Characterization of TLX Expression in Neural Stem Cells and Progenitor Cells in Adult Brains

TLX has been shown to play an important role in regulating the self-renewal and proliferation of neural stem cells in adult brains. However, the cellular distribution of endogenous TLX protein in adult brains remains to be elucidated. In this study, we used immunostaining with a TLX-specific antibody to show that TLX is expressed in both neural stem cells and transit-amplifying neural progenitor cells in the subventricular zone (SVZ) of adult mouse brains. Then, using a double thymidine analog labeling approach, we showed that almost all of the self-renewing neural stem cells expressed TLX. Interestingly, most of the TLX-positive cells in the SVZ represented the thymidine analog-negative, relatively quiescent neural stem cell population. Using cell type markers and short-term BrdU labeling, we demonstrated that TLX was also expressed in the Mash1+ rapidly dividing type C cells. Furthermore, loss of TLX expression dramatically reduced BrdU label-retaining neural stem cells and the actively dividing neural progenitor cells in the SVZ, but substantially increased GFAP staining and extended GFAP processes. These results suggest that TLX is essential to maintain the self-renewing neural stem cells in the SVZ and that the GFAP+ cells in the SVZ lose neural stem cell property upon loss of TLX expression.Understanding the cellular distribution of TLX and its function in specific cell types may provide insights into the development of therapeutic tools for neurodegenerative diseases by targeting TLX in neural stem/progenitors cells.     

Stroke Increases Neural Stem Cells and Angiogenesis in the Neurogenic Niche of the Adult Mouse

The unique cellular and vascular architecture of the adult ventricular-subventricular zone (V/SVZ) neurogenic niche plays an important role in regulating neural stem cell function. However, the in vivo identification of neural stem cells and their relationship to blood vessels within this niche in response to stroke remain largely unknown. Using whole-mount preparation of the lateral ventricle wall, we examined the architecture of neural stem cells and blood vessels in the V/SVZ of adult mouse over the course of 3 months after onset of focal cerebral ischemia. Stroke substantially increased the number of glial fibrillary acidic protein (GFAP) positive neural stem cells that are in contact with the cerebrospinal fluid (CSF) via their apical processes at the center of pinwheel structures formed by ependymal cells residing in the lateral ventricle. Long basal processes of these cells extended to blood vessels beneath the ependymal layer. Moreover, stroke increased V/SVZ endothelial cell proliferation from 2% in non-ischemic mice to 12 and 15% at 7 and 14 days after stroke, respectively. Vascular volume in the V/SVZ was augmented from 3% of the total volume prior to stroke to 6% at 90 days after stroke. Stroke-increased angiogenesis was closely associated with neuroblasts that expanded to nearly encompass the entire lateral ventricular wall in the V/SVZ. These data indicate that stroke induces long-term alterations of the neural stem cell and vascular architecture of the adult V/SVZ neurogenic niche. These post-stroke structural changes may provide insight into neural stem cell mediation of stroke-induced neurogenesis through the interaction of neural stem cells with proteins in the CSF and their sub-ependymal neurovascular interaction.     

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.     

Phosphatidylglucoside: a novel marker for adult neural stem cells

We investigated the expression of a novel glycophospholipid, phosphatidylglucoside (PtdGlc), in adult mouse brains. Immunohistochemical analysis with DIM21 antibody, a monoclonal anti-PtdGlc antibody, revealed robust PtdGlc staining in the two primary neurogenic regions of the adult rodent brain, the subventricular zone (SVZ) lining the lateral ventricle and the subgranular zone of the dentate gyrus. Intriguingly, the staining pattern of PtdGlc appeared to overlap that of glial fibrillary acidic protein, an adult neural stem cell marker in these regions. Further immunohistochemical analysis revealed that PtdGlc expression on the cell membranes of adult SVZ neural stem cells significantly overlapped with other proposed adult neural stem cell markers. Moreover, PtdGlc(+) cells isolated from adult mouse SVZs by fluorescence-activated cell sorting with anti-PtdGlc antibody efficiently generated neurospheres in cell culture. These cells differentiated into neurons, astrocytes, and oligodendrocytes in vitro, directly demonstrating that PtdGlc-expressing cells possessed multipotency. Our data suggest that PtdGlc could be a useful adult stem cell marker.     

