Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3β/β-catenin pathway

Autism is a spectrum of neurodevelopmental disorders characterized by social isolation and lack of interaction. Anatomically, autism patients often show macrocephaly and high neuronal density. To investigate the mechanism underlying the higher neuronal populations seen in ASD, we subcutaneously injected VPA (400 mg/kg) into pregnant Sprague-Dawley rats on E12, an animal model often used in ASD study. Alternatively, cultured rat neural progenitor cells were treated with VPA. Until E18, VPA induced NPC proliferation and delayed neurogenesis in fetal brain, but the subsequent differentiation of NPCs to neurons increased brain neuronal density afterward. Similar findings were observed with NPCs treated with VPA in vitro. At a molecular level, VPA enhanced Wnt1 expression and activated the GSK-3β/β-catenin pathway. Furthermore, inhibition of this pathway attenuated the effects of VPA. The findings of this study suggest that an altered developmental process underlies the macrocephaly and abnormal brain structure observed in the autistic brain.     

Comparison of Neurite Outgrowth Induced by Erythropoietin (EPO) and Carbamylated Erythropoietin (CEPO) in Hippocampal Neural Progenitor Cells

A previous animal study has shown the effects of erythropoietin (EPO) and its non-erythropoietic carbamylated derivative (CEPO) on neurogenesis in the dentate gyrus. In the present study, we sought to investigate the effect of EPO on adult hippocampal neurogenesis, and to compare the ability of EPO and CEPO promoting dendrite elongation in cultured hippocampal neural progenitor cells. Two-month-old male BALB/c mice were given daily injections of EPO (5 U/g) for seven days and were sacrificed 12 hours after the final injection. Proliferation assays demonstrated that EPO treatment increased the density of bromodeoxyuridine (BrdU)-labeled cells in the subgranular zone (SGZ) compared to that in vehicle-treated controls. Functional differentiation studies using dissociated hippocampal cultures revealed that EPO treatment also increased the number of double-labeled BrdU/microtubule-associated protein 2 (MAP2) neurons compared to those in vehicle-treated controls. Both EPO and CEPO treatment significantly increased the length of neurites and spine density in MAP2(+) cells. In summary, these results provide evidences that EPO and CEPO promote adult hippocampal neurogenesis and neuronal differentiation. These suggest that EPO and CEPO could be a good candidate for treating neuropsychiatric disorders such as depression and anxiety associated with neuronal atrophy and reduced hippocampal neurogenesis.     

α7 Nicotinic receptor agonist reactivates neurogenesis in adult brain

Reactivation of neurogenesis by endogenous Neural Stem/Progenitor Cells (NS/PC) in the adult brain or spinal cord holds the key for treatment of CNS injuries as well as neurodegenerative disorders, which are major healthcare issues for the world's aging population. Recent studies show that targeting the α7 nicotinic acetylcholine receptors (α7nAChR) with a specific TC-7020 agonist inhibits proliferation and stimulates neuronal differentiation of NS/PC in subventricular zone (SVZ) in the adult mouse brain. TC-7020-induced neuronogenesis is observed in different brain regions, including: (1) βIII Tubulin-expressing cortical neurons, (2) calretinin expressing hippocampal neurons and (3) cells in substantia nigra (SN) expressing predopaminergic Nurr1+phenotype. Reactivation of developmental integrative nuclear FGFR1 signaling (INFS), via gene transfection reinstates neurogenesis in the adult brain by promoting neuronal differentiation of brain NS/PC. TC-7020 neuronogenic effect is associated with a robust accumulation of endogenous FGFR1 in the nuclei of differentiating cells. Furthermore, direct in vitro stimulation of neural stem/progenitor cells with α7nAChR agonist activates INFS and neuronal-like differentiation and activation of neuronal genes. The α7nAChR upregulation of early neuronal βIII-Tubulin gene involves neurogenic FGFR1-Nur signaling and direct FGFR1 interaction with the gene promoter. The reactivation of developmental INFS and neurogenesis in adult brain by the α7nAChR agonist may offer new strategy to treat brain injuries, neurodegenerative and neurodevelopmental diseases.     

