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Titel: Characterization of the structural and functional impact of autism-associated SHANK3 missense mutations
Sonstige Titel: Charakterisierung der strukturellen und funktionellen Auswirkungen von Autismus-assoziierten SHANK3 Missense-Mutationen
Sprache: Englisch
Autor*in: Bucher, Michael
Schlagwörter: post-synaptic density (PSD); SHANK3 protein; missense mutation; structural perturbation; cellular consequences
GND-Schlagwörter: Molekulare BiophysikGND
Exzitatorische SynapseGND
Erscheinungsdatum: 2022-03
Tag der mündlichen Prüfung: 2022-07-01
Autism-spectrum disorders (ASDs) constitute a set of complex neurodevelopmental disorders associated with characteristic social cognition and communication impairments as well as repetitive behaviors, restricted interests, or intellectual disabilities. These phenotypic traits may additionally be accompanied by other neurological or psychiatric co-morbidities such as epilepsy or depression and generally show a strong variance in their occurrence and severity. It is estimated that approximately 1% of the worldwide population may be affected by ASD, underlining the clinical relevance and the need to understand the mechanisms and origins of ASDs. Due to the high frequency of autism-associated mutations occurring in genes coding for synaptic proteins, ASDs are increasingly understood as synaptopathies. Thereby, synapses constitute the central neuronal communication sites whose structure and function strongly depends on their molecular integrity. One of the central molecular regulators of excitatory glutamatergic synapses is the post-synaptic scaffolding protein SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), which links ionotropic and metabotropic glutamate receptors in the post-synaptic plasma membrane to the synaptic actin cytoskeleton. This functional versatility is reflected by the interaction of SHANK3 with multiple synaptic signaling proteins covering a variety of different pathways. Consequently, mutations in the SHANK3 gene have been implicated in the development of both syndromic and idiopathic ASDs. Besides copy-number variants (CNVs) and truncating mutations, numerous autistic patients were described carrying SHANK3 missense mutations for which several different pathogenic processes including modified dendritic spine morphologies, altered SHANK3 ligand binding affinities, epigenetic mechanisms or altered SHANK3 phosphorylation states have been described. A comprehensive study linking the molecular impact of such missense mutations on the structure and dynamics of SHANK3 itself to the corresponding synaptic phenotype is however largely missing. The present work therefore aims to provide insight into i) structural perturbations of SHANK3 elicited by two selected missense-mutations, R12C and L68P, previously identified in autistic patients, both located within the same domain ii) resulting alterations in the synaptic interactome, subcellular localization and dynamics of SHANK3 to identify new signaling pathways and cellular alterations relevant for the development of ASD and iii) the cellular and network impact of the L68P mutation in a corresponding knock-in mouse model. Using a broad range of techniques including small-angle X-ray scattering (SAXS), nano differential scanning fluorimetry (nanoDSF), circular dichroism (CD) and fluorescence spectroscopy as well as molecular dynamics (MD) simulations, structural alterations within SHANK3 were characterized and revealed changes in the folding, structural stability, and nanosecond peptide backbone dynamics of SHANK3 mutants. Furthermore, a novel concentration-dependent mode of SHANK3 homodimerization was identified by SAXS, which is not impaired by the studied mutations. Overall, mutation-induced structural changes are highlighted as initial breaking points for pathogenic processes. Thereby, the clear distinguishability of structural alterations induced by the respective mutation serves as potential explanation for the broad range of observed mutation-induced pathological alterations. These include, among others, a rearrangement of the synaptic interactome as mass spectrometry demonstrated both gain- and loss-of-function for several SHANK3 interaction partners, from which a potentially new ASD-associated pathogenic pathway could be identified involving the interaction of SHANK3 with SynGAP. On a cellular level, total internal reflection-fluorescence recovery after photobleaching (TIRF-FRAP) experiments showed that the R12C mutation significantly increases the diffusional mobility of SHANK3 in primary hippocampal neurons and revealed the ability of both mutants to alter the mobility of individual interaction partners such as cortactin. An increased tendency to form dendritic clusters, which partially represent active synapses, was additionally evident especially for the L68P mutation. Finally, morphological abnormalities of the post-synaptic density (PSD) nanostructure as well as reduced numbers of excitatory and inhibitory post-synaptic puncta were identified in a humanized mouse model of ASD carrying the L68P mutant human SHANK3 knock-in. These cellular alterations were ultimately found to result in synaptic transmission impairments of heterozygous and homozygous SHANK3 L68P mice. Collectively, the present work provides a comprehensive set of data on a molecular, cellular and functional level to correlate missense mutation-induced alterations in the structure and dynamics of SHANK3 to the corresponding synaptic phenotype. This puts ASD elicited by SHANK3 missense mutations in the context of conformational diseases and might thus contribute to the design and development of new therapeutic strategies involving the use of pharmacological chaperones aiming to reconstitute the native protein fold to alleviate the associated disease phenotype.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/9698
URN: urn:nbn:de:gbv:18-ediss-101689
Dokumenttyp: Dissertation
Betreuer*in: Mikhaylova, Marina
Brune, Wolfram
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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