Research ArticleDEVELOPMENTAL NEUROSCIENCE

NAD+-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior

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Science Advances  09 Oct 2020:
Vol. 6, no. 41, eabb9766
DOI: 10.1126/sciadv.abb9766
  • Fig. 1 NAD+-mediated rescue of prenatal forebrain angiogenesis and morphological defects in the Gabrb3ECKO telencephalon.

    (A to C) Fewer isolectin B4+ vessels in E18 saline-treated Gabrb3ECKO telencephalon [yellow asterisks, (B)] compared to saline-treated Gabrb3fl/fl telencephalon (A) and NAD+-treated Gabrb3ECKO telencephalon [white asterisks, (C)]. DAPI, 4′,6-diamidino-2-phenylindole. (D) Morphometric analysis revealed significant rescue in vessel densities in NAD+-treated Gabrb3ECKO telencephalon; data represent means ± SD [n = 10, *P < 0.05, one-way analysis of variance (ANOVA)]. (E1 to G5) Hematoxylin and eosin stainings revealed morphological abnormalities in E18 saline-treated Gabrb3ECKO telencephalon [red asterisks, (F1) to (F5)], while NAD+-treated Gabrb3ECKO telencephalon (G1 to G5) showed significant improvement in morphology, similar to saline-treated controls (E1 to E5). Yellow dotted half circles and arrows point to a NAD+-mediated cellular target in the medial ganglionic eminence (MGE) (G2 to G4). (H) Quantification of the MGE NAD+ target site (n = 10, *P < 0.0001, one-way ANOVA). (I to K) Well-formed vascular plexus in saline-treated Gabrb3fl/fl ganglionic eminence (GE) [white arrows, (I)] and NAD+-treated Gabrb3ECKO ganglionic eminence (K) that is lost in saline-treated Gabrb3ECKO ganglionic eminence [green arrows, (J)]. (L to N) Abnormal PHH3+ profiles were observed in saline-treated Gabrb3ECKO telencephalon [green arrows, (M)] when compared to saline [white arrows, (L)] and NAD+-treated Gabrb3ECKO telencephalon (N). (O) Quantification of PHH3+ cells in the ventral telencephalon. Data represent means ± SD (n = 10, *P < 0.05, one-way ANOVA). Scale bars, (A) 100 μm [applies to (B), (C), (E1) to (G5), and (I) to (N)]; CP, cortical plate; LV, lateral ventricle.

  • Fig. 2 Cellular mechanisms of NAD+ rescue in the Gabrb3ECKO telencephalon.

    (A to K) Robust NKX2.1 (A to C), PROX1 (E to G), and GABA (I to K) expression profiles in saline-treated Gabrb3fl/fl (A, E, and I) and NAD+-treated Gabrb3ECKO (C, G, and K) telencephalon versus abnormal or reduced expression in saline-treated Gabrb3ECKO telencephalon [red arrows, (B); (F) and (J)]. (D, H, and L) Quantification of NKX2.1+ cells in MGE, PROX1+ cells in ventral telencephalon, and GABA+ neurons in dorsal telencephalon (dotted box), (n = 10, *P < 0.05, one-way ANOVA). (M to O) Schematic of neuronal migration assay. (N1 to N3) Robust neuronal migration in saline-control and NAD+-Gabrb3ECKO groups unlike saline-Gabrb3ECKO group (red asterisk). (O) Quantification of cell migration (n = 3, *P < 0.05, one-way ANOVA). (P to S) Low-magnification (P and Q) and high-magnification (R and S) images of E18 telencephalic GAD65-GFP+ cells with (Q and S) or without (P and R) NAD+ treatment. MZ, marginal zone. (T) Quantification of GAD65-GFP+ cells (n = 10, *P < 0.05, Student’s t test). (U to Y) Concurrent reduction of vessels and GABAergic neurons in saline-treated Gabrb3ECKO prefrontal cortex (V, X, and Y) compared to the other groups (U, W, X, and Y). Quantification depicted in (X) and (Y) (n = 10, *P < 0.05; one-way ANOVA). All data represent means ± SD. Scale bars, 100 μm [(A) to (C), (E) to (G), (I) to (K), (N1) to (N3), (P), (Q), and (U) to (W)]; 50 μm [(R) and (S)]. IZ, intermediate zone; VZ, ventricular zone.

  • Fig. 3 Rescue of gene expression profiles in NAD+-treated Gabrb3ECKO telencephalon.

    (A) Schematic depicting microdissection of MGE and striatal region from the subcortical telencephalon of saline-treated Gabrb3fl/fl, saline-treated Gabrb3ECKO, and NAD+-treated Gabrb3ECKO groups (n = 3), cell isolation, RNA preparation, and subsequent microarray hybridization. (B and C) Heatmap clusters depicting down-regulation (B) or up-regulation (C) of genes in saline-treated Gabrb3ECKO telencephalon versus saline-treated Gabrb3fl/fl telencephalon and NAD+-treated Gabrb3ECKO telencephalon. (D to G) Violin plot comparison of top 10 down-regulated (D and E) and up-regulated (F and G) genes in “angiogenesis” and “GABAergic neuronal” categories in saline-treated Gabrb3ECKO telencephalon versus saline-treated Gabrb3fl/fl telencephalon and NAD+-treated Gabrb3ECKO telencephalon.

