Prenatal Origins of Neurometabolic Consequences

1篇摘要 2 3神经发育障碍（NDD）的发病率越来越高。NDD的原因包括缺血性 4例胎盘疾病（IPD）伴胎儿/宫内生长受限（FGR/IUGR）、围产期窒息，以及 5例低血糖。对异常胎盘健康和NDD之间的联系进行了探索。除了 6子宫内环境（IUE），基因突变也有助于NDD的子集，形成IUE基因 7范式。我们集中在神经元葡萄糖转运蛋白亚型3（GLUT 3;基因：GLUT 3）。Glut 3基因 8个突变报告了NDD/认知障碍。暴露于不利IUE的Glut 3突变可能 9描绘了对NDD内表型的扩展效应。诊断和早期饮食的发展 10项干预措施非常必要。我们已经证明，胎儿宫内发育迟缓和缺氧缺血扰乱发育中的脑组织， 11表达扰乱神经行为。我们还创建了小鼠的Rx 3基因缺失， 12葡萄糖转运导致出生后NDD（兴奋性自闭症谱系障碍），其中小细胞外 13囊泡（sEV）燃料诊断和生酮饮食干预正在探索中。我们接下来脱离了 来自神经特异性的14个胎盘B13基因突变，以破译独立的神经机制 15、落后于国家。我们还从诱导的多能干细胞中创建了表达人脑类器官的BMP 3 16（iPSC）。评估针对NDD的临床前生酮饮食效应将产生新的结果。实现这一 17目标，我们将测试假设，即IUE和神经源性β 3突变/依赖性导致NDD， 18扰乱神经发育，具有改善的潜力。目标是：1）a。调查变化 在具有神经祖细胞的大脑皮质（CC）中，每种细胞类型的细胞数量和细胞特异性转录组学为19 通过使用巢蛋白驱动的条件性无效出生后小鼠，观察到NAP 3的20细胞（NPC）缺失。这将是完成 21 × 10 X基因组学单细胞（sc）RNA测序和生物信息学分析，随后进行原位杂交 22（ISH）/免疫组织化学（IHC）检测特定细胞中关键转录/翻译产物的主要变化 23种细胞类型B.评估产前与产后生酮饮食管理作为早期干预， 24例改善NDD。2)a.探讨CC伴或不伴MoMCR/IUGR的神经过程和细胞特征， 25使用Emx 1驱动的条件刺激，在兴奋性神经元/NPC中靶向缺失或过表达（OE） 26只裸小鼠和OE小鼠从胚胎期、出生后到成年的整个生命过程。这将需要去卷积 27批量CC RNA-seq，ISH/IHC，非侵入性检测循环中的干扰转录组/蛋白质 28辆sEV。B.检查对成人CSF/血浆代谢组学、神经元功能和神经行为的影响 29个孩子3)a.从具有G403 OE和/或G403缺失的对照iPSC发育皮质类器官，以及 30通过用scRNA-seq和ISH/IHC去卷积类器官RNA-seq来检查细胞谱。B.审问 图31具有/不具有酮/乳酸盐的缺氧和低血糖对iPSC、NPC和皮质类器官的影响。

1 ABSTRACT 2 3 There is growing incidence of neurodevelopmental disorders (NDD). Causes for NDDs include ischemic 4 placental disorders (IPD) with fetal/intra-uterine growth restriction (FGR/IUGR), perinatal asphyxia, and 5 hypoglycemia. Exploration of connections between aberrant placental health and NDDs has occurred. Besides 6 the intra-uterine environment (IUE), genetic mutations also contribute to a subset of NDDs, forming an IUE-gene 7 paradigm. We have focused on the neuronal glucose transporter isoform 3 (GLUT3; gene: glut3). Glut3 gene 8 mutations are reported with NDDs/cognitive disabilities. Glut3 mutations with exposure to an adverse IUE may 9 portray expansive effects upon NDD endophenotypes. Development of diagnostics and early dietary 10 interventions is much needed. We have shown that IUGR and hypoxia-ischemia perturb developing brain glut3 11 expression perturbing neurobehavior. We also created murine glut3 deletions, that reduced trans-placental 12 glucose transport leading to postnatal NDD (excitatory autism spectrum disorders), where small extracellular 13 vesicles (sEVs) fueling diagnostics, and ketogenic dietary intervention are being explored. We next disengaged 14 placental glut3 gene from neural-specific glut3 mutations towards deciphering independent neural mechanisms 15 behind NDDs. We also created glut3 expressing human brain organoids from induced pluripotent stem cells 16 (iPSCs). Assessing pre-clinical ketogenic dietary effects targeting NDDs, will yield novel results. To achieve this 17 goal, we will test the hypothesis, that IUE and neural glut3 mutations/dependency cause NDDs by 18 perturbing neurodevelopment with a potential for amelioration. The aims are: 1) a. To investigate changes 19 in cell numbers per cell type and cell-specific transcriptomics in cerebral cortices (CC) with neural progenitor 20 cellular (NPC) absence of glut3 by using nestin-driven conditional null postnatal mice. This will be accomplished 21 by 10X genomics single cell (sc) RNA-sequencing and bioinformatic analyses, followed by in-situ hybridization 22 (ISH)/immunohistochemical (IHC) detection of major changes in key transcribed/translated products in specific 23 cell types. b. To assess administration of prenatal versus postnatal ketogenic diet as an early intervention in 24 ameliorating NDD. 2) a. To explore neural processes and cellular profile in CC with or without MoMCR/IUGR in 25 targeted absence or overexpression (OE) of glut3 in excitatory neurons/NPCs using Emx1-driven conditional 26 null and OE mice during the life course from embryonic and postnatal to the adult. This will entail deconvoluting 27 bulk CC RNA-seq with ISH/IHC, with non-invasive detection of perturbed transcriptome/proteins in circulating 28 sEVs. b. To examine the impact on CSF/plasma metabolomics, neuronal function and neurobehavior in the adult 29 offspring. 3) a. To develop cortical organoids from control iPSCs with glut3 OE and/or glut3 deletions, and 30 examine cellular profiles by deconvoluting organoid RNA-seq with scRNA-seq and ISH/IHC. b. To interrogate 31 the effect of hypoxia and hypoglycemia with/without ketones/lactate on iPSCs, NPCs and cortical organoids.