Hyperconnectivity of iPSC-derived SHANK2 Neurons and Networks is Rescued by mGluR5 Agonism in an in vitro Model of Autism Spectrum Disorder

Autism spectrum disorder (ASD) is etiologically complex and is associated with a multitude of genetic mutations, many of which disrupt genes that encode proteins important for proper synaptic function such as SHANK2. SHANK2 is a scaffolding protein that organizes important protein-protein interactions at the postsynaptic density (PSD) of excitatory synapses. In the first part of this thesis, I utilized neurons differentiated from human induced pluripotent stem cells (iPSCs) to model SHANK2 ASD, and characterized how ASD-associated mutations in SHANK2 impact neuronal morphology and connectivity. SHANK2 neurons were found to have increased synapse numbers and enhanced dendrite length and complexity when compared to isogenic controls. I found that this cellular phenotype is accompanied by a significant perturbation of the neuronal transcriptome, including the upregulation of genes related to synapse assembly, neurite development, glutamate receptor signaling, and the regulation of synaptic plasticity. I further demonstrated that dendrite length increases observed in cultures of SHANK2 neurons could be prevented by chronic treatment with the group 1 metabotropic glutamate receptor (mGluR) agonist (S)-3,5-Dihydroxyphenylglycine (DHPG), and show that signaling downstream of group 1 mGluRs may be disrupted in these cells. In the second half of this thesis, I focus on the use of in vitro multielectrode array (MEA) technology for characterizing network activity in iPSC models of neurodevelopmental disorders. I first developed a novel computational method for detecting a stereotypic pattern of network activity deemed reverberating superbursts (RSBs) in MEA recordings. Using this method, I showed that the characterization of RSBs is a useful phenotypic metric for separating networks of MECP2 null neurons from controls, and found that the presence of RSBs is dependent on intracellular calcium. Next, I utilized in vitro MEAs to thoroughly characterize a SHANK2 network phenotype. I found that firing activity within SHANK2 networks is hypersynchronous, with significantly increased functional connectivity strength observed between recording electrodes. In addition, I showed that spontaneous bursts of network-wide activity occur significantly more frequently in SHANK2 networks, and these events are significantly shorter in duration compared to controls. Importantly, chronic treatment of cultures with DHPG was also able to partially rescue important aspects of the SHANK2 network phenotype. Finally, I analyzed RSBs in SHANK2 network recordings and found a substantial reduction in the presence of calcium-dependent RSBs in SHANK2 cultures compared to controls. This deficit in RSB activity was significantly improved in SHANK2 networks treated with DHPG. In summary, this work presents a comprehensive characterization of iPSC-derived SHANK2 neurons and reveals a SHANK2 phenotype that is primarily denoted by neuronal hyperconnectivity. Moreover, it presents evidence that alterations in mGluR signaling are important to the SHANK2 phenotype, and provides novel analysis tools for the broader MEA phenotyping field to use in future studies.

Keywords: Autism spectrum disorders
in vitro neuronal networks
induced pluripotent stem cells
multielectrode arrays
Rett syndrome
SHANK2
Advisor: Ellis, James