Toll-like receptors modulate adult hippocampal neurogenesis

Neurogenesis - the formation of new neurons in the adult brain - is considered to be one of the mechanisms by which the brain maintains its lifelong plasticity in response to extrinsic and intrinsic changes. The mechanisms underlying the regulation of neurogenesis are largely unknown. Here, we show that Toll-like receptors (TLRs), a family of highly conserved pattern-recognizing receptors involved in neural system development in Drosophila and innate immune activity in mammals, regulate adult hippocampal neurogenesis. We show that TLR2 and TLR4 are found on adult neural stem/progenitor cells (NPCs) and have distinct and opposing functions in NPC proliferation and differentiation both in vitro and in vivo. TLR2 deficiency in mice impaired hippocampal neurogenesis, whereas the absence of TLR4 resulted in enhanced proliferation and neuronal differentiation. In vitro studies further indicated that TLR2 and TLR4 directly modulated self-renewal and the cell-fate decision of NPCs. The activation of TLRs on the NPCs was mediated via MyD88 and induced PKCalpha/beta-dependent activation of the NF-kappaB signalling pathway. Thus, our study identified TLRs as players in adult neurogenesis and emphasizes their specified and diverse role in cell renewal.     

Aging brain microenvironment decreases hippocampal neurogenesis through Wnt-mediated survivin signaling

Accumulating evidence suggests that adult hippocampal neurogenesis relies on the controlled and continued proliferation of neural progenitor cells (NPCs). With age, neurogenesis decreases through mechanisms that remain unclear but are believed to involve changes in the NPC microenvironment. Here, we provide evidence that NPC proliferation in the adult brain is in part regulated by astrocytes via Wnt signaling and that this cellular cross-talk is modified in the aging brain, leading to decreased proliferation of NPCs. Furthermore, we show that astrocytes regulate the NPC cell cycle by acting on the expression levels of survivin, a known mitotic regulator. Among cell cycle genes found down-regulated in aged NPCs, survivin was the only one that restored NPC proliferation in the aged brain. Our results provide a mechanism for the gradual loss of neurogenesis in the brain associated with aging and suggest that targeted modulation of survivin expression directly or through Wnt signaling could be used to stimulate adult neurogenesis.     

Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway

In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called ‘vascular niche’. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.     

GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.     

Blockade of TrkB but not p75NTR activates a subpopulation of quiescent neural precursor cells and enhances neurogenesis in the adult mouse hippocampus

Brain‐derived neurotrophic factor (BDNF) signaling plays a major role in the regulation of hippocampal neurogenesis in the adult brain. While the majority of studies suggest that this is due to its effect on the survival and differentiation of newborn neurons, it remains unclear whether this signaling directly regulates neural precursor cell (NPC) activity and which of its two receptors, TrkB or the p75 neurotrophin receptor (p75^NTR) mediates this effect. Here, we examined both the RNA and protein expression of these receptors and found that TrkB but not p75^NTR receptors are expressed by hippocampal NPCs in the adult mouse brain. Using a clonal neurosphere assay, we demonstrate that pharmacological blockade of TrkB receptors directly activates a distinct subpopulation of NPCs. Moreover, we show that administration of ANA‐12, a TrkB‐selective antagonist, in vivo either by systemic intraperitoneal injection or by direct infusion within the hippocampus leads to an increase in the production of new neurons. In contrast, we found that NPC‐specific knockout of p75^NTR had no effect on the proliferation of NPCs and did not alter neurogenesis in the adult hippocampus. Collectively, these results demonstrate a novel role of TrkB receptors in directly regulating the activity of a subset of hippocampal NPCs and suggest that the transient blockade of these receptors could be used to enhance adult hippocampal neurogenesis.     

Elevated Homocysteine by Levodopa Is Detrimental to Neurogenesis in Parkinsonian Model

Background

Modulation of neurogenesis that acts as an endogenous repair mechanism would have a significant impact on future therapeutic strategies for Parkinson’s disease (PD). Several studies demonstrated dopaminergic modulation of neurogenesis in the subventricular zone (SVZ) of the adult brain. Levodopa, the gold standard therapy for PD, causes an increase in homocysteine levels that induces neuronal death via N-methyl-D-aspartate (NMDA) receptor. The present study investigated whether elevated homocysteine by levodopa treatment in a parkinsonian model would modulate neurogenesis via NMDA receptor signal cascade and compared the effect of levodopa and pramipexol (PPX) on neurogenic activity.

