MicroRNA-17-92 cluster mediates the proliferation and survival of neural progenitor cells after stroke.

The role of microRNAs (miRNAs) in mediating adult neurogenesis after stroke has not been extensively studied. The present study investigated the function of the miR17-92 cluster in adult neural progenitor cells after experimental stroke. We found that stroke substantially up-regulated miR17-92 cluster expression in neural progenitor cells of the adult mouse. Overexpression of the miR17-92 cluster either in cultured ischemic neural progenitor cells or in the subventricular zone (SVZ) of ischemic animals significantly increased cell proliferation, whereas inhibition of individual members of the miR17-92 cluster, miR-18a and miR-19a, suppressed cell proliferation and increased cell death. The miR17-92 cluster mediated PTEN (phosphatase and tensin homolog) expression, which is a predicted target of the miR17-92 cluster. Addition of Sonic hedgehog (Shh) protein up-regulated miR17-92 expression and elevated c-Myc protein in ischemic neural progenitor cells, whereas blockade of the Shh signaling pathway down-regulated miR17-92 cluster expression and reduced c-Myc levels. Overexpression of c-Myc up-regulated miR17-92 cluster expression. Intraventricular infusion of Shh and a Shh receptor inhibitor, cyclopamine, to ischemic animals further elevated and suppressed, respectively, miR17-92 cluster expression in the SVZ. These data indicate that the miR17-92 cluster plays an important role in mediating neural progenitor cell function and that the Shh signaling pathway is involved in up-regulating miR17-92 cluster expression.

Tissue plasminogen activator and plasminogen activator inhibitor 1 contribute to sonic hedgehog-induced in vitro cerebral angiogenesis.

The molecular mechanisms underlying cerebral angiogenesis have not been fully investigated. Using primary mouse brain endothelial cells (MBECs) and a capillary-like tube formation assay, we investigated whether the sonic hedgehog (Shh) signaling pathway is coupled with the plasminogen/plasmin system in mediating cerebral angiogenesis. We found that incubation of MBECs with recombinant human Shh (rhShh) substantially increased the tube formation in naïve MBECs. This was associated with increases in tissue plasminogen activator (tPA) activation and reduction of plasminogen activator inhibitor 1 (PAI-1). Blockage of the Shh pathway with cyclopamine abolished the induction of tube formation and the effect of rhShh on tPA and PAI-1. Addition of PAI-1 reduced rhShh-augmented tube formation. Genetic ablation of tPA in MBECs impaired tube formation and downregulated of vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang1). Addition of rhShh to tPA-/- MBECs only partially restored the tube formation and upregulated Ang1, but not VEGF, although rhShh increased VEGF and Ang1 expression on wild-type MBECs. Complete restoration of tube formation in tPA-/- MBECs was observed only when both exogenous Shh and tPA were added. The present study provides evidence that tPA and PAI-1 contribute to Shh-induced in vitro cerebral angiogenesis.

MicroRNA profiling in subventricular zone after stroke: MiR-124a regulates proliferation of neural progenitor cells through Notch signaling pathway.

BACKGROUND: The Notch signaling pathway regulates adult neurogenesis under physiological and pathophysiological conditions. MicroRNAs are small non-coding RNA molecules that regulate gene expression. The present study investigated the effect of miR-124a on the Notch signaling pathway in stroke-induced neurogenesis. METHODOLOGY AND PRINCIPAL FINDINGS: We found that adult rats subjected to focal cerebral ischemia exhibited substantial reduction of miR-124a expression, a neuron specific miRNA, in the neural progenitor cells of the subventricular zone (SVZ) of the lateral ventricle, which was inversely associated with activation of Notch signals. In vitro, transfection of neural progenitor cells harvested from the SVZ of adult rat with miR-124a repressed Jagged-1 (JAG1), a ligand of Notch, in a luciferase construct containing the JAG1 target site. Introduction of miR-124a in neural progenitor cells significantly reduced JAG1 transcript and protein levels, leading to inactivation of Notch signals. Transfection of neural progenitor cells with miR-124a significantly reduced progenitor cell proliferation and promoted neuronal differentiation measured by an increase in the number of Doublecortin positive cells, a marker of neuroblasts. Furthermore, introduction of miR-124a significantly increased p27Kip1 mRNA and protein levels, a downstream target gene of the Notch signaling pathway. CONCLUSIONS: Collectively, our study demonstrated that in vivo, stroke alters miRNA expression in SVZ neural progenitor cells and that in vitro, miR-124a mediates stroke-induced neurogenesis by targeting the JAG-Notch signaling pathway.

Multipotent mesenchymal stromal cells increase tPA expression and concomitantly decrease PAI-1 expression in astrocytes through the sonic hedgehog signaling pathway after stroke (in vitro study).

Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-ß1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.

MicroRNA-21 protects neurons from ischemic death.

