Pten deletion in adult hippocampal neural stem/progenitor cells causes cellular abnormalities and alters neurogenesis

Adult neurogenesis persists throughout life in restricted brain regions in mammals and is affected by various physiological and pathological conditions. The tumor suppressor gene Pten is involved in adult neurogenesis and is mutated in a subset of autism patients with macrocephaly; however, the link between the role of PTEN in adult neurogenesis and the etiology of autism has not been studied before. Moreover, the role of hippocampus, one of the brain regions where adult neurogenesis occurs, in development of autism is not clear. Here, we show that ablating Pten in adult neural stem cells in the subgranular zone of hippocampal dentate gyrus results in higher proliferation rate and accelerated differentiation of the stem/progenitor cells, leading to depletion of the neural stem cell pool and increased differentiation toward the astrocytic lineage at later stages. Pten-deleted stem/progenitor cells develop into hypertrophied neurons with abnormal polarity. Additionally, Pten mutant mice have macrocephaly and exhibit impairment in social interactions and seizure activity. Our data reveal a novel function for PTEN in adult hippocampal neurogenesis and indicate a role in the pathogenesis of abnormal social behaviors.     

Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice

In adult hippocampal neurogenesis, new neurons appear to originate from a cell with astrocytic properties expressing glial fibrillary acidic protein (GFAP). Also, new astrocytes are generated in the adult dentate gyrus. Whereas the putative astrocyte-like progenitor cells are consistently S-100beta-negative, many new astrocytes are S-100beta-positive. Thus, it is unclear whether the GFAP-positive progenitor cells are astrocytes in a general sense or rather neural progenitor cells with certain astrocytic characteristics. We therefore investigated the development of GFAP-expressing cells in the context of adult hippocampal neurogenesis. Proliferating cells could be either GFAP-positive or doublecortin-positive (DCX), but never both, indicating two independent populations of dividing cells in the glial and neuronal lineages. Two distinct populations of cells with astroglial properties were detected-one expressing GFAP, the other co-expressing GFAP and S-100beta. We never found S-100beta-cells to be in S-phase. No overlap between neuronal and glial markers was seen at any time point. Thus, astrogenesis occurred in parallel and to some degree independent of adult neurogenesis. The uninterrupted GFAP expression in this lineage, and neuronal markers in the other lineage, argue against a late common precursor for neurogenesis and gliogenesis in the adult hippocampus. Very few newly generated microglia and no new oligodendrocytes were detected. Environmental enrichment and voluntary wheel running-two experimental paradigms with robust stimulatory effects on adult hippocampal neurogenesis-affected hippocampal astrogenesis differentially: Running, but not enrichment, strongly induced net astrogenesis (GFAP/S-100beta), but also GFAP-positive S-100beta-negative cells, which thus appear to be a transiently amplifiable intermediate population within the glial lineage.     

Enriched Environment Promotes Adult Hippocampal Neurogenesis through FGFRs

The addition of new neurons to existing neural circuits in the adult brain remains of great interest to neurobiology because of its therapeutic implications. The premier model for studying this process has been the hippocampal dentate gyrus in mice, where new neurons are added to mature circuits during adulthood. Notably, external factors such as an enriched environment (EE) and exercise markedly increase hippocampal neurogenesis. Here, we demonstrate that EE acts by increasing fibroblast growth factor receptor (FGFR) function autonomously within neurogenic cells to expand their numbers in adult male and female mice. FGFRs activated by EE signal through their mediators, FGFR substrate (FRS), to induce stem cell proliferation, and through FRS and phospholipase Cγ to increase the number of adult-born neurons, providing a mechanism for how EE promotes adult neurogenesis.SIGNIFICANCE STATEMENT How the environment we live in affects cognition remains poorly understood. In the current study, we explore the mechanism underlying the effects of an enriched environment on the production of new neurons in the adult hippocampal dentate gyrus, a brain area integral in forming new memories. A mechanism is provided for how neural precursor cells in the adult mammalian dentate gyrus respond to an enriched environment to increase their neurogenic output. Namely, an enriched environment acts on stem and progenitor cells by activating fibroblast growth factor receptor signaling through phospholipase Cγ and FGF receptor substrate proteins to expand the pool of precursor cells.     

