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

  1. Xue Chen
  2. Deng Pan
  3. Jia-Jia Liu
  4. Yanrui Yang

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    eLife Assessment

    This study presents a valuable finding on the molecular mechanisms that govern GABAergic inhibitory synapse function. The authors propose that Endophilin A1 serves as a novel regulator of GABAergic synapses by acting as a component of the inhibitory postsynaptic density. The authors have added substantial new analyses that take a wide range of approaches to provide solid support for their conclusions, although one of the reviewers concludes that the premise that gephyrin and endophilin A1 interact requires more robust analysis. The findings are likely to interest a broad audience of scientists focusing on inhibitory synaptic transmission, the excitation-inhibition balance, and its disruption in disorders such as epilepsy.

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Abstract

Abstract

The assembly and operation of neural circuits in the brain rely on the coordination and balance of excitatory and inhibitory activities. Inhibitory synapses are key regulators of the functional balance of neural circuits. However, due to the diversity of inhibitory presynaptic neurons, the complex composition of postsynaptic receptor subunits and the lack of typical postsynaptic dense structure, there are relatively few studies on the regulatory mechanisms for inhibitory synaptic structure and function, and insufficient understanding of the cellular and molecular abnormalities of inhibitory synapses in neurological and neuropsychiatric disorders. Here, we report a crucial role for endophilin A1 in inhibitory synapses. We show that endophilin A1 directly interacts with the inhibitory postsynaptic scaffold protein gephyrin in excitatory neurons, and promotes organization of the inhibitory postsynaptic density and synaptic recruitment/stabilization of the γ-aminobutyric acid type A receptors via its plasma membrane association and actin polymerization promoting activities. Loss of endophilin A1 by gene knockout in mouse hippocampal CA1 pyramidal cells weakens inhibitory synaptic transmission and causes imbalance in the excitatory/inhibitory function of neural circuits, leading to increased susceptibility to epilepsy. Our findings identify endophilin A1 as an iPSD component and provide new insights into the organization and stabilization of inhibitory postsynapses to maintain E/I balance as well as the pathogenesis of epilepsy.

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  1. eLife

    eLife Assessment

    This study presents a valuable finding on the molecular mechanisms that govern GABAergic inhibitory synapse function. The authors propose that Endophilin A1 serves as a novel regulator of GABAergic synapses by acting as a component of the inhibitory postsynaptic density. The authors have added substantial new analyses that take a wide range of approaches to provide solid support for their conclusions, although one of the reviewers concludes that the premise that gephyrin and endophilin A1 interact requires more robust analysis. The findings are likely to interest a broad audience of scientists focusing on inhibitory synaptic transmission, the excitation-inhibition balance, and its disruption in disorders such as epilepsy.

  2. eLife

    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. In the revised version of the manuscript, the authors have increased the number of samples analyzed and have significantly improved the statistical analysis, thereby substantially strengthening the conclusions of their study.

    Comments on revised version:

    The authors have addressed all of my concerns, and the manuscript has been substantially improved.

  3. eLife

    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.

    Comments on revised version:

    The authors addressed the concerns adequately. The three remaining concerns are:

    (1) The use of one-way ANOVA is not well justified.

    (2) The use of superplots to show culture to culture variability would make it more transparent.

    (3) Change EEN1 in Figure 8B to EndoA1.

  4. eLife

    Reviewer #3 (Public review):

    Summary:

    The authors investigated a possible role of Endophilin A1 in the inhibitory postsynaptic density.

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

    Specific comments:

    The authors have made a substantial effort to improve their manuscript. A number of issues, related to numbers of observations mentioned by the reviewers, are clarified in the revised manuscript. The authors have also clarified some of the other questions from the reviewers. The long list of issues brought up by the reviewers and the many corrections needed still raise questions about data quality in this manuscript.
    In response to my comments (Point 2), the added experiment with PSD95.FingR and GPN.FingR in cultured neurons (Fig. S5A-D) is a good addition; the in vivo data using FingRs in Figure S3 look less convincing however. In response to my Point 5, the authors have added a cell-free binding assay (Figure 5I). This is a useful addition, but to convincingly make the point of interaction between Gephyrin and EndoA1, more rigorous biophysical quantitation of binding is needed. The legend in Figure 5I states that 4 independent experiments were performed, but the graph only shows 3 dots. This needs to be corrected.

  5. eLife

    Author response:

    The following is the authors’ response to the original reviews

    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.

    Recommendations for the authors:

    Reviewer #1 (Recommendations for the authors):

    (1) For all of the electrophysiology experiments, only the number of neurons recorded is stated, but not the number of independent animals that these neurons were obtained from. The number of independent animals used should be stated for each panel. At least 3 independent animals should be used in each group, otherwise, more data needs to be added.

    We apologize for missing the information in the original manuscript. For all electrophysiological experiments, data were obtained from more than 3 experimental animals. The figure legends were updated to include the number of independent animals used for each panel.

    (2) For the cell culture experiments analyzing dendritic puncta at GABAergic synapses, the number of data points analysed appears to be the number of dendritic segments quantified, regardless of whether they originate from the same neuron or not. This analysis method is not valid, since dendritic segments from the same neuron cannot be counted as statistically independent samples. The authors need to average the values for all dendritic segments from one neuron, such that one neuron equals one data point. This alteration should be made for Figures 2B, 2D, 4H, 4J, 5B, 5C, 5E, 5J, 5L, 6B, 6D, 6F, 6H, 6J, 6K,7B, and 7D. In addition, the number of independent cultures from which the neurons were obtained should be stated for each panel. At least 3 independent cultures should be used in each group, otherwise, more data need to be added.

