Regulation of hippocampal mossy fiber-CA3 synapse function by a Bcl11b/C1ql2/Nrxn3(25b+) pathway

The transcription factor Bcl11b has been linked to neurodevelopmental and neuropsychiatric disorders associated with synaptic dysfunction. Bcl11b is highly expressed in dentate gyrus granule neurons and is required for the structural and functional integrity of mossy fiber-CA3 synapses. The underlying molecular mechanisms, however, remained unclear. We show in mice that the synaptic organizer molecule C1ql2 is a direct functional target of Bcl11b that regulates synaptic vesicle recruitment and long-term potentiation at mossy fiber-CA3 synapses in vivo and in vitro. Furthermore, we demonstrate C1ql2 to exert its functions through direct interaction with a specific splice variant of neurexin-3, Nrxn3(25b+). Interruption of C1ql2-Nrxn3(25b+) interaction by expression of a non-binding C1ql2 mutant or by deletion of Nrxn3 in the dentate gyrus granule neurons recapitulates major parts of the Bcl11b as well as C1ql2 mutant phenotype. Together, this study identifies a novel C1ql2-Nrxn3(25b+)-dependent signaling pathway through which Bcl11b controls mossy fiber-CA3 synapse function. Thus, our findings contribute to the mechanistic understanding of neurodevelopmental disorders accompanied by synaptic dysfunction.


Introduction
Disruptions in synaptic structure and function have been identified as a major determinant in the manifestation of various neurodevelopmental and neuropsychiatric disorders, such as autism spectrum disorder, schizophrenia, and intellectual disability (Hayashi-Takagi, 2017;Lepeta et al., 2016;Zoghbi and Bear, 2012).The need to understand how synaptic function is compromised in these disorders has accentuated the importance of studying the regulatory mechanisms of physiological synaptic function.These mechanisms involve cell adhesion molecules at both the pre-and post-synaptic side that act as synaptic organizers, whose unique combination determines the structural and functional properties of the synapse.Many such proteins have already been identified and our understanding of their complex role in synapse assembly and function has significantly increased over the last years (de Wit and Ghosh, 2016;O'Rourke et al., 2012;Südhof, 2017).Furthermore, recent advances in the genetics of neurodevelopmental and neuropsychiatric disorders have implicated genes encoding for several of the known synaptic proteins, supporting a role for these molecules in the pathogenesis of corresponding disorders (Südhof, 2021;Torres et al., 2017;Wang et al., 2018).
The sensitivity of the functional specification of the synapse to the combination of distinct synaptic proteins and their relative expression levels suggests that genetically encoded programs define at least facets of the synaptic properties in a cell-type-specific manner (Südhof, 2017).Several of the synaptic proteins have been shown to promote formation of functional pre-and postsynaptic assemblies when presented in non-neuronal cells (Dalva et al., 2000;Dean et al., 2003;Scheiffele et al., 2000), showing that their ability to specify synapses is in part independent of signaling processes and neuronal activity and supporting the idea that synaptic function is governed by cues linked to cellular origin (Gomez et al., 2021).Thus, the investigation of synaptic organizers and their function in health and disease should be expanded to the transcriptional programs that regulate their expression.
Bcl11b (also known as Ctip2) is a zinc finger transcription factor that has been implicated in various disorders of the nervous system including Alzheimer's and Huntington's disease, and schizophrenia (Kunkle et al., 2016;Song et al., 2022;Whitton et al., 2018;Whitton et al., 2016).Patients with BCL11B mutations present with neurodevelopmental delay, overall learning deficits as well as impaired speech acquisition and autistic features (Eto et al., 2022;Lessel et al., 2018;Punwani et al., 2016;Yang et al., 2020).Bcl11b is expressed in several neuron types, including the dentate gyrus granule neurons (DGN) of the hippocampus.Expression of Bcl11b in the DGN starts during embryonic development and persists into adulthood (Simon et al., 2020).We have previously demonstrated that Bcl11b plays a crucial role in the development of the hippocampal mossy fiber system, eLife digest The human brain contains billions of neurons working together to process the vast array of information we receive from our environment.These neurons communicate at junctions known as synapses, where chemical packages called vesicles released from one neuron stimulate a response in another.This synaptic communication is crucial for our ability to think, learn and remember.
However, this activity depends on a complex interplay of proteins, whose balance and location within the neuron are tightly controlled.Any disruption to this delicate equilibrium can cause significant problems, including neurodevelopmental and neuropsychiatric disorders, such as schizophrenia and intellectual disability.
One key regulator of activity at the synapse is a protein called Bcl11b, which has been linked to conditions affected by synaptic dysfunction.It plays a critical role in maintaining specific junctions known as mossy fibre synapses, which are important for learning and memory.One of the genes regulated by Bcl11b is C1ql2, which encodes for a synaptic protein.However, it is unclear what molecular mechanisms Bcl11b uses to carry out this role.
To address this, Koumoundourou et al. explored the role of C1ql2 in mossy fibre synapses of adult mice.Experiments to manipulate the production of C1ql2 independently of Bcl11b revealed that C1ql2 is vital for recruiting vesicles to the synapse and strengthening synaptic connections between neurons.Further investigation showed that C1ql2's role in this process relies on interacting with another synaptic protein called neurexin-3.Disrupting this interaction reduced the amount of C1ql2 at the synapse and, consequently, impaired vesicle recruitment.
These findings will help our understanding of how neurodevelopmental and neuropsychiatric disorders develop.Bcl11b, C1ql2 and neurexin-3 have been independently associated with these conditions, and the now-revealed interactions between these proteins offer new insights into the molecular basis of synaptic faults.This research opens the door to further study of how these proteins interact and their roles in brain health and disease.
adult hippocampal neurogenesis as well as hippocampal learning and memory (Simon et al., 2016;Simon et al., 2012).In the mature hippocampus, Bcl11b is critical for the structural and functional integrity of mossy fiber synapses (MFS), the connections between DGN and CA3 pyramidal neurons (De Bruyckere et al., 2018).MFS have a critical role in learning and memory stemming from their unique structural and functional properties, such as an enormous pool of releasable synaptic vesicles (SV), and reliable presynaptic short-and long-term plasticity (Nicoll and Schmitz, 2005;Rollenhagen and Lübke, 2010).Conditional ablation of Bcl11b in murine DGN impairs presynaptic recruitment of SV and abolishes mossy fiber long-term potentiation (MF-LTP;De Bruyckere et al., 2018).The molecular mechanisms, however, through which the transcriptional regulator Bcl11b controls highly dynamic properties of the MFS remained elusive.
In the present study, we show that the secreted synaptic organizer molecule C1ql2, a member of the C1q-like protein family (Yuzaki, 2017), is a functional target of Bcl11b in murine DGN.Reintroduction of C1ql2 in Bcl11b mutant DGN rescued the localization and docking of SV to the active zone (AZ), as well as MF-LTP that was abolished upon Bcl11b ablation.Knock-down (KD) of C1ql2 in wildtype animals recapitulated a major part of the MFS phenotype observed in Bcl11b mutants.Furthermore, we show that C1ql2 requires direct interaction with a specific neurexin-3 isoform, Nrxn3(25b+), a member of a polymorphic family of presynaptic cell adhesion molecules (Reissner et al., 2013;Südhof, 2017), to recruit SV in vitro and in vivo.Finally, we observe that localization of C1ql2 along the mossy fiber tract depends on C1ql2-Nrxn3(25b+) interaction.Taken together, this study identifies a novel Bcl11b/C1ql2/Nrxn3(25b+)-dependent regulatory mechanism that is essential for the control of MFS function.Recent genetic studies suggested its single components to be associated with neurodevelopmental and neuropsychiatric disorders characterized by synaptic dysfunction.Our data, for the first time, demonstrate these molecules to be interconnected in one regulatory pathway.Thus, our findings provide new mechanistic insight into the pathogenesis of corresponding human disorders.