  • Fig. 4 Molecular mechanisms of NAD+ treatment on Gabrb3ECKO endothelial cells.

    (A to C) Isolectin B4 and GABA colabeling in endothelial cells from the three groups. (D) Quantification of GABA secretion measured by ELISA; data represent means ± SD (n = 6, *P < 0.05, one-way ANOVA). (E to N) RT-qPCR validation of Daxx and P2x4 mRNA expression [(E) and (J); n = 3, *P < 0.05, one-way ANOVA], DAXX (F to H) and P2X4 (K to M) protein expression and quantification [(I) and (N); n = 10, *P < 0.05, one-way ANOVA] in endothelial cells from the three groups. Data represent means ± SD. (O) High-magnification images of P2X4 expression in endothelial cells from the three groups. (P to R) Calcium imaging shows no increase in intracellular calcium following αβ-meATP application, in saline-treated Gabrb3ECKO endothelial cells (Q) when compared to the other two groups [red asterisks, (P) and (R)]. (S) Quantification of calcium imaging data; data represent means ± SD (n = 10, *P < 0.05, one-way ANOVA). (T and U) Quantification of Daxx mRNA expression (T) and GABA secretion measured by ELISA (U) after culturing wild-type endothelial cells in the presence of a P2X4 receptor agonist (αβ-meATP) or antagonist (+NP-1815-PX). Data represent means ± SD, (n = 6, *P < 0.05, one-way ANOVA). Scale bars, 100 μm [(A) to (C) and (P) to (R)]; 50 μm [(F) to (H) and (K) to (M)].

  • Fig. 5 Rescue of blood flow and abnormal behaviors in Gabrb3ECKO adult brain after the prenatal NAD+ treatment.

    (A) Schematic of blood flow velocity acquisition by MPLSM. Dark streaks (negative contrast) correspond RBCs moving along the central axis of the blood vessel. The slopes of these streaks correspond to the RBC velocities. (B to F) Violin plots showing distribution of capillary diameter [(B), n = 40, n = 31, n = 39 vessels], collecting venule diameter [(C), n = 35, n = 26, n = 38 vessels], RBC velocities (D) and blood flow (E) in capillaries (n = 40, n = 31, n = 39 vessels) and blood flow in collecting venules [(F), n = 35, n = 26, n = 38 vessels], across the three mouse groups, respectively. Data are presented in violin plots showing median, quartiles, and distribution (*P < 0.05, **P < 0.005, ***P < 0.0005 one-way ANOVA). (G to L) Quantification of self-grooming time (G), exploration time in light-dark test (H), spontaneous alteration in Y-maze (I), immobility time in tail suspension test (J), and interaction time in social interaction (K) and social novelty (L) tests all showed significant improvement in behavior in NAD+-treated Gabrb3ECKO mice when compared to saline-treated Gabrb3ECKO mice and was comparable to saline-treated Gabrb3fl/fl mice. For all behavioral tests (G to L), data represent means ± SD (n = 12, *P < 0.05, ****P < 0.0001, one-way ANOVA).

  • Fig. 6 The significance of NAD+-mediated rescue of telencephalic angiogenesis and brain development.

    (A) Summary schema depicting defects in the positive feedback GABA signaling pathway in Gabrb3ECKO endothelial cells, in which due to loss of the β3 subunit, GABAA receptors become dysfunctional. As a result, endothelial GABA is unable to activate GABAA receptors and cannot trigger Ca2+ influx and endothelial cell proliferation. Gabrb3 also regulates GABA expression via the transcriptional repressor Daxx. Daxx expression is up-regulated in Gabrb3ECKO endothelial cells; therefore, GABA expression is significantly reduced. This affects GABA secretion from Gabrb3ECKO endothelial cells and disturbs paracrine GABA signaling for neuronal migration and autocrine GABA signaling for angiogenesis. VGAT, vesicular GABA transporter. (B) The NAD+-mediated rescue of Gabrb3ECKO endothelial cells bypasses the GABAA receptor–GABA signaling autocrine pathway. It acts via purinergic receptor signaling that triggers Ca2+ influx and restores cell proliferation in Gabrb3ECKO endothelial cells. It causes direct changes to gene expression in Gabrb3ECKO endothelial cells. By down-regulating Daxx, it restores GABA expression and secretion in Gabrb3ECKO endothelial cells and thereby restores neuronal migration. (C) Our studies show that the NAD+ prenatal administration paradigm is of great significance for rescuing abnormal embryonic brain development. Rescue of prenatal angiogenesis and brain development in the Gabrb3ECKO model improves blood flow and prevents the origin of psychiatric symptoms.

Supplementary Materials

  • Supplementary Materials

    NAD+-mediated rescue of prenatal forebrain angiogenesis restores postnatal behavior

    Sivan Subburaju, Sarah Kaye, Yong Kee Choi, Jugajyoti Baruah, Debkanya Datta, Jun Ren, Ashwin Srinivasan Kumar, Gabor Szabo, Dai Fukumura, Rakesh K. Jain, Abdallah Elkhal, Anju Vasudevan

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