Prenatal Origins of Neurometabolic Consequences

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.