Research Project
10364300
PROJECT SUMMARY/ ABSTRACT The goal of the proposed research is to use proteomics on samples from patient-derived cells with defined disease genotypes to identify the disrupted molecular pathways that underpin phenotypes of autism spectrum disorder (ASD). Family history and twin studies suggest that, in at least some cases, these disorders share genetic roots, but the degree to which environmental and genetic factors account for individual differences within the spectrum is currently unknown. The possibility that there is a genetic antecedent to the onset of ASD stimulated GWAS studies, but these studies were only able to identify genetic risk factors and not specific causative genes. To gain an understanding of the underlying molecular mechanisms associated with ASD, we will use human induced pluripotent stem cells (hiPSC) from patients with diseases on the autism spectrum. We have been reprogrammed fibroblasts from these patients and differentiated them into cells to create organoids, thus allowing the proteomic study in human diseases that were previously unobtainable (e.g. brain diseases). In this study, we will use large-scale proteomics combined with single cell proteomics to identify protein changes in individual cells of two genotypes on the autism spectrum. Patients suffering from the monogenetic disease Rett Syndrome (RTT) exhibit clinical phenotypes that mirror elements of ASD. We have fibroblasts that contain the different mutations in the gene MECP2 that cause RTT. We also have cells that contain the copy number variant CNV 16p11.2, a genetic variant on the autism spectrum. The molecular changes underpinning the development of ASD are poorly understood, but patient-derived cerebral organoids recapitulate molecular and morphological changes by gene dosage changes from CNV 16p11.2. We propose to use powerful mass spectrometry multiplexing technologies (Tandem Mass Tags ? TMT) to measure commonalities and differences in proteome and phosphoproteome encoded by these genotypes. In addition, electrophysiology measurements will be combined with single cell proteomics to measure protein changes in neurons with electrophysiology disrupted by these genetic changes.
