylev</surname><given-names>MA</given-names></name><name><surname>Stagi</surname><given-names>M</given-names></name><name><surname>Vortmeyer</surname><given-names>A</given-names></name><name><surname>Wisniewski</surname><given-names>T</given-names></name><name><surname>Gunther</surname><given-names>EC</given-names></name><name><surname>Strittmatter</surname><given-names>SM</given-names></name></person-group><year>2012</year><article-title>Alzheimer amyloid-beta oligomer bound to postsynaptic prion protein activates Fyn to impair neurons</article-title><source>Nature Neuroscience</source><volume>15</volume><fpage>1227</fpage><lpage>1235</lpage><pub-id pub-id-type="doi">10.1038/nn.3178</pub-id></element-citation></ref><ref id="bib100"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vincent</surname><given-names>B</given-names></name><name><surname>Paitel</surname><given-names>E</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name><name><surname>Frobert</surname><given-names>Y</given-names></name><name><surname>Hartmann</surname><given-names>D</given-names></name><name><surname>De Strooper</surname><given-names>B</given-names></name><name><surname>Grassi</surname><given-names>J</given-names></name><name><surname>Lopez-Perez</surname><given-names>E</given-names></name><name><surname>Checler</surname><given-names>F</given-names></name></person-group><year>2001</year><article-title>The disintegrins ADAM10 and TACE contribute to the constitutive and phorbol ester-regulated normal cleavage of the cellular prion protein</article-title><source>The Journal of Biological Chemistry</source><volume>276</volume><fpage>37743</fpage><lpage>37746</lpage><pub-id pub-id-type="doi">10.1074/jbc.M003965200</pub-id></element-citation></ref><ref id="bib101"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weber</surname><given-names>S</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name></person-group><year>2012</year><article-title>Ectodomain shedding and ADAMs in development</article-title><source>Development</source><volume>139</volume><fpage>3693</fpage><lpage>3709</lpage><pub-id pub-id-type="doi">10.1242/dev.076398</pub-id></element-citation></ref><ref id="bib102"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weber</surname><given-names>S</given-names></name><name><surname>Wetzel</surname><given-names>S</given-names></name><name><surname>Prox</surname><given-names>J</given-names></name><name><surname>Lehmann</surname><given-names>T</given-names></name><name><surname>Schneppenheim</surname><given-names>J</given-names></name><name><surname>Donners</surname><given-names>M</given-names></name><name><surname>Saftig</surname><given-names>P</given-names></name></person-group><year>2013</year><article-title>Regulation of adult hematopoiesis by the a disintegrin and metalloproteinase 10 (ADAM10)</article-title><source>Biochemical and Biophysical Research Communications</source><volume>442</volume><fpage>234</fpage><lpage>241</lpage><pub-id pub-id-type="doi">10.1016/j.bbrc.2013.11.020</pub-id></element-citation></ref><ref id="bib103"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Westergard</surname><given-names>L</given-names></name><name><surname>Turnbaugh</surname><given-names>JA</given-names></name><name><surname>Harris</surname><given-names>DA</given-names></name></person-group><year>2011</year><article-title>A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation</article-title><source>The Journal of Biological Chemistry</source><volume>286</volume><fpage>44234</fpage><lpage>44242</lpage><pub-id pub-id-type="doi">10.1074/jbc.M111.286195</pub-id></element-citation></ref><ref id="bib104"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wik</surname><given-names>L</given-names></name><name><surname>Klingeborn</surname><given-names>M</given-names></name><name><surname>Willander</surname><given-names>H</given-names></name><name><surname>Linne</surname><given-names>T</given-names></name></person-group><year>2012</year><article-title>Separate mechanisms act concurrently to shed and release the prion protein from the cell</article-title><source>Prion</source><volume>6</volume><fpage>498</fpage><lpage>509</lpage><pub-id pub-id-type="doi">10.4161/pri.22588</pub-id></element-citation></ref><ref id="bib105"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yang</surname><given-names>P</given-names></name><name><surname>Baker</surname><given-names>KA</given-names></name><name><surname>Hagg</surname><given-names>T</given-names></name></person-group><year>2006</year><article-title>The ADAMs family: coordinators of nervous system development, plasticity and repair</article-title><source>Progress in Neurobiology</source><volume>79</volume><fpage>73</fpage><lpage>94</lpage><pub-id pub-id-type="doi">10.1016/j.pneurobio.2006.05.001</pub-id></element-citation></ref><ref id="bib106"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yuan</surname><given-names>J</given-names></name><name><surname>Zhan</surname><given-names>YA</given-names></name><name><surname>Abskharon</surname><given-names>R</given-names></name><name><surname>Xiao</surname><given-names>X</given-names></name><name><surname>Martinez</surname><given-names>MC</given-names></name><name><surname>Zhou</surname><given-names>X</given-names></name><name><surname>Kneale</surname><given-names>G</given-names></name><name><surname>Mikol</surname><given-names>J</given-names></name><name><surname>Lehmann</surname><given-names>S</given-names></name><name><surname>Surewicz</surname><given-names>WK</given-names></name><name><surname>Castilla</surname><given-names>J</given-names></name><name><surname>Steyaert</surname><given-names>J</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Kong</surname><given-names>Q</given-names></name><name><surname>Petersen</surname><given-names>RB</given-names></name><name><surname>Wohlkonig</surname><given-names>A</given-names></name><name><surname>Zou</surname><given-names>WQ</given-names></name></person-group><year>2013</year><article-title>Recombinant human prion protein inhibits prion propagation in vitro</article-title><source>Scientific Reports</source><volume>3</volume><fpage>2911</fpage><pub-id pub-id-type="doi">10.1038/srep02911</pub-id></element-citation></ref><ref id="bib107"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yusa</surname><given-names>S</given-names></name><name><surname>Oliveira-Martins</surname><given-names>JB</given-names></name><name><surname>Sugita-Konishi</surname><given-names>Y</given-names></name><name><surname>Kikuchi</surname><given-names>Y</given-names></name></person-group><year>2012</year><article-title>Cellular prion protein: from physiology to pathology</article-title><source>Viruses</source><volume>4</volume><fpage>3109</fpage><lpage>3131</lpage><pub-id pub-id-type="doi">10.3390/v4113109</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04260.020</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>De Strooper</surname><given-names>Bart</given-names></name><role>Reviewing editor</role><aff><institution>VIB Center for the Biology of Disease, KU Leuven</institution>, <country>Belgium</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “The sheddase ADAM10 is a potent modulator of prion disease” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Randy Schekman (Senior editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>Overall all three referees agree that this work is interesting and is in principle good material for <italic>eLife</italic>. However the work needs an in depth revision with regard to the conclusions made as they are not all supported by the provided data. Also rewriting the Results section and reorganisation of the way the data are presented is needed before the manuscript can be accepted. The referees are also concerned about the low number of animals used to support some of the conclusions.</p><p>Below follow the list of substantial comments that need to be fully addressed in a revised manuscript. We also urge the authors to address systematically the many other concerns of the referees as those will help to improve considerably this potentially very interesting manuscript.</p><p>Substantial comments:</p><p>1) The most important conclusion drawn by the authors is that suppression of PrP shedding by ADAM10 shortens the incubation time of the disease by ca. 30%, and therefore shedding of PrP by ADAM10 puts a brake on prion pathogenesis. This interpretation may be valid, but ADAM10 deficiency is highly pathogenic by itself, and the authors say that they couldn't even perform the experiment in CNS-specific conditional mutants because of embryonic lethality. It is therefore plausible (and indeed likely) that the accelerated death of prion-inoculated A10 cKO mice results nonspecifically from the combined pathology of prion infection plus absence of ADAM10. This possibility is rendered even more likely by the titer determinations which failed to demonstrate any difference in prion replication between A10 cKO and wt mice. The above is not necessarily a killer of this well-conducted study, but these alternative interpretations must by all means be discussed.