Adult Hippocampal Progenitor Cells Research Articles

Assessment of the Behaviors of an In Vitro Brain Model On-Chip under Shockwave Impacts.

Herein we report the assessment of the effects of shockwave (SW) impacts on adult rat hippocampal progenitor cell (AHPC) neurospheres (NSs), which are used as in vitro brain models, for enhancing our understanding of the mechanisms of traumatic brain injury (TBI). The assessment has been achieved by using culture dishes and a new microchip. The microchip allows the chemicals released from the brain models cultured inside the cell culture chamber under SW impacts to diffuse to the nanosensors in adjacent sensor chambers through built-in diffusion barriers, which are used to prevent the cells from entering the sensor chambers, thereby mitigating the biofouling issues of the sensor surface. Experiments showed the negative impact of the SW on the viability, proliferation, and differentiation of the cells within the NSs. A qPCR gene expression analysis was performed and appeared to confirm some of the immunocytochemistry (ICC) results. Finally, we demonstrated that the microchip can be used to monitor lactate dehydrogenase (LDH) released from the AHPC-NSs subjected to SW impacts. As expected, LDH levels changed when AHPC-NSs were injured by SW impacts, verifying this chip can be used for assessing the degrees of injuries to AHPC-NSs by monitoring LDH levels. Taken together, these results suggest the feasibility of using the chip to better understand the interactions between SW impacts and in vitro brain models, paving the way for potentially establishing in vitro TBI models on a chip.

An integrated microfluidic chip for studying the effects of neurotransmitters on neurospheroids.

To improve our understanding of how the central nervous system functions in health and disease, we report the development of an integrated chip for studying the effects of the neurotransmitters dopamine and serotonin on adult rat hippocampal progenitor cell (AHPC) neurospheroids. This chip allows dopamine or serotonin located in one chamber to diffuse to AHPC neurospheroids cultured in an adjacent chamber through a built-in diffusion barrier created by an array of intentionally misaligned micropillars. The gaps among the micropillars are filled with porous poly(ethylene glycol) (PEG) gel to tune the permeability of the diffusion barrier. An electrochemical sensor is also integrated within the chamber where the neurospheroids can be cultured, thereby allowing monitoring of the concentrations of dopamine or serotonin. Experiments show that concentrations of the neurotransmitters inside the neurospheroid chamber can be increased over a period of several hours to over 10 days by controlling the compositions of the PEG gel inside the diffusion barrier. The AHPC neurospheroids cultured in the chip remain highly viable following dopamine or serotonin treatment. Cell proliferation and neuronal differentiation have also been observed following treatment, revealing that the AHPC neurospheroids are a valuable in vitro brain model for neurogenesis research. Finally, we show that by tuning the permeability of diffusion barrier, we can block transfer of Escherichia coli cells across the diffusion barrier, while allowing dopamine or serotonin to pass through. These results suggest the feasibility of using the chip to better understand the interactions between microbiota and brain via the gut-brain axis.

The metabolic regulator USF-1 is involved in the control of affective behaviour in mice

Epidemiological studies indicate a bidirectional association between metabolic disturbances, including obesity and related pathological states, and mood disorders, most prominently major depression. However, the biological mechanisms mediating the comorbid relationship between the deranged metabolic and mood states remain incompletely understood. Here, we tested the hypothesis that the enhanced activation of brown fat tissue (BAT), known to beneficially regulate obesity and accompanying dysfunctional metabolic states, is also paralleled by an alteration of affective behaviour. We used upstream stimulatory factor 1 (USF-1) knock-out (KO) mice as a genetic model of constitutively activated BAT and positive cardiometabolic traits and found a reduction of depression-like and anxiety-like behaviours associated with USF-1 deficiency. Surgical removal of interscapular BAT did not impact the behavioural phenotype of USF-1 KO mice. Further, the absence of USF-1 did not lead to alterations of adult hippocampal neural progenitor cell proliferation, differentiation, or survival. RNA-seq analysis characterised the molecular signature of USF-1 deficiency in the hippocampus and revealed a significant increase in the expression of several members of the X-linked lymphocyte-regulated (xlr) genes, including xlr3b and xlr4b. Xlr genes are the mouse orthologues of the human FAM9 gene family and are implicated in the regulation of dendritic branching, dendritic spine number and morphology. The transcriptional changes were associated with morphological alterations in hippocampal neurons, manifested in reduced dendritic length and complexity in USF-1 KO mice. Collectively these data suggest that the metabolic regulator USF-1 is involved in the control of affective behaviour in mice and that this modulation of mood states is unrelated to USF-1-dependent BAT activation, but reflected in structural changes in the brain.