Reintroduction of C1ql2 into Bcl11b mutant dentate granule neurons restores synaptic vesicle recruitment at the mossy fiber-CA3 synapse
We demonstrated before that Bcl11b is critical for the structural and functional integrity of adult excitatory hippocampal MFS (De Bruyckere et al., 2018;Simon et al., 2016).The downstream regulatory mechanisms, however, through which Bcl11b exerts its complex functions at the MFS remained unclear.In a previous study, we carried out differential transcriptomic analyses on Bcl11b conditional knock-out (cKO) and wildtype (WT) DGN, to systematically screen for candidate transcriptional targets of Bcl11b (De Bruyckere et al., 2018).Among the differentially expressed candidate genes, we identified the synaptic organizer molecule C1ql2 (De Bruyckere et al., 2018), previously implicated in modulating MFS functions (Matsuda et al., 2016).C1ql2 transcript and protein levels are massively downregulated in Bcl11b mutant DGN (Figure 1 -figure supplement 1;De Bruyckere et al., 2018), and the Bcl11b protein directly binds to consensus sequences within the C1ql2 promotor (De Bruyckere et al., 2018), suggesting Bcl11b to act on MFS through C1ql2.To directly test this, we stereotaxically injected a C1ql2-expressing AAV (Figure 1a-b) into the dentate gyrus (DG) of Bcl11b cKO mice 2 weeks after induction of the mutation and compared them to control animals.To avoid potential interference of the AAV-mediated gene expression with the interpretability of observed phenotypes, we stereotaxically injected the DG of control animals as well, with AAV expressing EGFP only.AAV-mediated re-expression of C1ql2 in the Bcl11b mutant DGN completely restored C1ql2 protein expression (Figure 1c-d; Control +EGFP: 1±0.216,Bcl11b cKO +EGFP: 0.2±0.023,Bcl11b cKO +EGFP-2A-C1ql2: 2.44±0.745,mean ± SEM).Furthermore, the spatial distribution of the exogenous C1ql2 protein in mutants was indistinguishable from controls (Figure 1e).Using vGlut1 and Homer1 as pre-and postsynaptic markers, respectively, we observed exogenous C1ql2 protein to precisely localize at glutamatergic synapses within the stratum lucidum (SL) of CA3, confirming that reintroduced C1ql2 is correctly targeted to the MFS (Figure 1f).
MFS of Bcl11b cKO animals were characterized by a misdistribution of SV in relation to the AZ, with fewer SV being present in the vicinity of AZ, as reflected by a lower average synapse score (Figure 2a-b).The scoring system used in this study rates MFS based on the number of SV and their C1ql3, a different member of the C1ql subfamily, is co-expressed with C1ql2 in DGN and the two proteins have been shown to form functional heteromers (Matsuda et al., 2016) 1c-d).To exclude that the observed effects were influenced by the elevated C1ql2 expression in the Bcl11b cKO background above physiological levels, we over-expressed C1ql2 as well in control animals, which resulted in a strong increase of C1ql2 (Figure 2h).However, this did not affect the average synapse score (Figure 2i To analyze the C1ql2-dependent functions of Bcl11b on SV distribution in more detail, we quantified the number of SV docked on AZ in control animals, Bcl11b mutants, and upon the reintroduction of C1ql2.SV with a≤5 nm distance from the plasma membrane were considered docked (Kusick et al., 2022;Vandael et al., 2020).Bcl11b mutant animals had significantly fewer docked vesicles per 100 nm of AZ profile length compared to control animals and more AZ with no docked vesicles at all.Rescue of C1ql2 expression restored the number of docked SV to control levels, while the overexpression of C1ql3 did not affect the number of docked vesicles (Figure 2d and f-g Conditional deletion of Bcl11b in the adult hippocampus also leads to a loss of MFS, as well as reduced ultrastructural complexity of the remaining mossy fiber boutons (MFB;De Bruyckere et al., 2018), posing the question of whether these phenotypic features also depend on C1ql2.Interestingly, C1ql2 reintroduction in the DGN of Bcl11b cKO neither restored the loss of glutamatergic synapses, as quantified by the colocalization of pre-and postsynaptic markers, vGlut1 and Homer1 (Figure 2   1e-f; Control +EGFP: 0.0051±0.00031,Bcl11b cKO +EGFP: 0.0042±0.00014,Bcl11b cKO +EGFP-2A-C1ql2: 0.0037±0.00021,mean ± SEM).This suggests that Bcl11b acts on MFS through C1ql2dependent as well as -independent signaling pathways.
Reintroduction of C1ql2 into Bcl11b mutant dentate granule neurons rescues mossy fiber synapse long-term potentiation The ultrastructural changes at the MFS point towards potential alterations in synaptic function.Indeed, adult-induced Bcl11b cKO was previously found to result in a loss of MF-LTP (De Bruyckere et al., 2018).We therefore tested whether the reintroduction of C1ql2 in Bcl11b cKO DGN can rescue LTP at the mutant MFS, similarly to SV recruitment.We stimulated mossy fibers in acute slices and measured the resulting field potentials in the SL of CA3.Field responses were carefully validated for the specificity of mossy fiber signals by the presence of strong paired-pulse facilitation and block by the mGluR antagonist DCG-IV.Under these conditions, input-output curves of fEPSP slopes versus axonal fiber volleys revealed no significant differences between control and Bcl11b cKO mice (Figure 3 MF-LTP is known to be mediated by the second messenger cAMP, which is produced by adenylyl cyclase (AC) in response to Ca 2+ influx through voltage-gated Ca 2+ channels (Li et al., 2007) and kainate receptors (KAR) (Lauri et al., 2001;Schmitz et al., 2003).To test whether Bcl11b acts on LTP by interfering with presynaptic Ca 2+ dynamics, we directly activated the cAMP pathway in slices from control and Bcl11b cKO mice by applying the AC activator forskolin (Weisskopf et al., 1994).