    Thanks for the criticism. We reanalyzed the data throughout the manuscript as suggested and updated the figure legends accordingly. Moreover, we increased the number of neurons from independent experiments to further confirm the results in our revised manuscript.

    In the revised manuscript, we averaged the values for all dendritic segments from a single neuron and updated the data in Figure 3B, 3D, 4H, 4J, 5B, 5C, 5E, 5K, 5M, 6B, 6D, 6F, 6H, 6J, 6K,7B, and 7D.

    Neurons analyzed in each group were derived from at least 3 independent cultures. Due to very low efficiency of sparse transfection in primary cultured hippocampal neurons, multiple experimental repetitions were necessary to obtain the sufficient number of neurons for analysis. We described statistical analysis in “Material and Methods” section in the original manuscript as follows:

    “For all biochemical, cell biological and electrophysiological recordings, at least three independent experiments were performed (independent cultures, transfections or different mice).”

    (3) Individual data points should be shown on all graphs, particularly in Figures 2C, 2F, 2I, 3F, 3K, and 3L.

    Thank you for the suggestion. We replaced the original graphs with scatterplots and mean ± S.E.M. in new Figures.

    (4) For each experiment, the authors should state explicitly in the methods section whether that experiment was conducted blind to genotype.

    Thank you for the suggestion. We have modified the description of blind analysis for each experiment in methods section to “Seizure susceptibility was measured blindly by rating seizures on a scale of 0 to 7 as follows…”, “Quantification of immunostaining were carried out blindly…” in our revised manuscript.

    (5) For each experiment, the authors should state whether they used male or female mice, and what age the mice were at the time of the experiment

    Thanks a lot for the suggestion. We usually use male and female mice for neuron culture and behavioral test. We observed no sex-related differences in PTZ-induced behaviors, so the results were pooled together.

    For mice ages, P0 pups were used for hippocampal neuron cultures and virus injection in electrophysiological recording assays or FingR probes assays. P14-21 mice were used for electrophysiological recording, immunofluorescent staining and FingR probes detection in brain slice, while adult mice (P60) for behavioral tests, immunofluorescent staining in brain slice and biochemical assays. We have modified the description in genders and ages of mice in methods section to “To evaluate seizure susceptibility, 8-10-week-old male and female EndoA1+/+ or EndoA1-/- littermates or EndoA1fl/fl littermates were intraperitoneally administered… ”, “For virus injection, 8-9-week-old naive male and female littermates were anesthetized…”, “Male and female littermates (P21 or P60) were anesthetized and immediately perfused…”, “Hippocampi of female or male pups (P0) were rapidly dissected under sterile conditions…”, “PSD fractions from adult mouse brain were prepared as previously described…”, “Newborn EndoA1fl/fl littermates (male or female) were anesthetized on ice for 4-5 min…” in our revised manuscript.

    (6) For each experiment involving WT and KO mice, please state whether WTs and KOs were bred as littermates from heterozygous breeders

    Sorry for the confusion. In our study, EndoA1+/+ and EndoA1-/- mice were bred as littermates from heterozygous breeders. We added the information in methods section as follows in our revised manuscript, “EndoA1+/+ and EndoA1-/- mice were bred as littermates from heterozygous breeders…”, “To evaluate seizure susceptibility, 8-10-week-old male and female EndoA1+/+ or EndoA1-/- littermates or EndoA1fl/fl littermates…”, “For virus injection, 8-9-week-old naive male and female littermates were anesthetized…”, “Male and female littermates (P21 or P60) were anesthetized and immediately perfused…”, “For co-IP from brain lysates, the whole brain from 8-10-week-old WT and KO littermates were dissected…”, “Newborn EndoA1fl/fl littermates (male or female) were anesthetized on ice for 4-5 min…”.

    (7) For experiments comparing three or more groups, the authors claim in the methods section to have used a one-way ANOVA for statistical analysis. However, no ANOVA values are given, only the post-hoc tests. Please add the ANOVA values for each experiment before stating the values of the post-hoc analysis.

    Sorry for the missing information. We used one-way ANOVA for comparing three or more groups in the original manuscript and have changed to two-way ANOVA for behavior data analysis in our revised manuscript as suggested in Recommendations (18). We added the ANOVA values (F & p values) for each experiment in new figures. For example, see Figure 1C.

    (8) In Figure 1A-C, seizure susceptibility was compared in EEN+/+ and EEN-/- mice, but the methods section states that seizure susceptibility was evaluated in 8-10-week-old male C57BL/6N mice (line 513). Was this meant to indicate that the EEN+/+ and EEN-/- mice were on a C57BL/6N background? How does this match with the statement that EEN1 -/- mice were generated on a C57BL/6J background (line 467)?

    We apologize for the mistake. In our study, EEN1-/- mice were generated on a C57BL/6J background, as stated in our previously published papers (Yang et al., 2021; Yang et al., 2018) and in “Animals” in Material and Methods of our original manuscript. We had corrected the statement to “To evaluate seizure susceptibility, 8-10-week-old male and female EndoA1+/+ or EndoA1-/- littermates…” in Material and Methods of the revised manuscript.