The role of hypoxia in stem cell regulation of the central nervous system: From embryonic development to adult proliferation

Hypoxia is involved in the regulation of various cell functions in the body, including the regulation of stem cells. The hypoxic microenvironment is indispensable from embryonic development to the regeneration and repair of adult cells. In addition to embryonic stem cells, which need to maintain their self-renewal properties and pluripotency in a hypoxic environment, adult stem cells, including neural stem cells (NSCs), also exist in a hypoxic microenvironment. The subventricular zone (SVZ) and hippocampal dentate gyrus (DG) are the main sites of adult neurogenesis in the brain. Hypoxia can promote the proliferation, migration, and maturation of NSCs in these regions. Also, because most neurons in the brain are non-regenerative, stem cell transplantation is considered as a promising strategy for treating central nervous system (CNS) diseases. Hypoxic treatment also increases the effectiveness of stem cell therapy. In this review, we firstly describe the role of hypoxia in different stem cells, such as embryonic stem cells, NSCs, and induced pluripotent stem cells, and discuss the role of hypoxia-treated stem cells in CNS diseases treatment. Furthermore, we highlight the role and mechanisms of hypoxia in regulating adult neurogenesis in the SVZ and DG and adult proliferation of other cells in the CNS.     

Ablation of proliferating neural stem cells during early life is sufficient to reduce adult hippocampal neurogenesis

Environmental exposures during early life, but not during adolescence or adulthood, lead to persistent reductions in neurogenesis in the adult hippocampal dentate gyrus (DG). The mechanisms by which early life exposures lead to long‐term deficits in neurogenesis remain unclear. Here, we investigated whether targeted ablation of dividing neural stem cells during early life is sufficient to produce long‐term decreases in DG neurogenesis. Having previously found that the stem cell lineage is resistant to long‐term effects of transient ablation of dividing stem cells during adolescence or adulthood (Kirshenbaum, Lieberman, Briner, Leonardo, & Dranovsky, 2014 ), we used a similar pharmacogenetic approach to target dividing neural stem cells for elimination during early life periods sensitive to environmental insults. We then assessed the Nestin stem cell lineage in adulthood. We found that the adult neural stem cell reservoir was depleted following ablation during the first postnatal week, when stem cells were highly proliferative, but not during the third postnatal week, when stem cells were more quiescent. Remarkably, ablating proliferating stem cells during either the first or third postnatal week led to reduced adult neurogenesis out of proportion to the changes in the stem cell pool, indicating a disruption of the stem cell function or niche following stem cell ablation in early life. These results highlight the first three postnatal weeks as a series of sensitive periods during which elimination of dividing stem cells leads to lasting alterations in adult DG neurogenesis and stem cell function. These findings contribute to our understanding of the relationship between DG development and adult neurogenesis, as well as suggest a possible mechanism by which early life experiences may lead to lasting deficits in adult hippocampal neurogenesis.     

PACAP promotes neural stem cell proliferation in adult mouse brain

In recent years, it has become evident that neural stem cells in the adult mammalian brain continuously generate new neurons, mainly in the hippocampus and olfactory bulb. Although different growth factors have been shown to stimulate neurogenesis in the adult brain, very little is known about the role of neuropeptides in this process. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic effects acting through three receptors to which it has high affinity, namely, PACAP receptor 1 (PAC1), vasoactive intestinal peptide (VIP) receptor 1, and VIP receptor 2. We show that PAC1 is expressed in the neurogenic regions of the adult mouse brain, namely the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus. Cultured neural stem cells isolated from the lateral ventricle wall of adult mice express PAC1 and proliferate in vitro in response to two PAC1 agonists, PACAP and Maxadilan, but not VIP at physiologic concentrations, indicating PAC1 as a mediator of neural stem cell proliferation. Pharmacologic and biochemical characterization of PACAP-induced neural stem cell proliferation revealed the protein kinase C pathway as the principal signaling pathway, whereas addition of epidermal growth factor synergistically enhanced the proliferating effect of PACAP. Further in vitro characterization of the effect of PACAP on neural stem cells showed PACAP capable of stimulating ex novo in vitro formation of multipotent neurospheres with the capacity to generate both neuronal and glial cells. Finally, intracerebroventricular infusion of PACAP increases cell proliferation in the ventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus. We conclude that PACAP, through PAC1, is a potent mediator of adult neural stem cell proliferation.     