</p><p>2) The claim that soluble PrP<sup>C</sup> (generated by ADAM10) explains the spreading of the disease in the <italic>tga20</italic> (and the absence in ADAM) is only based on at best correlative evidence: the alleged reduced pathology in the ADAM may be due to reduced prion spread, but it may also pertain to secondary effects unrelated to prion spread (or to prion pathogenesis altogether), such as reduced processing of proinflammatory molecules, or impairment of neuron-microglia interaction by the lack of ADAM10. The authors have not directly investigated whether prion spread is modified in their mice (an admittedly difficult task, though not impossible). A knockout of ADAM10 in the <italic>tga20</italic> mice would allow investigating more precisely the role of cleaved PrP<sup>C</sup> in the spreading of the disease in these mice. It might also be possible to inject /transduce /transgenically express soluble PrP<sup>C</sup> in the ADAM10 cKO and rescue the spreading of the disease. As these experiments require substantial time, the authors might alternatively reconsider this conclusion and either tone down the interpretation of these findings, or at least mention the many possible alternative explanations.</p><p>3) In the Results section related to <xref ref-type="fig" rid="fig1">Figure 1 A and B</xref>, while mentioned in the Methods, the control for reintroducing ADAM10 cDNA into ADAM10 knockout cell lines (Lenti virus lacking ADAM10 cDNA) is not shown. It is important to show that the decrease in PrP<sup>C</sup> signal is independent of the virus treatment itself. It would be desirable to have n>2 for quantification of surface PrP<sup>C</sup> expression in A10 cKO+ADAM10.</p><p>4) The number of mice used for the crucial experiments in <xref ref-type="fig" rid="fig7">Figure 7A</xref> (incubation times) is meagre. I realize that there is little that can be done about this problem at this stage, but the authors would be well-advised to use numbers of mice appropriate for sufficient statistical power. The same applies to the titer determinations in <italic>tga20</italic> mice: just four recipient mice for each titration is pathetically low and will by necessity undermine the precision of the determination.</p><p>Other comments:</p><p><italic>Reviewer #1:</italic></p><p>1) In the Abstract a conclusion is made with regard to treatment options for other neurodegenerative diseases (apart from prion). In the absence of any data, I suggest to remove this claim from the Abstract and only mention this in the Discussion as an interesting speculation.</p><p>2) <xref ref-type="fig" rid="fig1 fig2">Figure 1 and 2</xref>: the paper contains a whole series of experiments (covering two full figures) to proof that in the absence of ADAM 10, prion expression is increased and shedding is decreased. I believe that these data should be summarized in one figure, especially because this is not entirely novel. Moreover, instead of many different data it is better to have a couple of experiments fully documented. For instance it is not clear what was quantified in Figure Panel 1A and this should be complemented with the blot or the figure on which the quantitation is based. Panel 1C should include for the ADAM10 KO a similar analysis as for the control with regard to PrP surface staining. In <xref ref-type="fig" rid="fig2">Figure 2B</xref>: the immunohistochemistry shows a particular strong staining in the mouse brains of the ADAM cKO, more than what is seen in the western blot and expression is very diffuse over all cells (while the ko is only in a subset of neurons). Additional controls e.g. immunostaining of the cerebellum and more precise details of the cellular stainings would help to understand this better and to demonstrate for instance that the slides of wild type and knock out were incubated for similar length of time. It is also important to show that PrP accumulation is only seen in the neurons where ADAM10 was knocked out, as this is an important point also with regard to the conclusions with regard to the propagation of the disease.</p><p>3) The very strong conclusion in the second paragraph of the Discussion: “and show for the first time that PrP<sup>C</sup> and ADAM10 co-localize at the plasma membrane” is based on a single piece of weak evidence (inset in Panel 1C). The resolution of this type of experiment is insufficient to make such a strong claim.</p><p>4) In the ninth paragraph of the Results section: “Taken together, these data indicate that at terminal prion disease, lack of PrP<sup>C</sup>-shedding does not influence local PrP<sup>Sc</sup> distribution while it does impact the appearance of TVS”. This observation is mentioned here, and not further discussed or explained. Why mention it?</p><p>5) <xref ref-type="fig" rid="fig5">Figure 5A</xref>: I find it puzzling that there is such a contrast in the accumulation of proteinase K resistant species in the ADAM10 ko versus the <italic>tga20</italic> mouse? In principle both animals have more uncleaved prion protein on the cell membrane. Thus the main difference is the shed prion in the <italic>tga20</italic> mouse. Would one conclude from this that the shed prion inhibits the generation of proteinase K resistant forms of the PrP<sup>Sc</sup>? As also indicated above, I think that it is necessary to study the ADAM10 KO effect in the <italic>tga20</italic> mouse to allow making real conclusions with regard to the precise effect of ADAM10.</p><p>6) <xref ref-type="fig" rid="fig5">Figure 5D</xref>: The activation of calpain is linked to membrane pores in the text. I suppose that it is linked to Calcium influx? This needs to be better formulated.</p><p><italic>Reviewer #2:</italic></p><p>1) In its current form the manuscript has a few areas of weakness that need to be improved. In particular, throughout the manuscript the PrP<sup>C</sup> and PrP<sup>Sc</sup> levels, the incubation periods, and the spongiosis levels are compared between different mouse lines (A10 cKO, wild-type, <italic>tga20</italic>), different time points (65d, 95d), and different brain regions (forebrain, cerebellum, whole brain), but at present these comparisons are a bit haphazard. It would be great to include a table to assemble all these scattered data into one location. This table would make it easier for the reader to keep track of these differences and better follow the arguments the manuscript makes.</p><p>2) Throughout the manuscript a comma is used as the decimal mark. For proper English a full stop should be used instead.</p><p>3) In several places the A10 cKO results are compared with those that were obtained with mice expressing anchorless PrP<sup>C</sup> (e.g. at the end of the third paragraph of the Introduction, eighth paragraph of the Discussion, etc.). At present the comparison is limited to an anchorless PrP<sup>C</sup> low-expressor mouse line (<xref ref-type="bibr" rid="bib28">Chesebro et al., 2005</xref>), but it would be desirable to also include high-expressor results into this discussion (Stohr et al., 2011). Particularly, since the latter animals express PrP at levels closer to the mouse lines that are used in this study.</p><p>4) The first paragraph of the Results section describes results from a complete ADAM10 knockout mouse line, according to the figure legend the corresponding figures (<xref ref-type="fig" rid="fig1">Figure 1C and D</xref>) show only A10 cKO data. This mismatch needs to be cleared up.</p><p>5) It is difficult to judge the alleged co-localization of PrP<sup>C</sup> and ADAM10 (<xref ref-type="fig" rid="fig1">Figure 1C</xref>), since the magnification is so low. Higher resolution images would be needed to make this a convincing argument.</p><p>6) The electron micrographs in <xref ref-type="fig" rid="fig4">Figure 4D</xref> show tubulovesicular structures (TVS), which are claimed to be increased in diseased A10 cKO mice. It would be worthwhile to document and quantify this increase, since TVS and their link to prion disease are controversial. Ultimately, the A10 cKO mice may help to demystify these structures and their role in the prion diseases.</p><p>7) The comparison of PrP<sup>Sc</sup> levels between different mouse lines is solely based on PK-resistance (<xref ref-type="fig" rid="fig5">Figure 5</xref>). While this surrogate marker is widely used to estimate prion infectivity levels, the possible presence of protease-sensitive forms of PrP<sup>Sc</sup> should be taken into consideration and discussed.