Knock-down of C1ql2 in dentate granule neurons perturbs synaptic vesicle recruitment and long-term potentiation at the mossy fiber-CA3 synapse
To further corroborate the observation that Bcl11b acts on MFS specifically through C1ql2, we knocked down C1ql2 expression in the DGN of adult WT mice by stereotaxically injecting an AAV carrying an shRNA cassette against C1ql2 (Figure 4a).Quantitative PCR (Figure 4b), western blot analysis (Figure 4c), as well as immunohistochemistry using C1ql2 antibodies on hippocampal tissue (Figure 4d), revealed that the shRNA-mediated KD resulted in a strong reduction of C1ql2 transcripts (Figure 4b;+shNS-EGFP: 1±0.07,+shC1ql2-EGFP: 0.23±0.059,mean ± SEM) as well as protein levels (Figure 4c-d C1ql2-Nrxn3(25b+) interaction recruits presynaptic vesicles in vitro and in vivo C1ql2 was previously shown to interact with a particular splice variant of Nrxn3β containing exon 25b sequences, Nrxn3(25b+), which was recombinantly expressed in HEK293 cells (Matsuda et al., 2016).
To explore the relevant epitope that mediates the binding of C1ql2 to Nrxn3(25b+) proteins, we analyzed the solvent accessible electrostatic surface properties of the C1ql-domain trimeric structure of C1ql2 (Ressl et al., 2015) (PDB_ID: 4QPY) and found that a change of lysine262 (K262) to glutamic acid renders a large area underneath the C1ql2-specific calcium and receptor binding loops negative (Figure 5g) and hypothesized that this would repel binding to Nrxn3(25b+).We generated a C1ql2.K262E variant, expressed it in HEK293 cells as before, and tested it for its ability to cluster Nrxn3α(25b+) as well as vGlut1 in contacting primary neurons (Figure 5a-d).In the presence of C1ql2.K262E, recruitment of Nrxn3α(25b+) was significantly lower compared to WT C1ql2 and indistinguishable from myc-tag control levels (Figure 5a-b; myc-K262E: 18.84±5.15).Moreover, the expression of C1ql2.K262E in HEK293 cells was unable to accumulate vGlut1 in contacting neurons expressing GFP-Nrxn3α(25b+) (Figure 5c-d; myc-K262E: 16.9±1.2,mean ± SEM).Together, these results provide in vitro evidence that the clustering of vGlut1 depends on an intact C1ql2-Nrxn3(25b+) interaction and that a single point mutation that creates a negative charge of that surface area underneath the C1ql2-specific calcium and receptor binding loops abolishes this binding activity and, thereby, the regulation of SV clustering.
To further explore whether binding to Nrxn3 is required for C1ql2-dependent regulation of SV recruitment, we stereotaxically injected an AAV expressing GFP-tagged Cre or inactive Cre into the DG of 2-month-old Nrxn1, 2 & 3 flox/flox mice (Figure 7a), which resulted in strong reduction of Nrxn3 mRNA levels in DGN 2 months later.Only mild reduction of Nrxn1 and unchanged expression of Nrxn2 was observed (Figure 7b   ;+inactive Cre: 1±0.23,+Cre: 0.8±0.11,mean ± SEM), suggesting that overall production of C1ql2 protein was not affected.To control for the specificity of this effect, we also determined the level of C1ql3 expression and found no overt changes in Nrxn cTKO (Figure 7-figure supplement 1c).To exclude that the reduced C1ql2 fluorescence intensity was simply a consequence of an overall loss of MFB, we used ZnT3 as a marker of MFB and found it unchanged in Nrxn cTKO compared to controls (Figure 7e).Remarkably, disruption of the C1ql2-Nrxn3(25b+) binding by ablation of Nrxn3 in Nrxn cTKO mutants not only led to reduced C1ql2 fluorescence intensity (Figure 7c-d), but recapitulated the phenotype observed upon Bcl11b ablation or by KD of C1ql2 as evidenced by a large reduction of the average synapse score in Nrxn cTKO (Figure 7f-g, Figure 7-figure supplement 1d;+inactive Cre: 3.11±0.06;+Cre:2.67±0.074,mean ± SEM).Also, similarly to the Bcl11b and C1ql2 mutant phenotypes, we observed the number of docked vesicles per 100 nm of AZ profile length in Nrxn cTKO to be diminished compared to controls (Figure 7h, Figure 7-figure supplement 1e-f;+inactive Cre: 0.404±0.035,+Cre:0.195±0.02,mean ± SEM), whereas the AZ length and the diameter of docked vesicles remained unchanged (Figure 7-figure supplement 1g-h; AZ length:+inactive Cre: 193.2±6.88;+Cre:188.44±11.43,mean ± SEM; Vesicle diameter:+inactive Cre: 38.38±0.44;+Cre:37.12±0.8,mean ± SEM).Thus, our results provide evidence that Bcl11b controls MFS organization through C1ql2/Nrxn3(25b+)-dependent signaling, explicating how Bcl11b, a transcription factor with a broad range of functions, can regulate highly specific processes in the brain.