Methodology/Principal Findings

Neurogenesis was assessed in vitro using neural progenitor cells (NPCs) isolated from the SVZ and in vivo with the BrdU-injected animal model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Modulation of homocysteine levels was evaluated using co-cultures of NPCs and astrocytes and PD animals. Immunochemical and Western blot analyses were used to measure neurogenesis and determine the cell death signaling. Levodopa treatment increased release of homocysteine on astrocytes culture media as well as in plasma and brain of PD animals. Increased homocysteine by levodopa led to increased apoptosis of NPCs through the NMDA receptor-dependent the extracellular signal-regulated kinase (ERK) signaling pathways. The administration of a NMDA antagonist significantly attenuated apoptotic cell death in levodopa-treated NPCs and markedly increased the number of BrdU-positive cells in the SVZ of levodopa-treated PD animals. Comparative analysis revealed that PPX treatment significantly increased the number of NPCs and BrdU-positive cells in the SVZ of PD animals compared to levodopa treatment. Our present study demonstrated that increased homocysteine by levodopa has a detrimental effect on neurogenesis through NMDA receptor-mediated ERK signaling pathway.

Conclusions/Significance

Modulation of levodopa-induced elevated homocysteine by NMDA antagonist or dopamine agonist has a clinical relevance for PD treatment in terms of adult neurogenesis.     

Knockout of Atg5 delays the maturation and reduces the survival of adult-generated neurons in the hippocampus

Autophagy is an evolutionarily conserved lysosomal degradation pathway that plays important roles in cell maintenance, expansion and differentiation. Removal of genes essential for autophagy from embryonic neural stem and precursor cells reduces the survival and inhibits neuronal differentiation of adult-generated neurons. No study has modified autophagy within the adult precursor cells, leaving the cell-autonomous role of autophagy in adult neurogenesis unknown. Here we demonstrate that autophagic flux exists in the adult dividing progenitor cells and their progeny in the dentate gyrus. To investigate the role of autophagy in adult hippocampal neurogenesis, we genetically deleted Autophagy-related gene 5 (Atg5) that reduced autophagic flux and the survival of the progeny of dividing progenitor cells. This significant reduction in survival of adult-generated neurons is accompanied by a delay in neuronal maturation, including a transient reduction in spine density in the absence of a change in differentiation. The delay in cell maturation and loss of progeny of the Atg5-null cells was not present in mice that lacked the essential pro-apoptotic protein Bax (Bcl-2-associated X protein), suggesting that Atg5-deficient cells die through a Bax-dependent mechanism. In addition, there was a loss of Atg5-null cells following exposure to running, suggesting that Atg5 is required for running-induced increases in neurogenesis. These findings highlight the cell-autonomous requirement of Atg5 in the survival of adult-generated neurons.In the adult brain, neurogenesis allows for the continuous development of adult-generated neurons in response to physiological and pathological stimuli. The neural progenitor cells (NPCs) within the neurogenic niche of the subventricular zone (SVZ) and subgranular zone (SGZ) give rise to adult-generated neurons within the olfactory bulb and dentate, respectively.^{1, 2, 3} The ability of the NPCs to proliferate, differentiate and integrate into circuitry to modify behavior makes understanding these cells and the factors that regulate these processes critical to develop new therapies. This is especially important for a number of diseases such as neurodegenerative diseases including Parkinson''s and Huntington''s diseases that are associated with reduced adult neurogenesis, as well as regenerative medicine strategies for recovery after stroke.^{4, 5, 6}Two groups have found that in vivo macroautophagy (hereafter referred to as autophagy) can regulate adult neurogenesis by examining the effect of deleting autophagy-related genes (Atgs). Yazdankhah et al.⁷ found that Ambra1 and Beclin1 heterozygous embryonic knockout mice have less proliferating NPCs in the SVZ and an associated reduction in neurogenesis in the olfactory bulb. Wang et al.⁸ found that conditional removal of FIP200 (focal adhesion kinase (FAK) family interacting protein of M_r 200 K, also known as ULK1, an Atg1 homologue-interacting protein) from embryonic NPCs progressively depletes the number of postnatal NPCs, as well as reduces neurogenesis and increases astrogenesis. In contrast in the embryo, Lv et al.⁹ showed that a specific knockdown of the Autophagy-related gene 5 (Atg5) increases proliferation and inhibits neuronal differentiation of embryonic NPCs during cortical development. These data suggest that embryonic and adult NPCs are altered when autophagy-related genes are deleted in the embryo. However, it remains unknown whether autophagy, independent of effects in the embryo, is directly required for NPCs and their progeny in the adult.Here we tested the functional role of autophagy specifically in the adult brain by removing Atg5 from dividing NPCs. We found that autophagic flux occurs in adult NPCs and that removal of Atg5 is associated with a reduction in autophagic flux. In addition, we find that Atg5-null cells have a significant reduction in survival, as well as a delay in neuronal maturation. The reduction in neurogenesis occurred in the absence of altering proliferation or cell lineage. Furthermore, removal of Bax (Bcl-2-associated X protein) restored neurogenesis in the absence of Atg5, implicating Bax functions downstream of Atg5 to regulate the survival of adult-generated neurons. Finally, we showed that Atg5-dependent signaling is required for running-induced increases in the survival of the adult developing NPCs.     