    (9) In the electrophysiology experiments in Figure 1E-O, it is not clear to me which neurons were recorded in the control group. The methods section states that "Whole-cell recordings were performed on an AAV-infected neuron and a neighboring uninfected neuron" (line 736). However, the figure legends states that recordings were obtained from "10 control (Ctrl, mCherry alone) and 10 EEN1 KO (mCherry and Cre) pyramidal neurons" (line 1079), which would indicate that the controls are not uninfected neurons from the same animal, but AAV-mCherry infected neurons from a different animal. Please clarify which of the two descriptions is accurate.

    Thanks for catching the error! In all electrophysiological experiments, a neighboring uninfected neuron was used as the control in Figure 1E-O. This was incorrectly stated in the figure legend of the original manuscript. In the revised manuscript, the information has been corrected in figure legends of new Figure 1 (E-F).

    (10) The authors show that in Endophilin A1 KO animals, eIPSCs are reduced, but mIPSC frequency and amplitude are unaltered. How do they explain this finding in the context of the fact that gephyrin and GABAAR1.

    We apologize for the confusion about the data of electrophysiological recording. Compared with eIPSC, which are recorded in the presence of electrically evoked action potential that elicited a substantial release of neurotransmitter, mIPSCs are small, spontaneous currents recorded in the presence of TTX during patch-clamp experiments, resulting from the release of neurotransmitters from presynaptic terminals in the absence of action potential. The amplitude of mIPSCs typically reflects the quantal release of neurotransmitters, while their frequency can vary depending on synaptic activity and the state of the neuron.

    A number of molecules fine-tune presynaptic neurotransmitter release and functions of inhibitory postsynaptic receptors. In our study, inhibitory postsynapses were partially affected in endophilin A1 knockout neurons, while presynaptic endophilin A1 remained intact during electrophysiological recordings. Conceivably, the observed deficits in endophilin A1 knockout mice were mild. Following endophilin A1 depletion, inhibitory postsynaptic receptors appeared sufficient to respond to spontaneous neurotransmitter release but may be inadequate to large amounts of neurotransmitter release evoked by action potential. Meanwhile, spontaneous synaptic activity and the state of the neuron were not obviously affected under basic state by endophilin A1 depletion during postnatal stages. Consequently, mIPSC frequency and amplitude remain unaltered but eIPSCs were reduced compared to the control neurons. This finding was consistent with behavioral experiments, where aggressive epileptic behaviors were induced by PTZ rather than spontaneous epilepsy in endophilin A1 knockout mice.

    (11) Distribution of gephyrin, VGAT, and GABAARg2 differs substantially between the different layers of hippocampal area CA1, and the same goes for the other regions of the hippocampus. However, in Figure 2, it is not clear to me from the sample images which layers of each subregion the authors quantified, or indeed whether they paid attention to which layers they included in their analysis. This can lead to a substantial skewing of the data if different layers were preferentially included in the two genotypes. Please clarify which layers were analysed, and how comparability between WTs and KOs was ensured. This is particularly important given the authors' claim that Endophilin A1 acts equally at all subtypes of GABAergic synapses (lines 373- 376).

    Thanks for the cautiousness! We distinguished each hippocampal subregion based on the anatomical structure in brain slices. Quantification of fluorescent mean intensity of each synaptic protein in all layers of each subregion, as shown in new Figure 2 and Figure S2A-F, revealed that GABAergic synaptic proteins were impaired in both P21 and P60 KO mice.

    We further analyzed the fluorescent signal of core postsynaptic component, gephyrin, in individual layers of each subregion in the hippocampus of mature WT and KO mice, as presented in new Figures S2G-H. Our findings demonstrated a decrease in gephyrin levels across all layers of each subregion in KO mice. Additionally, we examined gephyrin clustering across the soma, axon initial segment (AIS), and dendrites in cultured mature endophilin A1 knockout hippocampal neurons, as shown in new Figure S5E-H. The results showed that gephyrin was affected in all subcellular regions following endophilin A1 knockout.

    Collectively, these data suggest that endophilin A1 functions across all subtypes of GABAergic postsynapses.

    (12) In Figure 3E-F, the authors state that there was no change in the total level of synaptic neurons in EEN1 KO neurons (line 188). However, there is no quantification of the total level of synaptic neurons shown, and based on the immunoblot in Figure 3E, it looks like there is a substantial reduction in NR1, NL2, and g2. The authors should present a quantification of the total levels of these proteins and adjust their statement accordingly if necessary.

    Thanks a lot for your comments. We quantified the total protein levels in Figure 3E and added the result to new Figure 3F, showing that total protein levels were not obviously affected in cultured KO neurons. When normalized to total protein levels, the surface levels of GABAA receptors were significantly compromised compared to surface GluN1 and NL2. Furthermore, the total protein levels were not affected in brains of KO mice, as shown in Figures 3K (input) and 3L (S1). Collectively, there was no change in the total level of synaptic proteins in KO neurons.

    (13) In Figure 3G-I, the authors claim, based on super-resolution images as presented here, that Endophilin A1 colocalizes with gephyrin and g2. However, no quantification of this colocalization is presented. The authors should add this quantification to support their claim and indicate how many GABAergic synapses contain Endophilin A1.