Nuclear receptor TLX regulates cell cycle progression in neural stem cells of the developing brain

TLX is an orphan nuclear receptor that is expressed exclusively in vertebrate forebrains. Although TLX is known to be expressed in embryonic brains, the mechanism by which it influences neural development remains largely unknown. We show here that TLX is expressed specifically in periventricular neural stem cells in embryonic brains. Significant thinning of neocortex was observed in embryonic d 14.5 TLX-null brains with reduced nestin labeling and decreased cell proliferation in the germinal zone. Cell cycle analysis revealed both prolonged cell cycles and increased cell cycle exit in TLX-null embryonic brains. Increased expression of a cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin D1 provide a molecular basis for the deficiency of cell cycle progression in embryonic brains of TLX-null mice. Furthermore, transient knockdown of TLX by in utero electroporation led to precocious cell cycle exit and differentiation of neural stem cells followed by outward migration. Together these results indicate that TLX plays an important role in neural development by regulating cell cycle progression and exit of neural stem cells in the developing brain.     

惊厥后大鼠海马神经再生与凋亡的动态变化

探讨惊厥持续状态(status convulsion,SC)后大鼠海马神经再生与凋亡的动态变化。建立成年Wistar鼠30minSC模型,在SC后1天至56天的6个时间点上处死动物,处死前1天均腹腔注射5-溴2-脱氧尿嘧啶核苷(5-bromo-2-deoxyuridine,BrdU);采用免疫组织化学方法动态检测BrdU、nestin的表达,确定神经干细胞增殖水平;双重荧光染色标记nestin/TUNEL,确定新生神经干细胞存活时间。与对照组相比,BrdU阳性细胞数目于SC后第7天在CA1区达增殖高峰,28天降至正常水平;于SC后第28天在齿状回达增殖高峰,56天降至正常水平;在SC后第7天,CA3区有大量的BrdU阳性细胞;BrdU和nestin阳性细胞数目无统计学差异。在SC后的前3天,CA1区新增殖的神经细胞呈TUNEL阳性;齿状回新增殖细胞始终表现TUNEL阴性。上述结果提示:SC后能激活自体神经干细胞原位增殖,并且部分新生细胞向损伤区域迁移。     

Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.     

Development of Circadian Oscillators in Neurosphere Cultures during Adult Neurogenesis

Circadian rhythms are common in many cell types but are reported to be lacking in embryonic stem cells. Recent studies have described possible interactions between the molecular mechanism of circadian clocks and the signaling pathways that regulate stem cell differentiation. Circadian rhythms have not been examined well in neural stem cells and progenitor cells that produce new neurons and glial cells during adult neurogenesis. To evaluate circadian timing abilities of cells undergoing neural differentiation, neurospheres were prepared from the mouse subventricular zone (SVZ), a rich source of adult neural stem cells. Circadian rhythms in mPer1 gene expression were recorded in individual spheres, and cell types were characterized by confocal immunofluorescence microscopy at early and late developmental stages in vitro. Circadian rhythms were observed in neurospheres induced to differentiate into neurons or glia, and rhythms emerged within 3–4 days as differentiation proceeded, suggesting that the neural stem cell state suppresses the functioning of the circadian clock. Evidence was also provided that neural stem progenitor cells derived from the SVZ of adult mice are self-sufficient clock cells capable of producing a circadian rhythm without input from known circadian pacemakers of the organism. Expression of mPer1 occurred in high frequency oscillations before circadian rhythms were detected, which may represent a role for this circadian clock gene in the fast cycling of gene expression responsible for early cell differentiation.     

Additive or Synergistic Effects of Aluminum on the Reduction of Neural Stem Cells, Cell Proliferation, and Neuroblast Differentiation in the Dentate Gyrus of High-Fat Diet-Fed Mice

Aluminum is the most plentiful metal on the Earth’s crust, and its usage in cooking utensils, cosmetics, drinking containers, food additives, pharmaceutical products, and building materials provides many opportunities for potential aluminum consumption. However, its toxicity is low and harmful effects only develop with large-scale deposition of aluminum. In this study, we investigated the effects of subchronic exposure to aluminum (40 mg/kg/day) on neural stem cells, cell proliferation, neuroblast differentiation, and mature neurons in the dentate gyrus of the hippocampus. These experiments were performed in both high-fat diet and low-fat diet-fed C57BL/6J mice via immunohistochemistry using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN. Subchronic exposure to aluminum in both low-fat and high-fat diet-fed mice reduced neural stem cells, cell proliferation, and neuroblast differentiation without any changes in mature neurons. Furthermore, this reduction effect was exacerbated in high-fat diet-fed mice. These results suggest that aluminum accelerates the reduction of neural stem cells, cell proliferation, and neuroblast differentiation additively or synergistically in high-fat diet-fed mice without any harmful changes in mature neurons.     

