Project Summary: Protein synthesis is a fundamental process in all living cells and is highly regulated to accommodate the specific needs of each cell. Dysregulated protein synthesis has been demonstrated to underlie many of syndromic forms of autism such as Fragile X syndrome (FXS) and Tuberous Sclerosis Complex (TSC), both of which result from defects in genes that regulate protein synthesis. Moreover, mouse models of FXS and TSC exhibit defective synaptic function, and ASD-like behaviors. Recent studies have shown that Eukaryotic Elongation Factor 1A2 (EEF1A2), a protein responsible for GTP-dependent transport of aminoacyl-tRNAs to the elongating ribosome, is mutated in patients with autism, intellectual disability and epilepsy. Elongation Factor 1A has two isoforms, one that is ubiquitously expressed, EEF1A1, and another, EEF1A2 that is expressed only in neurons and myocytes. It is unclear why another isoform is needed in these specific cells; however, it has been found that EEF1A2 is critical for neuronal survival. The wasted mouse, a mouse model with a homozygous deletion of mouse Eef1a2, has been found to exhibit neuron degeneration, tremors, loss of muscle bulk and gait abnormalities after weaning. EEF1A2 has been also shown to bundle actin and microtubules independently of translation, a process known to be critical for neuronal development and migration. This evidence suggests a critical role played by EEF1A2 in neuronal development and function. This proposal aims to uncover how ASD-associated mutations in EEF1A2 results in deficits in neuronal development and autism pathophysiology. Using human iPSC (induced pluripotent stems cells) derived neurons as a model, the CRISPR-Cas9 system will be used to recapitulate patient mutations. These iPSCs will then be differentiated into neurons using neurogenin-2, a master transcription factor capable of inducing differentiation into excitatory neurons in under 2 weeks. Using this platform, the effect of ASD-associated mutations on neuronal function will be studied. The first aim examines the effect of ASD-associated EEF1A2 mutations on protein synthesis in neurons, given the central role that EEF1A2 plays in protein synthesis. Furthermore, the changes to the translatome profile, elongation rate and translational efficiency in these cells will be identified. The second aim will explore changes to neuronal morphology. function and development. After differentiation, induced neurons with ASD-associated EEF1A2 mutations will be examined for altered morphology using immunocytochemical analysis. During differentiation, live cell imaging will be used to track neurite growth and the aberrant signaling pathways involved in actin dynamics and cytoskeletal regulation will be studied. Finally, electrophysiology will used to assess synapse function and strength by measuring excitatory post synaptic currents. The proposed research will advance our understanding of the role translation control plays in neuron...