</p><p>8) In the conclusions, ADAM10 is being promoted as a potential therapeutic target for prion diseases. The finding that a reduction of ADAM10 leads to a shortening of the incubation period, while its normal function increases the spreading of PrP<sup>Sc</sup>, suggests a no-win scenario. Hence, a more refined and discerning discussion would be needed to support such a statement.</p><p>9) Lastly, grammatical errors can be found all the way through the manuscript, a thorough proofreading would benefit the readability of the text.</p><p><italic>Reviewer #3:</italic></p><p>1) In <xref ref-type="fig" rid="fig4">Figure 4A</xref>, WB analysis of protein expression was compared only in frontal brain homogenates. This needs to be controlled by including whole brain homogenates as well.</p><p>2) In panel C, the GFAP signal of CD1 mock ADAM10 cKO seems higher than the control. Is this image representative? If yes, is there an explanation for this?</p><p>3) In panel D: How was the quantification done and what is the relevance of higher numbers of tubulovesicular structures in ADAM10 cKO? Does this bear relevance for the paper at all?</p><p>4) Results related to <xref ref-type="fig" rid="fig5">Figure 5</xref>: It would be important to show that the increase of PrP<sup>Sc</sup> levels in forebrain homogenates of ADAM10 cKO is comparable to the overall PrP<sup>Sc</sup> content of wild type mouse brains. This could underline that the difference resides in the lack of shedding and spreading of PrP<sup>Sc</sup>, leading to local accumulation of PrP<sup>Sc</sup> in ADAM10 cKO. For this, whole brain homogenates should be compared.</p><p>5) As for the results shown in <xref ref-type="fig" rid="fig6">Figure 6</xref>, the comparison of infectivity tests of non-forebrain regions of control and ADAM10 cKO would be important, as it could identify any infectious but non-toxic PrP subpopulations which still might leave PrP<sup>Sc</sup> aggregates in ADAM10 cKO.</p><p>6) Discussion: The authors state that PrP “is a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer's disease”. This is a highly controversial contention propagated by Dr. Strittmatter, and there is more than half-dozen papers out there showing that this idea is, at best, limited to certain animal models. It is the authors' privilege to choose what they wish to believe, and I understand that the charitable interpretation of the Strittmatter paper might add to the perceived importance of the current study, but in fairness they should at least mention the evidence against PrP<sup>C</sup> being the Aβ receptor.</p><p>7) One problematic point is the following: It is stated that ADAM10 cKO mice show massive neurodegeneration in the forebrain, but not in the cerebellum and brainstem, while controls including <italic>tga20</italic> show diffuse neurodegeneration. If this is really true, how can one explain completely identical clinical phenotypes?</p><p>8) As for <xref ref-type="fig" rid="fig5">Figure 5</xref>, the total amount of calpain is most certainly irrelevant to neurodegeneration, since calpains are mostly inactive in the absence of raised intracellular calcium. What matters is the amount of activated calpains. This can be assessed by investigating the amount of cleavage of a calpain substrate, such as spectrin (alpha-fodrin). The mere detection of calpains by Western blot is irrelevant. The whole argument about calpains needs to be revisited by activity measurements, or else it should be deleted in toto. This becomes painfully evident by the authors' finding that on the one hand there is prion toxicity (allegedly through calpain) with profound neurodegeneration in the forebrain of ADAM10 cKO, but on the other hand the significantly faster disease in <italic>tga20</italic> mice goes along with significantly less calpain Also, a comparison of overall (not only forebrain) calpain activity levels to mock inoculated animals could help.</p><p>9) When mentioning the possible inhibition of PrP<sup>Sc</sup> formation by shedded PrP<sup>C</sup>, it would be desirable to prove this statement with PMCA + shed PrP<sup>C</sup>.</p><p>10) In <xref ref-type="fig" rid="fig1">Figure 1E</xref>, POM2 is named as the applied antibody, while the Methods section describes POM1 for this purpose.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.04260.021</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) The most important conclusion drawn by the authors is that suppression of PrP shedding by ADAM10 shortens the incubation time of the disease by ca. 30%, and therefore shedding of PrP by ADAM10 puts a brake on prion pathogenesis. This interpretation may be valid, but ADAM10 deficiency is highly pathogenic by itself, and the authors say that they couldn't even perform the experiment in CNS-specific conditional mutants because of embryonic lethality. It is therefore plausible (and indeed likely) that the accelerated death of prion-inoculated A10 cKO mice results nonspecifically from the combined pathology of prion infection plus absence of ADAM10. This possibility is rendered even more likely by the titer determinations which failed to demonstrate any difference in prion replication between A10 cKO and wt mice. The above is not necessarily a killer of this well-conducted study, but these alternative interpretations must by all means be discussed</italic>.</p><p>The mice we used for the in vivo experiments are CNS-specific conditional mutants with Cre-mediated deletion of ADAM10 occurring in forebrain neurons under the control of the <italic>Camk2a</italic> promoter. These mice display an almost normal life span once they have survived a critical weaning period, additionally they do not show any gross morphological abnormalities in the CNS (Prox et al., 2013a).</p><p>The ADAM10 KO mice showing perinatal lethality, the reviewing editor is referring to, were only used for the ex vivo experiments shown in <xref ref-type="fig" rid="fig1">Figure 1</xref> (determination of PrP surface levels in neuronally differentiated neural stem cells (<xref ref-type="fig" rid="fig1">Figure 1A and B</xref>) and analyses of PrP-shedding in primary neurons derived from embryonal day 14 mice (<xref ref-type="fig" rid="fig1">Figure 1E</xref>). In these mice, ADAM10 is deleted under the control of the Nestin promoter with ADAM10 deficiency occurring in progenitor cell types present in the neuroectoderm, the developing mesonephros, and the somites (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>). We have clarified this in the first paragraph of the Results section in the manuscript:</p><p>“To further investigate this we generated neural stem cells (NSCs) from embryonic day (E) 14 wild-type and ADAM10 conditional knockout mice with an inactivation of the <italic>Adam10</italic> gene in neuroectodermal progenitor cells (<sup>Nestin</sup>A10 KO mice) (<xref ref-type="bibr" rid="bib46">Jorissen et al., 2010</xref>).”</p><p>Nevertheless, we agree with the reviewing editor that effects on prion disease acceleration caused by lack of ADAM10 in forebrain neurons (<sup><italic>Camk2a</italic></sup>ADAM10 cKO) may not solely be caused by lack of ADAM10-mediated PrP shedding and increased neuronal PrP<sup>C</sup> levels. We have thus introduced a text fragment in the fourth paragraph of the Discussion section where we discuss this possibility in detail, with special emphasis on potential contribution by ADAM10 substrates of the immune system:</p><p>“We considered the possibility that non-PrP dependent effects resulting from ADAM10 deficiency may have contributed to disease acceleration. […] Finally, we consider that increased PrP<sup>Sc</sup> levels found in A10 cKO mice strongly argue in favor of PrP-dependent effects of ADAM10 depletion.”</p><p>Regarding the observed identical prion titer, we would like to clarify that titers of prion infectivity and presence of potentially neurotoxic PrP conformers are rather not congruent. In fact, a growing body of published reports indicates a clear dissociation between these. We have clarified and referenced this in paragraph seven of the Discussion section. As suggested by Reviewer #2 (point 7), we have also checked for the presence of aberrant prion species/fragments that could contribute to the uncoupling of infectivity and PrP<sup>Sc</sup> amounts yet did not find any evidence for those in our samples (at 95 dpi) as shown in new <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>.</p><p>“A10 cKO mice and controls showed identical prion titers […] while it does not influence production of ‘prions’.”