    Thank you for the thoughtful comments. The resolution of the images is significantly improved by super-resolution microscopy. As a result, the overlap between the two proteins will become smaller or even disappear. Since no two proteins can occupy the same physical space, they would show lower colocalization and instead exhibit proximal localization. As expected, in Figures 3G and 3H, we observed only small overlap or proximal localization of endophilin A1 with gephyrin or GABAAR γ2. To further confirm the localization of endophilin A1 in inhibitory synapses, we co-stained endophilin A1 with both pre- and post-synaptic proteins, gephyrin and Bassoon. Then we quantified the colocalization of endophilin A1 with gephyrin or with Bassoon using the method for super-resolution images described in the reference (Andrew D. McCall. Colocalization by cross-correlation, a new method of colocalization suited for super-resolution microscopy. McCall BMC Bioinformatics (2024) 25:55). The percentage of gephyrin or Bassoon puncta that were in close proximity with endophilin A1 was also calculated, as shown in new video 5 and new Figure S4B-G. These data have been added in the revised manuscript as follows, “We further detected the localization of endophilin A1 to inhibitory synapses by co-immunostaining with both pre- and post-synaptic markers (Figure. S4B and Video 5). Quantitative analysis of super-resolution localization maps revealed that ~ 47 % puncta of gephyrin or Bassoon were proximal to endophilin A1 (Figure. S4G, n = 14), with a mean distance between endophilin A1- and gephyrin-positive pixels of ∼ 120 nm, or between endophilin A1- and Bassoon-positive pixels of ∼ 130 nm (Figure. S4C-F).”

    (14) In the quantification shown in Figure 3K-L, there are no error bars in the WT data sets. This presumably means that all values were normalized to WT. However, since this artificially eliminates the variance in the WT group, a t-test is no longer valid, since this assumes a normal distribution and normal variance, which are no longer given. The authors should either change the way they normalize their data to maintain the variance in the WT group or perform a different statistical test that can account for the artificial lack of variance in one of the groups.

    Thank you for the suggestions! We modified our analysis approach. Specifically, we used mean value of WTs to normalize data to preserve the variance in the WT group and performed unpaired t-tests to assess statistical significance in Figure 3K-L. Additionally, we replaced the bar graphs with modified graphs showing individual data points. Please see Response to Recommendation (12).

    (15) What is the difference between the coIP experiment in Figure 4E and 3J, right panel? In both cases, an Endophilin A1 IP is performed, and gephyrin, GABAARg2, and GABAARa1 are assessed. However, Figure 3J's right panel indicates that Endophilin A1 does interact with the GABAAR subunits, whereas Figure 4E shows that it does not. How do the authors explain this discrepancy? Were these experiments performed more than once?

    Sorry for the confusion. Figure 3J and Figure 4E show data from immunoisolation assay and conventional co-immunoprecipitation (co-IP), respectively. Immunoisolation allows for the rapid and efficient separation of subcellular membrane compartments using antibodies conjugated to magnetic beads. In Figure 3J, we used antibodies against GABAAR α1 subunit or endophilin A1 to isolate the inhibitory postsynaptic membranes or endophilin A1-associated membranous compartments. In contrast, co-immunoprecipitation detects direct protein-protein interactions in detergent-solubilized lysates. For Figure 4E, we applied antibodies against endophilin A1 to precipitate its interaction partners. The results in Figure 3J and Figure 4E demonstrate that endophilin A1 is localized in the inhibitory postsynaptic compartment and directly interacts with gephyrin, but not with GABAARs. Detailed information regarding the methods used for co-IP and immunoisolation can be found in “GST-pull down, co-immunoprecipitation (IP), and immunoisolation” in the “Material and Methods” section of original manuscript.

    These experiments were repeated multiple times to ensure reliability. In fact, consistent data showing endophilin A1 localization in the inhibitory postsynaptic compartment were observed in Figure 3K, showing the quantified data as well.

    (16) For the colocalization analysis in Figure 5A-C, what percentage of gephyrin puncta contain g2 in the WT and Endophilin A1 KO? Currently, only a correlation coefficient is provided, but not the degree of overlap. Please add this information to the figure.

    Thanks for the comments on the colocalization analysis. We analyzed the percentage of gephyrin puncta overlapping with GABAAR γ2 and added the graphs in new Figure 5C.

    (17) Figure 6 investigates how actin depolarization affects GABAergic synapse function, but does not assess how Endophilin A1 contributes to this process. The authors then provide an extremely short statement in the discussion, stating that their data are contradictory to a previous study (lines 412 - 417). This section of the discussion should be expanded to address the specific role of Endophilin A1 in the consequences of actin depolymerization.

    Thanks a lot for the advice. In the original manuscript, we discussed the specific role of endophilin A1 at inhibitory postsynapses as follows in Discussion:

    “As membrane-binding and actin polymerization-promoting activities of endophilin A1 are both required for its function in enhancing iPSD formation and g2–containing GABAAR clustering to iPSD, we propose that membrane-bound endophilin A1 promotes postsynaptic assembly by coordinating the plasma membrane tethering of the postsynaptic protein complex and its stabilization with the actin cytomatrix”

    Following your advice, we added a statement in the revised manuscript addressing the role of endophilin A1 in actin polymerization at inhibitory postsynapses, shown as follows, “In the present study, the impaired clustering of gephyrin and GABAA γ2 by F-actin depolymerization underscores the essential role of F-actin in the assembly and stabilization of the inhibitory postsynaptic machinery. Membrane-bound endophilin A1 promotes F-actin polymerization beneath the plasma membrane through its interaction with p140Cap, an F-actin regulatory protein, thereby facilitating and/or stabilizing the clustering of gephyrin and γ2-containing GABAA ​receptors at postsynapses.”

