SUSD4 Controls Activity-Dependent Degradation of AMPA Receptor GLUA2 and Synaptic Plasticity

  1. I. González-Calvo
  2. K. Iyer
  3. M. Carquin
  4. A. Khayachi
  5. F.A. Giuliani
  6. J. Vincent
  7. M. Séveno
  8. S.M. Sigoillot
  9. M. Veleanu
  10. S. Tahraoui
  11. M. Albert
  12. O. Vigy
  13. Y. Nadjar
  14. A. Dumoulin
  15. A. Triller
  16. J.-L. Bessereau
  17. L. Rondi-Reig
  18. P. Isope
  19. F. Selimi

  • Curated by eLife

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    Evaluation Summary:

    The reviewers agreed that this is a very interesting paper that demonstrates the involvement of a specific protein degradation pathway in a form of synaptic plasticity in the cerebellum. The strength of the work results from its innovative character. The authors show that SUSD4 is expressed throughout the brain and is abundant in cerebellar dendrites and spines. Mice with deletion of SUSD4 have motor coordination and learning deficits, along with impaired LTD induction. This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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Abstract

At excitatory synapses, the choice between recycling or degradation of glutamate AMPA receptors controls the direction of synaptic plasticity. In this context, how the degradation machinery is targeted to specific synaptic substrates in an activity-dependent manner is not understood. Here we show that SUSD4, a complement-related transmembrane protein, is a tether for HECT ubiquitin ligases of the NEDD4 subfamily, which promote the degradation of a large number of cellular substrates. SUSD4 is expressed by many neuronal populations starting at the time of synapse formation. Loss-of-function of Susd4 in the mouse prevents activity-dependent degradation of the GLUA2 AMPA receptor subunit and long-term depression at cerebellar synapses, and leads to impairment in motor coordination adaptation and learning. SUSD4 could thus act as an adaptor targeting NEDD4 ubiquitin ligases to AMPA receptors during long-term synaptic plasticity. These findings shed light on the potential contribution of SUSD4 mutations to the etiology of neurodevelopmental diseases.

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  1. eLife

    Response to Reviewer #3 (Public Review):

    Specific comments:

    1. Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Fig. 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

    We agree with Reviewer 3 that it would be important to localize SUSD4 at this particular synapse. However, the morphology of this synapse is rather peculiar with big CF boutons contacting several thorny spines on proximal dendrites (cf. Sotelo and Dusart 2009). In our experience, it is very difficult to interpret immunofluorescence data for postsynaptic localization at this synapse as the thorny spines are very close to the dendrite, and most often the images are not very conclusive.

    1. Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

    The mean baseline surface GluA2 levels are not significantly different between WT and KO slices (results added in Figure 4-figure supplement 1). We agree with Reviewer 3 that we cannot exclude an effect on endocytosis of GluA2 given that, despite the same magnitude of change on average, the statistical test shows a significant effect for controls, but not for KO. We have changed the text accordingly page 7.

    1. Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

    We have changed the panel in Figure 4D to better highlight the partial colocalization between GluA2 and SUSD4 in cultured Purkinje cells.

    1. Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

    We agree with Reviewer 3 that PVRL3aplha used as a control in the experiment in figure 5D only controls for non-specific interactions with the anti-HA beads and not for non-specific interactions of highly overexpressed membrane proteins. To control for this, we have added an experiment (Figure 4-figure supplement 2) in which we co-expressed SEP-GluA2 together with HA-SUSD4 or PVRL3alpha but this time performed co-immunoprecipitation using anti-GFP beads to pull down SEP-GluA2. We probed the extracts by immunoblot for either GluA2, HA or PVRL3alpha. In both cases SEP-GluA2 was readily immunoprecipitated. PVRL3alpha was not co-immunoprecipitated at all, while HA-tagged SUSD4 was. This result further supports a specific interaction between SEP-GluA2 and HA-SUSD4 in transfected HEK293 cells.

    1. Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

    We have modified the graph to show GluA2 in the IP normalized to the input and relative to the HA-SUSD4 construct (Figure 5D). We have added the quantifications of the GluA2 input amounts in the different conditions (Figure 5-figure supplement 2, former S11). We have also modified the text to clarify the presentation of these co-immunoprecipitation results (page 9).

