Synapse Dysfunction Induced by Autism Spectrum Disorder Associated Shank3 Mutations
Autism spectrum disorders (ASD) comprise a range of neurodevelopmental disorders characterised by deficits in social interaction and communication, and by repetitive behaviours. Recent research has identified ASD-associated mutations in important synaptic proteins, and this has prompted investigations into understanding the roles of synapse dysfunction in the pathogenesis of ASD. Shank3 is a postsynaptic scaffolding protein that is essential to regulating both the function and structure of excitatory synapses and Shank3 genetic mutations have been described in people affected with ASD. However, currently little is known about the effects of ASD-associated mutations in Shank3 on synapse structure, function and plasticity. The work in this thesis has examined the structural and physiological synaptic changes that are induced by alterations in Shank3 expression levels or by the expression of ASD-associated mutations in Shank3. In these experiments, dissociated rat hippocampal neurons grown in vitro were transiently transfected with Shank3 wild-type (WT) to increase normal Shank3 levels, Shank3 short hairpin ribonucleic acid (shRNA) to down-regulate Shank3 expression, or mutant forms of Shank3 that have been identified in people with ASDs including R87C and R375C point mutations, and the InsG frameshift mutation. The first part of this thesis aimed to investigate the changes that occur in γ- aminobutyric acid (GABA)-ergic inhibitory synapses induced by up- or down-regulation of Shank3 expression or expression of ASD-associated mutations in Shank3. We specifically investigated changes in the expression level and density of GABAergic pre- and postsynaptic proteins by immunocytochemistry, and examined inhibitory synaptic transmission using whole cell patch clamp electrophysiology. To directly compare the results observed in GABAergic inhibitory synapses to glutamatergic excitatory synapses, the density of excitatory synapses and the properties of excitatory synaptic transmission were analysed in the same experimental system. Our data revealed that overexpression of Shank3 enhanced excitatory glutamatergic synapse function, while loss of Shank3 or expression of ASD-association mutations in Shank3 depressed excitatory glutamatergic synapse function, similar to previous work in our laboratory (Arons et al., 2012). Elevation in GABAergic inhibition was observed in neurons overexpressing Shank3, whereas GABAergic inhibitory synapses were not altered in neurons expressing shRNA-Shank3 or InsG-Shank3. Moreover, neurons expressing the R87C or R375C point mutations in Shank3 showed an enhancement in GABAergic inhibitory synapse function. These data indicate that ASD-associated mutations in Shank3 induce heterogeneous physiological consequences at GABAergic inhibitory synapses. In addition, the observed elevation in GABAergic function in neurons expressing the R87C or R375C Shank3 ASD mutations may underlie the depression in excitatory synapse function. The second part of this thesis aimed to investigate how the changes in Shank3 expression levels or ASD-associated mutations in Shank3 alter synaptic plasticity in cultured hippocampal neurons. To address this aim, we induced N-Methyl-D-aspartate receptor (NMDAR)-dependent long term potentiation (LTP), NMDAR-dependent long term depression (LTD), or metabotropic glutamate receptor (mGluR)-dependent LTD in neurons overexpressing Shank3, shRNA-Shank3 or ASD-associated mutant forms of Shank3. Neurons which lacked Shank3 or which expressed ASD-associated mutant versions of Shank3 displayed deficits in NMDAR-dependent LTP and mGluR-dependent LTD, but no effects on NMDARdependent LTD were observed. Moreover, these neurons expressed LTD under our NMDARdependent LTP-inducing paradigm. Faster NMDAR-mediated EPSCs deactivation kinetics were detected in neurons with Shank3 down-regulation, indicating a possible change to GluN2A-containing NMDARs. This may in part contribute to the deficit in NMDARdependent LTP observed in neurons with loss of Shank3 by altering the threshold for LTP induction. In contrast, we found that both the NMDAR and the mGluR-dependent forms of LTD were normal in neurons overexpressing Shank3, however NMDAR-dependent LTP was impaired. We hypothesise that changes in the expression level of Shank3 or ASD-associated mutations in Shank3 drive synapses to distinct states, in which the direction and mechanisms for undergoing subsequent synaptic plasticity are altered. Altogether, these results further support synapse dysfunction as a major pathogenic mechanism for the development of ASD and could underlie cognitive deficits found in people with ASDs.
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