SMPD4: Role of a microcephaly gene in ceramide biosynthesis and human brain development

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Brain development in mammals begins early in embryogenesis and continues after birth. Intricate patterns of neural progenitor proliferation, differentiation, and migration are established early in neural development and are essential for this process. Our interest lies in primary microcephaly, a disorder of development which results primarily in a decrease in dorsal cortex size in humans. Microcephaly is a congenital, often genetic, malformation which affects 25,000 U.S. children per year. Currently, there are no treatments for this disorder. We recently published a cohort of twenty- three pediatric patients from twelve unrelated families with damaging variants in the gene SMPD4 (neutral sphingomyelinase-3/nSMase3) whose primary clinical finding is microcephaly. SMPD4 hydrolyzes sphingomyelin in the cell membrane to produce phosphocholine and ceramide. Ceramide is required for primary ciliogenesis. The objective of this proposal is to elucidate the function of SMPD4 in the brain in order to recognize why loss of this gene causes disorder of brain development in human patients. Our central hypothesis is that SMPD4 expression is uniquely required for human cortical neurogenesis via regulating proper sphingolipid biosynthesis. We propose to 1) specify the impact of loss of SMPD4 upon forebrain development, 2) determine the cellular mechanism of SMPD4 microcephaly, and 3) investigate the role of sphingolipid biosynthesis pathway components in this disorder. In Aim 1, I will use human pluripotent stem cell-derived neural rosettes and a conditional genetic ablation in mice to confirm the pathogenicity of human SMPD4 variants upon forebrain development. In Aim 2, I will define the requirement for SMPD4 in specific neural cell types, hypothesizing that SMPD4 is required in neural progenitors in the ventricular zone and regulates primary ciliogenesis in these cells. In Aim 3, I will assess cellular levels of key sphingolipid molecules in control and SMPD4 loss of function models and modulate sphingolipid pathway components using exogenous chemical treatment. I hypothesize that loss of SMPD4 decreases the level of ceramide and its downstream products, and rescuing ceramide biogenesis is sufficient to improve brain phenotypes. The results of the proposed work will contribute to both our understanding of SMPD4 pathogenicity in human patients with microcephaly and the role of the sphingolipid pathway in human brain development.