Jagged1 signals in the postnatal subventricular zone are required for neural stem cell self-renewal

Neural stem cells (NSCs) in the postnatal mammalian brain self-renew and are a source of neurons and glia. To date, little is known about the molecular and cellular mechanisms regulating the maintenance and differentiation of these multipotent progenitors. We show that Jagged1 is required by mitotic cells in the subventricular zone (SVZ) and stimulates self-renewal of multipotent epidermal growth factor-dependent NSCs. Jagged1-expressing cells line the adult SVZ and are juxtaposed to Notch1-expressing cells, some of which are putative NSCs. In vitro, endogenous Jagged1 acts through Notch1 to promote NSC maintenance and multipotency. In vivo, reducing Jagged1/Notch1 signaling decreases the number of proliferating cells in the SVZ. In addition, soluble Jagged1 promotes self-renewal and neurogenic potential of multipotent neural progenitors in vitro. Our findings suggest a central role for Jagged1 in the NSC niche in the SVZ for maintaining a population of NSCs in the postnatal brain.     

The HDAC inhibitor, sodium butyrate, stimulates neurogenesis in the ischemic brain

In the healthy adult brain, neurogenesis normally occurs in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Cerebral ischemia enhances neurogenesis in neurogenic and non-neurogenic regions of the ischemic brain of adult rodents. This study demonstrated that post-insult treatment with a histone deacetylase inhibitor, sodium butyrate (SB), stimulated the incorporation of bromo-2'-deoxyuridine (BrdU) in the SVZ, DG, striatum, and frontal cortex in the ischemic brain of rats subjected to permanent cerebral ischemia. SB treatment also increased the number of cells expressing polysialic acid–neural cell adhesion molecule, nestin, glial fibrillary acidic protein, phospho-cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in various brain regions after cerebral ischemia. Furthermore, extensive co-localization of BrdU and polysialic acid–neural cell adhesion molecule was observed in multiple regions after ischemia, and SB treatment up-regulated protein levels of BDNF, phospho-CREB, and glial fibrillary acidic protein. Intraventricular injection of K252a, a tyrosine kinase B receptor antagonist, markedly reduced SB-induced cell proliferation detected by BrdU and Ki67 in the ipsilateral SVZ, DG, and other brain regions, blocked SB-induced nestin expression and CREB activation, and attenuated the long-lasting behavioral benefits of SB. Together, these results suggest that histone deacetylase inhibitor-induced cell proliferation, migration and differentiation require BDNF–tyrosine kinase B signaling and may contribute to long-term beneficial effects of SB after ischemic injury.     

Notch1 and its ligands Delta-like and Jagged are expressed and active in distinct cell populations in the postnatal mouse brain

Notch signaling plays a pivotal role in the regulation of vertebrate neurogenesis. However, in vitro experiments suggest that Notch1 may also be involved in the regulation of later stages of brain development. We have addressed putative roles in the central nervous system by examining the expression of Notch signaling cascade components in the postnatal mouse brain. In situ mRNA hybridization revealed that Notch1 is associated with cells in the subventricular zone, the dentate gyrus and the rostromigratory stream, all regions of continued neurogenesis in the postnatal brain. In addition, Notch1 is expressed at low levels throughout the cortex and olfactory bulb and shows striking expression in the cerebellar Purkinje cell layer. The Notch ligands, including Delta-like1 and 3 and Jagged1 and Jagged2, show distinct expression patterns in the developing and adult brain overlapping that of Notch1. In addition, the downstream targets of the Notch signaling cascade Hes1, Hes3, Hes5 and the intrinsic Notch regulatory proteins Numb and Numblike also show active signaling in distinct brain regions. Hes5 coincides with the majority of Notch1 expression and can be detected in the cerebral cortex, cerebellum and putative germinal zones. Hes3, on the other hand, shows a restricted expression in cerebellar Purkinje cells. The distribution of Notch1 and its putative ligands suggest distinct roles in specific subsets of cells in the postnatal brain including putative stem cells and differentiated neurons.     

