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.     

Absence of PTHrP Nuclear Localization and Carboxyl Terminus Sequences Leads to Abnormal Brain Development and Function

We assessed whether the nuclear localization sequences (NLS) and C terminus of parathyroid hormone-related protein (PTHrP) play critical roles in brain development and function. We used histology, immunohistochemistry, histomorphometry, Western blots and electrophysiological recordings to compare the proliferation and differentiation of neural stem cells, neuronal hippocampal synaptic transmission, and brain phenotypes including shape and structures, in Pthrp knock-in mice, which express PTHrP (1-84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein, and their wild-type littermates. Results showed that Pthrp knock-in mice display abnormal brain shape and structures; decreased neural cell proliferative capacity and increased apoptosis associated with up-regulation of cyclin dependent kinase inhibitors p16, p21, p27 and p53 and down-regulation of the Bmi-1 oncogene; delayed neural cell differentiation; and impaired hippocampal synaptic transmission and plasticity. These findings provide in vivo experimental evidence that the NLS and C-terminus of PTHrP are essential not only for the regulation of neural cell proliferation and differentiation, but also for the maintenance of normal neuronal synaptic transmission and plasticity.     

Lack of cyclooxygenase-2 inhibits growth of teratocarcinomas in mice

Two isoforms of cyclooxygenase (COX-1 or COX-2) have been identified in the prostanoid biosynthetic pathway. The constitutive form, COX-1, is thought to maintain cellular homeostasis and the inducible form, COX-2, is recognized as a primary response gene thought to be involved in modulating cell proliferation and differentiation. To further characterize the role of the cyclooxygenases in cell proliferation, differentiation, and tumorigenicity we developed embryonic stem (ES) cell lines which contain homozygous disruptions in either the COX-1 or the COX-2 gene. These lines were then examined in terms of their viability, proliferation, and in vitro differentiation potential. Our results demonstrate that the wild-type ES cells do not express either COX-1 or COX-2 until the cells undergo differentiation. And the lack of either cyclooxygenase has no apparent effect on ES cell proliferation in vitro. However, the absence of a functional COX-2 gene leads to a dramatic reduction in the formation and growth of teratocarcinomas that appear when ES cells are injected into syngeneic mice. Histological microscopy shows that the few very small tumors that were generated from ES cells lacking COX-2 appear more differentiated than tumors emerging from COX-1 -/- or wild-type cells by exhibiting greater keratinization in the areas of squamous epithelium and the ossification of bone-forming cartilage. We conclude that the presence of a functional COX-2 enzyme is necessary for the efficient growth of these teratocarcinomas in animals.     

Genetic ablation of caveolin-1 increases neural stem cell proliferation in the subventricular zone (SVZ) of the adult mouse brain

Adult neural stem cells are self-renewing multipotent cells that have the potential to replace dysfunctional and/or dying neuronal cells at the site of brain injury or degeneration. Caveolins are well-known tumor-suppressor genes that were recently found to be involved in the regulation of stem cell proliferation. For instance, ablation of the caveolin-1 (Cav-1) gene in mice markedly increases the proliferation of intestinal and mammary stem cells. However, the roles of caveolins in the proliferation of adult neural stem cells still remain unknown. In this study, dual-label immunofluorescence analysis of the proliferation marker, Ki67, and the stem cell markers, nestin and Sox2, was performed on brains of 8 week-old wild-type (WT) and Cav-1 knockout (KO) mice. Our results demonstrate an increased number of Ki67-positive nuclei in the subventricular zone (SVZ) of Cav-1 KO brains. Importantly, our dual-label immunofluorescence analyses demonstrate increased co-localization of Ki67 with both nestin and Sox2 in the SVZ of Cav-1 KO brains. Remarkably similar results were also obtained with Cav-2 and Cav-3 KO mouse brains as well, with increased proliferation of adult neural stem cells. Thus, the SVZ of caveolin KO mouse brains displays an increased proliferation of adult neural stem cells. Caveolin proteins might represent new crucial regulators of adult neural stem cell proliferation.     