Discussion
There is emerging evidence that the zinc finger transcription factor Bcl11b is involved in the pathogenesis of neurodevelopmental as well as neuropsychiatric disorders that are frequently associated with synaptic dysfunction.Previous work from our group demonstrated Bcl11b to be essential for synapse function in the mossy fiber circuit of the adult murine hippocampus.The underlying molecular mechanisms downstream of Bcl11b, however, remained elusive.In the present study, we uncover a novel C1ql2-dependent regulatory pathway through which Bcl11b controls the structural as well as functional integrity of hippocampal MFS in adult mice.We show that SV recruitment to the AZ of MFS, as well as the expression of MF-LTP, depend on C1ql2, which is a direct functional target of Bcl11b.Reintroduction of C1ql2 into Bcl11b mutant DGN restores defective SV recruitment and LTP expression.KD of C1ql2 in DGN recapitulates the impaired SV recruitment and loss of LTP observed in Bcl11b mutants.Finally, we show that C1ql2 controls SV recruitment through a direct interaction with presynaptic Nrxn3(25b+), while LTP depends on C1ql2 signals independent of Nrxn3 interaction.Recent studies suggested Nrxn3, as well as C1ql2, to be associated with neuropsychiatric disorders (Hishimoto et al., 2007;Hu et al., 2013;Huggett and Stallings, 2020a;Marballi et al., 2022).Our study for the first time identifies a Bcl11b/C1ql2/Nrxn3-dependent signaling pathway in the control of basic structural and functional properties of MFS.Analysis of this regulatory pathway in mice may provide important novel insights into the pathogenesis of neurodevelopmental and neuropsychiatric disorders.
We have previously shown that conditional ablation of Bcl11b in the adult hippocampus leads to structural and functional changes of MFS characterized by an overall reduction in synapse numbers, loss of bouton complexity, misdistribution of SV as well as loss of MF-LTP (De Bruyckere et al., 2018).Here, we found that reintroduction of the synaptic organizer protein C1ql2, which is a direct transcriptional target of Bcl11b and is downregulated in Bcl11b mutant DGN (De Bruyckere et al., 2018), was Source data 2. Original file for the western blot analysis in Figure 6b.
Source data 3. PDF containing Figure 6b and original scans of the relevant western blot analysis with highlighted bands and sample labels.Furthermore, KD of C1ql2 in WT DGN recapitulated the Bcl11b phenotype with impaired SV recruitment and loss of LTP, supporting the specificity of C1ql2 function.MF-LTP, which manifests as a longterm increase in presynaptic vesicle release probability (P r ) (Shahoha et al., 2022), directly depends on the distribution of SV in the proximity of the AZ.Recent studies have shown that the increase in P r involves the recruitment of new AZ and an increase in the number of docked and tethered vesicles, corresponding to the readily releasable pool of SV (Orlando et al., 2021;Vandael et al., 2020).The perturbed SV recruitment in both Bcl11b cKO and C1ql2 KD mice could thus potentially explain the loss of LTP in both conditions.Indeed, the reintroduction of C1ql2 in Bcl11b cKO DGN specifically rescued SV recruitment and LTP, while synapse numbers and ultrastructural complexity of MFB remained unchanged.Whether the regulation of SV distribution by C1ql2 and the expression of MF-LTP are directly and causally linked remains to be determined.Additional factors have been suggested to contribute to the increase in P r , including a tighter coupling between Ca 2+ channels and SV (Midorikawa and Sakaba, 2017) and the accumulation of Ca 2+ channels near release sites (Fukaya et al., 2021).It cannot, therefore, be excluded that C1ql2 regulates MF-LTP through one of these alternative mechanisms.Aiming to narrow in on the nature of the mechanism through which Bcl11b regulates MF-LTP, we used forskolin to induce LTP in Bcl11b cKO.MF-LTP relies on presynaptic mechanisms (Castillo, 2012;Zalutsky and Nicoll, 1990) and is mediated by the second messenger cAMP, which is produced by AC in response to Ca 2+ influx through voltage-gated Ca 2+ channels (Li et al., 2007) and KARs (Lauri et al., 2001;Schmitz et al., 2003).By using forskolin to directly activate AC, we bypassed these initial steps, and still found a reduction of LTP in Bcl11b cKO mice, similarly to HFS.These results strongly suggest that the loss of LTP is caused by a process downstream of the initial presynaptic Ca 2+ influx following stimulation.We note that, in the present experiments, we did not observe the decrease in input-output relation in Bcl11b cKO as reported in De Bruyckere et al., 2018.After excluding technical differences, e.g., different methods of data analysis, we conclude that the discrepancy is best explained by differences in the population of presynaptic fibers.In the present study, mossy fiber responses were specifically identified by testing for frequency facilitation and sensitivity to mGluR antagonists, whereas in the previous study, this purification was not done (De Bruyckere et al., 2018).It is not immediately obvious why the reduction in synapse numbers and misdistribution of SV in Bcl11b cKO animals does not affect basal synaptic transmission.While a modest displacement of SV might fail to noticeably influence synaptic transmission due to the low initial P r at MFS, causing only a fraction of release-ready vesicles to be initially released, the reduction in synapse numbers might indeed be expected to reflect in the input-output relationship.It might be that synapses that are preferentially eliminated in Bcl11b mutants are predominantly silent or have weak coupling strength, such that their loss has only a minimal effect on synaptic transmission.Further investigation is needed to elucidate this apparent discrepancy.Together, our results suggest Bcl11b to be an important synaptic regulator that controls the structure and function of adult MFS through both C1ql2-dependent, as well as -independent transcriptional programs.C1ql proteins are complement-related factors that are synthesized by the presynapse and secreted into the synaptic cleft.Within the hippocampus, C1ql2 and -3 protein expression overlaps and is   highly restricted to DGN (Iijima et al., 2010) and the corresponding mossy fiber system, including MF-CA3 synapses.C1ql2 and -3 were previously suggested to form functional heteromers that can cluster postsynaptic KAR on MFS.Selective deletion of either C1ql2 or -3 in mice was reported to have no overt mutant hippocampal phenotype, suggesting functional compensation for both proteins (Matsuda et al., 2016).Using shRNA-mediated selective KD of C1ql2 in DGN as well as rescue of the Bcl11b mutation by the reintroduction of C1ql2 into mutant DGN, we observed a novel, presynaptic function for C1ql2 in the recruitment of SV and the expression of LTP in MFS.This function was specific to C1ql2 since overexpression of C1ql3 in Bcl11b mutant DGN was unable to rescue the synapse phenotype.Furthermore, another study has identified all four C1ql proteins, including C1ql2, as ligands for the postsynaptic Brain-specific angiogenesis inhibitor 3 (BAI3).Addition of any of the four C1ql proteins to cultured hippocampal neurons led to a loss of excitatory synapses, a function inhibited by the presence of BAI3 (Bolliger et al., 2011).In our study, we show that neither loss of C1ql2 nor overexpression of C1ql2 affects the number of MFS, supporting the notion that synaptic organizers have synapse-specific functions.This highlights the role of C1ql2 as a synaptic organizer and adds a new layer of understanding to its function at the MFS.
Previous in vitro studies suggested that C1ql2 function at the MFS involves interaction with Nrxn3 isoforms containing the splice site 5 25b sequence (SS5 25b ) (Matsuda et al., 2016).Nrxns are synaptic cell adhesion molecules that mediate various synaptic properties (Reissner et al., 2013;Südhof, 2017), including the recruitment of SV and dense-core vesicles (Dean et al., 2003;Ferdos et al., 2021;Quinn et al., 2017;Rui et al., 2017).This prompted us to analyze, whether C1ql2dependent SV recruitment in MFS requires a direct interaction with Nrxn3(25b+) in vitro and in vivo.Expression of C1ql2 in HEK293 cells co-cultured with GFP-Nrxn3α(25+)-expressing hippocampal neurons was able to recruit Nrxn3α(25b+) and vGlut1 at contact points, while C1ql2.K262E, a C1ql2 variant with an amino-acid replacement that perturbs the interaction with Nrxn3(25b+), was no longer able to recruit neuronal vGlut1.Furthermore, clustering of vGlut1 by C1ql2-secreting HEK293 cells was reduced in neurons harboring a pan-neurexin mutation, a phenotype that was rescued by the selective reintroduction of Nrxn3α(25b+).Finally, the introduction of C1ql2.K262E in Bcl11b cKO DGN in vivo was unable to rescue SV recruitment, while the silencing of Nrxns in DGN in vivo perturbed SV recruitment to a similar extent as in Bcl11b cKO and C1ql2 KD.Based on these findings, we anticipated the overexpression of C1ql2.K262E in Bcl11b cKO DGN to be unable to rescue MF-LTP.Unexpectedly, the introduction of C1ql2.K262E into Bcl11b cKO fully rescued MF-LTP.This raises the possibility that C1ql2 can influence MF-LTP through additional, yet uncharacterized mechanisms, independent of SV recruitment or direct interaction with Nrxn3(25b+).We cannot exclude, however, that the expression of a mutant C1ql2 variant created an additional gainof-function effect that circumvented SV recruitment and allowed the rescue of MF-LTP in our experimental system.The latter is supported by the fact that within the first 10 min after HFS, fEPSP slopes for C1ql2.K262E were significantly elevated compared to controls, an effect that was not seen after C1ql2 re-expression.Together, our data provide comprehensive experimental evidence that the direct interaction of C1ql2 with Nrxn3(25b+) is essential for SV recruitment at the MFS.Finally, we observed that an abolished interaction between C1ql2 and Nrxn3(25b+) was associated with reduced localization of the C1ql2 protein along the MF tract.This raises the possibility that C1ql2-Nrxn3 interaction might be involved in surface presentation of C1ql2, transportation, or stabilization at the MFS.The C1q domain can form stable, higher order oligomers (Ressl et al., 2015).Neurexins, on the other hand, are highly mobile outside and inside of synaptic terminals (Klatt et al., 2021;Neupert et al., 2015).Thus, the interaction of C1ql2 with Nrxn3(25b+) may reciprocally augment the accumulation of both proteins at synaptic sites.
Neurexin mRNAs are subjected to extensive alternative splicing that leads to the expression of thousands of isoforms with differential expression patterns (Treutlein et al., 2014;Ullrich et al., 1995) that act in a type-specific manner on synaptic functions (Dai et al., 2019;Schreiner et al., 2014;Traunmüller et al., 2016).Nrxn3 splice variants have been shown to regulate the function and plasticity of glutamatergic and GABAergic synapses through various mechanisms (Aoto et al., 2013;Dai et al., 2019;Lloyd et al., 2023;Trotter et al., 2023).The Nrxn3 splice site SS5 is a major contributor to the high number of Nrxn3 isoforms (Schreiner et al., 2014).One such isoform was recently found to be highly expressed in GABAergic interneurons at the DG, where it regulates dendritic inhibition (Hauser et al., 2022).Our findings on the role of Nrxn3 isoforms containing SS5 25b in the recruitment of SV at the MFS through interaction with C1ql2 add to the understanding of the synapse-specific mechanisms of action of Nrxns.
Perturbations in synaptic structure and function are major determinants of various neuropsychiatric and neurodevelopmental disorders (Hayashi-Takagi, 2017;Lepeta et al., 2016;Zoghbi and Bear, 2012).Emerging evidence from recent genetic studies suggests such disorders to be linked to various genes encoding for synaptic proteins (Südhof, 2021;Torres et al., 2017;Wang et al., 2018).Decoding the molecular mechanisms of synaptic organization and stability and their transcriptional regulation would therefore be expected to contribute to the mechanistic understanding of neuropsychiatric and neurodevelopmental disorders.The transcription factor Bcl11b has been linked to neurodevelopmental (Lessel et al., 2018), neurodegenerative (Kunkle et al., 2016;Song et al., 2022) and neuropsychiatric disorders (Whitton et al., 2018;Whitton et al., 2016).BCL11B mutations in humans are associated with neurodevelopmental delay, overall learning deficits as well as impaired speech acquisition and autistic features (Eto et al., 2022;Lessel et al., 2018;Punwani et al., 2016;Yang et al., 2020).Moreover, conditional ablation of Bcl11b selectively in the adult murine hippocampus results in impaired learning and memory behavior (Simon et al., 2016).NRXN3 single-nucleotide polymorphisms (SNP) have been implicated in schizophrenia (Hu et al., 2013) and addiction (Hishimoto et al., 2007), with one recorded SNP located close to SS5 altering the expression of Nrxn3(25b+).Interestingly, recent studies have also associated C1QL2 with schizophrenia (Marballi et al., 2022) as well as cocaine addiction (Huggett and Stallings, 2020b).In this study we demonstrate that Bcl11b, through its transcriptional target C1ql2, modulates the synaptic organization of MFS by controlling the recruitment of SV at AZ.This regulatory mechanism depends on a direct interaction of C1ql2 with Nrxn3(25b+).Importantly, SV trafficking and altered release probability have been implicated in neurological and neuropsychiatric disorders (Egbujo et al., 2016;Lepeta et al., 2016;Zhu et al., 2021).Thus, the identification of the Bcl11b/C1ql2/Nrxn3(25b+)-dependent signaling module in this study provides a new entry point for future mechanistic analyses of synaptopathies.Moreover, the existence of such cell-type-specific signaling modules reveals how a fundamental transcription factor with diverse functions such as Bcl11b can be implicated in the pathogenesis of brain disorders characterized by synaptic dysfunction.