A Microfluidic Chip for Growth and Characterization of Adult Rat Hippocampal Progenitor Cell Neurospheroids

Adult hippocampal neural stem/progenitor cells (AHPCs), which are self-renewing multipotent progenitors that can differentiate into neurons, astrocytes, and oligodendrocytes, are suitable as a central nervous system (CNS) molecular model, and the formation of 3D AHPC neurospheroids are potentially suitable as an in vitro brain model. In this paper we report a new microfluidic chip to culture AHPC neurospheroids (NSs-AHPC) inside culture-chambers on chip. After cell fixation and immunostaining were conducted, the fluorescence images of NSs-AHPC were analyzed. It has been found that the AHPCs comprising neurospheroids remained highly viable. Cell proliferation and neuronal differentiation have also been observed, indicating the feasibility of NSs-AHPC as the in vitro brain model on chip. Given its simple-to-use, low-cost, and orderly arranged culture-chambers, this type of chip is particularly suitable for culturing and analyzing multiple in vitro brain models in an efficient manner. [2021-0199]

GSK-3β S9A overexpression leads murine hippocampal neural precursors to acquire an astroglial phenotype in vivo.

The addition of new neurons to the existing hippocampal circuitry persists in the adult dentate gyrus (DG). During this process, named adult hippocampal neurogenesis (AHN), adult hippocampal progenitor cells (AHPs) give rise to newborn dentate granule cells (DGCs). The acquisition of a neuronal lineage by AHPs is tightly regulated by numerous signaling molecules and transcription factors. In this regard, glycogen synthase kinase 3β (GSK-3β) is a master regulator of the maturation of AHPs in vitro. Here we analyzed the cell-autonomous effects of overexpressing a constitutively active form of GSK-3β (GSK-3β S9A) in AHPs in vivo. To this end, we stereotaxically injected a GSK-3β S9A-encoding retrovirus (GSK-3β-V5) into the DG of young adult C57BL6/J Ola Hsd female mice and studied the cell lineage acquisition, migratory and marker expression patterns, and the morphological maturation of the infected cells over time. Strikingly, GSK-3β S9A-transduced cells expressed glial fibrillary acidic protein (GFAP) and NG2, thereby acquiring an immature astroglial phenotype, which differed markedly from the neuronal phenotype observed in cells transduced with a control retrovirus that encoded GFP. Accordingly, the morphology and migration patterns of cells transduced by the two retroviruses are remarkably divergent. These observations support the role of GSK-3β as a cornerstone that regulates the balance between new astocytes/neurons generated in the adult murine DG.

Determination of Electrical Stimuli Parameters To Transdifferentiate Genetically Engineered Mesenchymal Stem Cells into Neuronal or Glial Lineages

