Vasculature of the paraventricular nucleus of the hypothalamus: influence of development, gamma-aminobutyric acid (GABA) receptors, and prenatal glucocorticoids, The

2014 Spring.The paraventricular nucleus of the hypothalamus (PVN) is a critical brain region that regulates many homeostatic and stress responses. In addition to its dense cytoarchitecture, it also contains a vast network of blood vessels. These blood vessels within the mouse PVN have a higher density than other brain regions, which develops postnatally. Loss of gamma aminobutyric acid (GABA) signaling or prenatal dexamethasone (dex) treatment decreased the blood vessel density. Dex also decreased blood brain barrier (BBB) competency while increasing desmin-immunoreactive pericytes at postnatal day (P)20. Long-term consequences included a decrease in GFAP contact with blood vessels selectively in dex-treated females, and an increase in depression-like behaviors in dex-treated males. Chapter 2 examines the blood vessel density within the PVN. Initially the blood vessel density is similar than surrounding brain regions, then after P8 there was an increase that resulted in a highly vascularized network around P20. The highest densities were restricted to the rostral and mid regions of the PVN, where the neuroendocrine neurons are housed. In addition, mice lacking a functional GABAB receptor had a significant decrease in blood vessel density in the mid region at P20. The protein endocan has been proposed to be a "tip cell" marker, indicating angiogenesis. To further characterize the postnatal angiogenic period within the PVN, recently developed antibodies against endocan were used. Chapter 3 provides evidence that endocan is normally expressed in the mouse brain but not restricted to tip cells. In addition, prior perfusion with fluorescein isothiocyanate (FITC) prevents endocan-immunoreactivity (ir) and provides a novel method for identifying non-functional blood vessels. Chapters 4 and 5 show that excess fetal glucocorticoids alters the BBB within the PVN at two time points. At P20, there was a loss of BBB integrity accompanied by an increase in desmin-ir pericytes on a reduced blood vessel network due to dex-treatment for both prepubertal males and females. In contrast at P50, the blood vessel density and BBB were no longer disrupted following fetal dex-treatment. However, there was a decrease in glial fibrillary acidic protein (GFAP)-ir astrocytes in dex-treated females and an increase in desmin-ir pericytes in dextreated males. In conclusion, the work set forth in this dissertation indicates that the dense vascular network within the PVN develops postnatally and is susceptible to regulation by both exogenous and endogenous factors

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms

<div><p>Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate <i>in vitro</i>. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.</p></div

Pravastatin decreases NSPC expansion <i>in vitro</i>.

<p>(A) Representative images of neurosphere cultures. Treatments include vehicle, 1 μM and 25 μM pravastatin (prava) on days 1, 3 and 5 after treatment. Bright field images were taken on an inverted microscope at 10x magnification. (B) Quantification was performed on images taken in triplicate using image J software on days 0, 1, 3 and 5 after treatment with vehicle, 1 μM or 25 μM prava (n = 5). (C) After imaging, NSPCs were collected for automated cell counting in duplicate (n = 5).</p

Statins alter mRNA expression in differentiating NSPCs through a non-CBP dependent mechanism.

<p>Average mRNA expression of the neuronal markers (A) <i>Map2</i> (n = 6), (B) Tuj1 (n = 6), astrocyte marker (C) GFAP (n = 6), and oligodendrocyte marker (D) Olig2 (n = 6) after four days of NSPC differentiation in 10 uM prava. Average mRNA expression of the neuronal markers (E) <i>Map2</i> (n = 6) and (F) <i>Tuj1</i> (n = 6) as well as the astrocyte marker (G) <i>GFAP</i> (n = 6) and oligodendrocyte marker (H) <i>Olig2</i> (n = 6) after four days of NSPC differentiation in 1uM simva. (I) Average mRNA expression of <i>GFAP</i> in cells after four days of differentiation in 10uM prava co-treated with 1 uM or 10 uM FPP (n = 5). (J) Average mRNA expression of <i>Ldlr</i> in cells after four days of differentiation in 10 uM prava co-treated with 1 uM or 10 uM FPP (n = 5). (*p<0.05, **p<0.01, ***p<0.001 compared to vehicle unless otherwise indicated by brackets).</p

Pravastatin activates SREBP2 in NSPCs.

<p>(A) Ingenuity pathway analysis identified top upstream regulators of transcriptional change induced by 10 μM prava. (B) Network of transcriptional regulation by the SCAP-INSIG-SREBP2 complex. (C) Representative western blot of cleaved SREBP2 (cSREBP2) from NSPCs treated with vehicle, 1 μM or 25 μM prava for 24 hours. The treatment group of each band matches the column of the histogram directly below. (D) Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 μM or 25 μM prava for cSREBP2 normalized to ß-Actin (n = 7). (*p<0.05, **p<0.01, ****p<0.0001 compared to controls, unless otherwise demonstrated with a horizontal bracket).</p

Pravastatin induces CBP gene expression in NSPCs.

<p>(A) Ingenuity pathway gene ontology analysis identified “Superpathway of Cholesterol Biosynthesis” and related pathways as the top regulated pathways in neurospheres after a 10 μM prava treatment for 24 hours.–LOG10 (p-value) and percentage overlap with identified pathways are reported (n = 6). (B) Ingenuity pathway gene ontology analysis identified “Cholesterol Biosynthesis” as the top toxicity function in neurosphere after 10 μM prava treatment for 24 hours (n = 6). (C) Average mRNA expression of cholesterol pathway and cholesterol metabolism genes in NSPCs after treatment with vehicle, 1 μM or 25 μM prava for 24 hours (n = 3–5). Expression was normalized to <i>GAPDH</i> and the vehicle treatment group for each gene. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to controls, unless otherwise demonstrated with a horizontal bracket).</p

FPP prevents pravastatin dependent SREBP2 activation and induction of cell death.

<p>(A) Representative western blot of cleaved SREBP2 (cSREBP2) from NSPCs treated with vehicle, 10 μM prava, 1 μM farnesyl pyrophosphate (FPP), 10 μM FPP, 10 μM prava and 1 μM FPP, or 10 μM prava and 10 μM FPP, respectively, for 24 hours. The treatment group of each band matches the column of the histogram directly below. (B) Average intensity of cSREBP2 bands normalized to ß-actin (n = 7). (C) Average mRNA expression of <i>Ldlr</i>. Expression was normalized to <i>GAPDH</i> (n = 5). (D) Representative western blot of cleaved PARP (cPARP). The treatment group of each band matches the column of the histogram directly below. (E) Average intensity of cPARP bands normalized to ß-actin (n = 7) (***p<0.001, ****p<0.0001 compared to vehicle; #p<0.05, ##p<0.01, ###p<0.001 compared to 10 μM prava).</p

Pravastatin increases cyclin D1 expression but prevents passage through S-Phase in NSPCs.

<p>A. Average expression of <i>Ccnd1</i> mRNA as assessed by qRT-PCR (n = 5). B. Representative western blot of cyclin D1 from neurospheres treated with vehicle, 1 uM or 25 uM prava for 24 hours C. Average intensity of bands corresponding to 24 hour treatment with vehicle, 1 uM or 25 uM prava for cyclin D1 normalized to ß-Actin (n = 5). D. Percentage of EdU+ NSPCs treated with 1 uM or 25 uM prava for three days with EdU incorporation during the final 4 hours, quantified by flow cytometry (n = 8). E. Representative image of dissociated neurospheres after three days treatment with vehicle for DAPI and EdU incorporation. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to vehicle, unless otherwise indicated by a horizontal bracket).</p