    1. Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

    This has been corrected.

  2. eLife

    Response to Reviewer #2 (Public Review):

    Major comments:

    In Figure 1 localization images are shown using exogenous protein. Can the authors visualize endogenous protein?

    Unfortunately, we have tested many antibodies against SUSD4, commercial ones and custom-made ones. None gave satisfactory results for the detection of the endogenous SUSD4 protein, as assessed by immunohistochemistry in sections from control and SUSD4 KO mice. This is a classical problem in our field and is stated in the text (line 167 page 4).

    It appears that SUSD4 is expressed in multiple brain regions, even at higher levels than the cerebellum. The authors should provide a good explanation for why deficits in the KO do not affect other functions, and seem to preferentially affect cerebellar functions.

    We never intended to convey the message that cerebellar functions are preferentially affected in this mouse mutant. We have chosen to analyze in depth the phenotype of Susd4 loss-of-function in the cerebellar system, because of the many advantages of this model (as stated in page 3 introduction) and the high expression of Susd4 in cerebellar Purkinje cells. We have changed the text to make this point clearer: 1) in the results section at the level of the transition between the behavior results and the rest (page 5); 2) in the discussion page 11.

    Figure 4: immunofluorescence data are not very convincing.

    We have changed the panel in figure 4D to better highlight the partial colocalization between GluA2 and SUSD4 in cultured Purkinje cells.

    Figure 5: The use of the word "could" is not supporting a strong conclusion. The authors should demonstrate whether SUSD4 DOES indeed regulate GluA2.

    We have changed the title of Figure 5 and changed the text in the result section accordingly.

  3. eLife

    Response to Reviewer #1 (Public Review):

    I have three relatively minor comments:

    1. Fig 2E: it is surprising that the potentiation shown in WT mice is not longer lasting. Under the experimental conditions used here, plasticity seems to be biased towards depression. In the methods, the authors state that they use 2mM Calcium and 1mM Magnesium in their external saline. A recent study (Titley et al., J. Physiol. 597, 2019) has demonstrated that under realistic conditions (incl. an ion milieu of 1.2 mM Calcium and 1mM Magnesium), LTP results under most conditions - even those involving climbing fiber co-stimulation - while LTD only results from prolonged complex spike firing. Optimally, the authors would establish a real LTP control in their WT mice (using conditions as described in Titley et al or similar) and test for changes in the mutants. As LTP is not the focus of this paper and this might be out of the scope of this work, it should be acceptable to leave it as it is, but this caveat should at least be discussed.

    The experimental conditions and the strain of mice used to test potentiation and LTD at PF/PCs are indeed different from the ones used in Titley et al.(2019), (divalent concentration, internal solution, holding potential, CF stimulation frequency). We acknowledge that more physiological conditions may have slightly shifted plasticity toward more potentiation. However, our main goal was to compare WT vs KO genotype and we think our results, albeit under non strict physiological conditions, demonstrate that depression mechanisms are strongly impaired in SUSD4 KO mice. We have amended the results section page 5.

    1. Fig. 3: The climbing fiber physiology is described in detail, but what is missing is a characterization of potential changes in the complex spike waveform, recorded in current-clamp mode. This should certainly be provided. This is important as it has been shown that changes in the complex spike waveform affect the probability for LTD induction (Mathy et al., Neuron 62, 2009). The CF-EPSC is a rather indirect measure.

    As in Mathy et al. 2009 we used repeated CF stimulation to ensure LTD induction. As requested, we have included in our supplementary data the analysis of the complex spike waveform recorded in current-clamp mode during this protocol (Figure 3-figure supplement 1 and results section page 6). We have measured the same parameter as in Titley et al. (2019) and Mathy et al. (2009), the number of spikelets, spikes induced by repeated CF stimulation, and did not find any difference between Susd4 WT controls and Susd4 KO Purkinje cells

    1. Is synaptic pruning at parallel fiber synapses impaired in the SUSD4 mutants? The LTD deficit is quite obvious. In the light of the role of autophagy in pruning, and the molecular similarity between LTD and pruning, it would be of interest to see whether activity-dependent pruning at these synapses is altered. This aspect is somewhat addressed by the VGLuT1 measures shown in Figure 2, but should be discussed in more depth.

