Unique Role of ASPM in Development of Large and Folded Cerebral Cortex - PROJECT SUMMARY/ABSTRACT Neurodevelopmental disorders affecting the large and folded (gyrencephalic) human cerebral cortex require disease mechanisms that are robust in humans but insignificant in mice. For example, loss of ASPM (assembly factor for spindle microtubules) in humans causes severe microcephaly with 50-75% reduction in cortical volume, simplified gyri, epilepsy, and intellectual disability. In contrast, Aspm KO mice show <10% reduction. ASPM encodes a conserved centrosomal protein that regulates progenitor proliferation, delamination, and differentiation. Thus, it is unclear how loss of a neurodevelopmental gene such as ASPM causes remarkably severe phenotypes in the large, gyrencephalic human cortex compared to the mouse cortex. The ferret is a genetically and surgically tractable, gyrencephalic carnivore that bridges the gap between human brain disorders and mouse models. Its cortex shows transcriptional patterns similar to human cortex, and contains numerous outer radial glia, a progenitor cell type abundant in humans but rare in mice. Our germline Aspm KO ferrets show severe microcephaly with a 25-40% reduction in cortical volume and simplified gyri. Moreover, Aspm KO ferrets, but not mice, show premature progenitor delamination and death. Importantly, ASPM KO human cerebral organoids show the same progenitor defects as Aspm KO ferrets, as reported by the Vanderhaeghen lab (Benthem et al., 2023). However, the molecular mechanisms by which only human and ferret progenitors are uniquely susceptible to loss of ASPM are unknown. Our LONG-TERM GOAL is to elucidate the unique developmental mechanisms of the large and gyrencephalic human cortex in health and disease. ASPM microcephaly is a neurodevelopmental disorder for which human organoid, ferret, and mouse models are available, making it an ideal candidate for investigating gyrencephalic mammalian-specific mechanisms. Our CENTRAL HYPOTHESIS is that loss of ASPM triggers similar molecular changes in humans and ferrets, but not in mice, via gyrencephalic mammalian-specific ASPM interactors. To test the hypothesis, we propose three Specific Aims: (1) To systematically examine ferret and mouse cortical development in the presence and absence of ASPM using spatial transcriptomics; (2) To determine molecular mediators of progenitor delamination and death in ASPM KO human cerebral organoids and ferrets by comparing KO progenitors at one time point and similarly delaminating WT progenitors at a later time point in human organoids and ferrets using single-cell RNA sequencing; (3) To define the ASPM interactome in human, ferret, and mouse progenitors using proximity labeling and centrosome proteome analysis. The roles of newly identified mediators of progenitor defects will be tested in rescue experiments in ASPM KO human organoids and ferrets. With our strong expertise and cross- species approach using human organoids, ferret and mouse models, we are uniquely qualified to rigorously investigate gyrencephalic cortical development in health and disease.
