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

Reviewer #1 (Public Review):

Koumoundourou et al., identify a pathway downstream of Bcl11b that controls synapse morphology and plasticity of hippocampal mossy fiber synapses. Using an elegant combination of in vivo, ex vivo, and in vitro approaches, the authors build on their previous work that indicated C1ql2 as a functional target of Bcl11b (De Bruyckere et al., 2018). Here, they examine the functional implications of C1ql2 at MF synapses in Bcl11b cKO mice and following C1ql2 shRNA. The authors find that Bcl11b KO and shRNA against C1ql2 significantly reduces the recruitment of synaptic vesicles and impairs LTP at MF synapses. Importantly, the authors test a role for the previously identified C1ql2 binding partner, exon 25b-containing Nrxn3 (Matsuda et al., 2016), as relevant at MF synapses to maintain synaptic vesicle recruitment. To test this, the authors developed a K262E C1ql2 mutant that disrupts binding to Nrxn3. Curiously, while Bcl11b KO and C1ql2 KD largely phenocopy (reduced vesicle recruitment and impaired LTP), only vesicle recruitment is dependent on C1ql2-Nrxn3 interactions. These findings provide new insight into the functional role of C1ql2 at MF synapses. While the authors convincingly demonstrate a role for C1ql2-Nrxn3(25b+) interaction for vesicle recruitment and a Nrxn3(25b+)-independent role for C1ql2 in LTP, the underlying mechanisms remain inconclusive. Additionally, a discussion of how these findings relate to previous work on C1ql2 at mossy fiber synapses and how the findings contribute to the biology of Nrxn3 would increase the interpretability of this work.

Reviewer #2 (Public Review):

This manuscript describes experiments that further investigate the actions of the transcription factor Bcl11b in regulating mossy fiber (MF) synapses in the hippocampus. Prior work from the same group had demonstrated that loss of Bcl11b results in loss of MF synapses as well as a decrease in LTP. Here the authors focus on a target of Bcl11b a secreted synaptic organizer C1ql2 which is almost completely lost in Bcl11b KO. Viral reintroduction of C1ql2 rescues the synaptic phenotypes, whereas direct KD of C1ql2 recapitulates the Bcl1 phenotype. C1ql2 itself interacts directly with Nrxn3 and replacement with a binding deficient mutant C1q was not able to rescue the Bcl11b KO phenotype. Overall there are some interesting observations in the study, however there are also some concerns about the measures and interpretation of data.

The authors state that they used a differential transcriptomic analysis to screen for candidate targets of Bcl11b, yet they do not present any details of this screen. This should be included and at the very least a table of all DE genes included. It is likely that many other genes are also regulated by Bcl11b so it would be important to the reader to see the rationale for focusing attention on C1ql2 in this study.

All viral-mediated expression uses AAVs which are known to ablate neurogenesis in the DG (Johnston DOI: 10.7554/eLife.59291) through the ITR regions and leads to hyperexcitability of the dentate. While it is not clear how this would impact the measurements the authors make in MF-CA3 synapses, this should be acknowledged as a potential caveat in this study.

The authors claim that the viral re-introduction "restored C1ql2 protein expression to control levels. This is misleading given that the mean of the data is 2.5x the control (Figure 1d and also see Figure 6c). The low n and large variance are a problem for these data. Moreover, they are marked ns but the authors should report p values for these. At the least, this likely large overexpression and variability should be acknowledged. In addition, the use of clipped bands on Western blots should be avoided. Please show the complete protein gel in primary figures of supplemental information.

Measurement of EM micrographs: As prior work suggested that MF synapse structure is disrupted the authors should report active zone length as this may itself affect "synapse score" defined by the number of vesicles docked. More concerning is that the example KO micrographs seem to have lost all the densely clustered synaptic vesicles that are away from the AZ in normal MF synapses e.g. compare control and KO terminals in Fig 2a or 6f or 7f. These terminals look aberrant and suggest that the important measure is not what is docked but what is present in the terminal cytoplasm that normally makes up the reserve pool. This needs to be addressed with further analysis and modifications to the manuscript.

The study also presents correlated changes in MF LTP in Bcl11b KO which are rescued by C1ql2 expression. It is not clear whether the structural and functional deficits are causally linked and this should be made clearer in the manuscript. It is also not apparent why this functional measure was chosen as it is unlikely that C1ql2 plays a direct role in presynaptic plasticity mechanisms that are through a cAMP/ PKA pathway and likely disrupted LTP is due to dysfunctional synapses rather than a specific LTP effect. The authors should consider measures that might support the role of Bcl11b targets in SV recruitment during the depletion of synapses or measurements of the readily releasable pool size that would complement their findings in structural studies.
Bcl11b KO reduces the number of synapses, yet the I-O curve reported in Supp Fig 2 is not changed. How is that possible? This should be explained.

Matsuda et al DOI: 10.1016/j.neuron.2016.04.001 previously reported that C1ql2 organizes MF synapses by aligning postsynaptic kainate receptors with presynaptic elements. As this may have consequences for the functional properties of MF synapses including their plasticity, the authors should report whether they see deficient postsynaptic glutamate receptor signaling in the Bcl11b KO and rescue in the C1ql2 re-expression.