</p><p><italic>2) The claim that soluble PrP</italic><sup><italic>C</italic></sup> <italic>(generated by ADAM10) explains the spreading of the disease in the</italic> tga20 <italic>(and the absence in ADAM) is only based on at best correlative evidence: the alleged reduced pathology in the ADAM may be due to reduced prion spread, but it may also pertain to secondary effects unrelated to prion spread (or to prion pathogenesis altogether), such as reduced processing of proinflammatory molecules, or impairment of neuron-microglia interaction by the lack of ADAM10. The authors have not directly investigated whether prion spread is modified in their mice (an admittedly difficult task, though not impossible). A knockout of ADAM10 in the</italic> tga20 <italic>mice would allow investigating more precisely the role of cleaved PrP</italic><sup><italic>C</italic></sup> <italic>in the spreading of the disease in these mice. It might also be possible to inject /transduce /transgenically express soluble PrP</italic><sup><italic>C</italic></sup> <italic>in the ADAM10 cKO and rescue the spreading of the disease. As these experiments require substantial time, the authors might alternatively reconsider this conclusion and either tone down the interpretation of these findings or at least mention the many possible alternative explanations.</italic></p><p>We agree with the reviewing editor that experiments directly investigating if lack of ADAM10 mediated shedding of PrP is responsible for the reduction of prion spread in our mice are difficult if not impossible to conduct. Although the proposed experiment of crossing ADAM10 cKO into <italic>tg</italic>a<italic>20</italic> mice or transgenic expression of soluble PrP<sup>C</sup> on an ADAM10 cKO background will likely provide important information on the correlation between shedding of PrP and spread of prion pathology, this will rather not get rid of non-PrP mediated effects. Additionally, we believe that presence of soluble PrP<sup>C</sup> will introduce another variable by potential interference with the PrP<sup>C</sup>-PrP<sup>Sc</sup> conversion process, as discussed in paragraph eight of the Discussion. Nevertheless, we are initiating these breedings which are, as stated by the reviewing editor, time consuming.</p><p>We also agree that our evidence at this point is merely correlative. Therefore, we have toned down our interpretation in the Results and Discussion sections and specifically mention secondary effects unrelated to prion spread:</p><p>“ADAM10-mediated shedding might contribute to spreading of prion pathology” (in the Results section) and “Non-PrP dependent effects resulting from ADAM10 […] efficiency of prion spread is possible (<xref ref-type="bibr" rid="bib35">Glatzel et al., 2004</xref>; <xref ref-type="bibr" rid="bib38">Grizenkova et al., 2014</xref>)” (Discussion section).</p><p><italic>3) In the Results section related to</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1 A and B</italic></xref><italic>, while mentioned in the Methods, the control for reintroducing ADAM10 cDNA into ADAM10 knockout cell lines (Lenti virus lacking ADAM10 cDNA) is not shown. It is important to show that the decrease in PrP</italic><sup><italic>C</italic></sup> <italic>signal is independent of the virus treatment itself. It would be desirable to have n>2 for quantification of surface PrP</italic><sup><italic>C</italic></sup> <italic>expression in A10 cKO+ADAM10</italic>.</p><p>We are thankful for making us aware of this oversight. We have now included the data from the empty vector control. As stated in the Method section, we used Nucleofector® technology instead of lenitiviral transduction (this term was mistakenly included in the previous version) to genetically introduce ADAM10 or the linearized vector only. Moreover, we have performed additional surface biotinylation experiments to increase “n” numbers of independent culture samples and now show representative Western blots (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). We also performed a new set of immunofluorescent stainings including the requested empty vector control (<xref ref-type="fig" rid="fig1">Figure 1B</xref>). The new data are mentioned in the Results section (first paragraph) as well as in the figure legend for <xref ref-type="fig" rid="fig1">Figure 1</xref>.</p><p><italic>4) The number of mice used for the crucial experiments in</italic> <xref ref-type="fig" rid="fig7"><italic>Figure 7A</italic></xref> <italic>(incubation times) is meagre. I realize that there is little that can be done about this problem at this stage, but the authors would be well-advised to use numbers of mice appropriate for sufficient statistical power. The same applies to the titer determinations in</italic> tga20 <italic>mice: just four recipient mice for each titration is pathetically low and will by necessity undermine the precision of the determination</italic>.</p><p>We are aware of our shortcomings in this respect and while it was not possible to increase the amount of A10 cKO and littermate controls for time reasons and limitations in breeding capacities, we have been able to perform new experiments to increase the number of <italic>tg</italic>a<italic>20</italic> reporter mice for prion titer determination. For the most interesting and important time point (95 dpi) we have inoculated more indicator mice per group (from four to six) and we have bioassayed additional samples (one additional inoculum for both A10 cKO and littermate control, into n=6 reporter mice). The data remain largely unchanged but have now become more robust. This is mentioned in the figure legend for <xref ref-type="fig" rid="fig7">Figure 7</xref> as well as in the Results and Methods sections.</p><p><italic>Other comments</italic>:</p><p>Reviewer #1:</p><p><italic>1) In the Abstract, a conclusion is made with regard to treatment options for other neurodegenerative diseases (apart from prion). In the absence of any data, I suggest to remove this claim from the Abstract and only mention this in the Discussion as an interesting speculation</italic>.</p><p>We understand the point of this reviewer and followed the advice and have removed this claim from the Abstract and carefully speculate on this interesting issue in the Discussion.</p><p><italic>2)</italic> <xref ref-type="fig" rid="fig1 fig2"><italic>Figure 1 and 2</italic></xref><italic>: the paper contains a whole series of experiments (covering two full figures) to proof that in the absence of ADAM 10, prion expression is increased and shedding is decreased. I believe that these data should be summarized in one figure, especially because this is not entirely novel. Moreover, instead of many different data it is better to have a couple of experiments fully documented. For instance it is not clear what was quantified in Figure Panel 1A and this should be complemented with the blot or the figure on which the quantitation is based. Panel 1C should include for the ADAM10 KO a similar analysis as for the control with regard to PrP surface staining. In</italic> <xref ref-type="fig" rid="fig2"><italic>Figure 2B</italic></xref><italic>: the immunohistochemistry shows a particular strong staining in the mouse brains of the ADAM cKO, more than what is seen in the western blot and expression is very diffuse over all cells (while the ko is only in a subset of neurons). Additional controls e.g. immunostaining of the cerebellum and more precise details of the cellular stainings would help to understand this better and to demonstrate for instance that the slides of wild type and knock out were incubated for similar length of time. It is also important to show that PrP accumulation is only seen in the neurons where ADAM10 was knocked out, as this is an important point also with regard to the conclusions with regard to the propagation of the disease</italic>.</p><p>We thank this reviewer for the helpful comments on <xref ref-type="fig" rid="fig1 fig2">Figures 1 and 2</xref>. In light of the fact that Reviewers #2 and #3 as well as the reviewing editor commented on <xref ref-type="fig" rid="fig1">Figure 1</xref>, and given that almost all of the data are novel and, as we consider, relevant to the rest of the manuscript, we decided to keep revised versions of both figures. We are convinced that <xref ref-type="fig" rid="fig1">Figure 1</xref> is a reasonable start into the overall theme of this paper by revealing effects of ADAM10 depletion on PrP<sup>C</sup> levels in different cellular lineages and thus generalizing those mechanisms. Although we previously reported lack of PrP<sup>C</sup> shedding in <sup>Nestin</sup>ADAM10 KO and increased shedding by <italic>tg</italic>a<italic>20</italic> neurons, we also tend to keep the hitherto unpublished <xref ref-type="fig" rid="fig1">Figure 1E</xref> to support our arguments with regard to inefficient PrP<sup>Sc</sup> production and potentially increased prion spread in infected <italic>tg</italic>a<italic>20</italic> mice as described in the Discussion. With <xref ref-type="fig" rid="fig2">Figure 2</xref> we then introduce the novel <italic>Camk2a</italic> ADAM10 cKO mouse model with special emphasize on its PrP<sup>C</sup> expression pattern.