Stereotaxic injections
For the expression of C1ql2 and C1ql3, the DG of 80 days old Bcl11b cKO mice were injected with AAV vectors expressing EGFP-2A-C1ql2 and EGFP-2A-C1ql3, respectively.As control, Bcl11b cKO and control mice were injected with an AAV expressing EGFP.For the KD of C1ql2, the DG of 60-dayold C57BL/6JRj mice were injected with AAV 4 x shC1ql2-EGFP, expressing 4 shRNAs against C1ql2 or control AAV 4xshNS-EGFP, expressing 4 x non-sense shRNAs.For pan-neurexin KO, the DG of 60 days old Nrxn1, 2 and 3 flox/flox mice were injected with an AAV expressing EGFP-Cre or a control AAV expressing EGFP-Cre.Y324F, an inactive Cre.All AAVs were produced by the Viral Vector Facility of the Neuroscience Center Zurich on request.The four selected non-sense shRNAs and the four shC1ql2 sequences were checked for and presented with no off-target bindings on the murine exome with up to two mismatches by siRNA-Check (http://projects.insilico.us/SpliceCenter/siRNACheck).The mice were anesthetized with 5% isoflurane and placed in a mouse stereotaxic apparatus.During the entire procedure, anesthesia was maintained by constant administration of 2.2% isoflurane.Eye ointment was applied to prevent eyes from drying.For electrophysiological experiments, mice were subcutaneously injected with buprenorphine hydrochloride (0.1 mg/kg, Temgesic, Indivior) 30 min before and 3 hr after each surgery.For all other experiments, Butorphanol (Livisto) and Meloxicam (Boehringer-Ingelheim; 5 μg/g) were injected subcutaneously and the local anesthetic Bupivacaine (Puren; 5 μg/g) was injected subcutaneously at the incision site.After 10 min the head of the mouse was shaved and disinfected and an incision was made in the skin.Targeted injection sites were identified and a small craniotomy was performed for each site.The injector was placed at the individual sites and the viral solution was injected at 100 nL/min, with a 5-10 min recovery before removing the injector.After injections at all sites the incision was sutured and the animal was monitored for recovery from anesthesia, after which it was returned to its home cage.For histological and EM analyses of MFS, AAV were injected at three sites per hemisphere with the following coordinates (Bregma: AP 0; ML: 0; DV:0): AP -2 mm; ML ±1 mm; DV -2 mm.AP -2.5 mm; ML ±1.5 mm; DV -1.8 mm.AP -3.1 mm; ML ±2, DV -2.2 mm.For electrophysiological analyses, AAV were injected at two dorsoventral coordinates per hemisphere: AP -3.0 mm; ML ±3.25 mm; DV -2.4 and -2.8 mm.200-300 nL of AAV (1e12 vg/mL) were injected in each location.

