Endophilin A1 facilitates organization of the GABAergic postsynaptic machinery to maintain excitation-inhibition balance

Reviewer #1 (Public review):

Summary:

In the present study, Chen et al. investigate the role of Endophilin A1 in regulating GABAergic synapse formation and function. To this end, the authors use constitutive or conditional knockout of Endophilin A1 (EEN1) to assess the consequences on GABAergic synapse composition and function, as well as the outcome for PTZ-induced seizure susceptibility. The authors show that EEN1 KO mice show a higher susceptibility to PTZ-induced seizures, accompanied by a reduction in the GABAergic synaptic scaffolding protein gephyrin as well as specific GABAAR subunits and eIPSCs. The authors then investigate the underlying mechanisms, demonstrating that Endophilin A1 binds directly to gephyrin and GABAAR subunits, and identifying the subdomains of Endophilin A1 that contribute to this effect. Overall, the authors state that their study places Endophilin A1 as a new regulator of GABAergic synapse function.

Strengths:

Overall, the topic of this manuscript is very timely, since there has been substantial recent interest in describing the mechanisms governing inhibitory synaptic transmission at GABAergic synapses. The study will therefore be of interest to a wide audience of neuroscientists studying synaptic transmission and its role in disease. The manuscript is well-written and contains a substantial quantity of data.

Weaknesses:

A number of questions remain to be answered in order to be able to fully evaluate the quality and conclusions of the study. In particular, a key concern throughout the manuscript regards the way that the number of samples for statistical analysis is defined, which may affect the validity of the data analysed. Addressing this weakness will be essential to providing conclusive results that support the authors' claims.

Reviewer #2 (Public review):

Summary:

The function of neural circuits relies heavily on the balance of excitatory and inhibitory inputs. Particularly, inhibitory inputs are understudied when compared to their excitatory counterparts due to the diversity of inhibitory neurons, their synaptic molecular heterogeneity, and their elusive signature. Thus, insights into these aspects of inhibitory inputs can inform us largely on the functions of neural circuits and the brain.

Endophilin A1, an endocytic protein heavily expressed in neurons, has been implicated in numerous pre- and postsynaptic functions, however largely at excitatory synapses. Thus, whether this crucial protein plays any role in inhibitory synapse, and whether this regulates functions at the synaptic, circuit, or brain level remains to be determined.

New Findings:

(1) Endophilin A1 interacts with the postsynaptic scaffolding protein gephyrin at inhibitory postsynaptic densities within excitatory neurons.

(2) Endophilin A1 promotes the organization of the inhibitory postsynaptic density and the subsequent recruitment/stabilization of GABA A receptors via Endophilin A1's membrane binding and actin polymerization activities.

(3) Loss of Endophilin A1 in CA1 mouse hippocampal pyramidal neurons weakens inhibitory input and leads to susceptibility to epilepsy.

(4) Thus the authors propose that via its role as a component of the inhibitory postsynaptic density within excitatory neurons, Endophilin A1 supports the organization, stability, and efficacy of inhibitory input to maintain the excitatory/inhibitory balance critical for brain function.

(5) The conclusion of the manuscript is well supported by the data but will be strengthened by addressing our list of concerns and experiment suggestions.

Weaknesses:

Technical concerns:

(1) Figure 1F and Figure 1H, Figures 7H,J:
Can the authors justify using a paired-pulse interval of 50 ms for eEPSCs and an interval of 200 ms for eIPSCs? Otherwise, experiments should be repeated using the same paired pulse interval.

(2) Figures 3G,H,I:
While 3D representations of proteins of interest bolster claims made by superresolution microscopy, SIM resolution is unreliable when deciphering the localization of proteins at the subsynaptic level given the small size of these structures (<1 micrometer). In order to determine the actual location of Endophilin A1, especially given the known presynaptic localization of this protein, the authors should complete SIM experiments with a presynaptic marker, perhaps an active zone protein, so that the relative localization of Endophilin A1 can be gleaned. Currently, overlapping signals could stem from the presynapse given the poor resolution of SIM in this context.

