Adolescent binge alcohol exposure alters hippocampal progenitor cell proliferation in rats: Effects on cell cycle kinetics

The endocannabinoid system is known to regulate neural progenitor (NP) cell proliferation and neurogenesis. In particular, CB(2) cannabinoid receptors have been shown to promote NP proliferation. As CB(2) receptors are not expressed in differentiated neurons, CB(2)-selective agonists are promising candidates to manipulate NP proliferation and indirectly neurogenesis by overcoming the undesired psychoactive effects of neuronal CB(1) cannabinoid receptor activation. Here, by using NP cells, brain organotypic cultures, and in vivo animal models, we investigated the signal transduction mechanism involved in CB(2) receptor-induced NP cell proliferation and neurogenesis. Exposure of hippocampal HiB5 NP cells to the CB(2) receptor-selective agonist HU-308 led to the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin complex 1 (mTORC1) pathway, which, by inhibiting its downstream target p27Kip1, induced NP proliferation. Experiments conducted with the CB(2) receptor-selective antagonist SR144528, inhibitors of the PI3K/Akt/mTORC1 axis, and CB(2) receptor transient-transfection vector further supported that CB(2) receptors control NP cell proliferation via activation of mTORC1 signaling. Likewise, CB(2) receptor engagement induced cell proliferation in an mTORC1-dependent manner both in embryonic cortical slices and in adult hippocampal NPs. Thus, HU-308 increased ribosomal protein S6 phosphorylation and 5-bromo-2'-deoxyuridine incorporation in wild-type but not CB(2) receptor-deficient NPs of the mouse subgranular zone. Moreover, adult hippocampal NP proliferation induced by HU-308 and excitotoxicity was blocked by the mTORC1 inhibitor rapamycin. Altogether, these findings provide a mechanism of action and a rationale for the use of nonpsychotomimetic CB(2) receptor-selective ligands as a novel strategy for the control of NP cell proliferation and neurogenesis.

We previously demonstrated that proliferation of endogenous neural progenitor cells is enhanced by cerebral ischemia and that phosphatidylinositol 3-kinase (PI3-K)/Akt-dependent glycogen synthase kinase (GSK)-3β signaling is involved in ischemia-induced neurogenesis. It is important to learn more about the regulation of proliferation and differentiation of neural progenitor cells under ischemic conditions, as such knowledge that may serve as the basis for the development of new therapeutic approaches for stroke. However, it remains to be addressed whether a change in that signaling pathway is induced in neural progenitor cells. We prepared neural progenitor cells by using the neurosphere method and conducted experiments to determine the relative contributions of the PI3-K/Akt-dependent GSK-3β signaling pathway to the proliferation and differentiation of neural progenitor cells under the hypoxic condition in vitro. We showed that hypoxic exposure induced the proliferation of neural progenitor cells. This proliferation was accompanied by phosphorylation of Akt and GSK-3β at its Ser9. Furthermore, treatment with a PI3-K inhibitor decreased the hypoxia-induced phosphorylation of GSK-3β and proliferation of neural progenitor cells. Furthermore, hypoxic exposure enhanced the differentiation of neural progenitor cells, and this increased differentiation was not affected by treatment with the PI3-K inhibitor. Although the expression of NeuroD1 mRNA during cell differentiation was also enhanced by hypoxic exposure, this increased expression was not affected by treatment with the PI3-K inhibitor. Our findings suggest that the PI3K/Akt-dependent GSK-3β signaling pathway was involved in the proliferation of neural progenitor cells under a pathologic condition, such as hypoxia and/or cerebral ischemia in vivo.