RNA isolation and quantitative real-time PCR
All procedures were performed in an RNase-free environment.Animals were sacrificed under deep CO 2 -induced anesthesia, brains were quickly dissected in ice-cold PBS, cryopreserved in 20% sucrose overnight, frozen in OCT compound (Polysciences), and stored at −80 °C.Twenty μm thick coronal sections were collected on UV-treated and 0.05% poly-L-lysine coated membrane-covered PEN slides (Zeiss), fixed for 1 min in ice-cold 70% EtOH, incubated for 45 sec in 1% cresyl violet acetate solution (Waldeck) and washed for 1 min each in 70% EtOH and 100% EtOH.Sections were briefly dried on a 37 °C warming plate and immediately processed.The granule cell layer of the DG was isolated by laser capture microdissection using a PALM MicroBeam Rel.4.2 (Zeiss).RNA was isolated from the collected tissue using Rneasy Micro Kit (Qiagen) and reverse transcribed using the SensiFast cDNA Synthesis Kit (Bioline).Quantitative real-time PCR was performed in triplets for each sample using the LightCycler DNA Master SYBR Green I Kit in a LightCycler 480 System (Roche).The relative copy number of Gapdh RNA was used for normalization.Data were analyzed using the comparative CT method (Schmittgen and Livak, 2008).

