Characterization of Neuronal Protein Complexes to Identify Molecular Underpinnings of Autism Spectrum Disorder

Abstract

Autism spectrum disorder (ASD), a complex neurodevelopmental disorder that affects brain development, social and communication skills in children and adults, poses a tremendous burden on caregivers and the healthcare system. Although advances in genetics has enabled the discovery of hundreds of ASD-associated genes, the biological context of how these ASD-linked risk genes contribute to the pathophysiology of the disorder remains unclear. The study of protein-protein interactions (PPI) offers a valuable framework for elucidating this biological context, so far only few human proteomics studies were targeted to ASD. Many cell-context-dependent human ASD interactions involved in neuronal processes are unknown. This study addresses this gap by systematically. Fourty six literature-validated ASD-linked proteins expressed in differentiated SH-SY5Y cells were studied using immunoprecipitation coupled with mass spectrometry (IP/MS) to generate a high-quality PPI network, outlining the organization of individual co-purified proteins in multiprotein complexes. Enrichment analysis confirmed the disease relevance of the PPI network and revealed significant enrichment for genes involved in gene expression, cellular transport and mitochondrial (mt) processes. From the high-confidence PPI network, two previously unreported interacting protein pairs of interest (i.e. FMRP-TWNK and DYRK1A-TRMT61B) linking mitochondria with ASD were selected for further characterization. Co-purifying proteins were validated experimentally by co-immunoprecipitation. Knockouts of the interacting protein pairs of interest were generated, with knockout efficacy confirmed by immunoblotting. The interaction between the ASD-linked fragile X mental retardation protein (FMRP) and the mt twinkle DNA helicase (TWNK) were found to play a role in maintaining mtDNA integrity. As well, a functional role between the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK1A) and the mt tRNA methyltransferase 61B (TRMT61B) in tRNA methylation were established. While additional work is needed to dissect the molecular mechanisms of these interactions, the findings from this study offers support to the validity of the molecular models that were proposed. In conclusion, through this Master’s thesis, I was able to demonstrate that the ASD-linked PPI network generated by IP/MS can offer a powerful means for discovering new ASD links and providing future directions for understanding the role of mt in ASD biology.