Sp8阳性细胞在健康及放射性脑损伤豚鼠SGZ区的表达

目的 1观察Sp8阳性细胞(Sp8+)在健康成年豚鼠海马齿状回颗粒细胞下区(SGZ区)的表达,并探讨其表达与年龄的相关性;2观察放射性脑损伤豚鼠放射侧SGZ区Sp8及DCX的表达变化,进一步探讨X线对海马齿状回神经干细胞群的影响。方法 1运用免疫组化染色及免疫荧光技术对脑组织切片进行染色。随机选取6个月龄健康成年豚鼠4只用于形态学观察,观察正常成年豚鼠SGZ区的Sp8表达;2 Sp8分别与Neun、CB、GFAP、PSA-NCAM、Sox2、Ki67抗体进行免疫双标;3新生、6个月龄、2岁以及3.5岁豚鼠各4只,用于观察Sp8在不同年龄组豚鼠SGZ区表达的差别;4建立单侧颅脑放射性脑损伤模型:6个月龄健康成年豚鼠20只分别在第1、3、7天接受左侧颅脑X线照射,每天照射2次,单次照射剂量为5Gy,于第14天进行断头处理。选取海马部位清晰完整的冠状位组织切片,分别进行Sp8及DCX的免疫组化染色,观察两者在放射侧SGZ区的表达变化。结果 1 Sp8+细胞在健康成年豚鼠SGZ区广泛表达;2 Sp8+细胞与神经干细胞标记物Sox2共存;3豚鼠SGZ区Sp8+细胞的表达随着年龄的增长呈逐渐下降趋势(P0.05);4放射侧SGZ区DCX阳性细胞数量较对照侧显著减少(P0.05),而两侧Sp8+细胞数量差异不明显(P0.05)。结论 1 Sp8+细胞在豚鼠海马齿状回神经发生区广泛表达;2 Sp8可能是海马齿状回神经发生区静息状态神经干细胞的一种标记物;3豚鼠SGZ区神经干/祖细胞的减少与年龄呈负相关性;4 X线抑制SGZ区新生神经元的生成,而Sp8+细胞可抵抗X线损伤。     

Neurogenesis in Talpha-1 tubulin transgenic mice during development and after injury

Talpha-1 tubulin promoter-driven EYFP expression is seen in murine neurons born as early as E9.5. Double labeling with markers for stem cells (Sox 1, Sox 2, nestin), glial progenitors (S100beta, NG2, Olig2), and neuronal progenitors (doublecortin, betaIII-tubulin, PSA-NCAM) show that Talpha-1 tubulin expression is limited to early born neurons. BrdU uptake and double labeling with neuronal progenitor markers in vivo and in vitro show that EYFP-expressing cells are postmitotic and Talpha-1 tubulin EYFP precedes the expression of MAP-2 and NeuN, and follows the expression of PSA-NCAM, doublecortin (Dcx), and betaIII-tubulin. Talpha-1 tubulin promoter-driven EYFP expression is transient and disappears in most neurons by P0. Persistent EYFP expression is mainly limited to scattered cells in the subventricular zone (SVZ), rostral migratory stream, and hippocampus. However, there are some areas that continue to express Talpha-1 tubulin in the adult without apparent neurogenesis. The number of EYFP-expressing cells declines with age indicating that Talpha-1 tubulin accurately identifies early born postmitotic neurons throughout development but less clearly in the adult. Assessment of neurogenesis after stab wound injuries in the cortex, cerebellum and spinal cord of adult animals shows no neurogenesis in most areas with an increase in BrdU incorporation in glial and other non neuronal populations. An up-regulation of Talpha-1 tubulin can be seen in certain areas unaccompanied by new neurogenesis. Our results suggest that even if stem cells proliferate their ability to generate neurons is limited and caution is warranted in attributing increased BrdU incorporation to stem cells or cells fated to be neurons even in neurogenic areas.     

Leptin restores adult hippocampal neurogenesis in a chronic unpredictable stress model of depression and reverses glucocorticoid-induced inhibition of GSK-3β/β-catenin signaling

Stress and glucocorticoid stress hormones inhibit neurogenesis, whereas antidepressants increase neurogenesis and block stress-induced decrease in neurogenesis. Our previous studies have shown that leptin, an adipocyte-derived hormone with antidepressant-like properties, promotes baseline neurogenesis in the adult hippocampus. This study aimed to determine whether leptin is able to restore suppression of neurogenesis in a rat chronic unpredictable stress (CUS) model of depression. Chronic treatment with leptin reversed the CUS-induced reduction of hippocampal neurogenesis and depression-like behaviors. Leptin treatment elicited a delayed long-lasting antidepressant-like effect in the forced swim behavioral despair test, and this effect was blocked by ablation of neurogenesis with X-irradiation. The functional isoform of the leptin receptor, LepRb, and the glucocorticoid receptor (GR) were colocalized in hippocampal neural stem/progenitor cells in vivo and in vitro. Leptin treatment reversed the GR agonist dexamethasone (DEX)-induced reduction of proliferation of cultured neural stem/progenitor cells from adult hippocampus. Further mechanistic analysis revealed that leptin and DEX converged on glycogen synthase kinase-3β (GSK-3β) and β-catenin. While DEX decreased Ser9 phosphorylation and increased Tyr216 phosphorylation of GSK-3β, leptin increased Ser9 phosphorylation and attenuated the effects of DEX at both Ser9 and Tyr216 phosphorylation sites of GSK-3β. Moreover, leptin increased total level and nuclear translocation of β-catenin, a primary substrate of GSK-3β and a key regulator in controlling hippocampal neural progenitor cell proliferation, and reversed the inhibitory effects of DEX on β-catenin. Taken together, our results suggest that adult neurogenesis is involved in the delayed long-lasting antidepressant-like behavioral effects of leptin, and leptin treatment counteracts chronic stress and glucocorticoid-induced suppression of hippocampal neurogenesis via activating the GSK-3β/β-catenin signaling pathway.     