Excessive alcohol consumption during adolescence remains a significant health concern as alcohol drinking during adolescence increases the likelihood of an alcohol use disorder in adulthood by fourfold. Binge drinking in adolescence is a particular problem as binge-pattern consumption is the biggest predictor of neurodegeneration from alcohol and adolescents are particularly susceptible to the damaging effects of alcohol. The adolescent hippocampus, in particular, is highly susceptible to alcohol-induced structural and functional effects, including volume and neuron loss. However, hippocampal structure and function may recover with abstinence and, like in adults, a reactive burst in hippocampal neurogenesis in abstinence may contribute to that recovery. As the mechanism of this reactive neurogenesis is not known, the current study investigated potential mechanisms of reactive neurogenesis in binge alcohol exposure in adolescent, male rats. In a screen for cell cycle perturbation, a dramatic increase in the number of cells in all phases of the cycle was observed at 7 days following binge ethanol exposure as compared to controls. However, the proportion of cells in each phase was not different between ethanol-exposed rats and controls, indicating that cell cycle dynamics are not responsible for the reactive burst in neurogenesis. Instead, the marked increase in hippocampal proliferation was shown to be due to a twofold increase in proliferating progenitor cells, specifically an increase in cells colabeled with the progenitor cell marker Sox2 and S-phase (proliferation) marker, BrdU, in ethanol-exposed rats. To further characterize the individual subtypes of neural progenitor cells (NPCs) affected by adolescent binge ethanol exposure, a fluorescent quadruple labeling technique was utilized to differentiate type 1, 2a, 2b, and 3 progenitor cells simultaneously. At one week into abstinence, animals in the ethanol exposure groups had an increase in proliferating type 2 (intermediate progenitors) and type 3 (neuroblast) progenitors but not type 1 neural stem cells. These results together suggest that activation of type 2 NPCs out of quiescence is likely the primary mechanism for reactive hippocampal neurogenesis following adolescent alcohol exposure.

Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. Using the ReNcell CX (Millipore, Billerica, MA) human neural progenitor cell line, the authors investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, the authors assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of inositol-1,4,5-trisphosphate or ryanodine receptor activation to these processes. Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate and promoted glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with inositol-1,4,5-trisphosphate or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane-preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation, and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized inositol-1,4,5-trisphosphate and/or ryanodine receptors.

Neural stem cells (NSCs) are undifferentiated, primitive cells with important potential applications including the replacement of neural tissue lost due to neurodegenerative diseases, including Parkinson's disease, as well as brain and spinal cord injuries, including stroke. We have developed methods to rapidly expand populations of mammalian stem and progenitor cells in neurosphere cultures. In the present study, flow cytometry was used in order to understand cell cycle activation and proliferation of neural stem and progenitor cells in suspension bioreactors. First, a protocol was developed to analyze the cell cycle kinetics of NSCs. As expected, neurosphere cells were found to cycle slowly, with a very small proportion of the cell population undergoing mitosis at any time. Large fractions (65-70%) of the cells were detected in G1, even in rapidly proliferating cultures, and significant fractions (20%) of the cells were in G0. Second, it was observed that different culturing methods influence both the proportion of neurosphere cells in each phase of the cell cycle and the fraction of actively proliferating cells. The results show that suspension culture does not significantly alter the cell cycle progression of neurosphere cells, while long-term culture (>60 days) results in significant changes in cell cycle kinetics. This suggests that when developing a process to produce neural stem cells for clinical applications, it is imperative to track the cell cycle kinetics, and that a short-term suspension bioreactor process can be used to successfully expand neurosphere cells.

α-1 Adrenergic receptors stimulation induces the proliferation of neural progenitor cells in vitro

LXR agonists promote the proliferation of neural progenitor cells through MEK-ERK pathway

Valproic acid is a clinically used mood stabilizer and antiepileptic drug. Valproic acid has been suggested as a teratogen associated with the manifestation of neurodevelopmental disorders, such as fetal valproate syndrome and autism spectrum disorders, when taken during specific time window of pregnancy. Previous studies proposed that prenatal exposure to valproic acid induces abnormal proliferation and differentiation of neural progenitor cells, presumably by inhibiting histone deacetylase and releasing the condensed chromatin structure. Here, we found valproic acid up-regulates the transcription of T-type calcium channels by inhibiting histone deacetylase in neural progenitor cells. The pharmacological blockade of T-type calcium channels prevented the increased proliferation of neural progenitor cells induced by valproic acid. Differentiated neural cells from neural progenitor cells treated with valproic acid displayed increased levels of calcium influx in response to potassium chloride-induced depolarization. These results suggest that prenatal exposure to valproic acid up-regulates T-type calcium channels, which may contribute to increased proliferation of neural progenitor cells by inducing an abnormal calcium response and underlie the pathogenesis of neurodevelopmental disorders.