Regulation of neurogenesis by neurotrophins: implications in hippocampus-dependent memory

Neurogenesis, the generation of new neurons from neural precursor cells (NPCs), is a multi-step process that includes the proliferation of NPCs, fate determination, migration, and neuronal maturation. Neurogenesis is regulated by several extrinsic factors,such as enriched environment, physical exercise, hormones and stress, many of which also induce the expression of neurotrophins.In this review, we summarize studies on the role of neurotrophins in neurogenesis during development and in adults.We discuss the functional significance of neurogenesis in learning and memory, and how neurotrophins regulate this process.In this context, we describe recent experiments linking adult neurogenesis to long-term synaptic plasticity in the hippocampal dentate gyrus. Further study of the relationship between neurotrophins, adult neurogenesis and dentate synaptic plasticity might provide new insights into the mechanisms by which gene-environment interactions control cognition and brain plasticity.     

Aging neural progenitor cells have decreased mitochondrial content and lower oxidative metabolism

Although neurogenesis occurs in discrete areas of the adult mammalian brain, neural progenitor cells (NPCs) produce fewer new neurons with age. To characterize the molecular changes that occur during aging, we performed a proteomic comparison between primary-cultured NPCs from the young adult and aged mouse forebrain. This analysis yielded changes in proteins necessary for cellular metabolism. Mitochondrial quantity and oxygen consumption rates decrease with aging, although mitochondrial DNA in aged NPCs does not have increased mutation rates. In addition, aged cells are resistant to the mitochondrial inhibitor rotenone and proliferate in response to lowered oxygen conditions. These results demonstrate that aging NPCs display an altered metabolic phenotype, characterized by a coordinated shift in protein expression, subcellular structure, and metabolic physiology.     

Lithium enhances the neuronal differentiation of neural progenitor cells in vitro and after transplantation into the avulsed ventral horn of adult rats through the secretion of brain-derived neurotrophic factor

This study was undertaken to elucidate the molecular mechanisms by which lithium regulates the development of spinal cord-derived neural progenitor cells (NPCs) in vitro and after transplanted in vivo . Our results show that lithium at the therapeutic concentration significantly increases the proliferation and neuronal differentiation of NPCs in vitro. Specific ELISAs, western blotting, and quantitative real-time RT-PCR assays demonstrate that lithium treatment significantly elevates the expression and production of brain-derived neurotrophic factor (BDNF) by NPCs in culture. Application of a BDNF neutralizing antibody in culture leads to a marked reduction in the neurogenesis of lithium-treated NPCs to the control level. However, it shows no effects on the proliferation of lithium-treated NPCs. These findings suggest that the BDNF pathway is possibly involved in the supportive role of lithium in inducing NPC neurogenesis but not proliferation. This study also provides evidence that lithium is able to elevate the neuronal generation and BDNF production of NPCs after transplantation into the adult rat ventral horn with motoneuron degeneration because of spinal root avulsion, which highlights the therapeutic potential of lithium in cell replacement strategies for spinal cord injury because of its ability to promote neuronal differentiation and BDNF production of grafted NPCs in the injured spinal cord.     