Neuropeptide Y promotes neurogenesis and protection against methamphetamine-induced toxicity in mouse dentate gyrus-derived neurosphere cultures

Methamphetamine (METH) is a psychostimulant drug of abuse that causes severe brain damage. However, the mechanisms responsible for these effects are poorly understood, particularly regarding the impact of METH on hippocampal neurogenesis. Moreover, neuropeptide Y (NPY) is known to be neuroprotective under several pathological conditions. Here, we investigated the effect of METH on dentate gyrus (DG) neurogenesis, regarding cell death, proliferation and differentiation, as well as the role of NPY by itself and against METH-induced toxicity. DG-derived neurosphere cultures were used to evaluate the effect of METH or NPY on cell death, proliferation or neuronal differentiation. Moreover, the role of NPY and its receptors (Y(1), Y(2) and Y(5)) was investigated under conditions of METH-induced DG cell death. METH-induced cell death by both apoptosis and necrosis at concentrations above 10 nM, without affecting cell proliferation. Furthermore, at a non-toxic concentration (1 nM), METH decreased neuronal differentiation. NPY's protective effect was mainly due to the reduction of glutamate release, and it also increased DG cell proliferation and neuronal differentiation via Y(1) receptors. In addition, while the activation of Y(1) or Y(2) receptors was able to prevent METH-induced cell death, the Y(1) subtype alone was responsible for blocking the decrease in neuronal differentiation induced by the drug. Taken together, METH negatively affects DG cell viability and neurogenesis, and NPY is revealed to be a promising protective tool against the deleterious effects of METH on hippocampal neurogenesis.     

Valproic acid perturbs hematopoietic homeostasis by inhibition of erythroid differentiation and activation of the myelo-monocytic pathway

As a histone deacetylase inhibitor, valproic acid (VPA) is a candidate for anticancer therapy. Besides, VPA exhibits various mechanisms of action and its effects on the molecular basis of hematopoiesis remain unclear. To study the effects of VPA on the hematopoietic system, we performed microarray analysis using K562 cells treated with 1 mM VPA over a 72 h time course. The association between gene ontology (GO) terms and the lists of differentially expressed genes was tested using the Bioconductor package GOstats. Enrichment analysis for cellular differentiation pathways was performed based on manually curated gene lists. Results from microarray analysis were confirmed by studying cell differentiation features at the molecular and cellular levels using other hematopoietic cell lines as well as hematopoietic stem/progenitor CD34⁺ cells. Microarray analysis revealed 3440 modulated genes in the presence of VPA. Genes involved in the granulo-monocytic differentiation pathway were up-regulated while genes of the erythroid pathway were down-regulated. This was confirmed by analyzing erythrocytic and myeloid membrane markers and lineage-related gene expression in HEL, MEG01, HL60 as well as CD34⁺ cells. Moreover, GATA-1 and its co-factors (FOG1, SP1) were down-regulated, while myelopoiesis activator PU.1 was up-regulated, in agreement with an inhibition of erythropoiesis. Our functional profiling and cell phenotyping approach demonstrates that VPA is able to alter hematopoietic homeostasis by modifying the cell population balance in the myeloid compartment. This may lead to a potential failure of erythropoiesis in patients with cancer or chronic inflammatory diseases having a well-described propensity to anemia.     

Gene expression profiles of multiple brain regions in rats differ between developmental and postpubertal exposure to valproic acid

We have previously reported that the valproic acid (VPA)-induced disruption pattern of hippocampal adult neurogenesis differs between developmental and 28-day postpubertal exposure. In the present study, we performed brain region-specific global gene expression profiling to compare the profiles of VPA-induced neurotoxicity between developmental and postpubertal exposure. Offspring exposed to VPA at 0, 667, and 2000 parts per million (ppm) via maternal drinking water from gestational day 6 until weaning (postnatal day 21) were examined, along with male rats orally administered VPA at 0, 200, and 900 mg/kg body weight for 28 days starting at 5 weeks old. Four brain regions—the hippocampal dentate gyrus, corpus callosum, cerebral cortex, and cerebellar vermis—were subjected to expression microarray analysis. Profiled data suggested a region-specific pattern of effects after developmental VPA exposure, and a common pattern of effects among brain regions after postpubertal VPA exposure. Developmental VPA exposure typically led to the altered expression of genes related to nervous system development (Msx1, Xcl1, Foxj1, Prdm16, C3, and Kif11) in the hippocampus, and those related to nervous system development (Neurod1) and gliogenesis (Notch1 and Sox9) in the corpus callosum. Postpubertal VPA exposure led to the altered expression of genes related to neuronal differentiation and projection (Cd47, Cyr61, Dbi, Adamts1, and Btg2) in multiple brain regions. These findings suggested that neurotoxic patterns of VPA might be different between developmental and postpubertal exposure, which was consistent with our previous study. Of note, the hippocampal dentate gyrus might be a sensitive target of developmental neurotoxicants after puberty.     

