Patterns and dynamics of subventricular zone neuroblast migration in the ischemic striatum of the adult mouse.

The migratory behavior of neuroblasts after a stroke is poorly understood. Using time-lapse microscopy, we imaged migration of neuroblasts and cerebral vessels in living brain slices of adult doublecortin (DCX, a marker of neuroblasts) enhanced green fluorescent protein (eGFP) transgenic mice that were subjected to 7 days of stroke. Our results show that neuroblasts originating in the subventricular zone (SVZ) of adult mouse brain laterally migrated in chains or individually to reach the ischemic striatum. The chains were initially formed at the border between the SVZ and the striatum by neuroblasts in the SVZ and then extended to the striatum. The average speed of DCX-eGFP-expressing cells within chains was 28.67+/-1.04 microm/h, which was significantly faster (P<0.01) than the speed of the cells in the SVZ (17.98+/-0.57 microm/h). Within the ischemic striatum, individual neuroblasts actively extended or retracted their processes, suggestive of probing the immediate microenvironment. The neuroblasts close to cerebral blood vessels exhibited multiple processes. Our data suggest that neuroblasts actively interact with the microenvironment to reach the ischemic striatum by multiple migratory routes.

Lengthening the G(1) phase of neural progenitor cells is concurrent with an increase of symmetric neuron generating division after stroke.

The proportion of neural progenitors that remain in (P fraction) and exit from (Q fraction) the cell cycle determines the degree of neurogenesis. Using S-phase labeling with 5-bromo-2'-deoxyuridine and a double nucleoside analog-labeling scheme, we measured the cell-cycle kinetics of neural progenitors and estimated the proportion of P and Q fractions in the subventricular zone (SVZ) of adult rats subjected to stroke. Stroke increased SVZ cell proliferation, starting 2 days, reaching a maximum 4 and 7 days after stroke. The cell-cycle length (T(C)) of SVZ cells changed dynamically over a period of 2 to 14 days after stroke, with the shortest length of 11 h at 2 days after stroke. The reduction of the T(C) resulted from a decrease of the G(1) phase because the G(2), M, and S phases were unchanged. In addition, during this period, reduction of the G(1) phase was concomitant with an increase in the P fraction, whereas an augmentation of the Q fraction was associated with lengthening of the G(1) phase. Furthermore, approximately 90% of cells that exited the cell cycle were neurons and the population of a pair of dividing daughter cells with a neuronal marker increased from 9% at 2 days to 26% at 14 days after stroke. These data suggest that stroke triggers early expansion of the progenitor pool via shortening the cell-cycle length and retaining daughter cells within the cell cycle, and the lengthening of G(1) leads to daughter cells exiting the cell cycle and differentiating into neurons.

The Sonic hedgehog pathway mediates carbamylated erythropoietin-enhanced proliferation and differentiation of adult neural progenitor cells.

Carbamylated erythropoietin (CEPO), a well characterized erythropoietin (EPO) derivative, does not bind to the classical EPO receptor and does not stimulate erythropoiesis. Using neural progenitor cells derived from the subventricular zone of the adult mouse, we investigated the effect of CEPO on neurogenesis and the associated signaling pathways in vitro. We found that CEPO significantly increased neural progenitor cell proliferation and promoted neural progenitor cell differentiation into neurons, which was associated with up-regulation of Sonic hedgehog (Shh), its receptor ptc, and mammalian achaete-scute homolog 1 (Mash1), a pro-neuron basic helix-loop-helix protein transcription factor. Blockage of the Shh signaling pathway with a pharmacological inhibitor, cyclopamine, abolished the CEPO-induced neurogenesis. Attenuation of endogenous Mash1 expression by short-interfering RNA blocked CEPO-promoted neuronal differentiation. In addition, recombinant mouse Shh up-regulated Mash1 expression in neural progenitor cells. These results demonstrate that the Shh signaling pathway mediates CEPO-enhanced neurogenesis and Mash1 is a downstream target of the Shh signaling pathway that regulates CEPO-enhanced neuronal differentiation.