Reviewer #3 (Public Review):

Overall, this is a strong manuscript that uses multiple current techniques to provide specific mechanistic insight into prior discoveries of the contributions of the Bcl11b transcription factor to mossy fiber synapses of dentate gyrus granule cells. The authors employ an adult deletion of Bcl11b via Tamoxifen-inducible Cre and use immunohistochemical, electron microscopy, and electrophysiological studies of synaptic plasticity, together with viral rescue of C1ql2, a direct transcriptional target of Bcl11b or Nrxn3, to construct a molecular cascade downstream of Bcl11b for DG mossy fiber synapse development. They find that C1ql2 re-expression in Bcl11b cKOs can rescue the synaptic vesicle docking phenotype and the impairments in MF-LTP of these mutants. They also show that C1ql2 knockdown in DG neurons can phenocopy the vesicle docking and plasticity phenotypes of the Bcl11b cKO. They also use artificial synapse formation assays to suggest that C1ql2 functions together with a specific Nrxn3 splice isoform in mediating MF axon development, extending these data with a C1ql2-K262E mutant that purports to specifically disrupt interactions with Nrxn3. All of the molecules involved in this cascade are disease-associated and this study provides an excellent blueprint for uncovering downstream mediators of transcription factor disruption. Together this makes this work of great interest to the field. Strengths are the sophisticated use of viral replacement and multi-level phenotypic analysis while weaknesses include the linkage of C1ql2 with a specific Nrxn3 splice variant in mediating these effects.

Here is an appraisal of the main claims and conclusions:

1. C1ql2 is a downstream target of Bcl11b which mediates the synaptic vesicle recruitment and synaptic plasticity phenotypes seen in these cKOs. This is supported by the clear rescue phenotypes of synapse anatomy (Fig.2) and MF synaptic plasticity (Fig.3). One weakness here is the absence of a control assessing over-expression phenotypes of C1ql2. It's clear from Fig.1D that viral rescue is often greater than WT expression (totally expected). In the case where you are trying to suppress a LoF phenotype, it is important to make sure that enhanced expression of C1ql2 in a WT background does not cause your rescue phenotype. A strong overexpression phenotype in WT would weaken the claim that C1ql2 is the main mediator of the Bcl11b phenotype for MF synapse phenotypes.

2. Knockdown of C1ql2 via 4 shRNAs is sufficient to produce the synaptic vesicle recruitment and MF-LTP phenotypes. This is supported by clear effects in the shRNA-C1ql2 groups as compared to nonsense-EGFP controls. One concern (particularly given the use of 4 distinct shRNAs) is the potential for off-target effects, which is best controlled for by a rescue experiment with RNA-insensitive C1ql2 cDNA as opposed to nonsense sequences, which may not elicit the same off-target effects.

3. C1ql2 interacts with Nrxn3(25b+) to facilitate MF terminal SV clustering. This claim is theoretically supported by the HEK cell artificial synapse formation assay (Fig.5), the inability of the K262-C1ql2 mutation to rescue the Bcl11b phenotype (Fig.6), and the altered localization of C1ql2 in the Nrxn1-3 deletion mice (Fig.7). Each of these lines of experimental evidence has caveats that should be acknowledged and addressed. Given the hypothesis that C1ql2 and Nrxn3b(25b) are expressed in DG neurons and work together, the heterologous co-culture experiment seems strange. Up till now, the authors are looking at pre-synaptic function of C1ql2 since they are re-expressing it in DGNs. The phenotypes they are seeing are also pre-synaptic and/or consistent with pre-synaptic dysfunction. In Fig.5, they are testing whether C1ql2 can induce pre-synaptic differentiation in trans, i.e. theoretically being released from the 293 cells "post-synaptically". But the post-synaptic ligands (Nlgn1 and and GluKs) are not present in the 293 cells, so a heterologous synapse assay doesn't really make sense here. The effect that the authors are seeing likely reflects the fact that C1ql2 and Nrxn3 do bind to each other, so C1ql2 is acting as an artificial post-synaptic ligand, in that it can cluster Nrxn3 which in turn clusters synaptic vesicles. But this does not test the model that the authors propose (i.e. C1ql2 and Nrxn3 are both expressed in MF terminals). Perhaps a heterologous assay where GluK2 is put into HEK cells and the C1ql2 and Nrxn3 are simultaneously or individually manipulated in DG neurons?

4. K262-C1ql2 mutation blocks the normal rescue through a Nrxn3(25b) mechanism (Fig.6). The strength of this experiment rests upon the specificity of this mutation for disrupting Nrxn3b binding (presynaptic) as opposed to any of the known postsynaptic C1ql2 ligands such as GluK2. While this is not relevant for interpreting the heterologous assay (Fig.5), it is relevant for the in vivo phenotypes in Fig.6. Similar approaches as employed in this paper can test whether binding to other known postsynaptic targets is altered by this point mutation.

5. Altered localization of C1ql2 in Nrxn1-3 cKOs. These data are presented to suggest that Nrx3(25b) is important for localizing C1ql2 to the SL of CA3. Weaknesses of this data include both the lack of Nrxn specificity in the triple a/b KOs as well as the profound effects of Nrxn LoF on the total levels of C1ql2 protein. Some measure that isn't biased by this large difference in C1ql2 levels should be attempted (something like in Fig.1F).