Chronic Lithium Treatment Enhances the Number of Quiescent Neural Progenitors but Not the Number of DCX-Positive Immature Neurons

Background:

The term adult neurogenesis constitutes a series of developmental steps including the birth, survival, differentiation, maturation, and even death of newborn progenitor cells within neurogenic niches. Within the hippocampus progenitors reside in the neurogenic niche of the subgranular zone in the dentate gyrus subfield. At the different stages, designated type-I, type-IIa, type-IIb, type-III, and granule cell neurons, the cells express a series of markers enabling their identification and visualization. Lithium has been shown to increase hippocampal cell proliferation in the subgranular zone of the hippocampal dentate gyrus subfield of adult rodents and to stimulate the proliferation of hippocampal progenitor cells in vitro, but data regarding lithium’s ability to increase neuronal differentiation and survival is equivocal.

Methods:

To clarify the effect of lithium on adult hippocampal neurogenesis, we identified the effect of chronic lithium treatment on distinct stages of hippocampal progenitor development using adult Nestin-green fluorescent protein transgenic mice and immunofluorescent techniques.

Results:

The present observations confirm that lithium targets the initial stages of progenitor development enhancing the turnover of quiescent neural progenitors/putative stem-cells, corroborating previous reports. However, the enhanced quiescent neural progenitor-turnover does not translate into an increased number of immature neurons. We also observed a steep decline in the number of type-III immature neurons with complex tertiary-dendrites, suggesting that lithium alters the morphological maturation of newborn neurons.

Conclusions:

Our results do not corroborate previous reports of lithium-induced enhanced numbers of newly generated neurons.     

Capsaicin impairs proliferation of neural progenitor cells and hippocampal neurogenesis in young mice

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is a major pungent ingredient in hot peppers and induces apoptosis in malignant carcinoma cell lines. However, the adverse effects of capsaicin on neuronal development have not been fully explored. The aim of this study was to determine whether capsaicin affected murine-derived cerebellar multi-potent neural progenitor cells (NPC) or adult hippocampal neurogenesis in vivo. Capsaicin dose-dependently suppressed NPC proliferation, and higher concentrations were cytotoxic. Capsaicin decreased the activation of extracellular signal-regulated kinases (ERK) without markedly affecting p38 kinases. Capsaicin reduced the number of newly generated cells in the dentate gyrus of the hippocampus but did not significantly alter learning and memory performance in young adult mice. Interestingly, capsaicin decreased ERK activation in the hippocampus, suggesting that reduced ERK signaling may be involved in the capsaicin-mediated regulation of hippocampal neurogenesis.     

Lysine deacetylase inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptor instead of histone acetylation in spontaneously hypertensive rats

Inhibition of lysine deacetylase (KDAC) attenuated development of hypertension in spontaneously hypertensive rats (SHRs). We hypothesized that KDAC inhibition attenuates hypertension and is accompanied by acetylation of mineralocorticoid receptors (MR) instead of histone acetylation in SHRs. Valproate (VPA, 0.71 % wt/vol), an inhibitor of class I KDACs, was administered in drinking water to 7-week-old SHRs and Wistar Kyoto rats for 11 weeks. MR acetylation was determined by immunoprecipitation with anti-MR antibody followed by western blot with anti-acetyl-lysine antibody. Expression levels of acetylated histone H3, KDACs, MR target genes, or MR corepressors in the kidney cortex were measured by using western blot analysis or real-time PCR. Recruitment of MR and RNA polymerase II (Pol II) and histone modifications on promoters of target genes were analyzed by performing a chromatin immunoprecipitation (ChIP) assay. Treatment of SHR with VPA increased MR acetylation without affecting MR expression, which attenuated development of hypertension in SHR VPA decreased expression of KDAC class I but globally increased acetylated histone H3. Although VPA treatment increased histone 3 acetylation (H3Ac) and trimethylation of the fourth lysine (H3K4me3) in the promoter regions of MR target genes, it decreased the expression of target genes as well as recruitment of MR and Pol II. These results suggest that KDAC inhibition attenuates the development of hypertension in SHRs and is accompanied by acetylation of MR that is independent of histone acetylation.     