Manuscript consistency:

(1) Figure 2:
The authors looked at VGAT and noticed a reduction of signals in hippocampal regions in their P21 slices, indicating that the proposed postsynaptic organization/stabilization functions of Endophilin A1 extend to the inhibitory presynapse, perhaps via Neuroligin 2-Neurexin. Simultaneously, hippocampal regions in P21 slices showed a reduction in PSD-95 signals, indicating that excitatory synapses are also affected. It would be crucial to also look at excitatory presynapses, via VGLUT staining, to assess whether EndoA1 -/- also affects presynapses. Given the extensive roles of Endophilin A1 in presynapses, especially in excitatory presynapses, this should be investigated.

(2) Figure 7C:
The authors do not assess whether p140Cap overexpression rescues GABAAR receptor loss exhibited in Endophilin A1 KO, as they did for Gephryin. This would be an important data point to show, as p140Cap may somehow rescue receptor loss by another pathway. In fact, it is mentioned in the text that this experiment was done, "Consistently, neither p140Cap nor the endophilin A1 loss-of-function mutants could rescue the GABAAR clustering phenotype in EEN1 KO neurons (Figure 7C, D)" yet the data for p140Cap overexpression seem to be missing. This should be remedied.

Reviewer #3 (Public review):

Summary:

Chen et al. identify endophilin A1 as a novel component of the inhibitory postsynaptic scaffold. Their data show impaired evoked inhibitory synaptic transmission in CA1 neurons of mice lacking endophilin A1, and an increased susceptibility to seizures. Endophilin can interact with the postsynaptic scaffold protein gephyrin and promote assembly of the inhibitory postsynaptic element. Endophilin A1 is known to play a role in presynaptic terminals and in dendritic spines, but a role for endophilin A1 at inhibitory postsynaptic densities has not yet been described.

Strengths:

The authors used a broad array of experimental approaches to investigate this, including tests of seizure susceptibility, electrophysiology, biochemistry, neuronal culture, and image analysis.

Weaknesses:

Many results are difficult to interpret, and the data quality is not always convincing, unfortunately. The basic premise of the study, that gephyrin and endophilin A1 interact, requires a more robust analysis to be convincing.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

In the present study, Chen et al. investigate the role of Endophilin A1 in regulating GABAergic synapse formation and function. To this end, the authors use constitutive or conditional knockout of Endophilin A1 (EEN1) to assess the consequences on GABAergic synapse composition and function, as well as the outcome for PTZ-induced seizure susceptibility. The authors show that EEN1 KO mice show a higher susceptibility to PTZ-induced seizures, accompanied by a reduction in the GABAergic synaptic scaffolding protein gephyrin as well as specific GABAAR subunits and eIPSCs. The authors then investigate the underlying mechanisms, demonstrating that Endophilin A1 binds directly to gephyrin and GABAAR subunits, and identifying the subdomains of Endophilin A1 that contribute to this effect. Overall, the authors state that their study places Endophilin A1 as a new regulator of GABAergic synapse function.

Strengths:

Overall, the topic of this manuscript is very timely, since there has been substantial recent interest in describing the mechanisms governing inhibitory synaptic transmission at GABAergic synapses. The study will therefore be of interest to a wide audience of neuroscientists studying synaptic transmission and its role in disease. The manuscript is well-written and contains a substantial quantity of data.

Weaknesses:

A number of questions remain to be answered in order to be able to fully evaluate the quality and conclusions of the study. In particular, a key concern throughout the manuscript regards the way that the number of samples for statistical analysis is defined, which may affect the validity of the data analysed. Addressing this weakness will be essential to providing conclusive results that support the authors' claims.

We would like to thank the reviewer for appreciation of the value of our study and careful critics to help us improve the manuscript. We will correct the way that the number of samples for statistical analysis is defined throughout the manuscript as suggested and update figures, figure legends, and Materials and Methods accordingly. For example, we will average the values for all dendritic segments from one neuron, so that each data point represents one neuron in the graphs.

Reviewer #2 (Public review):

Summary:

The function of neural circuits relies heavily on the balance of excitatory and inhibitory inputs. Particularly, inhibitory inputs are understudied when compared to their excitatory counterparts due to the diversity of inhibitory neurons, their synaptic molecular heterogeneity, and their elusive signature. Thus, insights into these aspects of inhibitory inputs can inform us largely on the functions of neural circuits and the brain.

