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

哺乳动物的大脑发育在胚胎发育早期开始，并在出生后继续。错综复杂 神经祖细胞增殖、分化和迁移的模式在早期就已确立， 神经发育，是这个过程的关键。我们的兴趣在于原发性小头畸形， 一种主要导致人类背部皮质体积减小的发育障碍。 小头畸形是一种先天性的，通常是遗传性的畸形，每年影响25，000名美国儿童。 年目前，没有治疗这种疾病的方法。我们最近发表了一组20人的研究结果- 来自12个无关家族的3名儿童患者，在基因SMPD 4中存在破坏性变异， （中性鞘磷脂酶-3/nSM酶3），其主要临床表现为小头畸形。SMPD 4 水解细胞膜中的鞘磷脂以产生磷酸胆碱和神经酰胺。 神经酰胺是初级纤毛发生所必需的。本提案的目的是阐明 SMPD 4在大脑中的功能，以识别为什么该基因的缺失会导致 人类患者的大脑发育。我们的中心假设是，SMPD 4表达是 通过调节适当的鞘脂生物合成，这是人类皮质神经发生所特有的。 我们建议1）明确SMPD 4缺失对前脑发育的影响，2）确定 SMPD 4小头畸形的细胞机制; 3）研究鞘脂的作用 生物合成途径的组成部分在这种疾病。在目标1中，我将使用人类多能干细胞 细胞源性神经病变和小鼠中的条件性遗传消融以确认致病性 人SMPD 4变体在前脑发育中的作用。在目标2中，我将定义以下要求： SMPD 4在特定神经细胞类型中的作用，假设SMPD 4在神经祖细胞中是必需的 并调节这些细胞中的初级纤毛发生。在目标3中，我将评估 控制和SMPD 4功能丧失模型中关键鞘脂分子的细胞水平， 使用外源性化学处理调节鞘脂途径组分。我假设 SMPD 4的缺失降低了神经酰胺及其下游产物的水平， 神经酰胺生物合成足以改善脑表型。拟议工作的结果 将有助于我们理解SMPD 4在人类患者中的致病性， 小头畸形和鞘脂途径在人脑发育中的作用。

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.