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  1. Evaluation Summary:

    This study is of great interest to a broad group of neuroscientists including those studying plasticity in the nervous system and in neurodevelopmental disorders such as autism. The current work illustrates the importance of protein phosphorylation in regulating a form of homeostatic plasticity known as synaptic scaling, which has been associated with different neurodevelopmental disorders. In particular, the authors provide compelling evidence that the phosphorylation state of one synaptic scaffolding protein, Shank 3, is a necessary part of a complex signaling pathway mediating synaptic scaling and thus could be therapeutically useful for certain associated disorders.

    (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. Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    A strength of the manuscript is the phospho-proteomic analysis related to homeostatic synaptic scaling and the direct examination of Shank3 phosphorylation using phosphomimietic and phosphodeficient mutations. Concerns include place these results into context with previous work from this group regarding synaptic scaling requiring neuronal gene expression or new protein expression; the possible protein kinase involved in Shank3 phosphorylation; and the possible link to the key cell autonomous role for MeCP2 in synaptic scaling and Shank3 phosphorylation based on previous work by this group.

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  3. Reviewer #2 (Public Review):

    Strengths:

    1. Comprehensive/rigorous phospho-proteomic analyses in both rat and mouse cultured cortical neurons are transparently reported. This large data set will made publicly available via a web browser and should a valuable resource to many other investigators studying synaptic regulation.

    2. Analyses of the role of Shank3 phosphorylation have been carefully done. Newly developed phosphosite-specific antibodies were used to confirm changes detected in the proteomics dataset, and phospho-mimetic and phospho-null Shank3 mutations at this site to demonstrate the functional impact of these changes.

    3. Taken together the combined data provide compelling new insights into the changes in protein phosphorylation that play a role in short-term homeostatic plasticity that will be of interest to a wide range of investigators.

    Weaknesses:

    1. The phospho-proteomic data reported are obtained using a conceptually similar approach to that recently reported by Desch et al., 2021, but only rat cortical neurons. They detected a total of ~45K phosphorylation events (vs. ~32K here), with ~3,300 sites (1285 proteins) regulated during homeostatic scaling up to 24 hrs, similar to what was detected here. Readers would benefit from additional discussion of the similarities and differences in the two data sets, and the reasons for the differences.

    2. The authors rely on a pharmacological approach to identify protein phosphatases 2A (PP2A) as a key mediator of the changes in Shank3 phosphorylation. These experiments appear to have been carefully done, but the interpretations would be more robust if additional molecular approaches were used to manipulate PP2A activity.

    3. No data are presented to identify the protein kinase responsible for Shank3 phosphorylation at these sites. The discussion mentions several kinases that phosphorylate various site in Shank3. Only one appears to target S1586/1511 - CaMKII. Indeed, the Desch 2021 study reported changes in CaMKII activation, earlier studies also implicated CaMKII (e.g., DOI: 10.1016/s0896-6273(02)01049-8), and multiple labs have shown that CaMKII can interact with Shank3. It is curious that more is not made of this.

    4. The data in Fig. 7 (sGluA2 and Shank3 clustering) are not very compelling.

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  4. Reviewer #3 (Public Review):

    Homeostatic synaptic scaling is critical for regulating synaptic strength when circuits are challenged - scaling up when activity is blocked and scaling down when activity is enhanced. Scaling has been associated with neurodevelopmental disorders such as autism spectrum disorder. In the current study the authors report that scaling protocols (24 hr treatment with TTX/upscaling or bicuculline/downscaling) carried out on cortical rodent cultures have only modest changes in the proteome, but that significant changes are observed in the phosphorylation state of many proteins. They focus on the phosphorylation state of a synaptic scaffolding protein Shank 3, that they had previously implicated in synaptic upscaling, and which was associated with autism. The study does a nice job of showing that upscaling protocols are associated with Shank3 dephosphorylation and Shank3 synaptic localization. The report goes on to show that the maintained dephosphorylation of Shank3 during upscaling is mediated by PP2A and is necessary for the synaptic localization and the physiological/anatomical expression of scaling. Similar results are presented for downscaling, but in the opposite direction - Shank 3 phosphorylation and removal of Shank 3 from the synapse. This study therefore uncovers a critical role of Shank 3 phosphorylation state in the biochemical pathway that allows for scaling, and therefore could be important in designing a therapeutic strategy for certain neurodevelopmental disorders.

    Strengths:

    The observation that the phosphoproteome, more so than the proteome, is highly dynamic during scaling protocols, and they show this in both rat and mouse.

    Recognizing that phosphorylation of Shank 3 by itself does not drive scaling (not sufficient), but is permissive or necessary for scaling, suggesting a complexity of the biochemical pathways that mediate scaling.

    The compelling demonstration that Shank 3 phosphorylation is a bidirectional mediator of scaling and the further demonstration of PP2As role in upscaling establishes a significant part of the pathway that underlies homeostatic synaptic plasticity. The results therefore represent significant progress in a field that is trying to advance the mechanistic underpinnings of the pathways of homeostatic plasticity, which is thought to contribute to the establishment of neuronal excitability. This was possible by taking advantage of the accessibility of the culture system.

    Weaknesses:

    While the study is carefully executed and results are clear, a few results appear to be contrary to what would be expected. However, these results do not diminish the main findings of the study.

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