</p><p>Following the suggestions of this reviewer we now:</p><p>a) Show representative blots (including the empty vector control) and the ADAM10 expression for PrP<sup>C</sup> membrane levels experiment in neuronally differentiated neural stem cells (<xref ref-type="fig" rid="fig1">Figure 1A</xref>);</p><p>b) Provide new images for the PrP<sup>C</sup> surface staining including the empty vector control cells (<xref ref-type="fig" rid="fig1">Figure 1B</xref>);</p><p>c) Show detailed, higher resolution images of the colocalization between ADAM10 and PrP<sup>C</sup> on the plasma membrane of control MEF and also included this magnification for the ADAM10 KO MEF (<xref ref-type="fig" rid="fig1">Figure 1C</xref>);</p><p>d) Include a more detailed panel of the PrP immunohistochemistry of control and ADAM10 cKO brains with higher magnifications now also showing details of the cerebellum (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Moreover, we provide a new figure with co-staining of a neuronal marker (NeuN) and PrP (<xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>) to demonstrate the regional variation in PrP immunohistochemistry in the brain and differences between ADAM10 cKO mice and wild-type littermates. This analysis reveals that the signal in PrP immunohistochemistry is increased in regions where ADAM10 is deleted thus conforming previous data on the <italic>Camk2a</italic> driven deletion strategy (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; Prox et al., 2013a). This is mentioned in the Results (fifth paragraph) as follows:</p><p>“In addition, a coronal brain section showing co-staining of PrP<sup>C</sup> with the neuronal marker NeuN correlates with the <italic>Camk2a</italic> driven ADAM10 knockout strategy (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>) by showing increased PrP<sup>C</sup> expression in hippocampal and cortical areas as well as in the striatum (<xref ref-type="fig" rid="fig2s1">Figure 2–figure supplement 1</xref>).” Moreover, this is now also illustrated for the prospective readers' convenience and orientation in a new scheme (<xref ref-type="fig" rid="fig3">Figure 3A</xref>).</p><p>The new figures are described in the figure legends. Additionally, we have added experimental details on the highly standardized immunostaining with an automated system guaranteeing identical incubation times and staining conditions (“Experimental groups were stained in one run thus providing identical conditions”, paragraph nine of Materials and methods section).</p><p>With regard to the last point we agree that our immunohistochemical staining for PrP<sup>C</sup> shows a rather general staining yet with differences in intensities between genotypes. We aimed at showing that increased PrP<sup>C</sup> staining is restricted to neurons depleted of ADAM10. Since neurons are highly branched cells and PrP is highly expressed on the neuronal cell surface, single neuron analysis in 4 μ m thick paraffin sections was not feasible. Moreover, all other cell types in the CNS (though not affected by the knockout strategy) contribute to the overall staining. Finally, to the best of our knowledge there is no published reliable antibody against ADAM10 working in immnunohistochemical staining of mouse brain sections. We have nevertheless tried different ADAM10 antibodies (rabbit anti-ADAM10 (B42.1); rabbit anti-ADAM10 amino acids 608-627 (kindly provided by Dr. A. Chalaris, Kiel); rat anti-ADAM10 ectodomain antibody (MAB946)) under different staining conditions in formalin-fixed and cryo-fixed brain samples yet were not able to detect specific signals. Thus, we could not perform co-stainings of ADAM10 and PrP<sup>C</sup> which would help to answer this point of the reviewer.</p><p><italic>3) The very strong conclusion in the second paragraph of the Discussion: “and show for the first time that PrP</italic><sup><italic>C</italic></sup> <italic>and ADAM10 co-localize at the plasma membrane” is based on a single piece of weak evidence (inset in Panel 1C). The resolution of this type of experiment is insufficient to make such a strong claim</italic>.</p><p>We agree with this reviewer, have provided higher resolution figures, have deleted this strong claim and modified the sentence. Moreover, we became aware of a report published in the meantime also showing colocalization of ADAM10 and PrP<sup>C</sup>. We have now added this reference (Chen et al.,, 2014) to the second paragraph of the Discussion section.</p><p><italic>4) In the ninth paragraph of the Results section: “Taken together, these data indicate that at terminal prion disease, lack of PrP</italic><sup><italic>C</italic></sup><italic>-shedding does not influence local PrP</italic><sup><italic>Sc</italic></sup> <italic>distribution while it does impact the appearance of TVS”. This observation is mentioned here, and not further discussed or explained</italic>. <italic>Why mention it?</italic></p><p>We have to admit that TVS are not in the focus of our study and thus we did not discuss these structures in great detail. However, we think that the observation of increased TVS amounts in our model might be interesting to the prion field. Since Reviewer #2 suggested a more detailed workup of TVS including a quantification of these structures, we decided to keep these data including a quantification of TVS as a supplementary figure (<xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2</xref>). Additionally, we have slightly extended the Results section providing a description and references on TVS in prion disease (paragraph eleven) and added experimental details (eleventh paragraph of Materials and methods):</p><p>“Remarkably, prion-diseased ADAM10 cKO mice presented with extraordinarily high abundance of tubulovesicular structures (TVS) (<xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2</xref>) (<xref ref-type="bibr" rid="bib45">Jeffrey and Fraser, 2000</xref>; <xref ref-type="bibr" rid="bib61">Liberski et al., 2010</xref>). These are spherical structures of approx. 25-37 nm in diameter that are specific for prion diseases though devoid of PrP (<xref ref-type="bibr" rid="bib45">Jeffrey and Fraser, 2000</xref>; <xref ref-type="bibr" rid="bib61">Liberski et al., 2010</xref>; <xref ref-type="bibr" rid="bib62">Liberski et al., 2008</xref>). Thus, our model may allow purification of these structures and could contribute to unravel the nature and relevance of TVS in prion diseases.”</p><p><italic>5)</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5A</italic></xref><italic>: I find it puzzling that there is such a contrast in the accumulation of proteinase K resistant species in the ADAM10 ko versus the</italic> tga20 <italic>mouse? In principle both animals have more uncleaved prion protein on the cell membrane. Thus the main difference is the shed prion in the</italic> tga20 <italic>mouse. Would one conclude from this that the shed prion inhibits the generation of proteinase K resistant forms of the PrP</italic><sup><italic>Sc</italic></sup><italic>? As also indicated above, I think that it is necessary to study the ADAM10 KO effect in the</italic> tga20 <italic>mouse to allow making real conclusions with regard to the precise effect of ADAM10</italic>.</p><p>We thank Reviewer #1 for the advice to better clarify this point. In fact, the suggested conclusion of this reviewer is exactly what we think. In addition to our analysis of PrP<sup>C</sup> surface levels of neuronally differentiated neural stem cells derived from ADAM10 cKO and littermate control mice (showing an approx. 1.6-fold increase when ADAM10 is lacking; <xref ref-type="fig" rid="fig1">Figure 1A</xref>), we now also analyzed PrP<sup>C</sup> membrane levels in primary neurons of <italic>tg</italic>a<italic>20</italic> and C57BL/6 control mice (<xref ref-type="fig" rid="fig10">Author response image 1</xref>).<fig id="fig10" position="float"><label>Author response image 1.</label><caption><p>Increased PrP<sup>C</sup> surface levels in primary neurons of <italic>tg</italic>a<italic>20</italic> mice. Western blot analysis of lysates (on the left) and biotinylated surface proteins (on the right) of primary neurons from E14 embryos of <italic>tg</italic>a<italic>20</italic> and C57BL/6 wild-type mice. Flotillin served as a loading control. Quantification of densitometric analysis of surface PrP<sup>C</sup> is shown below. C57BL/6 set to 1 (+/-0.1 SEM); <italic>tg</italic>a<italic>20</italic>: 3.1 +/-0.5; n=6 samples per genotype from 2 independent experiments; **p=0,007).