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Title: Investigation into the autism-associated gene SHANK3 in human stem cell derived neurons and the impact of its deletion on synapses
Author: Taylor, Samuel Ellis
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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Autism spectrum disorder (ASD) includes a broad group of neurodevelopmental disorders that are defined by deficits in social communication and interaction alongside repetitive behaviours. ASD affects more than 1% of the UK population. To date there is no cure and the precise underlying mechanisms are not fully understood. The SH3 and multiple ankyrin repeat domains 3 (SHANK3) protein forms part of the postsynaptic scaffolding complex in excitatory synapses, and mutations/deletions in the SHANK3 gene are strongly associated with ASD. Indeed, mice lacking shank3 exhibit both behavioural deficits associated with ASD as well as a reduced synaptic transmission in excitatory synapses. One hypothesis regarding the underlying pathophysiology of ASD is that affected individuals may have an imbalance between excitation and inhibition in the brain. Here, neurons with deletions in SHANK3 derived from human stem cells were studied in vitro. Differentiation (into cortical neurons) and long-term culture protocols were first optimised. Single molecule fluorescence in situ hybridisation (smFISH) was used to fluorescently label single SHANK3 mRNA molecules in human stem cell derived neurons. A developmental increase in neuronal SHANK3 mRNA expression was observed, as well as direct visualisation of SHANK3 mRNA in neuronal processes. This local expression was reduced in human induced pluripotent stem cell (hiPSC) derived neurons from an individual who had been diagnosed with ASD and had a single heterozygous SHANK3 gene deletion. A characterisation of synapse structure and function was then carried out in both hiPSC and human embryonic stem cell (hESC) derived neurons with deletions in SHANK3 using immunohistochemistry and electrophysiology. Both hiPSC and hESC derived neurons with the SHANK3 deletion typically exhibited a reduced level of excitatory synaptic activity, measured through the frequency and amplitude of miniature postsynaptic currents (mEPSCs). Synaptic scaling, a mechanism for homeostatic plasticity, was assessed in both hiPSC and hESC derived neurons to investigate how the decrease or absence of SHANK3 affected the ability of neurons to adapt to changes in activity. Finally, a second ASD syndrome, Chromosome 15q11.2-q13.1 Duplication (Dup15q) syndrome, was studied. Dup15q syndrome is a result of the multigene duplication of the 15q11.2-q13.1 loci. Dup15q syndrome patients suffer from symptoms including muscle hypotonia, a delay in motor and language skills, intellectual disabilities, and seizures. The differentiation of the Dup15q syndrome patient hiPSCs into cortical neurons was optimised, and the synaptic composition and function analysed compared to a single control line.
Supervisor: Andreae, Laura Christiana ; Lieberam, Ivo Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available