This study investigated the effect of electrical stimuli parameters using graphene-based devices for the transdifferentiation of genetically engineered brain-derived neurotrophic factor (BDNF) hypersecreting mesenchymal stem cells (BDNF-MSCs) into neuronal or glial lineages. The results suggest that BDNF-MSCs have the tendency to transdifferentiate into both neuronal and Schwann cell (SC)-like phenotypes at lower voltages (25–50 mV). However, as the applied voltage changed from 25 to 100 mV at 50 Hz, the transdifferentiation of BDNF-MSCs yielded more into SC-like phenotypes and resulted in complete transdifferentiation into SC-like phenotypes at 100 mV and 50 Hz. With an increase in voltage to 100 mV, the complete transdifferentiation to SC-like phenotypes also resulted in enhanced paracrine activity leading to total secretion of nerve growth factor (NGF) up to 50 ng/mL with pronounced biological activity, causing neurite extension of 4 μm/cell on PC12-TrkB cells. Moreover, 90% of the transdifferentiated cells demonstrated significant myelination potential. The contact co-culture of BDNF-MSCs with adult hippocampal progenitor cells (AHPCs) in the presence of electrical stimuli resulted in differentiation of BDNF-MSCs into SC-like phenotypes accompanied by synergistic neurite extension of AHPCs. Overall, this study demonstrates the possibility of controlling simultaneous and spatial differentiation of MSCs into selected neuronal and glial lineages at desired ratios via changes in electrical stimuli through graphene-based devices and can contribute to the development of novel cell-based strategies for nervous system rescue and repair. This work evaluates the effect of different electrical stimuli conditions applied through inkjet-printed and laser-annealed graphene-based interdigitated circuits on the differentiation behavior of mesenchymal stem cells. Our results suggested that it is possible to spatially and locally control the differentiation of mesenchymal stem cells into final lineage type (glial or neuronal) by manipulating the electrical stimuli. The future work will include the control of stem cell differentiation and fate commitment in an in vivo model using electrical stimuli.

Cell-based immunofluorescence assay for screening the neurogenesis potential of new drugs in adult hippocampal neural progenitor cells

Preclinical studies have suggested that increased adult neurogenesis in the hippocampus might have potential therapeutic effects for Alzheimer's disease and depression; therefore, it is a target for the treatment of some brain diseases. In this technical communication, we propose a cell-based fluorescence assay to study the neurogenesis of adult hippocampal progenitor cells that can be used for high-throughput screening of drugs promoting neurogenesis. Three fluorescent dyes (DAPI, Alexa Fluor 488, and Alexa Fluor 594) and a fluorescence spectrophotometry reader were used, which confirmed that the mutual interference of the three fluorescent dyes is very low. We used this cell-based fluorescence assay to evaluate the effects of three neurotrophic factors, ciliary neurotrophic factor (CNTF), insulin-like growth factor 1 (IGF-1), and IGF-2 on the promotion of neurogenesis in adult hippocampal neural progenitor cells. The fluorescence intensity ratio of the neuronal marker, class III β-tubulin, to the housekeeping protein, glyceraldehyde 3-phosphate dehydrogenase, or nuclear staining dye, DAPI, in CNTF-treated cells was significantly higher than in control cells. The ratios in IGF-1 and IGF-2-treated cells were slightly higher under higher cell density conditions. These results are consistent with those in previous reports; therefore, this report proved the efficacy of this method. Taken together, the results showed that this simple, rapid, and economical cell-based immunofluorescence assay could be a powerful tool for the rapid screening of drugs that promote adult neurogenesis.

Conditioned media from AICAR-treated skeletal muscle cells increases neuronal differentiation of adult neural progenitor cells

Exercise has profound benefits for brain function in animals and humans. In rodents, voluntary wheel running increases the production of new neurons and upregulates neurotrophin levels in the hippocampus, as well as improving synaptic plasticity, memory function and mood. The underlying cellular mechanisms, however, remain unresolved. Recent research indicates that peripheral organs such as skeletal muscle, liver and adipose tissue secrete factors during physical activity that may influence neuronal function. Here we used an in vitro cell assay and proteomic analysis to investigate the effects of proteins secreted from skeletal muscle cells on adult hippocampal neural progenitor cell (aNPC) differentiation. We also sought to identify the relevant molecules driving these effects. Specifically, we treated rat L6 skeletal muscle cells with the AMP-kinase (AMPK) agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or vehicle (distilled water). We then collected the conditioned media (CM) and fractionated it using high-performance liquid chromatography (HPLC). Treatment of aNPCs with a specific fraction of the AICAR-CM upregulated expression of doublecortin (DCX) and Tuj1, markers of immature neurons. Proteomic analysis of this fraction identified proteins known to be involved in energy metabolism, cell migration, adhesion and neurogenesis. Culturing differentiating aNPCs in the presence of one of the factors, glycolytic enzyme glucose-6-phosphate isomerase (GPI), or AICAR-CM, increased the proportion of neuronal (Tuj1+) and astrocytic, glial fibrillary acidic protein (GFAP+) cells. Our study provides further evidence that proteins secreted from skeletal muscle cells may serve as a critical communication link to the brain through factors that enhance neural differentiation.