</p></caption><graphic xlink:href="elife04260f010"/></fig></p><p>Our analysis revealed that <italic>tg</italic>a<italic>20</italic> mice (which overexpress PrP<sup>C</sup> approx. 7-fold (Fischer, 1996)) show a ∼3-fold increase in PrP<sup>C</sup> surface levels. Thus, while we think that PrP<sup>C</sup> membrane levels are decisive for prion-associated neurotoxicity and disease duration (with <italic>tg</italic>a<italic>20</italic> showing much shorter incubation times than ADAM10 cKO mice), elevated total as well as surface levels of PrP<sup>C</sup> (<italic>tg</italic>a<italic>20</italic> > ADAM10 cKO > wild-type controls) do not explain the differences in PrP<sup>Sc</sup> formation. In fact, we think that shed PrP<sup>C</sup> inhibits the conversion to PrP<sup>Sc</sup> in vivo. As discussed in the main text (Discussion section) there is also some evidence from other studies showing that anchorless versions of PrP<sup>C</sup> act as antagonists of the conversion process. We also added a reference showing this inhibitory effect in PMCA approaches (<xref ref-type="bibr" rid="bib49">Kim et al., 2009</xref>). Soluble PrP<sup>C</sup> generated by ADAM10-mediated shedding could represent the physiological correlate of this. We speculate that lack of the protease is responsible for efficient PrP<sup>Sc</sup> production in ADAM10 cKO mice whereas increased production of shed PrP<sup>C</sup> in <italic>tg</italic>a<italic>20</italic> mice (partially) explains their poor conversion rate (a known phenomenon of the latter mouse model that, to our knowledge, has not been solved to date). Fitting to this concept, PrP<sup>Sc</sup> production was decreased in mice moderately overexpressing ADAM10 (<xref ref-type="bibr" rid="bib31">Endres et al., 2009</xref>). We have newly added this reference in this context in the main text (paragraph six of the Discussion section). As mentioned in our response to the reviewing editor (major point 2) we agree that the crossing of ADAM10 cKO and <italic>tg</italic>a<italic>20</italic> mice is an interesting experiment to gain deeper insight.</p><p><italic>6)</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5D</italic></xref><italic>: The activation of calpain is linked to membrane pores in the text. I suppose that it is linked to Calcium influx? This needs to be better formulated</italic>.</p><p>We have realized that our workup and statements regarding the membrane pore model was not clearly formulated and experimentally addressed. This point is addressed in detail below (Reviewer #3, point 8). To answer this specific point: Yes, if correct, our model would suggest that membrane pore formation leads to Ca<sup>2+</sup> influx. This is now clarified in the Discussion (eighth paragraph) and in the figure legend of <xref ref-type="fig" rid="fig9">Figure 9</xref>.</p><p>Reviewer #2:</p><p><italic>1) In its current form the manuscript has a few areas of weakness that need to be improved. In particular, throughout the manuscript the PrP</italic><sup><italic>C</italic></sup> <italic>and PrP</italic><sup><italic>Sc</italic></sup> <italic>levels, the incubation periods, and the spongiosis levels are compared between different mouse lines (A10 cKO, wild-type,</italic> tga20<italic>), different time points (65d, 95d), and different brain regions (forebrain, cerebellum, whole brain), but at present these comparisons are a bit haphazard. It would be great to include a table to assemble all these scattered data into one location. This table would make it easier for the reader to keep track of these differences and better follow the arguments the manuscript makes</italic>.</p><p>We thank this reviewer for the advice of providing a better structure of presented data. For this purpose we have prepared a new Figure (<xref ref-type="fig" rid="fig3">Figure 3</xref>) aiming to clarify the distribution of ADAM10 depletion within the brain and to explain our experimental strategy (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). As requested by this reviewer we have also included a table (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) summarizing our most important findings with comparison of prion relevant changes for different genotypes, brain regions and time points. Moreover, this figure provides direct reference to the relevant figures presenting original data. We hope that <xref ref-type="fig" rid="fig3">Figure 3</xref> improves understanding of our study and makes it more convenient for readers to follow our arguments. The figure is mentioned in the Results section (paragraphs six and seven) as follows:</p><p>“Based on previous reports (<xref ref-type="bibr" rid="bib24">Casanova et al., 2001</xref>; Prox et al., 2013a) […] chosen for immunohistochemical or biochemical analyses.”</p><p>and:</p><p>“For better orientation, <xref ref-type="fig" rid="fig3">Figure 3B</xref> […] showing the original data described below.”</p><p><italic>2) Throughout the manuscript a comma is used as the decimal mark. For proper English a full stop should be used instead</italic>.</p><p>We have now corrected this and use a full stop instead.</p><p><italic>3) In several places the A10 cKO results are compared with those that were obtained with mice expressing anchorless PrP</italic><sup><italic>C</italic></sup> <italic>(e.g. at the end of the third paragraph of the Introduction, eighth paragraph of the Discussion, etc.). At present the comparison is limited to an anchorless PrP</italic><sup><italic>C</italic></sup> <italic>low-expressor mouse line (</italic><xref ref-type="bibr" rid="bib28"><italic>Chesebro et al., 2005</italic></xref><italic>), but it would be desirable to also include high-expressor results into this discussion (Stohr et al., 2011). Particularly, since the latter animals express PrP at levels closer to the mouse lines that are used in this study</italic>.</p><p>We have mentioned these mice and referenced this study in the Introduction (end of the third paragraph).</p><p>“High expression of anchorless PrP<sup>C</sup> leads to formation of prions and a late onset neurological disease (Stohr et al., 2011).”</p><p>We also think that it is a good suggestion to mention these mice since this study contributes to our understanding of roles played by anchorless PrP. However, we have refrained from an in depth discussion of these mice since spread of prion pathology in the CNS was not the focus of that study.</p><p><italic>4) The first paragraph of the Results section describes results from a complete ADAM10 knockout mouse line, according to the figure legend the corresponding figures (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1C and D</italic></xref><italic>) show only A10 cKO data. This mismatch needs to be cleared up</italic>.</p><p>We are thankful for this hint. Indeed, <xref ref-type="fig" rid="fig1">Figure 1C and D</xref> show data derived from murine embryo fibroblasts (MEF) of a complete ADAM10 knockout (KO) mouse line. We have corrected this in the figure legend and in the labelling of the figure itself. Moreover, the term “ADAM10 cKO” (or “A10 cKO”) is now only used when referring to <italic>Camk2a</italic>-Cre ADAM10 conditional knockout mice.</p><p><italic>5) It is difficult to judge the alleged co-localization of PrP</italic><sup><italic>C</italic></sup> <italic>and ADAM10 (</italic><xref ref-type="fig" rid="fig1"><italic>Figure 1C</italic></xref><italic>), since the magnification is so low. Higher resolution images would be needed to make this a convincing argument</italic>.</p><p>As mentioned in our response to Reviewer #1, we now provide higher resolution images of both wild-type control and ADAM10 KO murine embryonic fibroblasts.</p><p><italic>6) The electron micrographs in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4D</italic></xref> <italic>show tubulovesicular structures (TVS), which are claimed to be increased in diseased A10 cKO mice. It would be worthwhile to document and quantify this increase, since TVS and their link to prion disease are controversial. Ultimately, the A10 cKO mice may help to demystify these structures and their role in the prion diseases</italic>.</p><p>As mentioned in our response to Reviewer #1, we did not put a special focus on TVS in our study. Nevertheless, our observation of high abundance of these structures in terminally prion diseased ADAM10 cKO mice (compared to wild-type controls in this study as well as to prion disease models published elsewhere) might be of interest to the prion field. Thus, we decided to devote an entire supplementary figure to the topic of TVS (<xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2</xref>). We now also provide quantification, a short description on TVS and more references (paragraphs eleven and twelve of the Results section). We agree with this reviewer that our model “may help to demystify these structures” and ultimately solve their true significance. However, we are fully aware that this would require a much more refined way of analysis.