    (18) Which statistical analysis was conducted in Figure 7F? Given the nature of the data, a repeated measures ANOVA would be necessary to accurately assess the statistical accuracy.

    Sorry for the confusion. We conducted one-way ANOVA followed by Tukey post hoc test at each time point in original Figure 7F. We have employed the method of repeated measures ANOVA followed by Tukey post hoc test as suggested in new Figure 7F. Meanwhile, we reanalyzed data in new Figure 1C with the same method. We also modified the description in “Statistical analysis” and Figure legends for new Figure1C and 7F in revised manuscript.

    Reviewer #2 (Recommendations for the authors):

    Data presentation:

    (1) Figures 2A, B, D, E, G, H. Figures S2A, B, D:

    Add P21 or P60 labels to these figures so that the difference between similarly stained samples (e.g. Figures 2A, B) is obvious to the reader.

    Thanks! We added “P21” or “P60” labels in new Figure 2 and Figure S2 as suggested.

    (2) Figures 4C, D:

    The authors must make their coIP data annotation consistent. In Figure 4C, they use actual microgram amounts when, e.g., describing how much input was present, yet in Figure 4D they use + and -. The authors should pick one.

    Thanks for the comments. We labeled the consistent data annotation in new Figure 4C and 4D, we also changed the label in 4F for the consistent data annotation.

    (3) Figure 5A

    GFP is gray in this figure, but in all other figures, it is blue. Consider changing for presentation reasons.

    Thanks a lot for pointing out the problem. We replaced gray with blue color to indicate GFP in new Figure 5A.

    (4) Figures 6A, C, E, G

    Label graphs as either short-term or long-term drug treatment.

    Thanks for the suggestion. We labeled the graphs as 60 min for short-term or 120 min for long-term drug treatment in new Figure 6A, C, E, G for convenient reading.

    Annotation, grammar, spelling, typing errors:

    (1) Figure 4G:

    Merge and GFP labels are seemingly swapped.

    Thanks a lot for sharp eye. We corrected the labels in new Figure 4G.

    (2) Fig 4I:

    The authors use "Gephryin" instead of GPN. They should be consistent and choose one.

    Sorry for the mistake. We changed the label consistent with other figures in new Figure 4I and rearranged the images in figures for good looking.

    (3) "One-hour or two-hour treatment of mature neurons with nocodazole..."

    Thanks for your advice. We modified the sentence to “Treatment of mature neurons with nocodazole, a microtubule depolymerizing reagent, for one hour (short-term) or two hours (long-term), caused…”.

    (4) The authors should indicate that one-hour is their short-term treatment and that two-hour is their long-term treatment so that when these terms are used later to describe LatA experiments, it is clearer to the reader.

    Thanks for your comments. We modified the statement as seen in Response to Recommendation (3), it is clearer to the reader.

    (5) EEA1. The authors should use a more conventional term EndoA1 so that the manuscript can be searched easily.

    Thanks a lot for the suggestion. We replaced all of the term “EEN1” with “EndoA1” in the revised manuscript.

    Reviewer #3 (Recommendations for the authors):

    Major Points

    (1) The number of observations for the electrophysiology experiments in Figure 1 (dots are neurons) is very low and it is not clear whether the data shown is derived from different mice. The same criticism applies to the data shown in Figures 7G-K.

    We apologize for the low neuron number in electrophysiology experiments. In the patch-clamp experiments, the number of neurons recorded was higher than what is shown in the figures. However, neurons with a membrane resistance (Rm) below 500 MΩ, indicating unstable seals or poor conditions, were excluded from the analysis. Additionally, we added the number of mice from which the data derived in each group in the figure legends for Figure 1, 7 and S1, this point was also raised by Reviewer #1 (Please see Response to Recommendation (1)).

    (2) Images in Figure 2 are shown at low magnification, statements on changes in intensity of inhibitory synaptic markers in the hippocampal region are impossible to interpret. Analysis of inhibitory synapses in vivo would require sparse neuronal labeling and 3D reconstruction, for instance using gephyrin-FingRs (Gross et al., Neuron 2013).

    Thanks for your insightful suggestion. We obtained pCAG_PSD95.FingR-eGFP-CCR5TC and pCAG_GPN.FingR-eGFP-CCR5TC constructs from Addgene (plasmid # 46295 & #46296). We attempted in utero electroporation (IUE) to introduce the DNAs into cortical neurons or hippocampal neurons at E14.5, unfortunately with no success. Following the repetitive operation for numerous times, we could eventually obtain newborn pups of ICR mice after IUE. However, we failed to obtain any newborn pups of C57BL/6J mice due to abortion following the procedure. Furthermore, pregnant C57BL/6J mice (WTs or KOs) did not survive or remained in a poor state of health after surgery. Therefore, we were unable to analyze synapses through sparse labeling and 3D reconstruction by IUE. Alternatively, we obtained commercial AAVs carrying rAAV-EF1a-PSD95.FingR-eGFP-CCR5TC and rAAV-EF1a-mRuby2-Gephyrin.FingR-IL2RGTC, then injected into the CA1 region of EndoA1fl/fl mice at P0. Mice were fixed and detected the fluorescent signals in CA1 regions at P21. Consistent with immunostaining with antibodies, decreased mRuby2-Gephyrin.FingR or PSD95.FingR-eGFP was observed in dendrites of KO neurons at P21, as shown in new Figure S3. In combination with electrophysiological recording, PSD fractionation and immunoisolation from brains, these data support our conclusion regarding the effects of endophilin A1 knockout on the inhibitory synapses.

