Deletion of Calsyntenin-3, an atypical cadherin, suppresses inhibitory synapses but increases excitatory parallel-fiber synapses in cerebellum
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Evaluation Summary:
This study reports that calsyntenin-3, a synaptic adhesion molecule, is expressed in cerebellar Purkinje cells and that acute knockout of calsyntenin-3 increases excitatory synapse density and decreases inhibitory synapse density. The manuscript provides compelling evidence that elimination of calsyntenin-3 from cells in the cerebellar cortex alters the E/I balance for Purkinje cells by increasing the strength of excitatory parallel fiber inputs and decreasing the strength of inhibitory inputs. However, it would have been far better to selectively target Purkinje cells, but that was not done. This is the first study showing that a particular synaptic adhesion molecule regulates excitatory and inhibitory synapse in opposite directions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Cadherins contribute to the organization of nearly all tissues, but the functions of several evolutionarily conserved cadherins, including those of calsyntenins, remain enigmatic. Puzzlingly, two distinct, non-overlapping functions for calsyntenins were proposed: As postsynaptic neurexin ligands in synapse formation, or as presynaptic kinesin adaptors in vesicular transport. Here, we show that, surprisingly, acute CRISPR-mediated deletion of calsyntenin-3 in mouse cerebellum in vivo causes a large decrease in inhibitory synapse, but a robust increase in excitatory parallel-fiber synapses in Purkinje cells. As a result, inhibitory synaptic transmission was suppressed, whereas parallel-fiber synaptic transmission was enhanced in Purkinje cells by the calsyntenin-3 deletion. No changes in the dendritic architecture of Purkinje cells or in climbing-fiber synapses were detected. Sparse selective deletion of calsyntenin-3 only in Purkinje cells recapitulated the synaptic phenotype, indicating that calsyntenin-3 acts by a cell-autonomous postsynaptic mechanism in cerebellum. Thus, by inhibiting formation of excitatory parallel-fiber synapses and promoting formation of inhibitory synapses in the same neuron, calsyntenin-3 functions as a postsynaptic adhesion molecule that regulates the excitatory/inhibitory balance in Purkinje cells.
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Evaluation Summary:
This study reports that calsyntenin-3, a synaptic adhesion molecule, is expressed in cerebellar Purkinje cells and that acute knockout of calsyntenin-3 increases excitatory synapse density and decreases inhibitory synapse density. The manuscript provides compelling evidence that elimination of calsyntenin-3 from cells in the cerebellar cortex alters the E/I balance for Purkinje cells by increasing the strength of excitatory parallel fiber inputs and decreasing the strength of inhibitory inputs. However, it would have been far better to selectively target Purkinje cells, but that was not done. This is the first study showing that a particular synaptic adhesion molecule regulates excitatory and inhibitory synapse in opposite directions.
(This preprint has been reviewed by eLife. We include the public reviews from the …
Evaluation Summary:
This study reports that calsyntenin-3, a synaptic adhesion molecule, is expressed in cerebellar Purkinje cells and that acute knockout of calsyntenin-3 increases excitatory synapse density and decreases inhibitory synapse density. The manuscript provides compelling evidence that elimination of calsyntenin-3 from cells in the cerebellar cortex alters the E/I balance for Purkinje cells by increasing the strength of excitatory parallel fiber inputs and decreasing the strength of inhibitory inputs. However, it would have been far better to selectively target Purkinje cells, but that was not done. This is the first study showing that a particular synaptic adhesion molecule regulates excitatory and inhibitory synapse in opposite directions.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study reports the function of calsyntenin-3 in the cerebellum. The authors find that cerebellar Purkinje cells selectively express calsyntenin-3. Acute Crispr/Cas-mediated knockout (KO) of calsyntenin-3 in cerebellar Purkinje cells increases the density of excitatory parallel fiber synapses but not excitatory climbing fiber synapses. At the same time, inhibitory synaptic density is decreased in Purkinje cells. This conclusion is supported by multiple lines of morphological and functional evidence. This is the first study showing that a particular synaptic adhesion molecule regulates excitatory and inhibitory synapse toward opposite directions. Although it remains unclear how acute calsyntenin-3 deletion initiated at a juvenile stage leads to opposite changes in excitatory and inhibitory synapses, the …
Reviewer #1 (Public Review):
This study reports the function of calsyntenin-3 in the cerebellum. The authors find that cerebellar Purkinje cells selectively express calsyntenin-3. Acute Crispr/Cas-mediated knockout (KO) of calsyntenin-3 in cerebellar Purkinje cells increases the density of excitatory parallel fiber synapses but not excitatory climbing fiber synapses. At the same time, inhibitory synaptic density is decreased in Purkinje cells. This conclusion is supported by multiple lines of morphological and functional evidence. This is the first study showing that a particular synaptic adhesion molecule regulates excitatory and inhibitory synapse toward opposite directions. Although it remains unclear how acute calsyntenin-3 deletion initiated at a juvenile stage leads to opposite changes in excitatory and inhibitory synapses, the current findings are quite comprehensive and set a new stage for follow-up studies.
