Prenatal Origins of Neurometabolic Consequences

1摘要 2. 3神经发育障碍(NDD)发病率呈上升趋势。NDDS的原因包括缺血性 4胎盘紊乱(IPD)合并胎儿/宫内生长受限(FGR/IUGR)、围产期窒息和 5低血糖。对异常胎盘健康与NDDS之间的联系的探索已经发生。此外 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。我们重点研究了神经元葡萄糖转运蛋白3(GLUT3；基因：GLUT3)。GLUT3基因 据报道，有8个突变患有NDDS/认知障碍。暴露于不良IUE的GLUT3突变可能 9表现为扩张效应对NDD内表型的影响。诊断学和早期饮食的发展 10项干预措施非常必要。我们已经证明，IUGR和缺氧缺血扰乱了大脑GLUT3的发育 11扰乱神经行为的表达。我们还创造了小鼠GLUT3缺失，减少了胎盘 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 正在探索13种囊泡(SEV)，以促进诊断和生酮饮食干预。我们下一步就离开了 14个胎盘GLUT3基因从神经特异性GLUT3突变到破译独立的神经机制 15%落后于NDDS。我们还从诱导的多能干细胞中创造了表达人脑器官的GLUT3 16(IPSCs)。评估针对NDDS的临床前生酮饮食效应，将产生新的结果。要做到这一点 17目标，我们将检验假设，IUE和神经性GLUT3突变/依赖通过以下方式导致NDDS 18干扰神经发育，具有改善的潜力。目标是：1)调查变化 每种细胞类型的细胞数和具有神经前体的大脑皮层(CC)的细胞特异性转录产物中的19 20利用巢蛋白驱动的条件空白出生后小鼠的GLUT3缺失的细胞(NPC)。这将会实现的 21×10倍基因组学单细胞(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)检测特定基因关键转录/翻译产物的主要变化 23种细胞类型。B.评估产前和产后生酮饮食的管理作为早期干预 24改善新城疫。2)a.探讨伴有或不伴有MoMCR/IUGR的CC的神经过程和细胞形态。 利用Emx1驱动的条件性作用于兴奋性神经元/神经前体细胞中GLUT3的靶向缺失或过度表达(OE) 26只Null和OE小鼠从胚胎、出生后到成年的整个生命过程。这将需要去卷积 27采用ISH/IHC的散装CC RNA-SEQ，非侵入性检测循环中受干扰的转录组/蛋白质 28辆SEV。B.检测对成人脑脊液/血浆代谢组学、神经功能和神经行为的影响 29个后代。3)a.从GLUT3OE和/或GLUT3缺失的对照IPSCs中开发皮质类有机化合物，以及 30通过用scRNA-seq和ISH/IHC去卷积有机RNA-seq来检查细胞图谱。B.审问 31低氧和低血糖加/无酮/乳酸对IPSCs、神经前体细胞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.