Terminal Differentiation of Adult Hippocampal Progenitor Cells Is a Step Functionally Dissociable from Proliferation and Is Controlled by Tis21, Id3 and NeuroD2.

Cell proliferation and differentiation are interdependent processes. Here, we have asked to what extent the two processes of neural progenitor cell amplification and differentiation are functionally separated. Thus, we analyzed whether it is possible to rescue a defect of terminal differentiation in progenitor cells of the dentate gyrus, where new neurons are generated throughout life, by inducing their proliferation and/or their differentiation with different stimuli appropriately timed. As a model we used the Tis21 knockout mouse, whose dentate gyrus neurons, as demonstrated by us and others, have an intrinsic defect of terminal differentiation. We first tested the effect of two proliferative as well as differentiative neurogenic stimuli, one pharmacological (fluoxetine), the other cognitive (the Morris water maze (MWM) training). Both effectively enhanced the number of new dentate gyrus neurons produced, and fluoxetine also reduced the S-phase length of Tis21 knockout dentate gyrus progenitor cells and increased the rate of differentiation of control cells, but neither factor enhanced the defective rate of differentiation. In contrast, the defect of terminal differentiation was fully rescued by in vivo infection of proliferating dentate gyrus progenitor cells with retroviruses either silencing Id3, an inhibitor of neural differentiation, or expressing NeuroD2, a proneural gene expressed in terminally differentiated dentate gyrus neurons. This is the first demonstration that NeuroD2 or the silencing of Id3 can activate the differentiation of dentate gyrus neurons, complementing a defect of differentiation. It also highlights how the rate of differentiation of dentate gyrus neurons is regulated genetically at several levels and that a neurogenic stimulus for amplification of neural stem/progenitor cells may not be sufficient in itself to modify this rate.

Running-Induced Systemic Cathepsin B Secretion Is Associated with Memory Function

Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. Invivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition.

The Mineralocorticoid Agonist Fludrocortisone Promotes Survival and Proliferation of Adult Hippocampal Progenitors.

Glucocorticoid receptor (GR) activation has been shown to reduce adult hippocampal progenitor cell proliferation and neurogenesis. By contrast, mineralocorticoid receptor (MR) signaling is associated with neuronal survival in the dentate gyrus of the hippocampus, and impairment of hippocampal MR has been linked to pathological conditions, such as depression or neurodegenerative disorders. Here, we aimed to further clarify the protective role of MR in adult hippocampal neurons by studying the survival and proliferative effects of the highly potent MR agonist fludrocortisone (Fludro) in adult rat hippocampal progenitor cells (AHPs), along with the associated signaling mechanisms. Fludro, which upregulated MR but not GR expression, increased survival and proliferation and prevented apoptosis in AHPs cultured in growth factor-deprived medium. These effects were blunted by the MR antagonist spironolactone and by high doses of the GR agonist dexamethasone. Moreover, they involved signaling through cAMP/protein kinase A (PKA)/cAMP response element-binding protein, phosphoinositide 3-kinase (PI3K)/Akt and its downstream targets glycogen synthase kinase-3β (GSK-3β) and mammalian target of rapamycin. Furthermore, Fludro attenuated the detrimental effects of amyloid-β peptide 1–42 (Aβ1–42) on cell survival, proliferation, and apoptosis in AHPs, and increased the phosphorylation of both PI3K/Akt and GSK-3β, which was reduced by Aβ1–42. Finally, Fludro blocked Aβ1–42-induced hyperphosphorylation of Tau protein, which is a main feature of Alzheimer’s disease. Overall, these results are the first to show the protective and proliferative role of Fludro in AHPs, suggesting the potential therapeutic importance of targeting MR for increasing hippocampal neurogenesis and for treating neurodegenerative diseases.

