Within the brain's neocortex originates our most evolutionarily advanced complex cognitive functions. The diverse subpopulations of neural stem cells and post-mitotic neurons underlying these intricate circuits are molecularly defined by their patterns of gene expression, where functional gene expression culminates in protein synthesis via mRNA translation at the level of the ribosome. Therefore, mRNA translation must be tightly regulated in development for specific mRNAs to generate molecularly defined subpopulations of neocortical neurons. Autism Spectrum Disorders (ASDs) are clinically heterogeneous disorders of complex cognitive functions. In the past decade, genomic and transcriptomic analyses have implicated genes and pathways that converge on protein synthesis in the cortex as a target in ASD etiology, reinforced by multiple syndromic ASD subtypes where translation abnormalities are central, such as Fragile-X Syndrome (FXS). While the regulation of core translation components has been implicated in ASDs, how abnormal translation of specific mRNAs leads to neocortical dysfunction remains unanswered. Furthermore, it is not known if ribosomal components are dynamic during neocortical development, nor whether this relates to how neocortical neurons are molecularly defined from the translation of specific mRNA transcripts. Our preliminary data suggest that components of the core translation machinery and the mRNAs that associate with them in the fetal neocortex are indeed dynamic, with a particular transition occurring at mid- neurogenesis - a susceptibility period highly implicated in ASD genomic meta-analyses. Our data show that this mid-neurogenic transition is marked by a dramatic increase in the phosphorylation of eukaryotic elongation factor 2 (eEF2), putatively modulating elongation activity via its kinase, eEF2k. Interestingly, eEF2k has been associated with ASDs as a variable genomic locus in multiple studies, and a unique microdeletion syndrome spanning this locus is characterized by severe cognitive deficits. eEF2k also partners with FMRP to regulate the translation of specific mRNAs in FXS. We found that dynamic eEF2 phosphorylation occurs in both cycling neural stem cells and differentiated neurons spanning neocorticogenesis, and loss of eEF2k results in abnormal neocortical neuron differentiation. We hypothesize that eEF2k regulates mRNA translation, neural stem cell cycling, and differentiation in fetal neocortical development and dysfunction. This will be tested by first analyzing the dynamic candidate protein components and mRNA cargo of actively translating ribosomes (polysomes) identified by our preliminary studies in eEF2k KO and WT neocortices throughout development. Second, neural stem cell cycle and differentiation will be analyzed in developing eEF2k KO vs. WT neocortices to extend our preliminary finding of abnormal neocortical circuits with eEF2k depletion. This project aims to advance our understanding of ASDs from previous studies of the genomic and transcriptomic levels towards an ASD proteome, employing advanced in vivo techniques to identify specific translational therapeutic targets.
Defective protein synthesis in the brain's cortex is a key feature of many Autism Spectrum Disorders (ASDs). However, it is unknown if core ribosomal proteins in the cortex change during the fetal brain development, nor how abnormalities in these proteins lead to a dysfunctional cortex in ASDs. This project aims to elucidate the molecular mechanisms underlying dynamic protein synthesis in the normal and ASD fetal cortex, a key step towards the ASD proteome as a novel therapeutic target.