Neural progenitor cell (NPC) proliferation is fundamental for population expansion and brain development. G1 phase control determines the cell cycle duration of NPCs and thereby affects their proliferation efficiency. However, the molecular mechanisms governing G1 phase progression in NPCs remain unclear. Here, we show that AKT gain-of-function mutations and pharmacological inhibition exert opposing effects on NPC proliferation. Consistently, Emx1-Cre-mediated deletion of Akt1/2/3 in mice impairs NPC proliferation and disrupts cortical development. We find that AKT deficiency induces G1 phase arrest and prolongs the cell cycle of NPCs. Mechanistically, we demonstrate that AKT-mediated phosphorylation inhibits the activity of CRL4AMBRA1 E3 ubiquitin ligase to safeguard cyclin D2 (CCND2) stability. Specifically, AKT phosphorylates DDB1, the adaptor of CRL4AMBRA1, which disrupts its interaction with CCND2 and reduces its degradation. These findings reveal a post-translational mechanism impacting NPC cell cycle and cortical morphogenesis, providing insight into the etiology of malformations of cortical development.

Recent studies show that type II transmembrane serine proteases play important roles in diverse cellular activities and pathological processes. Their expression and functions in the central nervous system, however, are largely unexplored. In this study, we show that the expression of one such member, matriptase (MTP), was cell type-restricted and primarily expressed in neural progenitor (NP) cells and neurons. Blocking MTP expression or MTP activity prevented NP cell traverse of reconstituted basement membrane, whereas overexpression of MTP promoted it. The NP cell mobilization induced by either vascular endothelial growth factor or hepatocyte growth factor was also impaired by knocking down MTP expression. MTP acts upstream of matrix metalloproteinase 2 in promoting NP cell mobility. In embryonic stem cell differentiation to neural cells, MTP knockdown had no effect on entry of embryonic stem cells into the neural lineage. High MTP expression or activity, however, shifts the population dynamics from NP cells toward neurons to favor neuronal differentiation. This is the first report to demonstrate the direct involvement of type II transmembrane serine protease in NP cell function.

HIV/gp120 Decreases Adult Neural Progenitor Cell Proliferation via Checkpoint Kinase-Mediated Cell-Cycle Withdrawal and G1 Arrest

G1 Phase Regulation, Area-Specific Cell Cycle Control, and Cytoarchitectonics in the Primate Cortex

In neocortical neurogenesis, neural progenitor cells (NPCs) give rise to diverse types of neurons. During this process, the balance between proliferation and differentiation, cell division mode determination, and cell fate specification are all intimately linked to the cell cycle of the NPCs. The cell cycle length hypothesis states that G1-phase lengthening switches NPCs from proliferative to neurogenic divisions. Meanwhile, however, the importance of S-phase shortening in differentiating NPCs emerges. Mutations in the polycystin-1 (PC1)- and polycystin-2 (PC2)-encoding genes are causative for the development of autosomal dominant polycystic kidney disease (ADPKD), a prominent feature of which is unbalanced cell proliferation. Here, we examine the impact of PC1 and PC2 on cell cycle kinetics, cell cycle exit, and the neuronal differentiation of NPCs. Loss-of-function analysis and cell-based assays demonstrate that NPCs with reduced PC1 expression exhibit a longer cell cycle with an increased S-phase duration. The cell cycle exit and the neuronal differentiation of these cells are significantly delayed. A strong tendency towards similar phenotypes is observed when reducing PC2 expression in NPCs. Thus, decreasing PC1 expression expands the pool of slowly cycling NPCs. These results highlight the significance of S-phase shortening in neurogenesis and may contribute to a better understanding of ADPKD pathophysiology.

CHL1 negatively regulates the proliferation and neuronal differentiation of neural progenitor cells through activation of the ERK1/2 MAPK pathway

Cell cycle inhibition of neural stem and progenitor cells is critical for maintaining the stability of central nervous system in adults, but it may represent a significant hurdle for neural regeneration after injury. We have previously demonstrated that the cyclin-dependent kinase inhibitor (CKI) p21(cip1/waf1) (p21) maintains the quiescence of neural stem-like cells under cerebral ischemia, as similarly shown for the hematopoietic stem cells. Here, we report the distinct role of another CKI member, p27(kip1) (p27) in neural progenitor cells (NPCs) from adult brain (subventricular zone and hippocampal subgranular zone) under both homeostatic and ischemic conditions. The basal level of NPC proliferation in the p27-/- mice was higher than that in p27+/+ mice. Upon ischemia, the overall proliferation of NPCs continued to be higher in p27-/- mice than that in p27+/+ mice. Moreover, the increase of NPC proliferation in p27-/- mice remained until 2 weeks after ischemia, whereas it resumed back to the basal level in p27+/+ mice. As a result, newly generated neuronal cells in the granular layer of p27-/- brain were more abundant compared with p27+/+ controls. These new data demonstrate that p27 functions as a distinct inhibitor for NPC proliferation under homeostatic as well as ischemic conditions.