Bone marrow-derived mesenchymal stem cells (MSCs) stimulate neurite outgrowth from differentiating adult hippocampal progenitor cells

Abstract Background: Bone marrow-derived mesenchymal stem cells (MSCs) have emerged as beneficial cellular vehicles for nervous system rescue and repair. A better understanding how MSCs are involved in mediating neural

Obestatin promotes proliferation and survival of adult hippocampal progenitors and reduces amyloid-β-induced toxicity

The ghrelin gene-derived peptide obestatin promotes survival in different cell types through a yet undefined receptor; however, its potential neuroprotective activities are still unknown. Here, obestatin effects were investigated on proliferation and survival of adult rat hippocampal progenitor cells (AHPs). Obestatin immunoreactivity was found in AHPs; moreover, obestatin binding to AHPs was displaced by the GLP-1R agonist Ex-4 and antagonist Ex-9. Furthermore, obestatin increased cell proliferation and survival in growth factor deprived medium and inhibited apoptosis; these effects were blocked by Ex-9. The underlying mechanisms involved Gαs/cAMP/PKA/CREB signaling, phosphorylation of ERK1/2 and PI3K/Akt, and the PI3K targets GSK-3β/β-catenin and mTOR. Obestatin also counteracted Aβ1-42-induced detrimental effects through inhibition of GSK-3β activity and Tau hyperphosphorylation, main hallmarks of neuronal death in Alzheimer's disease. These findings indicate a novel protective role for obestatin in AHPs and candidate this peptide as potential therapeutic target for increasing neurogenesis and for approaching neurodegenerative disorders.

Melatonin attenuates methamphetamine-induced inhibition of proliferation of adult rat hippocampal progenitor cells in vitro.

Methamphetamine (METH) is an extremely addictive stimulatory drug. A recent study suggested that METH may cause an impairment in the proliferation of hippocampal neural progenitor cells, but the underlying mechanism of this effect remains unknown. Blood and cerebrospinal levels of melatonin derive primarily from the pineal gland, and that performs many biological functions. Our previous study demonstrated that melatonin promotes the proliferation of progenitor cells originating from the hippocampus. In this study, hippocampal progenitor cells from adult Wistar rats were used to determine the effects of METH on cell proliferation and the mechanisms underlying these effects. We investigated the effects of melatonin on the METH-induced alteration in cell proliferation. The results demonstrated that 500 μm METH induced a decrease (63.0%) in neurosphere cell proliferation and altered the expression of neuronal phenotype markers in the neurosphere cell population. Moreover, METH induced an increase in the protein expression of the tumor suppressor p53 (124.4%) and the cell cycle inhibitor p21(CIP) (1) (p21) (128.1%), resulting in the accumulation of p21 in the nucleus. We also found that METH altered the expression of the N-methyl-d-aspartate (NMDA) receptor subunits NR2A (79.6%) and NR2B (126.7%) and Ca(2+) /calmodulin-dependent protein kinase II (CAMKII) (74.0%). In addition, pretreatment with 1 μm melatonin attenuated the effects induced by METH treatment. According to these results, we concluded that METH induces a reduction in cell proliferation by upregulating the cell cycle regulators p53/p21 and promoting the accumulation of p21 in the nucleus and that melatonin ameliorates these negative effects of METH.

Multipotent adult hippocampal progenitor cells maintained as neurospheres favor differentiation toward glial lineages.

Adult hippocampal progenitor cells (AHPCs) are generally maintained as a dispersed monolayer population of multipotent neural progenitors. To better understand cell-cell interactions among neural progenitors and their influences on cellular characteristics, we generated free-floating cellular aggregates, or neurospheres, from the adherent monolayer population of AHPCs. Results from in vitro analyses demonstrated that both populations of AHPCs were highly proliferative under maintenance conditions, but AHPCs formed in neurospheres favored differentiation along a glial lineage and displayed greater migrational activity than the traditionally cultured AHPCs. To study the plasticity of AHPCs from both populations in vivo, we transplanted green fluorescent protein (GFP)-expressing AHPCs via intraocular injection into the developing rat eyes. Both AHPC populations were capable of surviving and integrating into developing host central nervous system, but considerably more GFP-positive cells were observed in the retinas transplanted with neurosphere AHPCs, compared to adherent AHPCs. These results suggest that the culture configuration during maintenance for neural progenitor cells (NPCs) influences cell fate and motility in vitro as well as in vivo. Our findings have implication for understanding different cellular characteristics of NPCs according to distinct intercellular architectures and for developing cell-based therapeutic strategies using lineage-committed NPCs.