Transmission electron microscopy
Animals were sacrificed through CO 2 -inhalation and immediately perfused transcardially with 0.9% NaCl for 1 min, followed by a fixative solution of 1.5% glutaraldehyde (Carl Roth) and 4% PFA in 0.1 M PB pH 7.2 for 13 min.Brains were dissected and postfixed in the fixative solution for 4 hr at 4 °C.Ultrathin sections (60 nm) were prepared and stained with lead citrate.Images were acquired using a transmission electron microscope LEO 906 (Zeiss) with a sharp-eye 2 k CCD camera and processed with ImageSP (Tröndle).Synapse score (De Bruyckere et al., 2018) was calculated according to the following criteria: 0-5 vesicles above the active zone = 0; 5-20 vesicles = 1; small group of vesicles (≤200,000 nm 2 ) with distance between density and closest vesicle >100 nm=2; small group of vesicles (≤200,000 nm 2 ) with distance between density and closest vesicle ≤100 nm=3; big group of vesicles (>200,000 nm 2 ) with distance between density and closest vesicle >100 nm=4; big group of vesicles (>200,000 nm 2 ) with distance between density and closest vesicle ≤100 nm=5.Synapses from approximately 30 MFB per animal were analyzed.Vesicles with a distance ≤5 nm from the plasma membrane were considered docked (Kusick et al., 2022;Vandael et al., 2020).Approximately 100 AZ per animal were analyzed.
Recordings were carried out by placing a glass micropipette (tip diameter 3-5 μm) filled with ACSF in the SL of the CA3b area.To induce MF field excitatory post-synaptic potentials (fEPSP), a bipolar electrode (Science Products) was placed within the hilus region of the DG.0.1 ms pulses were delivered with an Iso-Flex stimulus isolator (AMPI) at 20 s intervals.Putative mossy fiber signals were preliminarily identified using a 25 Hz train of five pulses.Input-output relationships were obtained by measuring the fiber volley amplitude and fEPSP slope in response to stimulations with intensities ranging from 3 to 40 V.For LTP recordings, stimulation intensity for each slice was adjusted to obtain a slope value of 20% (30% in the case of forskolin (Biomol) experiments) of the maximum fEPSP slope.LTP was induced by three trains of 100 stimulation pulses at 100 Hz (high-frequency stimulation, HFS), repeated every 8 s. 3 μM DCG-IV (Tocris Bioscience) was applied after each experiment, and only recordings displaying >70% reduction in putative MF-fEPSP slopes were used for analysis.fEPSPs were amplified 100 x with an EXT 10-2 F amplifier (npi electronics).Signals were low-pass filtered at 2 kHz and high-pass filtered at 0.3 Hz, digitized at 20 kHz with an analog-to-digital converter (Cambridge Electronic Design [CED]) and stored for offline analysis using Spike2 (v7) software (CED).Offline data analysis was performed on raw traces using Spike2.Slope values were measured from the linear part of the fEPSP rising phase by manually placing vertical cursors.Changes in fEPSP slopes were calculated as a percentage of the average baseline fEPSP ((average fEPSP slope in a given time interval after HFS -average fEPSP slope before HFS)/ (average fEPSP slope before HFS)).

DNA constructs
For expression, C1ql2 was cloned from mouse cDNA.A 6xHis-myc tag or GFP was attached to the N-terminus and the construct was cloned into the pSecTag2A vector (Invitrogen) in frame with the N-terminal IgK signal sequence.A stop codon was introduced directly after C1ql2.The K262E point mutation was introduced with the Q5 Site-Directed Mutagenesis Kit (New England Biolabs).pSecTag2A was used for control experiments.Rat Nrxn3α(25b+) cDNA (Ushkaryov and Südhof, 1993) was inserted into an pSyn5 vector with human Synapsin promoter (Neupert et al., 2015) using BamHI and BglII.For the pan-neurexin KO and the control experiments, vectors with NLS-GFP-Cre or NLS-GFP-Cre.Y324F were used (Klatt et al., 2021;Wang et al., 2016).Expression vector for GluK2 was purchased from OriGene.All vectors were validated by sequencing (Eurofins Genomics).

Primary hippocampal cultures
Hippocampi were dissected from P0 mice in HBSS media, digested for 15 min with HBSS containing 0.1% Trypsin (Gibco) at 37 °C, dissociated in plating media (MEM supplemented with 0.6% glucose, 10% FBS, 1% penicillin/streptomycin, DnaseI 4 U/mL) and seeded on poly-L-Lysin precoated coverslips placed inside 12-well plates at 1.5x10 5 cells/mL.After 3 hr, the plating media was replaced with neuronal growth media (Neurobasal A supplemented with 2% B27, 2 mM L-Glutamine, 1% penicillin/ streptomycin, 1% N2 and 0.005% NGF).Cultures were kept at 37 °C under 5% CO 2 atmosphere.The day of plating was considered as 0 days in vitro (DIV).At DIV3 and DIV7 80% of the medium was exchanged with fresh growth medium.At DIV9 the medium was exchanged with penicillin/ streptomycin-free growth medium and at DIV10 neurons were transfected using Lipofectamine 2000 (Invitrogen).Briefly, a total of 200 μL transfection mix per well was prepared by first mixing 100 μL Opti-MEM with 4 μL Lipofectamine 2000 in one tube and 100 μL Opti-MEM with 3 μg DNA in a different tube.After 5 min both volumes were combined and the mixture was incubated for 20 min at RT.The transfection mix was then added dropwise to the neurons.After 3 hr of incubation, the medium was exchanged with fresh growth medium.

HEK293 cell culture
Human embryonic Kidney (HEK) 293 cells were obtained from ATCC and were maintained in DMEM supplemented with 10% fetal calf serum and 1% penicillin/streptomycin at 37 °C under 5% CO 2 atmosphere.Cells were transfected using Lipofectamine 2000 according to the manufacturer's instructions on the same day the neurons were transfected.Cells were incubated for at least 24 hr before being used in co-culture experiments.

Neuronal and HEK293 co-culture and immunostaining
Transfected HEK293 cells were washed, dissociated, and resuspended in neuronal growth medium.15x10 3 cells were added in each well containing DIV11 transfected neurons.HEK293 cells were co-cultured with the hippocampal primary neurons 2 days (DIV13 for neurons) before proceeding with immunostaining.Coverslips with cultured neurons and HEK293 cells were first fixed with 4% PFA in 1 x PBS for 10 min at 4 °C, then washed 3 x with 1 mL 1 x PBS and blocked with 1 x PBS containing 0.1% Triton X-100 and 10% horse serum for 1 hr at RT.Primary antibodies were incubated overnight at 4 °C, followed by a 90 min incubation with secondary antibodies.Cells were counterstained with DAPI (Molecular Probes).The following primary antibodies were used: rabbit anti-myc-tag (1:2000; Abcam), guinea pig anti-vGlut1 (1:250; Synaptic Systems), chicken anti-GFP (1:2000; Abcam).All fluorescent secondary antibodies were purchased from Jackson ImmunoResearch and used at 1:500 dilution.For each condition, 25 cells per experiment were analyzed.

