Effects of Treadmill Exercise on Neural Stem Cells, Cell Proliferation, and Neuroblast Differentiation in the Subgranular Zone of the Dentate Gyrus in Cyclooxygenase-2 Knockout Mice

Cyclooxygenase-2 (COX-2) function has been implicated in a number of physiological processes, including inflammatory responses, synaptic transmission, and synaptic plasticity in the brain. However, the specific role of COX-2 in exercise-induced neurogenesis is still debatable. Here, we assessed the role of COX-2 in exercise-induced plasticity by comparing COX-2 knockout mice to wild-type control littermates. We investigated the number of neural stem cells, and the degree of cell proliferation and neuronal differentiation in COX-2 knockout and its wild-type mice that either exercised or remained inactive. Wild-type and COX-2 knockout mice were put on a treadmill and were either sedentary or were forced to run 1 h/day for five consecutive days at a pace of 10–12 m/min for 5 weeks. Loss of COX-2 expression in the knockout mice was confirmed with two measures: (1) COX immunolabeling in the hippocampus, and (2) the identification of abnormal kidney development using hematoxylin and eosin staining, including subcapsular glomerular hypoplasia and hypertrophy of the deeper cortical glomeruli. Compared to wild-type mice, COX-2 knockout mice exhibited a significant reduction in the neural stem cells (nestin-positive cells), cell proliferation (Ki67-positive cells), and neuroblast differentiation (doublecortin-positive cells). In contrast, exercise significantly increased the neural stem cells, cell proliferation, and neuroblast differentiation in both the wild-type and COX-2 knockout mice although the NeuN-immunoreactive neurons were similar in all groups. Expression of phosphorylated cAMP-response element binding protein was decreased in knockout mice. Exercise increased its expression in the subgranular zone of the dentate gyrus in both wild-type and knockout mice. These results suggest that the COX-2 pathway is one of important factors on neural stem cells, cell proliferation and neuroblast differentiation in sedentary mice. The ability of exercise to increase these types of neural plasticity, regardless of COX-2 signaling, suggests that the effects of exercise on neural stem cells, cell proliferation, and neuroblast differentiation are induced via a pathway that is independent of COX-2.     

AlCl3 exposure regulates neuronal development by modulating DNA modification

BACKGROUNDAs the third most abundant element, aluminum is widespread in the environment. Previous studies have shown that aluminum has a neurotoxic effect and its exposure can impair neuronal development and cognitive function.AIMTo study the effects of aluminum on epigenetic modification in neural stem cells and neurons. METHODSNeural stem cells were isolated from the forebrain of adult mice. Neurons were isolated from the hippocampi tissues of embryonic day 16-18 mice. AlCl₃ at 100 and 200 μmol/L was applied to stem cells and neurons. RESULTSAluminum altered the differentiation of adult neural stem cells and caused apoptosis of newborn neurons while having no significant effects on the proliferation of neural stem cells. Aluminum application also significantly inhibited the dendritic development of hippocampal neurons. Mechanistically, aluminum exposure significantly affected the levels of DNA 5-hydroxy-methylcytosine, 5-methylcytosine, and N⁶-methyladenine in stem cells and neurons. CONCLUSIONOur findings indicate that aluminum may regulate neuronal development by modulating DNA modifications.     

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.     

Chronic Treatment with the GLP1 Analogue Liraglutide Increases Cell Proliferation and Differentiation into Neurons in an AD Mouse Model

Neurogenesis is a life long process, but the rate of cell proliferation and differentiation decreases with age. In Alzheimer''s patients, along with age, the presence of Aβ in the brain inhibits this process by reducing stem cell proliferation and cell differentiation. GLP-1 is a growth factor that has neuroprotective properties. GLP1 receptors are present on neuronal progenitor cells, and the GLP-1 analogue liraglutide has been shown to increase cell proliferation in an Alzheimer''s disease (AD) mouse model. Here we investigated acute and chronic effects of liraglutide on progenitor cell proliferation, neuroblast differentiation and their subsequent differentiation into neurons in wild type and APP/PS-1 mice at different ages. APP/PS1 and their littermate controls, aged 3, 6, 12, 15 months were injected acutely or chronically with 25 nmol/kg liraglutide. Acute treatment with liraglutide showed an increase in cell proliferation in APP/PS1 mice, but not in controls whereas chronic treatment increased cell proliferation at all ages (BrdU and Ki67 markers). Moreover, numbers of immature neurons (DCX) were increased in both acute and chronic treated animals at all ages. Most newly generated cells differentiated into mature neurons (NeuN marker). A significant increase was observed with chronically treated 6, 12, 15 month APP/PS1 and WT groups. These results demonstrate that liraglutide, which is currently on the market as a treatment for type 2 diabetes (Victoza^TM), increases neurogenesis, which may have beneficial effects in neurodegenerative disorders like AD.     