Glucocorticoid-Related Molecular Signaling Pathways Regulating Hippocampal Neurogenesis

Stress and glucocorticoid hormones regulate hippocampal neurogenesis, but the molecular mechanisms underlying their effects are unknown. We, therefore, investigated the molecular signaling pathways mediating the effects of cortisol on proliferation, neuronal differentiation, and astrogliogenesis, in an immortalized human hippocampal progenitor cell line. In addition, we examined the molecular signaling pathways activated in the hippocampus of prenatally stressed rats, characterized by persistently elevated glucocorticoid levels in adulthood. In human hippocampal progenitor cells, we found that low concentrations of cortisol (100 nM) increased proliferation (+16%), decreased neurogenesis into microtubule-associated protein 2 (MAP2)-positive neurons (−24%) and doublecortin (Dcx)-positive neuroblasts (−21%), and increased differentiation into S100β-positive astrocytes (+23%). These effects were dependent on the mineralocorticoid receptor (MR) as they were abolished by the MR antagonist, spironolactone, and mimicked by the MR-agonist, aldosterone. In contrast, high concentrations of cortisol (100 μM) decreased proliferation (−17%) and neuronal differentiation into MAP2-positive neurons (−22%) and into Dcx-positive neuroblasts (−27%), without regulating astrogliogenesis. These effects were dependent on the glucocorticoid receptor (GR), blocked by the GR antagonist RU486, and mimicked by the GR-agonist, dexamethasone. Gene expression microarray and pathway analysis showed that the low concentration of cortisol enhances Notch/Hes-signaling, the high concentration inhibits TGFβ-SMAD2/3-signaling, and both concentrations inhibit Hedgehog signaling. Mechanistically, we show that reduced Hedgehog signaling indeed critically contributes to the cortisol-induced reduction in neuronal differentiation. Accordingly, TGFβ-SMAD2/3 and Hedgehog signaling were also inhibited in the hippocampus of adult prenatally stressed rats with high glucocorticoid levels. In conclusion, our data demonstrate novel molecular signaling pathways that are regulated by glucocorticoids in vitro, in human hippocampal progenitor cells, and by stress in vivo, in the rat hippocampus.     

HDAC inhibition delays cell cycle progression of human bladder cancer cells in vitro

Our aim was to analyze the impact of the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on bladder cancer cell growth in vitro. RT-4, TCCSUP, UMUC-3, and RT-112 bladder cancer cells were treated with VPA (0.125-1 mmol/l) without and with preincubation periods of 3 and 5 days. Controls remained untreated. Tumor cell growth, cell cycle progression, and cell cycle-regulating proteins were investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and western blotting, respectively. Effects of VPA on histone H3 and H4 acetylation and HDAC3 and HDAC4 were also determined. Without preincubation, no tumor cell growth reduction was observed with 0.125 and 0.25 mmol/l VPA in TCCSUP, UMUC-3, and RT-112 cells, whereas 0.5 and 1 mmol/l VPA diminished the cell number significantly. VPA (0.25 mmol/l) did exert tumor growth-blocking effects after a 3-day preincubation. To achieve antitumor effects with VPA (0.125 mmol/l), a 5-day preincubation was necessary. A 3-day or 5-day preincubation was also necessary to distinctly delay cell cycle progression, with maximum effects at VPA (1 mmol/l). After the 5-day preincubation, the cell cycle-regulating proteins cdk1, cdk2, cdk4, and cyclins B, D1, and E were reduced, whereas p27 was enhanced. Diminished HDAC3 and 4 expression induced by VPA was accompanied by elevated acetylation of H3 and H4. VPA exerted growth-blocking properties on a panel of bladder cancer cell lines, commensurate with dose and exposure time. Long-term application induced much stronger effects than did shorter application and should be considered when designing therapeutic strategies for treating bladder carcinoma.     

Endocannabinoids: a new family of lipid mediators involved in the regulation of neural cell development

The endocannabinoids (eCBs) anandamide and 2-arachidonoylglycerol are important retrograde messengers that inhibit neurotransmitter release via presynaptic CB1 receptors. In addition, cannabinoids are known to modulate the cell death/survival decision of different neural cell types, leading to different outcomes that depend on the nature of the target cell and its proliferative/differentiation status. Thus, cannabinoids protect primary neurons, astrocytes and oligodendrocytes from apoptosis, whereas transformed glial cells are prone to apoptosis by cannabinoid challenge. Moreover, a potential role of the eCB system in neurogenesis and neural differentiation has been proposed. Recent research shows that eCBs stimulate neural progenitor proliferation and inhibit hippocampal neurogenesis in normal adult brain. Cannabinoids inhibit cortical neuron differentiation and promote glial differentiation. On the other hand, experiments with differentiated neurons have shown that cannabinoids also regulate neuritogenesis, axonal growth and synaptogenesis. These new observations support that eCBs constitute a new family of lipid signaling cues responsible for the regulation of neural progenitor proliferation and differentiation, acting as instructive proliferative signals through the CB1 receptor.