Understanding the Role of Autism Related Presynaptic NRXN1 and Postsynaptic SHANK3 in Regulation of Activity Dependent Plasticity Using hiPSC Derived Cortical Neurons

Background: Autism Spectrum Conditions (ASC) are a set of complex heterogeneous neurodevelopmental conditions characterised by impairments in social cognition, communication development alongside unusually narrow interests and sensory hypersensitivity. Genetic studies suggest that mutations, deletions and copy number variations in synaptic genes affect neural communication and synapse formation/pruning thereby altering brain development and connectivity. The homeostatic equilibrium of synaptic connections and the dynamic changes in neurites and dendritic spines in response to environmental changes in the early brain is a longstanding scientific question in autism research. NRXN1 and SHANK3 are presynaptic and postsynaptic genes respectively and are known to be highly penetrant for syndromic autism. However, the contribution of NRXN1 and SHANK3 towards autism pathophysiology through the regulation of activity dependent plasticity is not well characterised in human neurons.

Objectives: The aim is to study NRXN1 and SHANK3 deletion-mediated differences in the structure and function of synapses and to investigate how the dynamics of actin cytoskeleton might be in response to environmental changes. The emerging cellular phenotypes will be amenable to hypothesis-driven high content screen-based experiments aiming to rescue the synaptic deficits.

Methods: In this study we are using iPSCs from individuals with deletions in SHANK3 (n=2, 1 male, 1 female), NRXN1 (n=2, 1 male, 1 female) and healthy controls (n=2). GFP tagged NGN2 transcription factor was introduced in the iPSCs using lentiviral vectors to generate induced cortical neurons. The glutamatergic excitatory neurons thus produced were grown for three weeks (Day 21) in culture for the downstream assays. The healthy controls were also tested for siRNA mediated knockdown. The neurons were then treated with KCl, NBQX, TTX and tested for expression of MAP2 and synaptic markers such as Synaptophysin and Homer. The expression of immediate early genes such as Arc and BDNF were measured with immunoblots and RT PCR. Neurite formation was assayed using live imaging of GFP-tagged NGN2 neurons. The spontaneous and activity dependent firing was measured with calcium imaging under non-stimulating and stimulating conditions. The activity of neurons was further measured using extracellular (multielectrode arrays) and intracellular (patch clamp) recordings.

Results: The knockdown experiment demonstrate downregulation of early immediate genes such as Arc and BDNF in response channel blockers NBQX and TTX. The colocalization of presynaptic and postsynaptic markers also show marked decrease on such treatment conditions. The calcium imaging revealed impairment in synaptic connectivity induced by channel blockers and was more severe in knockdown conditions. Moreover, the neurite outgrowth assay on patient lines demonstrate impaired neuronal migration due to changes in the ratio of filamentous to globular actin. Currently we are generating isogenic hiPSC lines by introduction of deletions of NRXN1 and/or SHANK3 gene in wildtype hiPSC lines with CRISPR-Cas9 gene editing technique to validate the results.

Conclusions: Transient knockdown of autism related synaptic genes such as SHANK3 and NRXN1 affects activity dependent transcription of early immediate genes and synaptic connectivity under different stimulating conditions. Patient lines with deletions in these genes are affected severely due to deficits in neuronal migration and inability to form optimal synaptic connections.