Combination Effects of Sodium Butyrate and Pyridoxine Treatment on Cell Proliferation and Neuroblast Differentiation in the Dentate Gyrus of d-Galactose-Induced Aging Model Mice

We previously reported that sodium butyrate (SB), a histone deacetylase inhibitor, robustly increased pyridoxine-induced cell proliferation and neuroblast differentiation in the dentate gyrus of the adult mouse. In this study, we investigated the effects of treatment with SB combined with pyridoxine on cell proliferation and neuroblast differentiation in the dentate gyrus of a mouse model of aging induced by d-galactose (d-gal). d-gal was administered to 20-week-old male mice (d-gal mice) for 10 weeks to induce changes that resemble natural aging in animals. Seven weeks after d-gal (100 mg/kg) treatment, vehicle (physiological saline; d-gal-vehicle mice) and SB (300 mg/kg) combined with pyridoxine (Pyr; 350 mg/kg) were administered to the mice (d-gal-Pyr-SB mice) for 3 weeks. Escape latency under water maze in the d-gal mice was longer than that in the control mice. In the d-gal-Pyr-SB mice, escape latency was similar to that in the control mice. In the d-gal mice, many cells in the granule cell layer of the dentate gyrus showed pyknosis and condensation of the cytoplasm. However, in the d-gal-Pyr-SB mice, such cellular changes were rarely found. Furthermore, the d-gal mice showed a great reduction in cell proliferation (Ki67-positive cells) and neuroblast differentiation (doublecortin-positive neuroblasts) in the dentate gyrus compared to control mice. However, in the d-gal-Pyr-SB mice, cell proliferation and neuroblast differentiation were markedly increased in the dentate gyrus. Furthermore, the administration of pyridoxine with sodium butyrate significantly increased Ser133-phosphorylated cyclic AMP response element binding protein in the dentate gyrus. These results indicate that the combination treatment of Pyr with SB in d-gal mice ameliorated the d-gal-induced reduction in cell proliferation, neuroblast differentiation, and memory deficits.     

Insights into neural stem cell biology from flies

Drosophila neuroblasts are similar to mammalian neural stem cells in their ability to self-renew and to produce many different types of neurons and glial cells. In the past two decades, great advances have been made in understanding the molecular mechanisms underlying embryonic neuroblast formation, the establishment of cell polarity and the temporal regulation of cell fate. It is now a challenge to connect, at the molecular level, the different cell biological events underlying the transition from neural stem cell maintenance to differentiation. Progress has also been made in understanding the later stages of development, when neuroblasts become mitotically inactive, or quiescent, and are then reactivated postembryonically to generate the neurons that make up the adult nervous system. The ability to manipulate the steps leading from quiescence to proliferation and from proliferation to differentiation will have a major impact on the treatment of neurological injury and neurodegenerative disease.     

Changes in cell migration and survival in the olfactory bulb of the pcd/pcd mouse

Postnatally, the Purkinje cell degeneration mutant mice lose the main projecting neurons of the main olfactory bulb (OB): mitral cells (MC). In adult animals, progenitor cells from the rostral migratory stream (RMS) differentiate into bulbar interneurons that modulate MC activity. In the present work, we studied changes in proliferation, tangential migration, radial migration patterns, and the survival of these newly generated neurons in this neurodegeneration animal model. The animals were injected with bromodeoxyuridine 2 weeks or 2 months before killing in order to label neuroblast incorporation into the OB and to analyze the survival of these cells after differentiation, respectively. Both the organization and cellular composition of the RMS and the differentiation of the newly generated neurons in the OB were studied using specific markers of glial cells, neuroblasts, and mature neurons. No changes were observed in the cell proliferation rate nor in their tangential migration through the RMS, indicating that migrating neuroblasts are only weakly responsive to the alteration in their target region, the OB. However, the absence of MC does elicit differences in the final destination of the newly generated interneurons. Moreover, the loss of MC also produces changes in the survival of the newly generated interneurons, in accordance with the dramatic decrease in the number of synaptic targets available.     