    As noted by Reviewer #1, our morphological measurements do not reveal any significant difference in the number of PF synapses in Susd4 WT versus Susd4 KO Purkinje cells. This is also supported by the input-output curve in Figure 2C that does not reveal any difference in Purkinje cell responses to PF stimulation in the absence of Susd4. However, we agree that our data do not exclude a transient effect of Susd4 mutations on synaptic pruning during development, in particular at earlier stages when PF pruning is likely more important (third postnatal week, Ichikawa et al. PNAS 2016).

  4. eLife

    Reviewer #3 (Public Review):

    In this study from the Selimi lab, Gónzalez-Calvo and colleagues investigate the role of the uncharacterized complement family protein SUSD4. SUSD4 is expressed at the time of cerebellar synaptogenesis and localizes to dendritic spines of Purkinje cells. Susd4 KO mice show impaired motor learning, a cerebellum-dependent task. Susd4 KO mice display impaired LTD and facilitated LTP at parallel fiber (PF)-Purkinje cell (PC) synapses, indicating altered synaptic plasticity in the absence of Susd4. Climbing fiber (CF)-Purkinje cell synapses show largely normal basal transmission, with the exception of larger quantal EPSCs. Immunohistochemical analysis shows a small decrease in the proportion of CF/PC synapses lacking GluA2. As their data indicates a role for SUSD4 in regulation of postsynaptic GluA2 content at cerebellar synapses, they next explored the molecular mechanism by which SUSD4 might do so. Activity-dependent degradation of GluA2 does not occur in the absence of SUSD4. Affinity purification of proteins associated with recombinant SUSD4 identifies ubiquitin ligases as well as several proteins involved in AMPAR turnover. Finally, the authors show that SUSD4 can bind GluA2 in HEK cells, and that SUSD4 can bind the ubiquitin ligase NEDD4, but that these two interactions are not dependent on each other.

    This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

    Specific comments:

    1. Localization of SUSD4. The authors demonstrate localization to spines in distal PC dendrites (Fig. 1C). Does SUSD4 also localize to CF/PC synapses? This is important to establish given the phenotype of increased quantal EPSCs and decreased proportion of synapses without GluA2 at the CF/PC synapse.

    2. Figure 4B: There seems to be considerably less surface GluA2 in Susd4 KO cerebellar slices. Is the difference in surface GluA2 between WT and KO significant? Although the mean reduction in surface GluA2 in Susd4 KO following cLTD is similar to WT, the difference with control is not significant. This should be pointed out in the text because it can not be definitively concluded that endocytosis of GluA2 is not altered in Susd4 KO on the basis of this experiment.

    3. Figure 4D: The colocalization of SUSD4 with GluA2 is difficult to see in this image. An inset with higher zoom could help. Quantification of colocalization using e.g. Manders coefficient would help.

    4. Figure 5B: The negative control used here, PVRL3alpha, lacks an HA tag. This therefore does not control for non-specific interactions of highly overexpressed membrane proteins in co-transfected HEK cells. The authors should use an HA-tagged membrane protein as a control here to demonstrate that the interaction of SUSD4 and GluA2 is specific for SUSD4.

    5. Figure 5D: The level of GluA2 recovered in the IP appears normalized to HA-SUSD4. This does not control for the variations in GluA2 input levels shown in Fig. S11. Statements on amounts of GluA2 recovered for various SUSD4 mutants should be adjusted to take this into account.

    6. Line 357: binding of SUSD4=is likely meant to be binding of NEDD4.

  5. eLife

    Reviewer #2 (Public Review):

    The authors show that SUSD4 is expressed throughout the brain and is abundant in cerebellar dendrites and spines. Mice with deletion of SUSD4 have motor coordination and learning deficits, along with impaired LTD induction. The also attempt to show that GluA2 AMPA subunits are misregulated, but that is not as convincing. They find Nedd1, along with many other proteins in a proteomic screen for SUSD4 interactors, and try to explain the phenotypes through the regulation of GluA2 degradation by Nedd4 through SUSD4. These are potentially interesting findings, but very preliminary at this point. While the electrophysiology is good, the mechanistic studies are incomplete.