    Additionally, we transfected DIV12 cultured hippocampal neurons with pCAG_PSD95.FingR-eGFP-CCR5TC or pCAG_GPN.FingR-eGFP-CCR5TC and observed fluorescent signals on DIV16. Both the signal intensity and number of GPN.FingR-eGFP clusters were also significantly attenuated, with no obvious changes in PSD95.FingR-eGFP clusters in dendrites of mature neurons, as shown in new Figure S5A-D. We are very pleased that the result further strengthened our original conclusion. We have added the new pieces of data in our revised manuscript.

    (3) Figure 3: surface labeling of GluA1 or the GABAAR gamma 2 subunit is difficult to interpret: the patterns are noisy and the numerous puncta appear largely non-synaptic although this is difficult to judge in the absence of additional synaptic markers. It appears statistics are done on dendritic segments rather than the number of neurons. The legend does not mention how many independent cultures this data is derived from. In their previous study (Yang et al., Front Mol Neurosci 2018), the authors noted a decrease in surface GluA1 levels in the absence of endophilin A1. How do they explain the absence of an effect on surface GluA1 levels in the current study?

    Sorry for the concern and thanks for your comments. First, we assessed changes in the surface levels of excitatory and inhibitory receptors by co-immunostaining in cultured WT and KO hippocampal neurons. Given the very low transfection efficiency of neurons in high density culture, numerous puncta of receptors from adjacent non-transfected neurons were also detected. This approach may contribute to the noisy pattern observed in Figure 3A. Besides, the projections of z-stack for higher magnified dendrites may likely introduced higher background signals. We have now replaced the original images with the newest repeat in new Figure 3A. Moreover, we confirmed a decrease in the surface expression of GABAAR γ2 by the biotinylation assay, as shown in Figure 3E. Indeed, we agree that some puncta for surface labeling of receptors seemed to be non-synaptic localization. In order to reflect the decrease in synaptic proteins at synapses, we isolated PSD fraction by biochemical assay and found that gephyrin and GABAAR γ2, two major inhibitory postsynaptic components, were reduced in the PSD fraction from KO brains, as shown in Figure 3L. Their colocalization was also attenuated in the absence of endophilin A1, as shown in Figure 5A-C. Combined with electrophysiological recording, these data from multiple assays indicate GluA1 at synapses was not obviously affected but GABAAR γ2 at synapses was impaired in endophilin A1 KO neurons in the present study.

    We have corrected the way that the number of samples is defined for statistical analysis as suggested. This point was also raised by Reviewer #1 (Recommendation (2)). We averaged the values from all dendritic segments of a single neuron, such that one neuron equaled one data point. We had replaced the original Figure 3B and 3D (please see Response to Recommendation (2) by Reviewer #1). Additionally, we added the number of independent cultures these data were derived from to figure legends in revised manuscript.

    Previously, we observed a small decrease in surface GluA1 levels in spines under basal conditions and a more pronounced suppression of surface GluA1 accumulation in spines upon chemical LTP in endophilin A1 KO neurons from EndoA1-/- mice that knockout endophilin A1 since embryonic development stages (Figure 5C,H. Yang et al., Front Mol Neurosci, 2018). In Figure 3A and B in current study, we analyzed surface receptor levels in GFP-positive dendrites, rather than spines, under basal conditions when endophilin A1 was depleted at the later developmental stage. We found a decrease in surface GABAAR γ2 levels but no significant effects on surface GluA1 levels in dendrites. These findings indicate that endophilin A1 primarily affects excitatory synaptic proteins in spines during synaptic plasticity and inhibitory synaptic proteins in dendrites under basal conditions in mature neurons.

    (4) Super-resolution images in Figure 3G, H, I: endophilin A1 puncta look different in panel 3I compared to 3G and 3H, which are very noisy. It is difficult to interpret how specific these EEN1 puncta are. Previous images showing EEN1 distribution in dendrites look different (Yang et al., Front Mol Neurosci 2018); is the same KO-verified antibody being used here? Colocalization of EEN1 with gephyrin or the GABAAR gamma 2 subunit is difficult to interpret; gephyrin mostly does not seem to colocalize with EEN1 in the example shown.

    Sorry for your concerns. As stated previously in Major Points (3), transfection efficiency was very low in cultured neurons and our cultured neurons were at relative high density. As a result, numerous puncta of proteins located in the adjacent non-transfected neurons were also detected, which may contribute to noisy signals observed in Figure 3G-I.

    In our previous paper, we confirmed the specificity of the antibody against endophilin A1 (5A,B. Yang et al., Front Mol Neurosci, 2018). We used the same antibody (rabbit anti-endophilin A1, Synaptic Systems GmbH, Germany) in the current study. While the previous images were obtained using confocal microscopy, the current images in Figures 3G, H, and I were acquired using super-resolution microscopy (SIM). The different patterns observed in the dendrites may be attributed to the difference in image resolution, antibodies dilution and reaction time.

    Reviewer #1 also points out the quantification of colocalization of gephyrin and GABAAR γ2 with endophilin A1. Please see Response to Recommendation (13) by Reviewer #1.

    (5) The interaction of gephyrin and endophilin A1 is based on coIP experiments in cells and brain tissue. To convincingly demonstrate that these proteins interact, biophysical experiments with purified proteins are necessary.