Effects of Sensitive to Apoptosis Gene Protein on Cell Proliferation, Neuroblast Differentiation, and Oxidative Stress in the Mouse Dentate Gyrus

Sensitive to apoptosis gene (SAG) protein is a redox-inducible protein that protects cells against apoptosis induced by redox agents. In this study, we observed effects of SAG on cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus (DG) using Ki67 and doublecortin (DCX), respectively. For easy penetration into neurons, Tat-SAG expression vector was constructed by ligation with SAG and expression vector, Tat, in-frame with six histidine open-reading frames to generate the expression vector, and cloned into E. coli DH5α cells. One or 5?mg/kg Tat-SAG fusion protein (Tat-SAG) was intraperitoneally administered to mice once a day for 3?weeks. The administration of Tat-SAG significantly increased the number of 5-bromodeoxyuridine positive cells, Ki67 positive cells and DCX immunoreactive neuroblast in the mouse DG: Especially, in the 5?mg/kg Tat-SAG-treated mice, DCX positive neuroblasts showed a well-developed arborization of tertiary dendrites in the DG. On the other hand, we examined that the administration of Tat-SAG significantly reduced the DNA damage and lipid peroxidation judging from 8-hydroxy-2'-deoxyguanosine and 4-hydroxynonenal immunohistochemistry: The decrease was much more distinct in the 5?mg/kg Tat-SAG-treated mice than 1?mg/kg Tat-SAG-treated mice. This result suggests that SAG significantly increases cell proliferation, neuroblast differentiation and oxidative stress in normal states.     

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.     

Synergistic Effects of Sodium Butyrate,a Histone Deacetylase Inhibitor,on Increase of Neurogenesis Induced by Pyridoxine and Increase of Neural Proliferation in the Mouse Dentate Gyrus

We previously observed that pyridoxine (vitamin B₆) significantly increased cell proliferation and neuroblast differentiation without any neuronal damage in the hippocampus. In this study, we investigated the effects of sodium butyrate, a histone deacetylase (HDAC) inhibitor which serves as an epigenetic regulator of gene expression, on pyridoxine-induced neural proliferation and neurogenesis induced by the increase of neural proliferation in the mouse dentate gyrus. Sodium butyrate (300 mg/kg, subcutaneously), pyridoxine (350 mg/kg, intraperitoneally), or combination with sodium butyrate were administered to 8-week-old mice twice a day and once a day, respectively, for 14 days. The administration of sodium butyrate significantly increased acetyl-histone H3 levels in the dentate gyrus. Sodium butyrate alone did not show the significant increase of cell proliferation in the dentate gyrus. But, pyridoxine alone significantly increased cell proliferation. Sodium butyrate in combination with pyridoxine robustly enhanced cell proliferation and neurogenesis induced by the increase of neural proliferation in the dentate gyrus, showing that sodium butyrate treatment distinctively enhanced development of neuroblast dendrites. These results indicate that an inhibition of HDAC synergistically promotes neurogenesis induced by a pyridoxine and increase of neural proliferation.     

Effects of Adrenalectomy and Replacement Therapy of Corticosterone on Cell Proliferation and Neuroblast Differentiation in the Rat Dentate Gyrus

Newly generated neurons in the dentate gyrus differentiate into mature granule cells. In the present study, we observed the effects of adrenalectomy (ADX) and corticosterone replacement therapy (CRT) on cell death, cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus (SZDG). For this, the animals received vehicle or CRT after ADX, and were sacrificed 5 or 42 days later. Plasma corticosterone levels were very low in the adrenalectomized groups, whereas CRT after ADX significant increased serum corticosterone levels at 42 days, not 5 days, after ADX. ADX induced some neuronal damage in the dentate gyrus at 5 days post-ADX. CRT did not significantly reduce the neuronal damage at 5 days post-ADX; however, neuronal damage was not shown at 42 post-ADX with CRT. Ki67 (a marker for cell proliferation) and doublecortin (DCX, a marker for neuronal differentiation) immunoreaction was detected in the SZDG. ADX transiently increased cell proliferation and neuroblast differentiation 5 days after ADX, not 42 days, after ADX, and the CRT 42 days after ADX prominently decreased cell proliferation and neuroblast differentiation in the dentate gyrus. These results suggest that adrenal corticosteroid hormone is not essential for cell proliferation and neuroblast differentiation in long-term period after ADX.     