Endogenous expression of FAD-linked PS1 impairs proliferation, neuronal differentiation and survival of adult hippocampal progenitors

BackgroundAlzheimer’s disease (AD) is characterized by progressive memory loss and impaired cognitive function. Early-onset familial forms of the disease (FAD) are caused by inheritance of mutant genes encoding presenilin 1 (PS1) variants. We have demonstrated that prion promoter (PrP)-driven expression of human FAD-linked PS1 variants in mice leads to impairments in environmental enrichment (EE)-induced adult hippocampal neural progenitor cell (AHNPC) proliferation and neuronal differentiation, and have provided evidence that accessory cells in the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes, in vivo. While of significant interest, these latter studies relied on transgenic mice that express human PS1 variant transgenes ubiquitously and at high levels, and the consequences of wild type or mutant PS1 expressed under physiologically relevant levels on EE-mediated AHNPC phenotypes has not yet been tested.ResultsTo assess the impact of mutant PS1 on EE-induced AHNPC phenotypes when expressed under physiological levels, we exposed adult mice that constitutively express the PSEN1 M146V mutation driven by the endogenous PSEN1 promoter (PS1 M146V “knock-in” (KI) mice) to standard or EE-housed conditions. We show that in comparison to wild type PS1 mice, AHNPCs in mice carrying homozygous (PS1M146V/M146V) or heterozygous (PS1M146V/+) M146V mutant alleles fail to exhibit EE-induced proliferation and commitment towards neurogenic lineages. More importantly, we report that the survival of newborn progenitors are diminished in PS1 M146V KI mice exposed to EE-conditions compared to respective EE wild type controls.ConclusionsOur findings reveal that expression at physiological levels achieved by a single PS1 M146V allele is sufficient to impair EE-induced AHNPC proliferation, survival and neuronal differentiation, in vivo. These results and our finding that microglia expressing a single PS1 M146V allele impairs the proliferation of wild type AHNPCs in vitro argue that expression of mutant PS1 in the AHNPC niche impairs AHNPCs phenotypes in a dominant, non-cell autonomous manner.

Mutant presenilin 1 expression in excitatory neurons impairs enrichment-mediated phenotypes of adult hippocampal progenitor cells

Inheritance of mutant presenilin 1 genes (PSEN1) encoding presenilin 1 (PS1)variants causes autosomal dominant forms of familial Alzheimer's disease (FAD). We previously reported that ubiquitous expression of FAD-linked PS1 variants in mice impairs environmental enrichment (EE)-induced proliferation and neuronal commitment of adult hippocampal neural progenitor cells (AHNPCs). Notably, the self-renewal and differentiation properties of cultured AHNPCs expressing either human PS1 wild-type or PS1 variants were identical, suggesting that accessory cells within the hippocampal niche expressing PS1 variants may modulate AHNPC phenotypes in vivo. We now report that nontransgenic mouse AHNPCs transduced with retroviruses harboring cDNAs that encode either human PS1 wild-type or FAD-linked PS1 variants show no differences in EE-mediated proliferation and neuronal differentiation. Moreover, conditional inactivation of a mutant PS1 transgene in type-1 primary progenitor cells failed to rescue impairments of EE-induced proliferation, survival, or neurogenesis. In contrast, conditional inactivation of the mutant PS1 transgene in excitatory neurons of the mouse forebrain largely rescued the deficits in EE-induced proliferation and survival of AHNPCs, but not their differentiation into mature neuronal phenotypes. These results persuasively argue for a noncell autonomous effect of FAD-linked PS1 mutants on EE-mediated adult hippocampal neurogenesis.