MicroRNAs are small RNAs that attenuate protein expression by complementary binding to the 3'-UTR of a target mRNA. Currently, very little is known about microRNAs after cerebral ischemia. In particular, microRNA-21 (miR-21) is a strong antiapoptotic factor in some biological systems. We investigated the role of miR-21 after stroke in the rat. We employed in situ hybridization and laser capture microdissection in combination with real-time RT-PCR to investigate the expression of miR-21 after stroke. In situ hybridization revealed that miR-21 expression was upregulated in neurons of the ischemic boundary zone, and quantitative real-time RT-PCR analysis revealed that stroke increased mature miR-21 levels by approximately threefold in neurons isolated from the ischemic boundary zone by laser capture microdissection as compared with homologous contralateral neurons 2 days (n = 4; P < 0.05) and 7 days (n = 3; P < 0.05) after stroke. In vitro, overexpression of miR-21 in cultured cortical neurons substantially suppressed oxygen and glucose deprivation-induced apoptotic cell death, whereas attenuation of endogenous miR-21 by antisense inhibition exacerbated cell death after oxygen and glucose deprivation. Moreover, overexpression of miR-21 in neurons significantly reduced FASLG levels, and introduction of an miR-21 mimic into 293-HEK cells substantially reduced luciferase activity in a reporter system containing the 3'-UTR of Faslg. Our data indicate that overexpression of miR-21 protects against ischemic neuronal death, and that downregulation of FASLG, a tumor necrosis factor-α family member and an important cell death-inducing ligand whose gene is targeted by miR-21, probably mediates the neuroprotective effect. These novel findings suggest that miR-21 may be an attractive therapeutic molecule for treatment of stroke.

Angiopoietin 2 mediates the differentiation and migration of neural progenitor cells in the subventricular zone after stroke.

Ischemic stroke stimulates neurogenesis in the adult rodent brain. The molecules underlying stroke-induced neurogenesis have not been fully investigated. Using real-time reverse transcription-PCR, we found that stroke substantially up-regulated angiopoietin 2 (ANG2), a proangiogenic gene, expression in subventricular zone neural progenitor cells. Incubation of neural progenitor cells with recombinant human ANG2 significantly increased the number of beta-III tubulin-positive cells, a marker of immature neurons, but did not alter the number of glial fibrillary acidic protein (GFAP)-positive cells, a marker of astrocytes, suggesting that ANG2 promotes neuronal differentiation. Blockage of the ANG2 receptor, Tie2, with small interference RNA (siRNA)-Tie2 attenuated recombinant human ANG2 (rhANG2)-increased beta-III tubulin mRNA levels compared with levels in the progenitor cells transfected with control siRNA. Chromatin immunoprecipitation analysis revealed that CCAAT/enhancer-binding protein (C/EBP beta) up-regulated by rhANG2 bound to beta-III tubulin, which is consistent with published data that there are several C/EBP beta binding sites in the promoter of beta-III tubulin gene. In addition, rhANG2 enhanced migration of neural progenitor cells measured by single neurosphere assay. Blockage of Tie2 with siRNA-Tie2 and a Tie2-neutralizing antibody did not suppress ANG2-enhanced migration. However, inhibition of matrix metalloproteinases with GM6001 blocked ANG2-enhanced migration. Collectively, our data suggest that interaction of ANG2, a proangiogenic factor, with its receptor Tie2 promotes neural progenitor cell differentiation into neuronal lineage cells, whereas ANG2 regulates neural progenitor cell migration through matrix metalloproteinases, which do not require its receptor Tie2.

Gene profiles and electrophysiology of doublecortin-expressing cells in the subventricular zone after ischemic stroke.

Stroke increases neuroblasts in the subventricular zone (SVZ) of the lateral ventricle and these neuroblasts migrate toward the ischemic boundary to replace damaged neurons. Using brain slices from the nonischemic adult rat and transgenic mice that expressed enhanced green fluorescent protein (EGFP) concomitantly with doublecortin (DCX), a marker for migrating neuroblasts, we recorded electrophysiological characteristics while simultaneously analyzing the gene expression in single SVZ cells. We found that SVZ cells expressing the DCX gene from the nonischemic rat had a mean resting membrane potential (RMP) of -30 mV. DCX-EGFP-positive cells in the nonischemic SVZ of the transgenic mouse had a mean RMP of -25+/-7 mV and did not exhibit Na(+) currents, characteristic of immature neurons. However, DCX-EGFP-positive cells in the ischemic SVZ exhibited a hyperpolarized mean RMP of -54+/-18 mV and displayed Na(+) currents, indicative of more mature neurons. Single-cell multiplex RT-PCR analysis revealed that DCX-EGFP-positive cells in the nonischemic SVZ of the transgenic mouse expressed high neural progenitor marker genes, Sox2 and nestin, but not mature neuronal marker genes. In contrast, DCX-EGFP-positive cells in the ischemic SVZ expressed tyrosine hydroxylase, a mature neuronal marker gene. Together, these data indicate that stroke changes gene profiles and the electrophysiology of migrating neuroblasts.