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.     

CDK2 is Dispensable for Adult Hippocampal Neurogenesis

Granule neurons of the dentate gyrus (DG) of the hippocampus undergo continuous renewal throughout life. Among cell-cycle regulators, cyclin-dependent kinase 2 (Cdk2) is considered as a major regulator of S-phase entry. We used Cdk2-deficient mice to decipher the requirement of Cdk2 for the generation of new neurons in the adult hippocampus. The quantification of cell cycle markers first revealed that the lack of Cdk2 activity does not influence spontaneous or seizure-induced proliferation of neural progenitor cells (NPC) in the adult DG. Using bromodeoxyuridine incorporation assays, we showed that the number of mature newborn granule neurons generated de novo was similar in both wild-type (WT) and Cdk2-deficient adult mice. Moreover, the apparent lack of cell output reduction in Cdk2-/- mice DG did not result from a reduction in apoptosis of newborn granule cells as analyzed by TUNEL assays. Our results therefore suggest that Cdk2 is dispensable for NPC proliferation, differentiation and survival of adult-born DG granule neurons in vivo. These data emphasize that functional redundancies between Cdks also occur in the adult brain at the level of neural progenitor cell cycle regulation during hippocampal neurogenesis.     

猕猴神经干细胞的诱导分化、干性维持及同种脑内移植

为探索猕猴神经干细胞分化及特性维持,推进神经干细胞临床应用研究,该实验以绿色荧光蛋白(green fluorescence protein,GFP)为标记探讨猕猴胚胎干细胞向玫瑰花环(rosettes)结构神经干细胞的分化及其碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和表皮生长因子(epidermal growth factor,EGF)的扩增培养。结果表明:1)建立了稳定高效的猕猴神经干细胞分化体系,在该分化体系下,GFP标记猕猴胚胎干细胞在分化的第12天时,95%以上的细胞分化为神经干细胞;2)分化得到的Rosettes结构神经干细胞经bFGF/EGF扩增后,能够较好地维持其Rosettes结构;3)经bFGF/EGF扩增后的rosettes结构神经干细胞移植到猕猴脑内后能够较好的存活并向神经元分化,即bFGF/EGF扩增培养能较好地维持Rosettes结构的神经干细胞,且移植到猕猴脑内的该细胞亦能够较好地存活并向神经元分化,该结果为神经干细胞应用于临床提供了基础理论依据。     

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.     

Distinct response of adult neural stem cells to low versus high dose ionising radiation

Radiosusceptibility is the sensitivity of a biological organism to ionising radiation (IR)-induced carcinogenesis, an outcome of IR exposure relevant following low doses. The tissue response is strongly influenced by the DNA damage response (DDR) activated in stem and progenitor cells. We previously reported that in vivo exposure to 2 Gy X-rays activates apoptosis, proliferation arrest and premature differentiation in neural progenitor cells (transit amplifying cells and neuroblasts) but not in neural stem cells (NSCs) of the largest neurogenic region of the adult brain, the subventricular zone (SVZ). These responses promote adult quiescent NSC (qNSC) activation after 2 Gy. In contrast, neonatal (P5) SVZ neural progenitors continue proliferating and do not activate qNSCs. Significantly, the human and mouse neonatal brain is radiosusceptible.Here, we examine the response of stem and progenitor cells in the SVZ to low IR doses (50–500 mGy). We observe a linear dose-response for apoptosis but, in contrast, proliferation arrest and neuroblast differentiation require a threshold dose of 200 or 500 mGy, respectively. Importantly, qNSCs were not activated at doses below 500 mGy. Thus, full DDR activation in the neural stem cell compartment in vivo necessitates a threshold dose, which can be considered of significance when evaluating IR-induced cancer risk and dose extrapolation.