Brain ischemia, neurogenesis, and neurotrophic receptor expression in primates

Generation of new neurons persists in the normal adult mammalian brain, with neural stem/progenitor cells residing in at least two brain regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Adult neurogenesis is well documented in the rodent, and has also been demonstrated in vivo in nonhuman primates and humans. Brain injuries such as ischemia affect neurogenesis in adult rodents as both global and focal ischemic insults enhance the proliferation of progenitor cells residing in SGZ or SVZ. We addressed the issue whether an injury triggered activation of endogenous neuronal precursors also takes place in the adult primate brain. We found that the ischemic insult increased the number of progenitor cells in monkey SGZ and SVZ, and caused gliogenesis in the ischemia-prone hippocampal CA1 sector. To better understand the mechanisms regulating precursor cell division and differentiation in the primate, we analyzed the expression at protein level of a panel of potential regulatory molecules, including neurotrophic factors and their receptors. We found that a fraction of mitotic progenitors were positive for the neurotrophin receptor TrkB, while immature neurons expressed the neurotrophin receptor TrkA. Astroglia, ependymal cells and blood vessels in SVZ were positive for distinctive sets of ligands/receptors, which we characterized. Thus, a network of neurotrophic signals operating in an autocrine or paracrine manner may regulate neurogenesis in adult primate SVZ. We also analyzed microglial and astroglial proliferation in postischemic hippocampal CA1 sector. We found that proliferating postischemic microglia in adult monkey CA1 sector express the neurotrophin receptor TrkA, while activated astrocytes were labeled for nerve growth factor (NGF), ligand for TrkA, and the tyrosine kinase TrkB, a receptor for brain derived neurotrophic factor (BDNF). These results implicate NGF and BDNF as regulators of postischemic glial proliferation in adult primate hippocampus.     

Conditional depletion of neurogenesis inhibits long-term recovery after experimental stroke in mice

We reported previously that ablation of doublecortin (DCX)-immunopositive newborn neurons in mice worsens anatomical and functional outcome measured 1 day after experimental stroke, but whether this effect persists is unknown. We generated transgenic mice that express herpes simplex virus thymidine kinase under control of the DCX promoter (DCX-TK transgenic mice). DCX-expressing and recently divided cells in the rostral subventricular zone (SVZ) and hippocampus of DCX-TK transgenic mice, but not wild-type mice, were specifically depleted after ganciclovir (GCV) treatment for 14 days. Focal cerebral ischemia was induced by permanent distal middle cerebral artery occlusion (MCAO) on day 14 of vehicle or GCV treatment, and mice were killed 12 weeks after MCAO. Infarct volume was significantly increased and neurologic deficits were more severe in GCV- compared to vehicle-treated DCX-TK transgenic mice at first 8 weeks, after depletion of DCX- and bromodeoxyuridine-immunoreactive cells in the SVZ and dentate gyrus following focal ischemia. Our results indicate that endogenous neurogenesis in a critical period following experimental stroke influences the course of long-term recovery.     

Ischemia-induced neurogenesis is preserved but reduced in the aged rodent brain

The adult mammalian brain retains the capacity for neurogenesis, by which new neurons may be generated to replace those lost through physiological or pathological processes. However, neurogenesis diminishes with aging, and this casts doubt on its feasibility as a therapeutic target for cell replacement therapy in stroke and neurodegenerative disorders, which disproportionately affect the aged brain. In previous studies, neurogenesis was stimulated by cerebral ischemia in young rodents, and the neurogenesis response of the aged rodent brain to physiological stimuli, such as hormonal manipulation and growth factors, was preserved. To investigate the effect of aging on ischemia-induced neurogenesis, transient (60 min) middle cerebral artery occlusion was induced in young adult (3-month) and aged (24-month) rats, who were also given bromodeoxyuridine to label newborn cells. As found in prior studies, basal neurogenesis in control, nonischemic rats was reduced with aging. Ischemia failed to stimulate neurogenesis in the dentate gyrus (DG) subgranular zone (SGZ), in contrast to results obtained previously after more prolonged (90-120 min) middle cerebral artery occlusion, but increased the number of BrdU-labeled cells in the forebrain subventricular zone (SVZ). This effect was less prominent in aged than in young adult rats, with fold-stimulation of BrdU incorporation reduced by approximately 20% and the total number of cells generated diminished by approximately 50%. BrdU-labeled cells in SVZ coexpressed neuronal lineage markers, consistent with newborn neurons. We conclude that ischemia-induced neurogenesis occurs in the aged brain, and that measures designed to augment this phenomenon might have therapeutic applications.     