Alcohol inhibition of neurogenesis: A mechanism of hippocampal neurodegeneration in an adolescent alcohol abuse model

Adolescents diagnosed with an alcohol use disorder show neurodegeneration in the hippocampus, a region important for learning, memory, and mood regulation. This study examines a potential mechanism by which excessive alcohol intake, characteristic of an alcohol use disorder, produces neurodegeneration. As hippocampal neural stem cells underlie ongoing neurogenesis, a phenomenon that contributes to hippocampal structure and function, we investigated aspects of cell death and cell birth in an adolescent rat model of an alcohol use disorder. Immunohistochemistry of various markers along with Bromo‐deoxy‐Uridine (BrdU) injections were used to examine different aspects of neurogenesis. After 4 days of binge alcohol exposure, neurogenesis was decreased by 33 and 28% at 0 and 2 days after the last dose according to doublecortin expression. To determine whether this decrease in neurogenesis was due to effects on neural stem cell proliferation, quantification of BrdU‐labeled cells revealed a 21% decrease in the dentate gyrus of alcohol‐exposed brains. Cell survival and phenotype of BrdU‐labeled cells were assessed 28 days after alcohol exposure and revealed a significant, 50% decrease in the number of surviving cells in the alcohol‐exposed group. Reduced survival was supported by significant increases in the number of pyknotic‐, FluoroJade B positive‐, and TUNEL‐positive cells. However, so few cells were TUNEL‐positive that cell death is likely necrotic in this model. Although alcohol decreased the number of newborn cells, it did not affect the percentage of cells that matured into neurons (differentiation). Thus, our data support that in a model of an adolescent alcohol use disorder, neurogenesis is impaired by two mechanisms: alcohol‐inhibition of neural stem cell proliferation and alcohol effects on new cell survival. Remarkably, alcohol inhibition of neurogenesis may outweigh the few dying cells per section, which implies that alcohol inhibition of neurogenesis contributes to hippocampal neurodegeneration in alcohol use disorders. © 2009 Wiley‐Liss, Inc.     

Prolonged voluntary wheel‐running stimulates neural precursors in the hippocampus and forebrain of adult CD1 mice

Voluntary wheel‐running induces a rapid increase in proliferation and neurogenesis by neural precursors present in the adult rodent hippocampus. In contrast, the responses of hippocampal and other central nervous system neural precursors following longer periods of voluntary physical activity are unclear and are an issue of potential relevance to physical rehabilitation programs. We investigated the effects of a prolonged, 6‐week voluntary wheel‐running paradigm on neural precursors of the CD1 mouse hippocampus and forebrain. Examination of the hippocampus following 6 weeks of running revealed two to three times as many newly born neurons and 60% more proliferating cells when compared with standard‐housed control mice. Among running mice, the number of newly born neurons correlated with the total running distance. To establish the effects of wheel‐running on hippocampal precursors dividing during later stages of the prolonged running regime, BrdU was administered after 3 weeks of running and the BrdU‐retaining cells were analyzed 18 days later. Quantifications revealed that the effects of wheel‐running were maintained in late‐stage proliferating cells, as running mice had two to three times as many BrdU‐retaining cells within the hippocampal dentate gyrus, and these yielded greater proportions of both mature neurons and proliferative cells. The effects of prolonged wheel‐running were also detected beyond the hippocampus. Unlike short‐term wheel‐running, prolonged wheel‐running was associated with higher numbers of proliferating cells within the ventral forebrain subventricular region, a site of age‐associated decreases in neural precursor proliferation and neurogenesis. Collectively, these findings indicate that (i) prolonged voluntary wheel‐running maintains an increased level of hippocampal neurogenesis whose magnitude is linked to total running performance, and (ii) that it influences multiple neural precursor populations of the adult mouse brain. © 2009 Wiley‐Liss, Inc.