</p><p>With regard to <xref ref-type="fig" rid="fig5">Figure 5</xref> (former <xref ref-type="fig" rid="fig4">Figure 4</xref>) we would like to clarify that we introduced a new EM picture showing vacuolization with membranous structures (A). In addition, we modified the scale bars (in the figure and figure legends of B and C) as we became aware of a mistake in the former version of this manuscript.</p><p><italic>7) The comparison of PrP</italic><sup><italic>Sc</italic></sup> <italic>levels between different mouse lines is solely based on PK-resistance (</italic><xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>). While this surrogate marker is widely used to estimate prion infectivity levels, the possible presence of protease-sensitive forms of PrP</italic><sup><italic>Sc</italic></sup> <italic>should be taken into consideration and discussed</italic>.</p><p>Since our data, as discussed above, support an uncoupling of PrP<sup>Sc</sup> amounts and prion infectivity, we followed the suggestion of this reviewer and analyzed our samples for the presence of aberrant, low-abundance or PK sensitive prion forms/fragments. However, we did not find any atypical banding following digestions with “cold PK” (i.e. digestion with 200 µg/ml PK at 4°C for 1 h) or PK dilutions and detection with PrP-specific 1E4 or POM1 antibodies. This is now shown in the new <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref> and mentioned in the Methods (under the subsection “Western blot analysis”) and in the subsection “Enhanced PrP<sup>Sc</sup> formation and calpain levels in A10 cKO mice” in Results:</p><p>“There were no atypical PrP patterns as assessed by PK digestion at 4°C (‘cold PK’) or with lower dilutions of PK (<xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref>).”</p><p>Moreover, we mentioned this issue in the revised Discussion (seventh paragraph):</p><p>“A10 cKO mice and controls showed identical prion titers, yet elevated levels of PrP<sup>Sc</sup> and no evidence for atypical protease-sensitive forms of PrP<sup>Sc</sup>.”</p><p><italic>8) In the conclusions, ADAM10 is being promoted as a potential therapeutic target for prion diseases. The finding that a reduction of ADAM10 leads to a shortening of the incubation period, while its normal function increases the spreading of PrP</italic><sup><italic>Sc</italic></sup><italic>, suggests a no-win scenario. Hence, a more refined and discerning discussion would be needed to support such a statement</italic>.</p><p>We agree with this reviewer that the dual role of ADAM10 in prion disease makes therapeutic approaches questionable. Thus, rather than embarking on a lengthy discussion of this topic we preferred to delete this claim in the conclusion of the Discussion (tenth paragraph):</p><p>“Understanding this dual role of ADAM10 in prion disease brings together current concepts of prion biology and might reveal mechanistic insight into important pathophysiological processes.”</p><p><italic>9) Lastly, grammatical errors can be found all the way through the manuscript, a thorough proofreading would benefit the readability of the text</italic>.</p><p>All co-authors have checked the final manuscript again carefully and we have engaged a professional proofreader to eliminate errors.</p><p>Reviewer #3:</p><p><italic>1) In</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4A</italic></xref><italic>, WB analysis of protein expression was compared only in frontal brain homogenates. This needs to be controlled by including whole brain homogenates as well</italic>.</p><p>As demonstrated in the new <xref ref-type="fig" rid="fig3">Figure 3A</xref>, we had chosen to take samples from frontal brain for biochemical analysis while the rest of the brain was taken for in-depth immunohistochemical (IHC) analysis. Thus, unfortunately we are unable to present the requested controls. However, our IHC analysis of PK-resistant material (now shown in <xref ref-type="fig" rid="fig5">Figure 5</xref>) shows similar staining pattern indicating that equal amounts of PrP<sup>Sc</sup> exist at a terminal state of disease in ADAM10 cKO (at ∼103 dpi) and littermate controls (at ∼146 dpi).</p><p><italic>2) In panel C, the GFAP signal of CD1 mock ADAM10 cKO seems higher than the control. Is this image representative? If yes, is there an explanation for this?</italic></p><p>This reviewer has correctly noticed that ADAM10 cKO mice indeed do show a mild gliosis. This has been published before (Prox et al., 2013a) and is indeed representative yet there is no one obvious explanation for this given the multitude of ADAM10 substrate in the brain discussed above. However, in both genotypes a much more dramatic increase in GFAP signal can be observed upon challenge with prions (<xref ref-type="fig" rid="fig5">Figure 5C</xref>).</p><p><italic>3) In panel D: How was the quantification done and what is the relevance of higher numbers of tubulovesicular structures in ADAM10 cKO? Does this bear relevance for the paper at all?</italic></p><p>As requested by this reviewer and already mentioned in our response to reviewers #2 and #3 we now provide quantification, description and references on TVS and more experimental detail (twelfth paragraph of the Results section). Although we are, at this stage, unable to explain the role of increased TVS abundance in ADAM10 cKO mice (which also show increased PrP<sup>Sc</sup> production), we think that this observation might have some relevance to the prion field. As suggested by Reviewer #2 a detailed analysis of these structures “may help to demystify these structures and their role in the prion diseases” in the future. However, since TVS are not the main focus of the current study, we now present this data in a supplementary figure (<xref ref-type="fig" rid="fig5s2">Figure 5–figure supplement 2</xref>).</p><p><italic>4) Results related to</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>: It would be important to show that the increase of PrP</italic><sup><italic>Sc</italic></sup> <italic>levels in forebrain homogenates of ADAM10 cKO is comparable to the overall PrP</italic><sup><italic>Sc</italic></sup> <italic>content of wild type mouse brains. This could underline that the difference resides in the lack of shedding and spreading of PrP</italic><sup><italic>Sc</italic></sup><italic>, leading to local accumulation of PrP</italic><sup><italic>Sc</italic></sup> <italic>in ADAM10 cKO. For this, whole brain homogenates should be compared</italic>.</p><p>We have to admit that this would be an interesting comparison. However, as stated in our response to point 1 and shown in <xref ref-type="fig" rid="fig3">Figure 3A</xref> we did not consider taking these samples for biochemical analysis when we planned our experimental strategy but rather decided for a detailed immunohistochemical assessment.</p><p><italic>5) As for the results shown in</italic> <xref ref-type="fig" rid="fig6"><italic>Figure 6</italic></xref><italic>, the comparison of infectivity tests of non-forebrain regions of control and ADAM10 cKO would be important, as it could identify any infectious but non-toxic PrP subpopulations which still might leave PrP</italic><sup><italic>Sc</italic></sup> <italic>aggregates in ADAM10 cKO</italic>.</p><p>We agree with this reviewer that the discrimination of infectious species, toxic entities and protease resistant PrP<sup>Sc</sup> in the framework of prion diseases is an important issue with relevance to the whole field. Although we believe that our observation of an uncoupling of PrP<sup>Sc</sup> and infectivity titers in forebrain samples at 95 dpi might contribute to this ongoing discussion, we again have to admit that our experimental design did not consider the requested experiment (see response to point 1 and 4; <xref ref-type="fig" rid="fig3">Figure 3A</xref>). Nevertheless we did not find evidence for existence of atypical PrP fragments that could correspond to infectivity and explain this uncoupling observed in forebrain samples (as shown in new <xref ref-type="fig" rid="fig6s1">Figure 6–figure supplement 1</xref> and mentioned in our response to Reviewer #2, point 7).