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.     

Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.     

Sustained up-regulation of semaphorin 3A, Neuropilin1, and doublecortin expression in ischemic mouse brain during long-term recovery

Strategies to provide neuroprotection and to promote regenerative axonal outgrowth in the injured brain are thwarted by the plethora of axon growth inhibitors and the ligand promiscuity of some of their receptors. Especially, new neurons derived from ischemia-stimulated neurogenesis must integrate this multitude of inhibitory molecular cues, generated as a result of cortical damage, into a functional response. More often than not the response is one of growth cone collapse, axonal retraction and neuronal death. Therefore, characterization of the expression of inhibitory molecules in long-term surviving ischemic brains following stroke is important for designing selective therapeutics. Here, we describe a long-term recovery mouse model for cerebral ischemia in which a brief transient occlusion of the middle cerebral artery (30 min) was followed by up to 30 days of long-term reperfusion. Significantly decreased grip strength motor function and increased expression of one of the major repulsive guidance cues, Semaphorin 3A (Sema3A) and its receptor Neuropilin1 (NRP1) occurred in brains of these mice. Interestingly, increased Doublecortin (DCX) expression occurred only in the lateral ventricular wall zone, but not in the dentate gyrus granule cell layer on the ischemic side of the brain. Importantly, no DCX positive cells were detected in the infarct core region after 30 d ischemic recovery. Collectively, these studies demonstrated the sustained elevation of Sema3A/NRP1 expression in the ischemic territory, which may contribute to the inhibitory microenvironment responsible for preventing new neurons from entering the infarct area. This model will be of use as a platform for testing anti-inhibitory therapies to stroke.     

Bcl-2 increases stroke-induced striatal neurogenesis in adult brains by inhibiting BMP-4 function via activation of β-catenin signaling

Our previous experiments suggest that treatment with Bcl-2 increases proliferation and differentiation of neuronal progenitors induced by ischemic injury and ameliorates neurological functional deficits after stroke. However, in addition to its traditional anti-apoptotic effect, little is known about the concrete molecular modulation mechanism. In this study, Bcl-2-expressing plasmids were injected into the lateral ventricle of rat brains immediately following a 30-min occlusion of the middle cerebral artery to determine the role of Bcl-2 in adult neurogenesis. Bcl-2 overexpression reduced ischemic infarct and astrogenesis, and enhanced ischemia-induced striatal neurogenesis. We further found that Bcl-2 increased β-catenin, a key mediator of canonical Wnt/β-catenin signaling pathway, and reduced bone morphogenetic proteins-4 (BMP-4) expression in the ipsilateral striatum following ischemia. Treatment of stroke with β-catenin siRNA (i.c.v.) showed that β-catenin siRNA antagonized Bcl-2 neuroprotection against ischemic brain injury. More interestingly, β-catenin siRNA simultaneously abolished Bcl-2-mediated reduction of BMP-4 expression and enhancement of neurogenesis in the ipsilateral striatum. This effect is independent of Noggin, the known BMP antagonist. These findings highlight a new regulatory mechanism that Bcl-2 elevates ischemia-induced striatal neurogenesis by down-regulating expression of BMP-4 via activation of the Wnt/β-catenin signaling pathway in adult rat brains.     

Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke

Mice transgenic for antisense Notch and normal mice treated with inhibitors of the Notch-activating enzyme gamma-secretase showed reduced damage to brain cells and improved functional outcome in a model of focal ischemic stroke. Notch endangers neurons by modulating pathways that increase their vulnerability to apoptosis, and by activating microglial cells and stimulating the infiltration of proinflammatory leukocytes. These findings suggest that Notch signaling may be a therapeutic target for treatment of stroke and related neurodegenerative conditions.     

