Titel: Omics Approaches for the Analysis of Shank1/3-associated Autism Spectrum Disorders and the Lessel-Kreienkamp syndrome
Sonstige Titel: Omics-Ansätze zur Analyse der Shank1/3-assoziierten Autismus-Spektrum-Störungen und des Lessel-Kreienkamp-Syndroms
Sprache: Englisch
Autor*in: Tibbe, Debora Rabea Uta
Schlagwörter: Neurologische Entwicklungsstörungen; Genetik; Biochemie; Argonaute-2; Shank
Erscheinungsdatum: 2023-07
Tag der mündlichen Prüfung: 2023-09-15
Neurodevelopmental disorders arise due to malfunctions in brain development and often are genetically caused. The disease-associated genes encode three major classes of proteins: transcriptional regulators, synaptic proteins, and RNA-binding proteins. It was proposed that the pathomechanisms may converge in shared molecular pathways. I functionally analyzed and compared the pathomechanisms of insufficiency for SHANK1 and SHANK3, two genes associated with autism spectrum disorders, with a variant of the AGO2 gene, encoding the RNA-binding protein Argonaute-2 and causing Lessel-Kreienkamp syndrome.
The SHANK genes encode Shank proteins, scaffolds of excitatory, glutamatergic synapses that indirectly connect postsynaptic glutamate receptors to F-actin via a protein network. The N-terminus of Shank3 interacts with Ras family G-proteins, connecting Shank to the MAPK pathway, which is involved in translational regulation. Altered translation has been implicated in the pathology of autism spectrum disorders. The hypothesis of this project was that loss of Shank may alter the regulation of translation. Omics were applied to test this hypothesis. The translatome and proteome of mouse models for Shank-associated autism spectrum disorders were investigated. Actively translated mRNAs were purified from hippocampal neurons of Shank3αβ knockout mice via RNA affinity purification and analyzed by RNA sequencing. Absence of Shank3αβ resulted in subtly altered translation of a subset of neuronal transcripts. Proteomics were performed with biochemically purified postsynaptic density fractions from hippocampi of Shank1 and Shank3αβ knockout mice. The loss of Shank1 and Shank3αβ severely altered the postsynaptic proteome. The abundance of active, phosphorylated CaMKIIα was increased in Shank knockout mice, which may contribute to misregulated neuronal signaling.
Argonaute-2 functions in RNA interference. To execute translational silencing, Argonaute-2 associates with microRNA and forms the RNA induced silencing complex. It was hypothesized that patient derived Argonaute-2 mutants may bind different microRNAs, altering RNA interference. To test this hypothesis, primary cortical neurons were infected with adeno-associated viruses to induce expression of the Argonaute-2 mutant L192P. Expression of the L192P variant induced increased occurrence of dendritic processing bodies, shown by immunocytochemistry and confocal microscopy. MicroRNA affinity purification was applied, and microRNAs were sequenced. The expressed and Argonaute-2-bound microRNAs were identified. Four different aspects of microRNA-dependent RNA interference were altered in neurons that expressed the Argonaute-2 mutant. A subset of microRNAs was differentially expressed, indicating an affected miRNome. Several microRNAs exhibited altered association to the RNA induced silencing complex. The Argonaute-2 mutant showed altered strand selectivity, which resulted in arm switching events between guide and passenger strands of microRNAs. The patient variant induced enhanced incorporation of isomers of microRNAs, termed isomiRs. A subset of isomiRs was exclusively bound to Argonaute-2 L192P, whereas none of the isomiRs associated exclusively with the wildtype protein. The Argonaute-2 variant resulted in deregulation of the miR379-409 genomic cluster. Deregulation of this cluster likely affects neurogenesis, neuronal migration, and synaptic function.
Analyses of translatome, proteome, miRNome, and Argonaute-2-bound microRNAs demonstrated variable deregulations contributing to the molecular defects in model systems for neurodevelopmental disorders. This suggests that the pathomechanisms of Shank-associated forms of autism spectrum disorders and Lessel-Kreienkamp syndrome do not converge in common pathways, but rather result in broad and gene-specific alterations of cellular functions.
URL: https://ediss.sub.uni-hamburg.de/handle/ediss/10535
URN: urn:nbn:de:gbv:18-ediss-112832
Dokumenttyp: Dissertation
Betreuer*in: Torda, Andrew
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen

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