Endophilin A1, an endocytic protein heavily expressed in neurons, has been implicated in numerous pre- and postsynaptic functions, however largely at excitatory synapses. Thus, whether this crucial protein plays any role in inhibitory synapse, and whether this regulates functions at the synaptic, circuit, or brain level remains to be determined.

New Findings:

(1) Endophilin A1 interacts with the postsynaptic scaffolding protein gephyrin at inhibitory postsynaptic densities within excitatory neurons.

(2) Endophilin A1 promotes the organization of the inhibitory postsynaptic density and the subsequent recruitment/stabilization of GABA A receptors via Endophilin A1's membrane binding and actin polymerization activities.

(3) Loss of Endophilin A1 in CA1 mouse hippocampal pyramidal neurons weakens inhibitory input and leads to susceptibility to epilepsy.

(4) Thus the authors propose that via its role as a component of the inhibitory postsynaptic density within excitatory neurons, Endophilin A1 supports the organization, stability, and efficacy of inhibitory input to maintain the excitatory/inhibitory balance critical for brain function.

(5) The conclusion of the manuscript is well supported by the data but will be strengthened by addressing our list of concerns and experiment suggestions.

We would like to thank the reviewer for their favorable impression of manuscript. We also appreciate the great experiment suggestions to help us improve the manuscript.

Weaknesses:

Technical concerns:

(1) Figure 1F and Figure 1H, Figures 7H,J:

Can the authors justify using a paired-pulse interval of 50 ms for eEPSCs and an interval of 200 ms for eIPSCs? Otherwise, experiments should be repeated using the same paired pulse interval.

We apologize for the confusion. As illustrated by the schematic current traces, the decay time constants of eEPSCs and eIPSCs in hippocampal CA1 neurons are different. The eEPSCs exhibit a faster channel closing rate, corresponding to a smaller time constant Tau. Thus, a shorter inter-stimulus interval (50 ms) was chosen for paired-pulse ratio recordings. In contrast, the eIPSCs display a slower channel closing rate, with a Tau value larger than that of eEPSCs, so a longer inter-stimulus interval (200 ms) was used for PPR. This protocol has been long-established and adopted in previous studies (please see below for examples).

Contractor, A., Swanson, G. & Heinemann, S. F. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus. Neuron 29, 209-216, doi:10.1016/s0896-6273(01)00191-x (2001).

Babiec, W. E., Jami, S. A., Guglietta, R., Chen, P. B. & O'Dell, T. J. Differential Regulation of NMDA Receptor-Mediated Transmission by SK Channels Underlies Dorsal-Ventral Differences in Dynamics of Schaffer Collateral Synaptic Function. Journal of neuroscience 37, 1950-1964, doi:10.1523/JNEUROSCI.3196-16.2017 (2017).

(2) Figures 3G,H,I:

While 3D representations of proteins of interest bolster claims made by superresolution microscopy, SIM resolution is unreliable when deciphering the localization of proteins at the subsynaptic level given the small size of these structures (<1 micrometer). In order to determine the actual location of Endophilin A1, especially given the known presynaptic localization of this protein, the authors should complete SIM experiments with a presynaptic marker, perhaps an active zone protein, so that the relative localization of Endophilin A1 can be gleaned. Currently, overlapping signals could stem from the presynapse given the poor resolution of SIM in this context.

Thanks for your suggestions. It is certainly preferable to investigate the relative localization of endophilin A1 using both presynaptic and postsynaptic markers. For SIM imaging in Figure 3G-I, to visualize neuronal morphology, we immunostained GFP as cell fill, leaving two other channels for detection of immunofluorescent signals of endophilin A1 and another protein. We will try co-immunostaining of endophilin A1, the active zone protein bassoon (presynaptic marker) and gephyrin without morphology labeling. Alternatively, we will do co-staining of endophilin A1 and bassoon in GFP-expressing neurons. We agree that overlapping signals or proximal localization of presynaptic endophilin A1 with gephyrin or GABAAR γ2 could not be ruled out. To note, if image resolution is improved with the use of a more advanced imaging system, the overlap between two proteins will become smaller or even disappear. With the ~110 nm lateral resolution of SIM microscopy, the degree of overlap between the two proteins of interest is much lower than in confocal microscopy. Given the presynaptic localization of endophilin, most likely we will observe a small overlap (presynatpic) or proximal localization (postsynaptic) of endophilin A1 with bassoon. Nevertheless, we will complete the SIM experiments as suggested to improve the manuscript.