局灶缺血性脑卒中大鼠侧脑室下带Shh信号通路成分的动态表达

通过研究Sonic hedgehog(Shh)信号通路成分在局灶缺血性脑卒中大鼠侧脑室下带(subventricular zone,SVZ)的动态表达,初步探讨该通路在局灶性缺血性脑卒中后神经再生的调控作用.将84只健康成年雄性SD大鼠随机分为正常组(n=12)、假手术组(n=12)、缺血6、12、24 h和3、7 d,共7组(n=12).采用线栓法制备大鼠右侧大脑中动脉阻断(middle cerebral artery occlussion,MCAO)模型.分别应用逆转录聚合酶链反应(RT-PCR)、免疫组化、免疫印迹法检测局灶脑缺血大鼠侧脑室下带Shh、Gli1 mRNA和蛋白变化.与正常组比较,Shh、Gli1mRNA和蛋白在假手术组表达变化不明显(P>0.05),模型组6 h表达增高(P<0.01),24 h达峰值(P<0.01),3 d时接近正常水平(P>0.05),7 d表达又升高(P<0.01).缺血性脑卒中可以上调Shh信号通路成分在SVZ区的表达,提示Shh信号通路可能参与卒中后神经再生机制的调控.     

miR-210 activates notch signaling pathway in angiogenesis induced by cerebral ischemia

The compensatory angiogenesis that occurs after cerebral ischemia increases blood flow to the injured area and limits extension of the ischemic penumbra. In this way, it improves the local blood supply. Fostering compensatory angiogenesis is an effective treatment for ischemic cerebrovascular disease. However, angiogenesis in the adult organism is a complex, multi-step process, and the mechanisms underlying the regulation of angiogenesis are not well understood. Although Notch signaling reportedly regulates the vascularization process that occurs in ischemic tissues, little is known about the role of Notch signaling in the regulation of ischemia-induced angiogenesis after ischemic stroke. Recent research has indicated that miR-210, a hypoxia-induced microRNA, plays a crucial role in regulating the biological processes that occur in blood vessel endothelial cells under hypoxic conditions. This study was undertaken to investigate the role of miR-210 in regulating angiogenesis in response to brain ischemia injury and the role of the Notch pathway in the body’s response. We found miR-210 to be significantly up-regulated in adult rat ischemic brain cortexes in which the expression of Notch1 signaling molecules was also increased. Hypoxic models of human umbilical vein endothelial cells (HUVE-12) were used to assess changes in miR-210 and Notch1 expression in endothelial cells. Results were consistent with in vivo findings. To determine the molecular mechanisms behind these phenomena, we transfected HUVE-12 cells with miR-210 recombinant lentiviral vectors. We found that miR-210 overexpression caused up-regulation of Notch1 signaling molecules and induced endothelial cells to migrate and form capillary-like structures on Matrigel. These data suggest that miR-210 is involved in the regulation of angiogenesis in response to ischemic injury to the brain. Up-regulation of miR-210 can activate the Notch signaling pathway, which may contribute to angiogenesis after cerebral ischemia.     

Expression of Notch1 and Jagged2 in the enteric nervous system.

The Notch signaling pathway is a vitally important pathway in regulating brain development. To explore the involvement of the Notch pathway in neuronal cells of adult rat gut, we investigated the expression of Notch1 and Jagged2 by in situ hybridization (ISH) and immunohistochemistry (IHC). In the enteric nervous system, Notch1 and Jagged2 were expressed in ganglia of the submucosal and myenteric plexus. Notch1 was preferentially expressed in cholinergic neurons lacking calretinin or nitric oxide synthase (NOS), whereas Jagged2 was present in most neuron subtypes. We propose that Notch1 and Jagged2 have a continuing role in the maintenance and function of neuronal cells in the adult enteric nervous system.     