Influence of endogenous ciliary neurotrophic factor on neural differentiation of adult rat hippocampal progenitors.

Ciliary neurotrophic factor is the only known neurotrophic factor that can promote differentiation of hippocampal neural progenitor cells to glial cells and neurons in adult rats. This process is similar to spontaneous differentiation. Therefore, ciliary neurotrophic factor may be involved in spontaneous differentiation of neural stem cells. To verify this hypothesis, the present study isolated neural progenitor cells from adult male rats and cultured them in vitro. Results showed that when neural progenitor cells were cultured in the absence of mitogen fibroblast growth factor-2 or epidermal growth factor, they underwent spontaneous differentiation into neurons and glial cells. Western blot and immunocytochemical staining showed that exogenous ciliary neurotrophic factor strongly induced adult hippocampal progenitor cells to differentiate into neurons and glial cells. Moreover, passage 4 adult hippocampal progenitor cells expressed high levels of endogenous ciliary neurotrophic factor, and a neutralizing antibody against ciliary neurotrophic factor prevented the spontaneous neuronal and glial differentiation of adult hippocampal progenitor cells. These results suggest that the spontaneous differentiation of adult hippocampal progenitor cells is mediated partially by endogenous ciliary neurotrophic factor.

Fibroblast growth factor-2 counteracts the effect of ciliary neurotrophic factor on spontaneous differentiation in adult hippocampal progenitor cells

Neural stem/progenitor cells (NSCs) can spontaneously differentiate into neurons and glial cells in the absence of mitogen fibroblast growth factor-2 (FGF-2) or epidermal growth factor (EGF) in medium and the spontaneous differentiation of NSCs is mediated partially by endogenous ciliary neurotrophic factor (CNTF). This study examined the relationship of FGF-2 and CNTF in the spontaneous differentiation of adult hippocampal progenitor cells (AHPs). AHPs were cultured in the medium containing different concentration of FGF-2 (1-100 ng/mL). Western blotting and immunofluorescence staining were applied to detect the expression of the astrocytic marker GFAP, the neuronal marker Tuj1, the oligodendrocytic marker CNPase and, Nestin, the marker of AHPs. The expression of endogenous CNTF in AHPs at early (passage 4) and late stage (passage 22) was also measured by Western blotting. The results showed that FGF-2 increased the expression of Nestin, dramatically inhibited the expression of GFAP and Tuj1 and slightly suppressed the expression of CNPase. FGF-2 down-regulated the expression of endogenous CNTF in AHPs at both early (passage 4) and late stage (passage 22). These results suggested that FGF-2 could inhibit the spontaneous differentiation of cultured AHPs by negatively regulating the expression of endogenous CNTF in AHPs.

Green tea epigallocatechin‐3‐gallate (EGCG) promotes neural progenitor cell proliferation and sonic hedgehog pathway activation during adult hippocampal neurogenesis

Adult hippocampal neurogenesis is a lifelong feature of brain plasticity that appears to be critically involved in adult brain function and neurological disease. Recent studies suggest that (-)-epigallocatechin-3-gallate (EGCG), which is the main polyphenolic constituent of green tea, may be used for the prevention and treatment of various neurodegenerative diseases. We hypothesized that EGCG promotes adult neurogenesis, which may be beneficial to hippocampus-dependent learning and memory. We show that EGCG treatment significantly increased the number of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in adult hippocampal neural progenitor cell (NPC) cultures and in the dentate gyrus of adult mice. Meanwhile, EGCG markedly improved spatial cognition in mice. These events are associated with the sonic hedgehog (Shh) signaling pathway. We observed that EGCG triggered a robust upregulation of Shh receptor (Patched) mRNA and protein expression in cultured NPCs as well as an upregulation of the downstream Shh transcriptional target Gli1. These changes were further confirmed in the hippocampus of mice administered EGCG. The blockage of the Shh signal with the pharmacological inhibitor cyclopamine attenuated EGCG-induced hippocampal neurogenesis. Our results provide strong evidence that EGCG enhances adult hippocampal neurogenesis.