Neuroblast division during migration toward the ischemic striatum: a study of dynamic migratory and proliferative characteristics of neuroblasts from the subventricular zone.

Ischemic stroke induces neurogenesis in the subventricular zone (SVZ), and newly generated neurons in the SVZ migrate toward the ischemic boundary. However, the characteristics of migrating SVZ cells have not been investigated after stroke. Using time-lapse imaging in both SVZ cells and organotypic brain slice cultures, we measured the dynamics of SVZ cell division and migration of adult rats subjected to stroke. In normal brain slices, SVZ cells primarily migrated dorsally and ventrally along the lateral ventricular surface. However, in stroke brain slices, SVZ cells migrated laterally toward the striatal ischemic boundary. Cultured stroke-derived SVZ cells exhibited a significant (p < 0.01) increase in the migration distance (212 +/- 21 microm) compared with the nonstroke-derived SVZ cells (97 +/- 12 microm). Migrating stroke-derived SVZ cells spent significantly (p = 0.01) less time in cytokinesis (0.63 +/- 0.04 h) compared with the time (1.09 +/- 0.09 h) for nonstroke-derived SVZ cells. Newborn cells with a single leading process exhibited fast migration (7.2 +/- 0.8 microm/h), and cells with multiple processes showed stationary migration (3.6 +/- 0.8 microm/h). Stroke SVZ daughter cells further divided during their migration. The morphology of doublecortin (DCX)-positive cells in fixed brain sections resembled those observed in cultured newborn cells, and the DCX-positive cells proliferated in the ischemic striatum. Collectively, the present study suggests that stroke promotes cytokinesis of migrating neuroblasts, and these cells migrate toward the ischemic striatum with distinct migratory behaviors and retain the capacity for cell division during migration.

Stroke induces ependymal cell transformation into radial glia in the subventricular zone of the adult rodent brain.

Adult ependymal cells are postmitotic and highly differentiated. Radial glial cells are neurogenic precursors. Here, we show that stroke acutely stimulated adult ependymal cell proliferation, and dividing ependymal cells of the lateral ventricle had genotype, phenotype, and morphology of radial glial cells in the rat. The majority of radial glial cells exhibited symmetrical division about the cell cleavage plane, and a radial fiber was maintained throughout each stage of cell mitosis. Increases of radial glial cells parallel expansion of neural progenitors in the subventricular zone (SVZ). Furthermore, after stroke radial glial cells derived from the SVZ supported neuron migration. These results indicate that adult ependymal cells divide and transform into radial glial cells after stroke, which could function as neural progenitor cells to generate new neurons and act as scaffolds to support neuroblast migration towards the ischemic boundary region.

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.

Neurogenin 1 mediates erythropoietin enhanced differentiation of adult neural progenitor cells.

Proneuronal basic helix-loop-helix (bHLH) transcription factor, neurogenin 1 (Ngn1), regulates neuronal differentiation during development of the cerebral cortex. Akt mediates proneuronal bHLH protein function to promote neuronal differentiation. Here, we show that recombinant human erythropoietin (rhEPO) significantly increased Akt activity and Ngn1 mRNA levels in neural progenitor cells derived from the subventricular zone (SVZ) of adult rat, which was coincident with increases of neural progenitor cell proliferation, differentiation, and neurite outgrowth. Inhibition of Akt activity by the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) inhibitor, LY294002, abolished rhEPO-increased Ngn1 mRNA levels and the effects of rhEPO on neural progenitor cells. In addition, reducing expression of endogenous Ngn1 by means of short-interfering RNA (siRNA) blocked rhEPO-enhanced neuronal differentiation and neurite outgrowth but not rhEPO-increased proliferation. Furthermore, treatment of stroke rat with rhEPO significantly increased Ngn1 mRNA levels in SVZ cells. These data suggest that rhEPO acts as an extracellular molecule that activates the PI3K/Akt pathway, which enhances adult neural progenitor cell proliferation, differentiation, and neurite outgrowth, and Ngn1 is required for Akt-mediated neuronal differentiation and neurite outgrowth.