Bringing target-matched PI3King from the bench to the clinic

Caveolin-1 (Cav-1) is a protein marker for caveolae organelles, and acts as a scaffolding protein to negatively regulate the activity of signaling molecules by binding to and releasing them in a timely fashion. We have previously shown that loss of Cav-1 promotes the proliferation of mouse embryo fibroblasts (MEFs) in vitro. Here, to investigate the in vivo relevance of these findings, we evaluated the turnover rates of small intestine crypt stem cells from WT and Cav-1 deficient mice. Interestingly, we show that Cav-1 null crypt stem cells display higher proliferation rates, as judged by BrdU and PCNA staining. In addition, we show that Wnt/?-catenin signaling, which normally controls intestinal stem cell self-renewal, is up-regulated in Cav-1 deficient crypt stem cells. Because the small intestine constitutes one of the main targets of radiation, we next evaluated the role of Cav-1 in radiation-induced damage. Interestingly, after exposure to 15 Gy of ?-radiation, Cav-1 deficient mice displayed a decreased survival rate, as compared to WT mice. Our results show that after radiation treatment, Cav-1 null crypt stem cells of the small intestine exhibit far more apoptosis and accelerated proliferation, leading to a faster depletion of crypts and villi. As a consequence, six days after radiation treatment, Cav-1 -/- mice lost all their crypt and villus structures, while WT mice still showed some crypts and intact villi. In summary, we show that ablation of Cav-1 gene expression induces an abnormal amplification of crypt stem cells, resulting in increased susceptibility to ?-radiation. Thus, our studies provide the first evidence that Cav-1 normally regulates the proliferation of intestinal stem cells in vivo.     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

Exendin-4对成年小鼠脑室下区神经干细胞分化作用的体外研究*

目的:研究艾塞那肽(Ex-4)对成年小鼠脑室下区(SVZ)神经干细胞(NSCs)分化的影响及机制。方法:提取5周龄C57BL/6J小鼠SVZ的NSCs,100 nmol/L Ex-4处理分化14 d观察细胞形态,用免疫荧光检测巢蛋白(nestin)和胰高糖素样肽-1受体(GLP-1R)的表达。用shRNA敲低GLP-1R,将研究分为四组:对照组,Ex-4组,GLP-1R敲低组,GLP-1R敲低+Ex-4组。100 nmol/L Ex-4处理14 d后免疫荧光标记β-微管蛋白3(β-tublin III)和胶质纤维酸性蛋白(GFAP)并统计β-tublin III阳性细胞比例,Western blot检测环磷腺苷效应元件结合蛋白(CREB)的活化。为进一步研究Ex-4对MAPK和PI3K通路的影响,分别以丝裂原活化蛋白激酶(MAPK)抑制剂U0126 0.07 μmol/L预处理细胞30 min、磷脂酰肌醇-3激酶(PI3K)抑制剂LY294002 50 μmol/预处理细胞2 h,将研究分为: 对照组,Ex-4组,U0126组,U0126+Ex-4组,LY294002组,LY294002+Ex-4组,Western blot检测各组CREB的活化,各组实验独立重复三次。结果:成功从C57BL/6J小鼠SVZ提取NSCs,免疫荧光提示NSCs中nestin以及GLP-1R阳性。相对于对照组,Ex-4组分化为神经元的比例更高。GLP-1R敲低+Ex-4组中神经元比例与对照组基本一致(P＜0.01),β-tublin III阳性的细胞显示出GLP-1R以及CREB活化阳性。Western blot显示Ex-4组中CREB显著活化,GLP-1R敲低+Ex-4组的CERB活化与对照组基本一致(P＜0.01)。U0126+Ex-4组与Ex-4组CERB活化水平一致,LY294002+Ex-4组与对照组CERB活化水平一致(P＜0.01)。 结论:Ex-4通过GLP-1R受体促进成年小鼠SVZ中NSCs分化为神经元,这一作用可能通过PI3K/ CREB通路来实现。     

Caveolin-1-Deficient Mice Have An Increased Mammary Stem Cell Population with Upregulation of Wnt/?-Catenin Signaling