Conditionally immortalised neural stem cells promote functional recovery and brain plasticity after transient focal cerebral ischaemia in mice

Cell therapy has enormous potential to restore neurological function after stroke. The present study investigated effects of conditionally immortalised neural stem cells (ciNSCs), the Maudsley hippocampal murine neural stem cell line clone 36 (MHP36), on sensorimotor and histological outcome in mice subjected to transient middle cerebral artery occlusion (MCAO).Adult male C57BL/6 mice underwent MCAO by intraluminal thread or sham surgery and MHP36 cells or vehicle were implanted into ipsilateral cortex and caudate 2 days later. Functional recovery was assessed for 28 days using cylinder and ladder rung tests and tissue analysed for plasticity, differentiation and infarct size.MHP36-implanted animals showed accelerated and augmented functional recovery and an increase in neurons (MAP-2), synaptic plasticity (synaptophysin) and axonal projections (GAP-43) but no difference in astrocytes (GFAP), oligodendrocytes (CNPase), microglia (IBA-1) or lesion volumes when compared to vehicle group.This is the first study showing a potential functional benefit of the ciNSCs, MHP36, after focal MCAO in mice, which is probably mediated by promoting neuronal differentiation, synaptic plasticity and axonal projections and opens up opportunities for future exploitation of genetically altered mice for dissection of mechanisms of stem cell based therapy.     

Effects of Scopolamine and Melatonin Cotreatment on Cognition,Neuronal Damage,and Neurogenesis in the Mouse Dentate Gyrus

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.     

Nitric oxide acts in a positive feedback loop with BDNF to regulate neural progenitor cell proliferation and differentiation in the mammalian brain

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.     

Resident stem cells are not required for exercise-induced fiber-type switching and angiogenesis but are necessary for activity-dependent muscle growth

Skeletal muscle undergoes active remodeling in response to endurance exercise training, and the underlying mechanisms of this remodeling remain to be defined fully. We have recently obtained evidence that voluntary running induces cell cycle gene expression and cell proliferation in mouse plantaris muscles that undergo fast-to-slow fiber-type switching and angiogenesis after long-term exercise. To ascertain the functional role of cell proliferation in skeletal muscle adaptation, we performed in vivo 5-bromo-2'-deoxyuridine (BrdU) pulse labeling (a single intraperitoneal injection), which demonstrated a phasic increase (5- to 10-fold) in BrdU-positive cells in plantaris muscle between days 3 and 14 during 4 wk of voluntary running. Daily intraperitoneal injection of BrdU for 4 wk labeled 2.0% and 15.4% of the nuclei in plantaris muscle in sedentary and trained mice, respectively, and revealed the myogenic and angiogenic fates of the majority of proliferative cells. Ablation of resident stem cell activity by X-ray irradiation did not prevent voluntary running-induced increases of type IIa myofibers and CD31-positive endothelial cells but completely blocked the increase in muscle mass. These findings suggest that resident stem cell proliferation is not required for exercise-induced type IIb-to-IIa fiber-type switching and angiogenesis but is required for activity-dependent muscle growth. The origin of the angiogenic cells in this physiological exercise model remains to be determined. endurance exercise; adaptation     

TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation

In the adult mammalian brain, neural stem cells in the subventricular zone continuously generate new neurons for the olfactory bulb. Cell fate commitment in these adult neural stem cells is regulated by cell fate-determining proteins. Here, we show that the cell fate-determinant TRIM32 is upregulated during differentiation of adult neural stem cells into olfactory bulb neurons. We further demonstrate that TRIM32 is necessary for the correct induction of neuronal differentiation in these cells. In the absence of TRIM32, neuroblasts differentiate slower and show gene expression profiles that are characteristic of immature cells. Interestingly, TRIM32 deficiency induces more neural progenitor cell proliferation and less cell death. Both effects accumulate in an overproduction of adult-generated olfactory bulb neurons of TRIM32 knockout mice. These results highlight the function of the cell fate-determinant TRIM32 for a balanced activity of the adult neurogenesis process.     

Persistent migration of neuroblasts from the subventricular zone to the injured striatum mediated by osteopontin following intracerebral hemorrhage

We investigated intracerebral hemorrhage (ICH)-induced lateral migration of neuroblasts and the mechanism underlying this migration. ICH model was induced by collagenase injection into the striatum of adult wild-type and osteopontin (OPN) knockout mice. In the wild-type mice, the lateral migration of neuroblasts from the ipsislateral subventricular zone (SVZ) towards the hematoma started at day 3 and continued up to day 28 after ICH. In addition to migrating towards the hematoma, neuroblasts also migrated to the area of ipsilateral striatum remote to the hematoma. The migrating neuroblasts were closely associated with activated astrocytes and blood vessels in the injured striatum. Following ICH, the expression of OPN was up-regulated in the ipsilateral striatum from day 1 to day 28. In vitro , OPN treatment did not affect the proliferation of neural progenitors, but enhanced the trans-well and radial migration of neural progenitors. In vivo , OPN deficiency did not affect the proliferation of neural progenitors in the SVZ. However, following ICH a significant decrease in lateral neuroblast migration was observed in the OPN knockout mice compared with the wild-type mice. These results suggest that increased OPN expression in the injured striatum plays a significant role in the lateral migration of neuroblasts following ICH.     