Manuscript consistency:

(1) Figure 2:

The authors looked at VGAT and noticed a reduction of signals in hippocampal regions in their P21 slices, indicating that the proposed postsynaptic organization/stabilization functions of Endophilin A1 extend to the inhibitory presynapse, perhaps via Neuroligin 2-Neurexin. Simultaneously, hippocampal regions in P21 slices showed a reduction in PSD-95 signals, indicating that excitatory synapses are also affected. It would be crucial to also look at excitatory presynapses, via VGLUT staining, to assess whether EndoA1 -/- also affects presynapses. Given the extensive roles of Endophilin A1 in presynapses, especially in excitatory presynapses, this should be investigated.

Thanks for the thoughtful comments. Given that the both VGAT and PSD95 signals are reduced in hippocampal regions in P21 slices, it is conceivable that the proposed postsynaptic organization/stabilization functions of endophilin A1 extend to the inhibitory presynapse via Neuroligin-2-Neurexin and the excitatory presynapse as well during development. Of note, endophilin A1 knockout did not impair the distribution of Neuroligin-2 in inhibitory postsynapses (immunoisolated with anti-GABAAR α1) in mature mice (Figure 3K), and endophilin A1 did not bind to Neuroligin-2 (Figure 4D), suggesting that endophilin A1 might function via other mechanisms. Nevertheless, as functions of endophilin A family members at the presynaptic site are well-established, the reduction of presynaptic signals in developmental hippocampal regions of EndoA-/- mice might result from the depletion of presynaptic endophilin A1. The presynaptic deficits can be compensatory by other mechanisms as neurons mature. Certainly, we will do VGLUT staining of EndoA1-/- brain slices as suggested to assess the role of endophilin A1 in excitatory presynapses in vivo.

(2) Figure 7C:

The authors do not assess whether p140Cap overexpression rescues GABAAR receptor loss exhibited in Endophilin A1 KO, as they did for Gephryin. This would be an important data point to show, as p140Cap may somehow rescue receptor loss by another pathway. In fact, it is mentioned in the text that this experiment was done, "Consistently, neither p140Cap nor the endophilin A1 loss-of-function mutants could rescue the GABAAR clustering phenotype in EEN1 KO neurons (Figure 7C, D)" yet the data for p140Cap overexpression seem to be missing. This should be remedied.

Thanks a lot for the thoughtful comment. We will determine whether p140Cap overexpression also rescues the GABAAR clustering phenotype in EndoA1-/- neurons by surface GABAAR γ2 staining in our revised manuscript.

Reviewer #3 (Public review):

Summary:

Chen et al. identify endophilin A1 as a novel component of the inhibitory postsynaptic scaffold. Their data show impaired evoked inhibitory synaptic transmission in CA1 neurons of mice lacking endophilin A1, and an increased susceptibility to seizures. Endophilin can interact with the postsynaptic scaffold protein gephyrin and promote assembly of the inhibitory postsynaptic element. Endophilin A1 is known to play a role in presynaptic terminals and in dendritic spines, but a role for endophilin A1 at inhibitory postsynaptic densities has not yet been described.

Strengths:

The authors used a broad array of experimental approaches to investigate this, including tests of seizure susceptibility, electrophysiology, biochemistry, neuronal culture, and image analysis.

Weaknesses:

Many results are difficult to interpret, and the data quality is not always convincing, unfortunately. The basic premise of the study, that gephyrin and endophilin A1 interact, requires a more robust analysis to be convincing.

We greatly appreciate the positive comment on our study and the very valuable feedback for us to improve the manuscript. We will conduct additional experiments to improve our data quality and strengthen our evidences according to these great constructive suggestions. To gain strong evidence for the interaction between endophilin A1 and gephyrin, we will perform in vitro pull-down assay with recombinant proteins from bacterial expression system.