The dark side of BrdU in neural stem cell biology: detrimental effects on cell cycle, differentiation and survival

5-Bromo-2'-deoxyuridin (BrdU) is frequently used in anaylsis of neural stem cell biology, in particular to label and to fate-map dividing cells. However, up to now, only a few studies have addressed the question as to whether BrdU labeling per se affects the cells to be investigated. Here, we focused on the potential impact of BrdU on neurosphere cultures derived from the adult rat brain and on proliferation of progenitors in vivo. In vitro, neurospheres were pulsed for 48?h with BrdU, and cell proliferation, cell cycle, differentiation, survival and adhesion properties were subsequently analyzed. BrdU inhibited the expansion of neural progenitors as assessed by MTS assay and increased the fraction of cells in the G0/G1-phase of the cell cycle. Moreover, BrdU increased cell death and dose-dependently induced adherence of NPCs. Cell adherence was accompanied by a reduced amount of active matrix-metalloproteinase-2 (MMP-2). Furthermore, BrdU repressed neuronal and oligodendroglial differentiation, whereas astroglial fate was not affected. In contrast to the in vitro situation, BrdU apparently did not influence endogenous proliferation of NPCs or neurogenesis in concentrations that are typically used for labeling of neural progenitors in vivo. Our results reveal so far uncharacterized effects of BrdU on adult NPCs. We conclude that, because of its ubiquitous use in stem cell biology, any potential effect of BrdU of NPCs has to be scrutinized prior to interpretation of data.     

局灶性脑缺血后海马齿状回神经发生及其与血管内皮生长因子关系的研究

目的研究成年大鼠局灶性脑缺血后海马齿状回（DG）神经发生的情况及其与血管内皮生长因子（VEGF）的关系,探讨脑缺血后神经发生及其调控机制。方法通过大脑中动脉阻断法（MCAO）建立大鼠局灶性脑缺血模型,以5-溴-2-脱氧尿核苷（BrdU）标记增殖的神经前体细胞（NPCs）,用免疫组化及免疫荧光双标记法动态检测脑缺血后不同时间DG神经细胞增殖及其分化,同时观察增殖细胞表达VEGF及其受体情况。结果与对照组相比,缺血侧DG的BrdU阳性细胞数在脑缺血后1d开始增加,7d达高峰,28d接近正常水平;BrdU/TuJ1、BrdU/MAP-2阳性双标细胞数在脑缺血后14d开始增加,28d达高峰;BrdU/GFAP阳性双标细胞数则无明显变化;增殖的BrdU阳性细胞同时表达VEGF及其受体FLK-1。结论大鼠局灶性脑缺血可激活DG自体NPCs原位增殖、分化,增殖的细胞同时表达VEGF及其受体可能是脑缺血后神经发生增强的调节机制之一。     

Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naïve cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.     

Functional convergence of neurons generated in the developing and adult hippocampus

The dentate gyrus of the hippocampus contains neural progenitor cells (NPCs) that generate neurons throughout life. Developing neurons of the adult hippocampus have been described in depth. However, little is known about their functional properties as they become fully mature dentate granule cells (DGCs). To compare mature DGCs generated during development and adulthood, NPCs were labeled at both time points using retroviruses expressing different fluorescent proteins. Sequential electrophysiological recordings from neighboring neurons of different ages were carried out to quantitatively study their major synaptic inputs: excitatory projections from the entorhinal cortex and inhibitory afferents from local interneurons. Our results show that DGCs generated in the developing and adult hippocampus display a remarkably similar afferent connectivity with regard to both glutamate and GABA, the major neurotransmitters. We also demonstrate that adult-born neurons can fire action potentials in response to an excitatory drive, exhibiting a firing behavior comparable to that of neurons generated during development. We propose that neurons born in the developing and adult hippocampus constitute a functionally homogeneous neuronal population. These observations are critical to understanding the role of adult neurogenesis in hippocampal function.     