    Major comments:

    In Figure 1 localization images are shown using exogenous protein. Can the authors visualize endogenous protein?

    It appears that SUSD4 is expressed in multiple brain regions, even at higher levels than the cerebellum. The authors should provide a good explanation for why deficits in the KO do not affect other functions, and seem to preferentially affect cerebellar functions.

    Figure 4: immunofluorescence data are not very convincing.

    Figure 5: The use of the word "could" is not supporting a strong conclusion. The authors should demonstrate whether SUSD4 DOES indeed regulate GluA2.

    Overall, while the electrophysiology seems fine, the mechanistic studies are preliminary and speculative at this point.

  6. eLife

    Reviewer #1 (Public Review):

    This is a highly interesting manuscript by Gonzalez-Calvo et al., describing the involvement of the CCP domain containing protein SUSD4 in the degradation of GluA4 receptors at cerebellar synapses. The novelty of this work lies in the specificity of this degradation pathway. In comparison, synaptic proteins involved in AMPA receptor endocytosis, such as GRIP1 and PICK1, play a role in multiple trafficking processes. In addition, CCP domain proteins play a role in synaptic pruning, which is closely related to LTD. We will return later to this point.

    The paper will certainly enrich the field and further our understanding of cellular plasticity in the cerebellum. These are exciting findings that should be published. I have three relatively minor comments:

    1. Figure 2E: it is surprising that the potentiation shown in WT mice is not longer lasting. Under the experimental conditions used here, plasticity seems to be biased towards depression. In the methods, the authors state that they use 2mM Calcium and 1mM Magnesium in their external saline. A recent study (Titley et al., J. Physiol. 597, 2019) has demonstrated that under realistic conditions (incl. an ion milieu of 1.2 mM Calcium and 1mM Magnesium), LTP results under most conditions - even those involving climbing fiber co-stimulation - while LTD only results from prolonged complex spike firing. Optimally, the authors would establish a real LTP control in their WT mice (using conditions as described in Titley et al or similar) and test for changes in the mutants. As LTP is not the focus of this paper and this might be out of the scope of this work, it should be acceptable to leave it as it is, but this caveat should at least be discussed.

    2. Figure 3: The climbing fiber physiology is described in detail, but what is missing is a characterization of potential changes in the complex spike waveform, recorded in current-clamp mode. This should certainly be provided. This is important as it has been shown that changes in the complex spike waveform affect the probability for LTD induction (Mathy et al., Neuron 62, 2009). The CF-EPSC is a rather indirect measure.

    3. Is synaptic pruning at parallel fiber synapses impaired in the SUSD4 mutants? The LTD deficit is quite obvious. In the light of the role of autophagy in pruning, and the molecular similarity between LTD and pruning, it would be of interest to see whether activity-dependent pruning at these synapses is altered. This aspect is somewhat addressed by the VGLuT1 measures shown in Figure 2, but should be discussed in more depth.

  7. eLife

    Evaluation Summary:

    The reviewers agreed that this is a very interesting paper that demonstrates the involvement of a specific protein degradation pathway in a form of synaptic plasticity in the cerebellum. The strength of the work results from its innovative character. The authors show that SUSD4 is expressed throughout the brain and is abundant in cerebellar dendrites and spines. Mice with deletion of SUSD4 have motor coordination and learning deficits, along with impaired LTD induction. This study provides novel insight in the uncharacterized role of SUSD4 and provides a detailed and well-performed analysis of the Susd4 loss of function phenotype in the cerebellar circuit. The exact mechanism by which SUSD4 affects GluA2 levels remains unclear. However, their findings provide leads for further functional follow-up studies of SUSD4.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  8. Version published to 10.1101/859587v2 on bioRxiv
  9. Version published to 10.1101/859587v1 on bioRxiv