    Thanks a lot for your great suggestions on the interaction of endophilin A1 with gephyrin. To convincingly demonstrate their interaction, we performed pull-down assay with purified recombinant proteins and the result shows that both G and E domains of gephyrin were involved in the interaction with endophilin A1. The data has been added to the revised manuscript as new Figure 5I. We also modified the statement about the data and figure legends in the revised manuscript.

    (6) Figure 4G: the gephyrin images are not convincing; the inhibitory postsynaptic element typically looks somewhat elongated; these puncta are very noisy and do not appear to represent iPSDs. The same criticism applies to the images shown in Figures 5 and 7.

    Thanks for the comment. The gephyrin puncta in our images exhibited heterogeneous shapes and sizes, with some appearing somewhat elongated. To address this, we compared the puncta pattern of gephyrin with that shown in the reference. As illustrated in the figure from the reference, gephyrin puncta also displayed distinct shapes and sizes, Figure 3A-F, Neuron 78, 971–985, June 19, 2013). Please note that the images were z-stack projections at higher magnification, as described in the "Materials and Methods" section. This approach may likely introduce higher background signals and may contribute to the much more heterogeneous appearance of the puncta in Figures 4, 5, and 7. As mentioned previously, the numerous gephyrin puncta located in the adjacent non-transfected neurons may also contribute to some of the noisy signals observed. We have replaced the original images with new images in new Figure 4G, 5 and 7.

    Moreover, in order to confirm the effects of endophilin A1 KO on the gephyrin clustering, we also detected the endogenous clusters of gephyrin or PSD95 visualized by GPN.FingR-eGFP or PSD95.FingR-eGFP in cultured mature neurons. The results were consistent with immunostaining with antibodies against gephyrin. Please see Response to Recommendation (2)

    (7) Figure 7E, F: the rescue (Cre + WT) appears to perform better than the control (mCherry + GFP) in the PTZ condition; how do the authors explain this? Mixes of viral vectors were injected, would this approach achieve full rescue?

    Thanks for the thoughtful comment. Mixed viruses were injected bilaterally into the hippocampal CA1 regions. The results showed a full rescue effect by WT endophilin A1 in knockout mice during the early days, with even a little bit better rescue effect than the control group in the later days under the PTZ condition, as shown in Figures 7E and 7F. In the current study, overexpression of endophilin A1 increased the clustering of gephyrin and GABAAR γ2 in cultured neurons, as shown in Figures 4I-J and 5D-E. Presumably, the slightly better rescue effects observed in the behavioral tests was likely attributed to the enhanced clustering and/or stabilization of gephyrin/GABAAR γ2 by WT endophilin A1 expression in KO neurons in vivo. Moreover, the electrophysiological recording also showed full rescue effects on eIPSC by WT endophilin A1 in KO neurons (Figure 7G-K).

    Minor Points

    (1) The authors mention that they previously found a decrease in eEPSC amplitude in EEN1 KO mice (Yang et al., Front Mol Neurosci 2018). The data in Fig. 1E suggests a decrease in eEPSC amplitude but is not significant here, likely due to the small number of observations. If both eEPSC and iEPSC amplitude are reduced in the absence of EEN1. Would the E/I ratio still be significantly changed?

    We apologize for the confusion. In our previous study, AMPAR-mediated excitatory postsynaptic currents (eEPSCs) were found to be slightly but significantly reduced compared to the control group, while NMDAR-mediated excitatory postsynaptic currents showed no significant difference (Figure 4N,O. Yang et al., Front Mol Neurosci, 2018). In the current study, we adopted a different recording protocol, simultaneously measuring eEPSCs and eIPSCs from the same neuron to calculate the E/I ratio. Unlike previous studies, we did not use inhibitors to suppress GABA receptor activity. As a result, the recorded signals did not distinguish AMPAR-mediated or NMDAR-mediated excitatory postsynaptic currents to reflect total eEPSCs, which may explain the non-significant reduction observed compared to control neurons in this study.

    It is possible that the eEPSC amplitude would show a significant reduction if a larger number of neurons were recorded. Nevertheless, the larger suppression of eIPSCs in the absence of endophilin A1 indicates that the E/I ratio is significantly altered.

    (2) Page 7: the authors mention they aim to exclude effects on presynaptic terminals of deleting endophilin A1 in cultured neurons, is this because of a sparse transfection approach?

    Please clarify.

    Sorry for the confusion. In cultured neurons, we always observed sparse transfection due to the very low transfection efficiency (~ 0.5%). Therefore, we could examine the effects of endophilin A1 knockout specifically in the specific CamKIIa promoter-driven Cre-expressing postsynaptic neurons, while endophilin A1 remained intact in the non-transfected presynaptic neurons.

    (3) The representative blot of the surface biotinylation experiment (Figure 3E) suggests that loss of endophilin A1 also affects GluN1 and Nlgn2 levels, and error bars in panel 3F (lacking individual data points) suggest these experiments were highly variable.

    Sorry for the confusion. Reviewer #1 also raised the question and we quantified the total level of GluN1 and NL2 in Figure 3E. And we replaced the original graphs with scatterplots and means ± S.E.M. Please see the Response to Recommendation (3) & (12) by Reviewer #1.

    (4) Have other studies analyzing inhibitory synapse composition identified endophilin A1 as a component? The rationale for this study seems to be primarily based on the presence of epileptic seizures and E/I imbalance.