Mutant α-synuclein and aging reduce neurogenesis in the acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease

Neurogenesis, the production of new neurons from less differentiated precursor cells, normally occurs in adult brains in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Neurogenesis declines with aging. In previous studies, neurogenesis was stimulated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP) in young animals. In this study, we examined the effect of acute MPTP administration and mutant α-synuclein A53T on neurogenesis and migration of newborn neurons in the aged (23-month) vs. young (2-month) rodent brain. Cell proliferation and neurogenesis were assessed via bromodeoxyuridine labeling and immunostaining for cell type-specific markers. In the aged brain, neural precursor cells in the rostral SVZ retained the capacity for proliferation and migration in response to MPTP-induced Parkinsonism, although the response is less robust than in younger animals. Furthermore, in transgenic mice that overexpress mutant α-synuclein (A53T), brains examined day 21 after MPTP administration showed markedly decreased olfactory bulb and substantia nigra neurogenesis. Our data suggest that in addition to aging effects associated with decline in the number of newly generated cells, mutant α-synuclein reduces MPTP-induced neurogenesis. This could provide a novel therapeutic target for chronic brain repair in this condition.     

Enhanced neurogenesis and cell migration following focal ischemia and peripheral stimulation in mice

Peripheral stimulation and physical therapy can promote neurovascular plasticity and functional recovery after CNS disorders such as ischemic stroke. Using a rodent model of whisker-barrel cortex stroke, we have previously demonstrated that whisker activity promotes angiogenesis in the penumbra of the ischemic barrel cortex. This study explored the potential of increased peripheral activity to promote neurogenesis and neural progenitor migration toward the ischemic barrel cortex. Three days after focal barrel cortex ischemia in adult mice, whiskers were manually stimulated (15 min x 3 times/day) to enhance afferent signals to the ischemic barrel cortex. 5-Bromo-2'-deoxyuridine (BrdU, i.p.) was administered once daily to label newborn cells. At 14 days after stroke, whisker stimulation significantly increased vascular endothelial growth factor and stromal-derived factor-1 expression in the penumbra. The whisker stimulation animals showed increased doublecortin (DCX) positive and DCX/BrdU-positive cells in the ipsilateral corpus of the white matter but no increase in BrdU-positive cells in the subventricular zone, suggesting a selective effect on neuroblast migration. Neurogenesis indicated by neuronal nuclear protein and BrdU double staining was also enhanced by whisker stimulation in the penumbra at 30 days after stroke. Local cerebral blood flow was better recovered in mice that received whisker stimulation. It is suggested that the enriched microenvironment created by specific peripheral stimulation increases regenerative responses in the postischemic brain and may benefit long-term functional recovery from ischemic stroke.     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Low Density Lipoprotein Receptor-related Protein-1 (LRP1) Regulates Thrombospondin-2 (TSP2) Enhancement of Notch3 Signaling

Intracellular trafficking of Notch and Notch ligands modulates signaling, suggesting that choreography of ligand and receptor translocation is essential for optimal Notch activity. Indeed, a major model for Notch signaling posits that Notch trans-endocytosis into the ligand-expressing (signal sending) cell is a key driving force for Notch signal transduction. The extracellular protein thrombospondin-2 (TSP2) enhances Notch signaling and binds to both Jagged1 and Notch3 ectodomains, potentially bridging two essential extracellular components of Notch signaling. We investigated the role of low density lipoprotein receptor-related protein-1 (LRP1), a TSP2 receptor, in the regulation of Notch3 signaling. TSP2 potentiation of Notch is blocked by the receptor-associated protein (an inhibitor of low density lipoprotein receptor-related protein function) and requires LRP1 expression in the signal-sending cell. TSP2 stimulates Notch3 endocytosis into wild type fibroblasts but not LRP1-deficient fibroblasts. Finally, recombinant Notch3 and Jagged1 interact with the LRP1 85-kDa B-chain, a subunit that lacks known ligand binding function. Our data suggest that LRP1 and TSP2 stimulate Notch activity by driving trans-endocytosis of the Notch ectodomain into the signal-sending cell and demonstrate a novel, non-cell autonomous function of LRP1 in cell-cell signaling.