</p><p><italic>6) Discussion: The authors state that PrP “is a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer’s disease”. This is a highly controversial contention propagated by Dr. Strittmatter, and there is more than half-dozen papers out there showing that this idea is, at best, limited to certain animal models. It is the authors' privilege to choose what they wish to believe, and I understand that the charitable interpretation of the Strittmatter paper might add to the perceived importance of the current study, but in fairness they should at least mention the evidence against PrP</italic><sup><italic>C</italic></sup> <italic>being the Aβ receptor.</italic></p><p>We agree with this reviewer that there is considerable controversy regarding the suggested role of PrP<sup>C</sup> as a receptor for Aβ. This controversy becomes even stronger with regard to the downstream effects of this interaction and their contribution to synaptic impairment and neuronal loss in Alzheimer's disease (AD). For this reason we have now mentioned this scientific dispute and referenced three studies and a close-up article covering this controversy in the Introduction (as we could not find the statement the reviewer referred to in our Discussion):</p><p>“Despite some degree of controversy regarding this receptor function and its relevance in AD (<xref ref-type="bibr" rid="bib9">Balducci et al., 2010</xref>; <xref ref-type="bibr" rid="bib14">Benilova and De Strooper, 2010</xref>; <xref ref-type="bibr" rid="bib22">Calella et al., 2010</xref>; <xref ref-type="bibr" rid="bib48">Kessels et al., 2010</xref>), binding of pathogenic oligomers […]” (second paragraph).</p><p>In addition, we have toned down statements in the Abstract (“Moreover, it has been suggested as a receptor mediating neurotoxicity in common neurodegenerative proteinopathies such as Alzheimer’s disease”) and in the conclusion (“In view of a potential role of PrP<sup>C</sup> in Alzheimer's disease…”; last paragraph of the Discussion section). Given that this issue is not of central relevance for our study, we decided to avoid a detailed discussion in the manuscript. However, to the best of our knowledge the majority of studies that have tackled this topic in the meantime (including a study from our own group investigating binding of Aβ to PrP<sup>C</sup> in AD brains; <xref ref-type="bibr" rid="bib30">Dohler et al., 2014</xref>) rather support the role of PrP<sup>C</sup> as a specific binding partner of Aβ.</p><p><italic>7) One problematic point is the following: It is stated that ADAM10 cKO mice show massive neurodegeneration in the forebrain, but not in the cerebellum and brainstem, while controls including</italic> tga20 <italic>show diffuse neurodegeneration. If this is really true, how can one explain completely identical clinical phenotypes?</italic></p><p>We are thankful for this question as it gives us the opportunity to clarify that we did not describe “completely identical phenotypes”. In fact, <italic>tg</italic>a<italic>20</italic> mice show a much more rapid progression of clinical signs (<xref ref-type="bibr" rid="bib33">Fischer et al., 1996</xref>). While most of those signs were indeed comparable between ADAM10 cKO, wild-type control and <italic>tg</italic>a<italic>20</italic> mice, we rarely observed kyphosis and ungroomed fur in the latter. In contrast to the other experimental groups, almost all <italic>tg</italic>a<italic>20</italic> mice developed a hind leg paresis. This might be related to the cerebellar pathology found in these mice (although this remains speculative). Nevertheless, we have added the data for <italic>tg</italic>a<italic>20</italic> mice in <xref ref-type="table" rid="tbl1">Table 1</xref> and comparisons between genotypes and brain regions are summarized in <xref ref-type="fig" rid="fig3">Fig. 3B</xref> (as requested by Reviewer #2).</p><p><italic>8) As for</italic> <xref ref-type="fig" rid="fig5"><italic>Figure 5</italic></xref><italic>, the total amount of calpain is most certainly irrelevant to neurodegeneration, since calpains are mostly inactive in the absence of raised intracellular calcium. What matters is the amount of activated calpains. This can be assessed by investigating the amount of cleavage of a calpain substrate, such as spectrin (alpha-fodrin). The mere detection of calpains by Western blot is irrelevant. The whole argument about calpains needs to be revisited by activity measurements, or else it should be deleted in toto. This becomes painfully evident by the authors' finding that on the one hand there is prion toxicity (allegedly through calpain) with profound neurodegeneration in the forebrain of ADAM10 cKO, but on the other hand the significantly faster disease in</italic> tga20 <italic>mice goes along with significantly less calpain Also, a comparison of overall (not only forebrain) calpain activity levels to mock inoculated animals could help.</italic></p><p>As mentioned above, we have performed additional experiments and changed the manuscript text to address the points raised here.</p><p>Importantly we showed that increase of calpain expression in ADAM10 cKO (<xref ref-type="fig" rid="fig6">Figure 6D</xref>) is specifically seen in prion disease since we could not detect differences in calpain level between non-infected ADAM10 cKO and controls. Thus, although calpain activity is of course the most relevant readout these specific differences deserve to be mentioned.</p><p>We used prion-infected mice in most experiments and their tissues have to be handled in a biosafety level 3* laboratory due to governmental regulation. Given this, sophisticated measurements to assess calpain activity (Ca<sup>2+</sup> imaging or detection of fluorogenic substrates) were not possible. As mentioned above and described in <xref ref-type="fig" rid="fig3">Figure 3</xref> all biochemical analyses had to be done on forebrain because the other reasons were used for morphological analysis.</p><p>Nevertheless, we followed the advice of this reviewer and detected spectrin. Additionally we decided to detect p35, another calpain substrate implicated in neurodegeneration (<xref ref-type="bibr" rid="bib58">Lee et al., 2000</xref>). Full-length versions of both proteins appeared reduced in ADAM10 cKO. This decrease was not accompanied by increased levels of cleavage products, which may be due to technical reasons.</p><p>Thus, this point remains to be further investigated. We have included these data in the Results section (seventeenth paragraph), in the new <xref ref-type="fig" rid="fig6s2">Figure 6–figure supplement 2B, C</xref>, and we have toned down our statements in the Abstract, Discussion and in the description of our model (<xref ref-type="fig" rid="fig9">Figure 9</xref>):</p><p>“This is specific for prion disease as no differences were detected in non-prion infected ADAM10 cKO mice (mean: 0.90 ±0.26 SEM; n=4) when compared to age-matched wild-type controls (set to 1 ±0.19; n=5; <italic>p</italic>=0.7) (<xref ref-type="fig" rid="fig6s2">Figure 6-figure supplement 2A</xref>). In prion disease, we observed a reduction of the described calpain substrates spectrin (<xref ref-type="bibr" rid="bib32">Falsig et al., 2012</xref>) and neuron-specific activator p35 (<xref ref-type="bibr" rid="bib58">Lee et al., 2000</xref>) suggesting that increased expression in ADAM10 cKO mice correlated with activity of calpain (<xref ref-type="fig" rid="fig6s2">Figure 6–figure supplement 2B,C</xref>). However, it should be noted that we failed to detect differences in specific cleavage products of spectrin and p35.”</p><p><italic>9) When mentioning the possible inhibition of PrP</italic><sup><italic>Sc</italic></sup> <italic>formation by shedded PrP</italic><sup><italic>C</italic></sup><italic>, it would be desirable to prove this statement with PMCA + shed PrP</italic><sup><italic>C</italic></sup>.</p><p>We agree that a PMCA-based assessment of the role of shed PrP<sup>C</sup> on PrP<sup>Sc</sup> formation would be a desirable addition to the paper. However, obtaining quantities of physiologically shed PrP<sup>C</sup> (and its purification) necessary for this method is not trivial. Usage of recombinant PrP has other disadvantages and does not fully recapitulate the in vivo situation (<xref ref-type="bibr" rid="bib49">Kim et al, 2009</xref>). This reference has now been added in the Discussion part covering the inhibition of PrP<sup>Sc</sup> formation by anchorless PrP versions (sixth paragraph). Thus, we have decided to address this in more detail in a separate study.</p><p><italic>10) In</italic> <xref ref-type="fig" rid="fig1"><italic>Figure 1E</italic></xref><italic>, POM2 is named as the applied antibody, while the Methods section describes POM1 for this purpose</italic>.</p><p>This mismatch has been corrected. As now stated in the figure legend for <xref ref-type="fig" rid="fig1">Figure 1</xref> and Methods section, POM2 was used for detection of shed PrP<sup>C</sup> in this experiment.</p></body></sub-article></article> |