Mutations affecting the N-terminal domains of SHANK3 point to different pathomechanisms in neurodevelopmental disorders
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Evaluation Summary:
This study identifies a novel Shank3 mutation from individuals with ADHD-like syndrome and tests the impacts of this mutation together with other known Shank3 mutations on inter- and intramolecular protein-protein interactions of Shank3 involving the N-terminal SPN and Ank repeats. The results indicate that Shank3 mutations have diverse impacts on the intramolecular SPN-Ank domains and the interaction of Shank3 with other proteins including delta-catenin, fodrin, and CaMKIIa. Overall, the results of the study are novel and of high quality. Considering the lack of detailed biochemical understanding on various Shank3 mutations associated with PMS, ASD, and schizophrenia, this study is a meaningful step forward in the basic understanding of Shank3 functions and related pathophysiology.
(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
Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology.
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Evaluation Summary:
This study identifies a novel Shank3 mutation from individuals with ADHD-like syndrome and tests the impacts of this mutation together with other known Shank3 mutations on inter- and intramolecular protein-protein interactions of Shank3 involving the N-terminal SPN and Ank repeats. The results indicate that Shank3 mutations have diverse impacts on the intramolecular SPN-Ank domains and the interaction of Shank3 with other proteins including delta-catenin, fodrin, and CaMKIIa. Overall, the results of the study are novel and of high quality. Considering the lack of detailed biochemical understanding on various Shank3 mutations associated with PMS, ASD, and schizophrenia, this study is a meaningful step forward in the basic understanding of Shank3 functions and related pathophysiology.
(This preprint has been reviewed …
Evaluation Summary:
This study identifies a novel Shank3 mutation from individuals with ADHD-like syndrome and tests the impacts of this mutation together with other known Shank3 mutations on inter- and intramolecular protein-protein interactions of Shank3 involving the N-terminal SPN and Ank repeats. The results indicate that Shank3 mutations have diverse impacts on the intramolecular SPN-Ank domains and the interaction of Shank3 with other proteins including delta-catenin, fodrin, and CaMKIIa. Overall, the results of the study are novel and of high quality. Considering the lack of detailed biochemical understanding on various Shank3 mutations associated with PMS, ASD, and schizophrenia, this study is a meaningful step forward in the basic understanding of Shank3 functions and related pathophysiology.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This study identifies a novel Shank3 mutation from individuals with ADHD-like syndrome and tests the impacts of this mutation together with other known Shank3 mutations on protein-protein interactions of Shank3 involving the N-terminal SPN and Ank repeats. The results indicate that Shank3 mutations have diverse impacts on the intramolecular SPN-Ank domains and the interaction of Shank3 with other proteins including delta-catenin, fodrin, and CaMKIIa. Overall, the results of the study are novel and of high quality. Considering the lack of detailed biochemical understanding on various Shank3 mutations with regard to their association with PMS, ASD, and schizophrenia, this study is a meaningful step forward.
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Reviewer #2 (Public Review):
In this paper Woike et al. identified a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. The biochemical analysis of the interaction partner of the mutant reveals that the mutated SHANK3 protein loses the binding to δ-catenin, the intramolecular interaction between N-terminal SPN domain and the Ank repeats and the binding to αCaMKII. Another variant of SHANK3, the p.P141A has the most dramatic effect on the function of SHANK3 protein, because it disrupts the interaction with all the binding partners tested and reduces dendritic spines number when transfected in neurons.
The paper provides some evidence of the possible altered functions of the mutated SHAKN3 gene, however the data provided are mostly limited to the analysis of the ability of mutated …
Reviewer #2 (Public Review):
In this paper Woike et al. identified a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. The biochemical analysis of the interaction partner of the mutant reveals that the mutated SHANK3 protein loses the binding to δ-catenin, the intramolecular interaction between N-terminal SPN domain and the Ank repeats and the binding to αCaMKII. Another variant of SHANK3, the p.P141A has the most dramatic effect on the function of SHANK3 protein, because it disrupts the interaction with all the binding partners tested and reduces dendritic spines number when transfected in neurons.
The paper provides some evidence of the possible altered functions of the mutated SHAKN3 gene, however the data provided are mostly limited to the analysis of the ability of mutated proteins to interact with some defined partners. No further functional data were provided. The possible synaptic alterations induced by p.P141A variant were studied in neurons by looking at the number of dendritic spines after the overexpression of SHANK3 mutated proteins. However it's unclear how dendritic spines were measured because the images in Figure 9 do not show neurons with dendritic spines.
In this form, the presented data are too preliminary to be considered sufficient to suggest a revision.
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Reviewer #3 (Public Review):
In this manuscript, Woike et al, investigated and compared the molecular effects of the several N-terminal (SPN-Ank) missense mutations of SHANK3, mainly using biochemical and biophysical analyses. The authors also identified a novel missense mutation (L270M) in the AnK repeats in patients with an ADHD-like phenotype, further expanding clinical diversity caused by SHANK3 variants.
The major strength of this work is that the authors could identify or dissect two major effects of the N-terminal mutations; interference with the SPN/Ank interaction and reduced binding to catenin. Meanwhile, the major weakness is that most experiments were performed in HEK 293T cells with overexpression of the small fragment, not full-length, of Shank3. Whether the two distinct groups of missense mutations have different synaptic …
Reviewer #3 (Public Review):
In this manuscript, Woike et al, investigated and compared the molecular effects of the several N-terminal (SPN-Ank) missense mutations of SHANK3, mainly using biochemical and biophysical analyses. The authors also identified a novel missense mutation (L270M) in the AnK repeats in patients with an ADHD-like phenotype, further expanding clinical diversity caused by SHANK3 variants.
The major strength of this work is that the authors could identify or dissect two major effects of the N-terminal mutations; interference with the SPN/Ank interaction and reduced binding to catenin. Meanwhile, the major weakness is that most experiments were performed in HEK 293T cells with overexpression of the small fragment, not full-length, of Shank3. Whether the two distinct groups of missense mutations have different synaptic or functional phenotypes in neurons also needs to be further investigated to better support the authors' conclusions.
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