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UBC Theses and Dissertations

Characterization of two ASD-associated genes in primary hippocampal neurons : SEMA5A and PTEN Dingwall, Riki


Autism spectrum disorder (ASD) is a pervasive neurodevelopmental disorder primarily characterized by stereotypic behaviors, deficits in social interaction and difficulties with communication. Extensive epidemiological studies suggest a major role for genetics in the etiology of ASD. To date, 600-1,200 human genes have putatively been linked to ASD, including SEMA5A and PTEN. A large number of these ASD-associated genes play a role in the formation, maintenance, elimination or stabilization of synapses, while others are involved in broader elements of neurodevelopment, such as dendrite arborization, dendritogenesis, and soma size. Consistent with neurological dysfunction in ASD are observations that individuals with ASD often have supernumerary synapses, disrupted excitatory/inhibitory balance, and patterns of hypo- and hyper-connectivity compared to the general population. Despite this, many of the neurological functions of ASD-associated genes or gene disruptions remain poorly elucidated. In this study, we examine the role of Sema5A in activity-mediated synapse elimination, notably hippocampal long-term potentiation (LTP) and long-term depression (LTD). We describe the enhanced trafficking of Sema5A to the surface membrane during LTD and the subsequent Sema5A-dependent elimination of excitatory synapses. Furthermore, we demonstrate that Sema5A selectively mediates excitatory—and not inhibitory—synapse elimination, suggesting a mechanism by which the dysregulation of Sema5A could disrupt excitatory/inhibitory balance. Secondly, we describe the role of PTEN in negatively regulating excitatory synapse density, total dendritic arbor length, and soma size. Moreover, we characterize alterations to the neurological functions of PTEN in mature hippocampal neurons following the introduction of ASD-associated single nucleotide variants (SNVs). We demonstrate that most of the ASD-associated PTEN SNVs tested are broadly loss of function, with two notable exceptions: P38H PTEN exhibits a single altered neurological function, while H123Q PTEN phenocopies wild type human PTEN across all measures, further stressing the importance of biological functionalization. Lastly, we establish a PTEN knockdown assay in which PTEN SNVs could be tested for synaptic, dendrite and somal phenotypes. Combined and integrated, the functionalization of ASD-associated genes and gene variants could permit greater accuracy in ASD diagnoses and prognoses, as well as the improved targeting of therapeutic interventions.

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