Non-cell-autonomous effects of presenilin 1 variants on enrichment-mediated hippocampal progenitor cell proliferation and differentiation

Presenilin 1 (PS1) regulates environmental enrichment (EE)-mediated neural progenitor cell (NPC) proliferation and neurogenesis in the adult hippocampus. We now report that transgenic mice that ubiquitously express human PS1 variants linked to early-onset familial Alzheimer's disease (FAD) neither exhibit EE-induced proliferation, nor neuronal lineage commitment of NPCs. Remarkably, the proliferation and differentiation of cultured NPCs from standard-housed mice expressing wild-type PS1 or PS1 variants are indistinguishable. On the other hand, wild-type NPCs cocultured with primary microglia from mice expressing PS1 variants exhibit impaired proliferation and neuronal lineage commitment, phenotypes that are recapitulated with mutant microglia conditioned media in which we detect altered levels of selected soluble signaling factors. These findings lead us to conclude that factors secreted from microglia play a central role in modulating hippocampal neurogenesis, and argue for non-cell-autonomous mechanisms that govern FAD-linked PS1-mediated impairments in adult hippocampal neurogenesis.     

Ion channels in postnatal neurogenesis

Neural stem and progenitor cells (NSC/NPCs) are unspecialized cells found in the adult peri-ventricular and sub-granular zones that are capable of self-renewal, migration, and differentiation into new neurons through the remarkable process of postnatal neurogenesis. We are now beginning to understand that the concerted action of ion channels, multi-pass transmembrane proteins that allow passage of ions across otherwise impermeable cellular membranes tightly regulate this process. Specific ion channels control proliferation, differentiation and survival. Furthermore, they have the potential to be highly selective drug targets due to their complex structures. As such, these proteins represent intriguing prospects for control and optimization of postnatal neurogenesis for neural regeneration following brain injury or disease. Here, we concentrate on ion channels identified in adult ventricular zone NSC/NPCs that have been found to influence the stages of neurogenesis. Finally, we outline the potential of these channels to elicit repair, and highlight the outstanding challenges.     

Ion channels in postnatal neurogenesis: potential targets for brain repair

Neural stem and progenitor cells (NSC/NPCs) are unspecialized cells found in the adult peri-ventricular and sub-granular zones that are capable of self-renewal, migration, and differentiation into new neurons through the remarkable process of postnatal neurogenesis. We are now beginning to understand that the concerted action of ion channels, multi-pass transmembrane proteins that allow passage of ions across otherwise impermeable cellular membranes tightly regulate this process. Specific ion channels control proliferation, differentiation and survival. Furthermore, they have the potential to be highly selective drug targets due to their complex structures. As such, these proteins represent intriguing prospects for control and optimization of postnatal neurogenesis for neural regeneration following brain injury or disease. Here, we concentrate on ion channels identified in adult ventricular zone NSC/NPCs that have been found to influence the stages of neurogenesis. Finally, we outline the potential of these channels to elicit repair, and highlight the outstanding challenges.     

BAG-1 is preferentially expressed in neuronal precursor cells of the adult mouse brain and regulates their proliferation in vitro

BAG-1 protein has been well characterized as necessary for proper neuronal development. However, little is known about the function of BAG-1 in the adult brain. In this work, the expression and localization of BAG-1 in the mature mouse brain was studied. The levels of both BAG-1 isoforms decrease significantly in the brain during development. BAG-1 was found preferentially expressed in Neuronal Precursor Cells (NPCs) in the two major niches of neurogenesis. Lentiviral mediated overexpression of BAG-1 increased the proliferation rate of cultured NPCs. In addition, depletion of BAG-1 from NPCs induced a decrease in NPCs proliferation in the presence of a stress hormone, corticosterone. These data suggest a role for BAG-1 in mechanisms of neurogenesis in the adult mouse brain.