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SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism

Abstract

De novo heterozygous mutations in activity-dependent neuroprotective protein (ADNP) cause autistic ADNP syndrome. ADNP mutations impair microtubule (MT) function, essential for synaptic activity. The ADNP MT-associating fragment NAPVSIPQ (called NAP) contains an MT end-binding protein interacting domain, SxIP (mimicking the active-peptide, SKIP). We hypothesized that not all ADNP mutations are similarly deleterious and that the NAPV portion of NAPVSIPQ is biologically active. Using the eukaryotic linear motif (ELM) resource, we identified a Src homology 3 (SH3) domain-ligand association site in NAP responsible for controlling signaling pathways regulating the cytoskeleton, namely NAPVSIP. Altogether, we mapped multiple SH3-binding sites in ADNP. Comparisons of the effects of ADNP mutations p.Glu830synfs*83, p.Lys408Valfs*31, p.Ser404* on MT dynamics and Tau interactions (live-cell fluorescence-microscopy) suggested spared toxic function in p.Lys408Valfs*31, with a regained SH3-binding motif due to the frameshift insertion. Site-directed-mutagenesis, abolishing the p.Lys408Valfs*31 SH3-binding motif, produced MT toxicity. NAP normalized MT activities in the face of all ADNP mutations, although, SKIP, missing the SH3-binding motif, showed reduced efficacy in terms of MT-Tau interactions, as compared with NAP. Lastly, SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a major autism gene product, interact with the cytoskeleton through an actin-binding motif to modify behavior. Similarly, ELM analysis identified an actin-binding site on ADNP, suggesting direct SH3 and indirect SHANK3/ADNP associations. Actin co-immunoprecipitations from mouse brain extracts showed NAP-mediated normalization of Shank3-Adnp-actin interactions. Furthermore, NAP treatment ameliorated aberrant behavior in mice homozygous for the Shank3 ASD-linked InsG3680 mutation, revealing a fundamental shared mechanism between ADNP and SHANK3.

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Fig. 1: Truncated ADNP proteins impair MT dynamics and assembly.
Fig. 2: Expression of truncated ADNP proteins reduces Tau-MT interactions.
Fig. 3: ADNP p.Lys408Valfs*31 with a disrupted SH3-binding site exhibits adverse effects on MT dynamics and Tau-MT association.
Fig. 4: The SH3-binding domain in NAP increases its protection against tauopathy so as to increase Tau-MT association.
Fig. 5: In vitro and in vivo SHANK3/ADNP/NAP/actin interactions.

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Acknowledgements

We are grateful to Tal Barak and Elad Malikov for their diligent work with the mutant Shank3 model mice and Prof. R. Frank Kooy for the ADNP patient-derived lymphoblastoid cell lines. This work was partially supported by grants to Prof. IG from the European Research Area Network (ERA-NET) Neuron ADNPinMED, Drs. Ronith and Armand Stemmer and Arthur Gerbi (French Friends of Tel Aviv University), Holly and Jonathan Strelzik (American Friends of Tel Aviv University) and Anne and Alex Cohen (Canadian Friends of Tel Aviv University). IG and BB are further supported by Sagol School of Neuroscience Interdisciplinary Partnership Grant (SNIP). IG is Director of the Elton Laboratory for Molecular Neuroendocrinology and the former first incumbent of the Lily and Avraham Gildor Chair for the Investigation of Growth Factors. This work is in partial fulfillment of Ph.D. thesis requirements at the Miriam and Sheldon G. Adelson Graduate School, Sackler Faculty of Medicine, Tel Aviv University (MG), who was also generously supported by a Neubauer Family Foundation Student Scholarship. This work is also in partial fulfillment of M.Sc. thesis requirements at the Miriam and Sheldon G. Adelson Graduate School (AL) and at the Sagol School of Neuroscience, Tel Aviv University (IB-H-H).

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Together with IG, YI-P planned and performed all the cell culture experiments (Figs. 14), and wrote an initial, in depth, draft. MG performed biochemical experiments outlined in Fig. 5. IB-H-H performed animal behavior experiments. AL performed biochemical experiments outlined in Fig. 5. NB helped with the biochemical experiments. IF, GL, SSr, GK, and EG helped with all animal experiments. SSh performed in silico analyses (Fig. 5). Homozygous Shank3 ASD-linked InsG3680 mutated mice were housed and tested under the supervision of BB. IG orchestrated and supervised the entire project and wrote the paper with data analysis and editorial remarks from all contributing authors.

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Correspondence to Illana Gozes.

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NAP (CP201, davunetide) use is under patent protection (US patent nos. US7960334, US8618043, and USWO2017130190A1) (IG), PCT/IL2020/051010 (IG) and provisional patent applications (IG inventor and contributing scientists, YI-P, MG, IB-H-H, SS, EG). Davunetide is exclusively licensed to ATED Therapeutics LTD (IG, Co-Founder and Chief Scientific Officer).

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Ivashko-Pachima, Y., Ganaiem, M., Ben-Horin-Hazak, I. et al. SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism. Mol Psychiatry (2022). https://doi.org/10.1038/s41380-022-01603-w

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