Functional response to SDF1 alpha through over-expression of CXCR4 on adult subventricular zone progenitor cells.

The chemokine receptor CXCR4 and its ligand, stromal cell derived factor-1 alpha (SDF1 alpha) regulate neuroblast migration towards the ischemic boundary after stroke. Using loss- and gain-function, we investigated the biological effect of CXCR4/SDF1 alpha on neural progenitor cells. Neural progenitor cells, from the subventricular zone (SVZ) of the adult rat, were transfected with rat CXCR4-pLEGFP-C1 and pSIREN-RetroQ-CXCR4-siRNA retroviral vectors. Migration assay analysis showed that inhibition of CXCR4 by siRNA significantly reduced cell migration compared to the empty vector, indicating that CXCR4 mediated neural progenitor cell motility. When neural progenitor cells were cultured in growth medium containing bFGF (20 ng/ml), over-expression of CXCR4 significantly reduced the cell proliferation as measured by the number of bromodeoxyuridine+ (BrdU+) cells (26.4%) compared with the number in the control group (54.0%). Addition of a high concentration of SDF1 alpha (500 ng/ml) into the progenitor cells with over-expression of CXCR4 reversed the cell proliferation back to the control levels (57.6%). Immunostaining analysis showed that neither over-expression nor inhibition of CXCR4 altered the population of neurons and astrocytes, when neural progenitor cells were cultured in differentiation medium. These in vitro results suggest that CXCR4/SDF1 alpha primarily regulates adult neural progenitor cell motility but not differentiation, while over-expression of CXCR4 in the absence of SDF1 alpha decreases neural progenitor cell proliferation.

The PI3K/Akt pathway mediates the neuroprotective effect of atorvastatin in extending thrombolytic therapy after embolic stroke in the rat.

OBJECTIVE: We tested the hypothesis that the phosphatidylinositol-3 kinase (PI3K)/Akt pathway mediates the neuroprotective effect of combination therapy of atorvastatin and tissue-type plasminogen activator (tPA) in rats after stroke. METHODS AND RESULTS: Combination of atorvastatin (20 mg/kg) and tPA (10 mg/kg) significantly reduced ischemic lesion volume, whereas monotherapy with atorvastatin and tPA did not reduce the lesion volume, when the treatments were initiated 4 hours after embolic middle cerebral artery occlusion (MCAo). Western blot analysis revealed that treatment with atorvastatin alone and in combination treatment with tPA significantly increased Akt phosphorylation compared with treatment with saline and tPA alone. Inhibition of the PI3K/Akt pathway with wortmannin completely abolished the reduction of lesion volume afforded by combination of atorvastatin and tPA. Real-time RT-PCR analysis of cerebral endothelial cells isolated by laser-capture microdissection from the ischemic boundary region showed that MCAo upregulated early growth response 1 (Egr-1) and vascular endothelial growth factor (VEGF) mRNA levels and tPA monotherapy further increased Egr-1 and VEGF mRNA levels. However, combination of atorvastatin and tPA significantly suppressed Egr-1 and VEGF mRNA levels in cerebral endothelial cells. CONCLUSIONS: Activation of Akt and downregulation of cerebral endothelial Egr-1 and VEGF gene expression by atorvastatin contribute to the neuroprotective effect of combination treatment with atorvastatin and tPA.

Matrix metalloproteinase 2 (MMP2) and MMP9 secreted by erythropoietin-activated endothelial cells promote neural progenitor cell migration.

We investigated the hypothesis that endothelial cells activated by erythropoietin (EPO) promote the migration of neuroblasts. This hypothesis is based on observations in vivo that treatment of focal cerebral ischemia with EPO enhances the migration of neuroblasts to the ischemic boundary, a site containing activated endothelial cells and angiogenic microvasculature. To model the microenvironment within the ischemic boundary zone, we used a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of the adult mouse. Treatment of MBECs with recombinant human EPO (rhEPO) significantly increased secretion of matrix metalloproteinase 2 (MMP2) and MMP9. rhEPO-treated MBEC supernatant as conditioned medium significantly increased the migration of neural progenitor cells. Application of an MMP inhibitor abolished the supernatant-enhanced migration. Incubation of neurospheres alone with rhEPO failed to increase progenitor cell migration. rhEPO activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2) in MBECs. Selective inhibition of the PI3K/Akt and ERK1/2 pathways significantly attenuated the rhEPO-induced MMP2 and MMP9, which suppressed neural progenitor cell migration promoted by the rhEPO-activated MBECs. Collectively, our data show that rhEPO-activated endothelial cells enhance neural progenitor cell migration by secreting MMP2 and MMP9 via the PI3K/Akt and ERK1/2 signaling pathways. These data demonstrate that activated endothelial cells can promote neural progenitor cell migration, and provide insight into the molecular mechanisms underlying the attraction of newly generated neurons to injured areas in brain.