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Reviewer #2 (Public Review):
Calsyntenins (Clstns) are evolutionarily conserved synaptogenic adhesion proteins of the cadherin superfamily, but how it functions remains not completely understood, partly due to the functional redundancy among Clstn members. In this paper, the authors examined roles of Clstn3 in cerebellar Purkinje cells, which predominantly expressed Clstn3, by CRISPR/Cas9-mediated deletion in vivo. They showed deletion of Clstn3 caused a large decrease in inhibitory synapses, but a robust increase in excitatory parallel-fiber synapses, indicating a unique postsynaptic function of Clstn3.
The major strength of this paper is that the authors have clarified the phenotype of Clstn3 deletion using a variety of techniques, including electrophysiology, immunohistochemistry, cell morphology and mouse behaviors. In addition, the …
Reviewer #2 (Public Review):
Calsyntenins (Clstns) are evolutionarily conserved synaptogenic adhesion proteins of the cadherin superfamily, but how it functions remains not completely understood, partly due to the functional redundancy among Clstn members. In this paper, the authors examined roles of Clstn3 in cerebellar Purkinje cells, which predominantly expressed Clstn3, by CRISPR/Cas9-mediated deletion in vivo. They showed deletion of Clstn3 caused a large decrease in inhibitory synapses, but a robust increase in excitatory parallel-fiber synapses, indicating a unique postsynaptic function of Clstn3.
The major strength of this paper is that the authors have clarified the phenotype of Clstn3 deletion using a variety of techniques, including electrophysiology, immunohistochemistry, cell morphology and mouse behaviors. In addition, the phenotype that excitatory and inhibitory synapses showed reciprocal changes is interesting.
A major concern is that the phenotype could not be solely attributable to the postsynaptic function of Clstn3 in Purkinje cells because cell type-specific promoters were not used to express gRNAs. Molecular-layer interneurons, which make inhibitory synapses onto Purkinje cells, likely express Clstn3. Thus, presynaptic functions of Clstn3 cannot be ruled out. Another concern is that although synaptic functions were carefully studied by electrophysiological analyses, it is not completely clear whether the number of inhibitory and excitatory synapses was affected by the deletion of Clstn3. Moreover, it is unclear whether Clstn3 is localized at parallel-fiber, but not climbing-fiber, synapses in wild-type Purkinje cells to explain the parallel fiber-specific phenotype in Clstn3 KO Purkinje cells. Because of these major weak points, the authors' claims and conclusions are not fully justified by their data at this point.
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Reviewer #3 (Public Review):
This study investigates the role of calsyntenin-3, an atypical cadherin, in controlling synaptic inputs to cerebellar Purkinje cells. The authors need to do a thorough job of dealing with all of the potential complications given that Purkinje cells were not selectively targeted. The Allen brain atlas shows that Clstn2 and Clstn3 are both expressed by Purkinje cells and that Clstn3 is also expressed by stellate cells and basket cells. The authors used a viral approach that did not select for Purkinje cells. As a result, the effects they see on synaptic transmission could arise from the elimination of all cells in a region rather than the selective elimination of calsyntenin-3 in Purkinje cells. This changes the interpretation of many of the experimental results.
It is difficult to understand such large …
Reviewer #3 (Public Review):
This study investigates the role of calsyntenin-3, an atypical cadherin, in controlling synaptic inputs to cerebellar Purkinje cells. The authors need to do a thorough job of dealing with all of the potential complications given that Purkinje cells were not selectively targeted. The Allen brain atlas shows that Clstn2 and Clstn3 are both expressed by Purkinje cells and that Clstn3 is also expressed by stellate cells and basket cells. The authors used a viral approach that did not select for Purkinje cells. As a result, the effects they see on synaptic transmission could arise from the elimination of all cells in a region rather than the selective elimination of calsyntenin-3 in Purkinje cells. This changes the interpretation of many of the experimental results.
It is difficult to understand such large effects on the total cerebellar mRNA levels and protein levels. The images show large regions of the cerebellum that do not have TdTomato fluorescence, as would be expected given the limitations of injections. This would be expected to be a bigger problem far from the midline. It is also difficult to understand how the protein levels could be reduced to a much larger extent than mRNA levels.
The behavioral data is not very compelling and far from complete.
The manuscript has a tendency to make statements that go beyond the data. For example, the distinction between CF and PF mEPSCs is not easy, and the rise time of 1 ms is unlikely to provide a clear-cut distinction between the two. It is suggestive but far from definitive. Similarly, the kinetics of large and small EPSCs are unlikely to provide a means of discriminating between large and small EPSCs. If the authors really want to make a distinction between basket cells and stellate cells, extracellular recording and rise-times and decay-times are not sufficient, but would require paired recordings and filling of presynaptic cells to better make this distinction.
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