    Thank you for your questions. To date, no other studies investigated endophilin A1 as an inhibitory postsynaptic component. We observed the proximal localization of endophilin A1 with inhibitory postsynaptic proteins using super-resolution microscopy (SIM) and quantification results showed ~ 47% puncta of gephyrin correlated with endophilin A1 (Figure 3G-I and S4B-G). We further immunoisolated the inhibitory postsynaptic fraction using GABAA receptors and found that endophilin A1 was present in the isolated fraction, and vice versa (Figure 3J). Additionally, we demonstrated that endophilin A1 directly interacted with gephyrin through co-IP and pull-down assays (Figure 5J-I). Together with data from immunolabeling, biochemical assays, electrophysiological recordings, and behavioral tests, these results identified endophilin A1 as an inhibitory postsynaptic component.

    (5) Figure 3J: what are S100 and P100 labels? Is Nlgn2 part of the EEN1 complex? If it is, why are Nlgn2 surface levels not affected by EEN1 loss (Figure 3E, F, K)? Why does EEN1 not interact with Nlgn2 in HEK cells (Figure 4D)?

    Sorry for the confusion. The detailed information regarding S100 and P100 can be found in the “GST-pull down, co-immunoprecipitation (IP), and immunoisolation” in the “Materials and Methods” section. S100 contains soluble proteins, while P100 refers to the membrane fraction after high speed (100,000xg) centrifugation.

    Figures 3J-K and 4C-F showed the data from immunoisolation and conventional co-immunoprecipitation assays, respectively. Immunoisolation, which uses antibodies coupled to magnetic beads, allows for the rapid and efficient separation of subcellular membrane compartments. In Figure 3J-K, we used antibodies against GABAAR α1 to isolate membrane protein complexes from the inhibitory postsynaptic fraction. In contrast, co-immunoprecipitation typically detects direct interactions between proteins solubilized by detergent treatment. For Figure 4C-F, FLAG beads were used in HEK293 lysates, or antibodies against endophilin A1 were employed in brain lysates to precipitate direct interaction partners. Combined with the results from Figure 3J-L, the data in 4C-F indicated that endophilin A1 was localized in the inhibitory postsynaptic compartment and directly bound to gephyrin but not to either GABAA receptors or Nlgn2 (NL2). This binding promoted the clustering of gephyrin and GABAAR γ2 at synapses, facilitating GABAAR assembly.

    Nlgn2 (NL2) is a key inhibitory postsynaptic component but does not directly bind to endophilin A1. Consequently, endophilin A1 failed to co-immunoprecipitate with NL2 in the presence of detergent in HEK293 cell lysates (Figure 4D). Furthermore, the surface levels of NL2 or its distribution in PSD fraction were unaffected by the loss of endophilin A1 (Figure 3E, F, K, L). This suggests that mechanisms independent of endophilin A1 orchestrate the surface expression and synaptic distribution of NL2.

    (6) How do the authors interpret the finding that endophilin A1, but not A2 or A3, binds gephyrin? What could explain these differences?

    Thanks for the thoughtful comment. Endophilin As contain BAR and SH3 domains. While the amino acid sequences in the BAR and SH3 domains are highly conserved, the intrinsically disordered loop region between BAR and SH3 domains is highly variable. A study by the Verstreken lab revealed that a human mutation in the unstructured loop region of endophilin A1 increases the risk of Parkinson's disease. They also demonstrated that the disordered loop region controls protein flexibility, which fine-tunes protein-protein and protein-membrane interactions critical for endophilin A1 function (Bademosi et al., Neuron 111, 1402–1422, May 3, 2023). Our previous study showed that endophilin A1 and A3, but not A2, bind to p140Cap through their SH3 domains, despite the high sequence homology in the SH3 domains among these proteins (Figure2A,B. Yang et al., Cell Research, 2015). These findings indicate that each endophilin A likely interacts with specific partners due to distinct key amino acids.

    Additionally, endophilin A1 is expressed at much higher levels than A2 and A3 in neurons, with distinct distribution of them across different brain regions. Our lab demonstrated that the function of A1 at postsynapses (both excitatory and inhibitory synapses) cannot be compensated by A2 or A3. Therefore, it is reasonable that endophilin A1, rather than A2 or A3, binds to gephyrin, even though the underlying mechanisms remain unclear.

    (7) Figure 4G: panels are mislabeled (GFP vs merge).

    Thanks for careful reading and sorry for the mistake. We corrected the label in new Figure 4G. Please see Response to Annotation, grammar, spelling, typing errors:(1) by Reviewer #2.

  6. Version published to 10.7554/elife.102792.2 on eLife
  7. Version published to 10.7554/elife.102792 on eLife
  8. Version published to 10.1101/2024.09.07.611815v3 on bioRxiv
  9. Version published to 10.1101/2024.09.07.611815v2 on bioRxiv
  10. Version published to 10.7554/elife.102792.1 on eLife
  11. eLife

    eLife Assessment

    This study presents a valuable finding on the molecular mechanisms that govern GABAergic inhibitory synapse function. The authors propose that Endophilin A1 serves as a novel regulator of GABAergic synapses by acting as a component of the inhibitory postsynaptic density. Although the study employs a variety of approaches to address this question, the current analysis is incomplete and requires further experiments to substantiate the claims fully. The findings are likely to interest a broad audience of scientists focusing on inhibitory synaptic transmission, the excitation-inhibition balance, and its disruption in disorders such as epilepsy.

  12. eLife

    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.

  13. eLife

    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.

  14. eLife

    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.

  15. eLife

    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.

  16. Version published to 10.1101/2024.09.07.611815v1 on bioRxiv