Here, we show that a caveolin-1 (Cav-1) deficiency leads to an amplification of the adult mammary stem cell population, both in vivo and in vitro. First, the expression of two stem cell markers, Sca-1 and Keratin 6, is dramatically increased in the hyperplastic mammary ducts of Cav-1 deficient mice, suggesting that loss of Cav-1 induces the accumulation of a progenitor cell population in the mammary gland. To independently validate these results, we reconstituted mammary acini formation in vitro via a 3D Matrigel assay system--using primary cultures of mammary epithelial cells derived from WT and Cav-1 deficient mice. We show that Cav-1 null 3D epithelial structures display an intense increase in the expression of three stem cell markers, i.e., Sca-1, keratin 6 and keratin 5. Overall, we observed a 2-to-3 fold increase in the number of Cav-1 KO acini that are positive for a given stem cell marker. Also, we show that such amplification of progenitor cells has functional consequences, as demonstrated by the abnormal presence of myoepithelial cells in the hyperplastic lesions of Cav-1 deficient mammary glands. Finally, we provide evidence that hyper-activation of Wnt/?-catenin signaling may constitute one of the down-stream mechanisms leading to mammary stem cell accumulation. The longevity and slow-dividing properties of mammary stem cells facilitates the accumulation of genetic alterations, and renders these progenitor cells the likely precursors of malignant derivatives. As such, we propose that loss of Cav-1 induces the accumulation of mammary stem cells, and that this event may be an initiating factor during mammary tumorigenesis.     

p75 NTR -Immunoreactivity in the subventricular zone of adult male rats: Expression by cycling cells

While the study of in vitro regulation of neural stem cell lineage from both embryonic and adult neurospheres is greatly advanced, much less is known about factors acting in situ for neural stem cell lineage in adult brain. We reported that neurotrophin low affinity receptor p75^NTR is present in the subventricular zone (SVZ) in adult male rats. We then characterized co-distribution of markers associated with precursor cells (nestin and PSA-NCAM) with growth factor receptors (p75^NTR, trkA, EGFr) and proliferation-associated antigens (Ki67 and BrDU-uptake) in adult male rat by immunocytochemistry and confocal laser scan microscopy. Distribution of p75^NTR-immunoreactivity (IR) was investigated using different mono- and polyclonal antisera. p75^NTR– is not co-distributed with glial fibrillary acid protein. It was found to be co-distributed with a small number of nestin-IR cells, whereas no coexistence with PSA-NCAM-IR was observed. Conversely, p75^NTR-IR was present in numerous dividing cells (Ki-67-positive) and co-distributed with EGFr. In order to verify the possible association between p75^NTR and cell death, we investigated co-distribution of p75^NTR-IR with nuclear condensation images as visualized by Hoechst 33258 staining. While few images indicating nuclear condensation were observed in the SVZ, no coexistence with p75^NTR was found. TrkA- and trkB-IR was not found in the SVZ. We also investigated p75^NTR immunostaining on post-natal day 1 and day 16, because of the dramatic reduction of proliferating cells in SVZ over this time-interval. p75^NTR-IR was not increased in the early post-natal phase. Thus, p75^NTR seems to be associated with cell cycle regulation in SVZ in adult rat brain.     

Subventricular zone astrocytes are neural stem cells in the adult mammalian brain.

Neural stem cells reside in the subventricular zone (SVZ) of the adult mammalian brain. This germinal region, which continually generates new neurons destined for the olfactory bulb, is composed of four cell types: migrating neuroblasts, immature precursors, astrocytes, and ependymal cells. Here we show that SVZ astrocytes, and not ependymal cells, remain labeled with proliferation markers after long survivals in adult mice. After elimination of immature precursors and neuroblasts by an antimitotic treatment, SVZ astrocytes divide to generate immature precursors and neuroblasts. Furthermore, in untreated mice, SVZ astrocytes specifically infected with a retrovirus give rise to new neurons in the olfactory bulb. Finally, we show that SVZ astrocytes give rise to cells that grow into multipotent neurospheres in vitro. We conclude that SVZ astrocytes act as neural stem cells in both the normal and regenerating brain.     

Telomere shortening and chromosomal instability abrogates proliferation of adult but not embryonic neural stem cells

Chromosome integrity is essential for cell viability and, therefore, highly proliferative cell types require active telomere elongation mechanisms to grow indefinitely. Consistently, deletion of telomerase activity in a genetically modified mouse strain results in growth impairments in all highly proliferative cell populations analyzed so far. We show that telomere attrition dramatically impairs the in vitro proliferation of adult neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of telomerase-deficient adult mice. Reduced proliferation of postnatal neurogenic progenitors was also observed in vivo, in the absence of exogenous mitogenic stimulation. Strikingly, severe telomere erosion resulting in chromosomal abnormalities and nuclear accumulation of p53 did not affect the in vitro proliferative potential of embryonic NSCs. These results suggest that intrinsic differences exist between embryonic and adult neural progenitor cells in their response to telomere shortening, and that some populations of tissue-specific stem cells can bypass DNA damage check points.