Examining the Physiological Effects of Zinc Supplementation on Shank-associated Mutations in Autism Spectrum Disorders

Abstract

Autism Spectrum Disorders (ASDs) are characterised by deficits in social interactions and repetitive behaviours, and can be caused by genetic mutations. ASD-associated mutations in the Shank genes lead to impaired synapse structure, weakened synapse function, and the presentation of ASD-associated behavioural deficits in mice. Zinc deficiency is a risk factor in ASD, and low zinc levels have been found in autistic children. At the synaptic level, zinc supplementation improves Shank protein stability and its recruitment to glutamatergic synapses, enhancing synapse function. At the behavioural level, dietary zinc supplementation in young adult mice can reverse ASD-associated behaviours by mediating synaptic function. The overall goal of the research in this thesis was therefore to examine the effect of in vitro and in vivo chronic zinc supplementation on synapse structure and function, as well as ASD behaviours caused by ASD-associated Shank2 single nucleotide variants (SNVs) and Shank3 deletion. In vitro studies in dissociated primary hippocampal cultures examined whether zinc supplementation could prevent the development of synaptic deficits caused by ASD-associated Shank2 SNVs. Regardless of the Shank2 protein domain affected by the ASD-associated SNV, zinc supplementation prevented impairments in synaptic density, and alterations in the density of presynaptic VGluT1 and postsynaptic Homer proteins. In vitro zinc supplementation also prevented a decrease in the frequency of miniature excitatory postsynaptic currents. This demonstrates that ASD-associated Shank2 mutations cause structural and functional deficits at excitatory glutamatergic synapses in hippocampal neurons, and zinc supplementation reduces these deficits in vitro. In in vivo models of ASD, we studied whether dietary zinc supplementation during pregnancy and lactation could prevent the development of ASD-associated behaviours in Shank3 knockout (Shank3-/-) offspring. Behavioural tests were performed in three, nine, and 16 week old Shank3-wildtype and Shank3-/- mice born from mothers fed a control (30 ppm) or supplemented (150 ppm) zinc diet. The supplemented maternal zinc diet normalised anxiety behaviours, and prevented deficits in sociability and social novelty recognition in Shank3-/- mice, and repetitive grooming was also prevented in adult Shank3-/- mice. Electrophysiological examination demonstrated that maternal zinc supplementation altered postsynaptic NMDA receptor-mediated currents and presynaptic function at glutamatergic synapses onto medium spiny neurons in the cortico-striatal pathway. Our data show that zinc supplementation from the beginning of brain development has the potential to prevent ASD-associated deficits in Shank3-/- mice long term. Taken together, data from this thesis demonstrate that zinc supplementation has potential as a therapeutic strategy in ASD.