Regulatory Role of the JNK-STAT1/3 Signaling in Neuronal Differentiation of Cultured Mouse Embryonic Stem Cells

Stem cell transplantation therapy has provided promising hope for the treatment of a variety of neurodegenerative disorders. Among challenges in developing disease-specific stem cell therapies, identification of key regulatory signals for neuronal differentiation is an essential and critical issue that remains to be resolved. Several lines of evidence suggest that JNK, also known as SAPK, is involved in neuronal differentiation and neural plasticity. It may also play a role in neurite outgrowth during neuronal development. In cultured mouse embryonic stem (ES) cells, we test the hypothesis that the JNK pathway is required for neuronal differentiation. After neural induction, the cells were plated and underwent differentiation for up to 5 days. Western blot analysis showed a dramatic increase in phosphorylated JNKs at 1–5 days after plating. The phosphorylation of JNK subsequently induced activation of STAT1 and STAT3 that lead to expressions of GAP-43, neurofilament, βIII-tubulin, and synaptophysin. NeuN-colabelled with DCX, a marker for neuroblast, was enhanced by JNK signaling. Neuronal differentiation of ES cells was attenuated by treatment with SP600125, which inhibited the JNK activation and decreased the activation of STAT1 and STAT3, and consequently suppressed the expressions of GAP-43, neurofilament, βIII-tubulin, and the secretion of VEGF. Data from immunocytochemistry indicated that the nuclear translocation of STAT3 was reduced, and neurites of ES-derived neurons were shorter after treatment with SP600125 compared with control cells. These results suggest that the JNK-STAT3 pathway is a key regulator required for early neuronal differentiation of mouse ES cells. Further investigation on expression of JNK isoforms showed that JNK-3 was significantly upregulated during the differentiation stage, while JNK-1 and JNK-2 levels decreased. Our study provided interesting information on JNK functions during ES cell neuronal differentiation.     

Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

Background

Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus.

Methodology/Principal Findings

To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting.

Conclusions/Significance

Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus.     

Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells

Low-intensity pulsed ultrasound (LIPUS) acting on induced pluripotent stem cells–derived neural crest stem cells (iPSCs–NCSCs) is considered a promising therapy to improve the efficacy of injured peripheral nerve regeneration. Effects of LIPUS on cell viability, proliferation and neural differentiation of iPSCs–NCSCs were examined respectively in this study. LIPUS at 500 mW cm^?2 enhanced the viability and proliferation of iPSCs–NCSCs after 2 days and, after 4 days, up-regulated gene and protein expressions of NF-M, Tuj1, S100β and GFAP in iPSCs–NCSCs whereas after 7 days expression of only NF-M, S100β and GFAP were up-regulated. LIPUS treatment at an appropriate intensity can, therefore, be an efficient and cost-effective method to enhance cell viability, proliferation and neural differentiation of iPSCs–NCSCs in vitro for peripheral nerve tissue engineering.     

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis,Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.     

肌卫星细胞在失重肌萎缩中的可塑性变化及机制

肌卫星细胞在骨骼肌生长发育和出生后骨骼肌损伤修复中起着重要的作用,但是有关肌萎缩中肌卫星细胞的可塑性变化、作用及其机制尚不清楚.本研究采用小鼠尾悬吊模拟失重效应诱导失重肌萎缩,动态分析了失重肌萎缩发生过程中不同类型肌纤维的肌卫星细胞数量和增殖、分化潜能可塑性的改变,发现在失重肌萎缩过程中,处于安静状态的肌卫星细胞显著增多、激活增殖的肌卫星细胞显著减少,而具有成肌分化潜能的肌卫星细胞有持续减少趋势.此外,在失重肌萎缩比目鱼肌单根肌纤维移出的体外培养中,证明了失重肌萎缩肌纤维肌卫星细胞可塑性降低的特征性变化.进一步,通过对比分析Smad3基因敲除及其同窝野生型小鼠,在失重肌萎缩中肌卫星细胞可塑性的差异性变化,揭示了Smad3在调控失重肌萎缩肌卫星细胞可塑性变化中的关键作用.