Structural protein modelling
The crystal structure of trimeric C1q-domains of mouse C1ql2 (Ressl et al., 2015) was used to predict a potential electrostatic binding site to splice insert 25 of Nrxn3α.An electrostatic surface map of the trimer was calculated using APBS (Jurrus et al., 2018).The K262E mutation was introduced using FoldX ( foldxsuite.crg.eu) and was chosen in order to generate a negatively charged surface that would potentially be repulsive to Nrxn3α binding.Final models were visualized with PyMOL (https://pymol.org/2/).

Co-immunoprecipitation
HEK293 cells were transfected using Lipofectamine 3000 according to the manufacturer's instructions and were incubated for at least 48 hr.Cells were harvested and proteins were extracted in lysis buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl 2 , 1% Igepal, 5% Glycerol, 1 x EDTA-free proteinase inhibitor, 0.5 mM DTT and 2.5 U/mL Benzonase.Protein A magnetic beads were washed 2 x with PBS including 0.02% Tween-20 and were incubated on a rotating wheel in RT for 2 hr with 2 μg of the following antibodies suspended in 200 μL 2 x with PBS/0.02%Tween-100: rabbit anti-IgG (Cell Signal), rabbit anti-flag (Sigma-Aldrich) or rabbit anti-C1ql2 (Sigma-Aldrich).Beads were washed 2 x with PBS/0.02%Tween-20, resuspended in 50 μL 2 x with PBS/0.02%Tween-20 and 40 μg protein extract was added.Beads were incubated o/n on a rotating wheel at 4 °C.Beads were thoroughly washed with 2 x with PBS/0.02%Tween-20, resuspended in 2 x SDS loading dye, and boiled for 10 min at 95 °C.Western blot (see above) was performed.Briefly, membranes were blocked with 5% non-fat milk (Sigma-Aldrich) and incubated with mouse anti-flag M2 (1:2000; Sigma-Aldrich).Three independent experiments were carried out.

Image acquisition and analysis
All fluorescent images of sectioned hippocampal tissue were examined on a TCS SP5II confocal microscope (Leica) using LAS-X software and processed with Fiji (Schindelin et al., 2012).Overview images were acquired with a 20 x objective.Synapse numbers and ZnT3 + puncta were quantified in the SL imaged with a 40 x objective at x2 zoom.C1ql2 fluorescence intensity was quantified in the SL imaged with a 40 x objective.Acquisition settings were kept constant for every sample and condition.All fluorescent images of co-cultured HEK293 cells were examined on a TCS SP8 confocal microscope (Leica) using LAS-X software and processed with Fiji.Images were acquired with a 40 x objective at 4 x zoom.As before, acquisition settings were kept constant for every sample and condition.Images were analyzed by masking transfected HEK293 cells and measuring the area of each mask covered by the chosen stain.

Quantification and statistical analysis
Statistical analysis and graph generation was done using Python 3. If samples met the criteria for normality, we used two-tailed unpaired t-test to compare two groups and one-way ANOVA for more than two groups.For non-normally distributed data Mann-Whitney u-test was used.Two-way ANOVA was used for examining the influence of two different categorical independent variables.If ANOVAs were significant, we used a post hoc Tukey's multiple-comparisons test to compare groups (structural and expression data) or a post hoc Bonferonni's comparison test (electrophysiological data).Data are presented as mean ± SEM.Significance levels were set as indicated in figures: *p<0.05,**p<0.01,***p<0.001.

Figure supplement 1 .
Figure supplement 1. C1ql2 mRNA and protein are lost upon Bcl11b cKO in DGN.

Figure supplement 1 .
Figure supplement 1. C1ql2 reintroduction in Bcl11b cKO DGN does not rescue MFS number and MFB complexity.
The online version of this article includes the following source data and figure supplement(s) for figure3: Source data 1.File containing the raw data for Figure 3, panels b & e and for Figure 3-figure supplement 1, panels b and d.

Figure supplement 1 .
Figure supplement 1. Bcl11b cKO and C1ql2 KD in DGN do not affect basal synaptic transmission.

Figure 4 .
Figure3 continued after HFS.Changes in fEPSP slope are shown as percentage of the mean baseline fEPSP.+shNS EGFP, 6 slices from 6 mice;+shC1ql2-EGFP, 7 slices from 7 mice.All data are presented as means; error bars indicate SEM.Mann-Whitney U-test for each time interval.10-20 min: **p=0.0012;20-30 min: **p=0.0023;30-40 min: **p=0.0023;ns, not significant.The online version of this article includes the following source data and figure supplement(s) for figure 4: Source data 1.File containing the raw data for Figure 4, panels b, e-f & h and for Figure 4-figure supplement 1, panels b-g & i.Source data 2. Original file for the western blot analysis in Figure4c.Source data 3. PDF containing Figure4cand original scans of the relevant western blot analysis with highlighted bands and sample labels.

Figure supplement 1 .
Figure supplement 1. C1ql2 KD in DGN of WT mice impairs SV recruitment.

Figure 6
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Figure supplement 1
Figure supplement 1-source data 1.Original file for the Western blot analysis in Figure 6-figure supplement 1k.

Figure supplement 1
Figure supplement 1-source data 2. PDF containing Figure 6-figure supplement 1k and original scans of the relevant Western blot analysis with highlighted bands and sample labels.

Figure 7
Figure 7 continued on next page Scale bar: 15 μm.(d) Integrated density of C1ql2 fluorescence in the SL of CA3.N=3.All data are presented as means; error bars indicate SEM.Unpaired t-test.*p=0.02.(e) Immunohistochemistry of ZnT3 (red) and GFP (green) in hippocampal sections.Scale bar: 200 μm.Upper right corner of ZnT3 panels depicts close-ups from the SL of CA3.Scale bar: 15 μm.(f) Electron microscope images of MFS and proximal SVs.White bars mark synapse length from postsynaptic side.Scale bar: 100 nm.(g) Average synapse score.N=3.Unpaired t-test.**p=0.009.(h) Number of docked vesicles per 100 nm AZ profile length.N=3.All data are presented as means; error bars indicate SEM.Points represent the individual examined AZ.Unpaired t-test.**p=0.007.The online version of this article includes the following source data and figure supplement(s) for figure 7: Source data 1.File containing the raw data for Figure 7, panels b, d, and g-h and for Figure 7-figure supplement 1, panels a-b and d-g.
Figure 7 continued