Zhao Ll

Metabotropic glutamate receptor 5 promotes proliferation of human neural stem/progenitor cells with activation of mitogen-activated protein kinases signaling pathway in vitro

Metabotropic glutamate receptors (mGluRs) regulate neurogenesis in brain, but the mechanisms remain unknown. In this study, we investigated the effect of mGluR5 on the proliferation of human embryonic neural stem/progenitor cells (NPCs), the expression of cyclin D1 and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that mGluR5 agonist (S)-3,5-dihydroxyphenylglycine hydrate (DHPG) increased the proliferation of NPCs by increasing cell activity, diameter of neurospheres and cell division, while mGluR5 siRNA and antagonist 6-methyl-2-(phenylethynyl) pyridine hydrochloride (MPEP) decreased the NPC proliferation. The mRNA and protein expressions of cyclin D1 increased with DHPG treatment and decreased after siRNA or MPEP treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal protein kinase (JNK) signaling pathways were involved in the proliferation of NPCs. After DHPG treatment, p-ERK1/2 and p-JNK2 levels increased, and meanwhile p-p38 level decreased; but p-ERK1/2 and p-JNK2 levels decreased after siRNA or MPEP treatment, and p-p38 level increased. Our findings demonstrated that mGluR5 promoted the proliferation of human embryonic cortical NPCs and increased cyclin D1 expression with the changes in phosphorylation of MAPKs signaling pathways in vitro, suggesting a novel mechanism for pharmacological study of treatment for ischemic brain injury and neurodegenerative disorders.

MicroRNA-25 Negatively Regulates Cerebral Ischemia/Reperfusion Injury-Induced Cell Apoptosis Through Fas/FasL Pathway.

MicroRNA-25 (miR-25) has been reported to be a major miRNA marker in neural cells and is strongly expressed in ischemic brain tissues. However, the precise mechanism and effect of miR-25 in cerebral ischemia/reperfusion (I/R) injury needs further investigations. In the present study, the oxygen-glucose deprivation (OGD) model was constructed in human SH-SY5Y and IMR-32 cells to mimic I/R injury and to evaluate the role of miR-25 in regulating OGD/reperfusion (OGDR)-induced cell apoptosis. We found that miR-25 was downregulated in the OGDR model. Overexpression of miR-25 via miRNA-mimics transfection remarkably inhibited OGDR-induced cell apoptosis. Moreover, Fas was predicted as a target gene of miR-25 through bioinformatic analysis. The interaction between miR-25 and 3′-untranslated region (UTR) of Fas mRNA was confirmed by dual-luciferase reporter assay. Fas protein expression was downregulated by miR-25 overexpression in OGDR model. Subsequently, the small interfering RNA (siRNA)-mediated knockdown of Fas expression also inhibited cell apoptosis induced by OGDR model; in contrast, Fas overexpression abrogated the protective effects of miR-25 on OGDR-induced cells. Taken together, our results indicate that the upregulation of miR-25 inhibits cerebral I/R injury-induced apoptosis through downregulating Fas/FasL, which will provide a promising therapeutic target.

The increased expression of metabotropic glutamate receptor 5 in subventricular zone neural progenitor cells and enhanced neurogenesis in a rat model of intracerebral hemorrhage

The metabotropic glutamate receptor 5 (mGluR5) is closely relative to the proliferation, survival, and differentiation of neural progenitor cells (NPCs). This study primarily examined the mGluR5 expression of NPCs in subventricular zone (SVZ) and the effects of mGluR5 on neurogenesis to intracerebral hemorrhage (ICH) rat. The experiment was designated as the following: (1) The ICH model was established by collagenase infusion into the right striatum of the rats, and the brain tissue was collected to assess the expression of mGluR5 in SVZ NPCs. (2) The rat brains were sampled for immunostaining of doublecortin (DCX) and 5-bromo-2-deoxyuridine (BrdU) to examine the effects of the (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG) on neurogenesis. (3) Behavioral testing was carried out to evaluate the effects of CHPG on neurofunctional recovery. The results of Western blot analysis showed that mGluR5 levels in the ipsilateral SVZ increased as early as at 3 days after ICH, peaked at 14 days. The change of mGluR5 mRNA level in the ipsilateral SVZ was generally similar to the pattern of Western blot analysis. The immunostaining also demonstrated that some nestin-positive cells were co-expressed with mGluR5. The injection of CHPG into ipsilateral ventricle increased DCX levels both in the ipsilateral striatum (STR) and the peri-lesion area of the striatum (PLA). Meanwhile, a significant difference in behavioral score was presented at 28 days after ICH between the CHPG-treated rats and the vehicle-treated or the non-treated rats. Our results demonstrated for the first time that the increased expression of mGluR5 in SVZ NPCs occurred in ICH rat. The CHPG promoted the neurogenesis and improved neurofunctional symptom induced by ICH. These results suggested that the increased expression of mGluR5 on NPCs in SVZ may play an important role in neurogenesis in ICH rat.

mGluR5 is involved in proliferation of rat neural progenitor cells exposed to hypoxia with activation of mitogen-activated protein kinase signaling pathway

Hypoxia/ischemia induces proliferation of neural progenitor cells (NPCs) in rodent and human brain; however, the mechanisms remain unknown. We investigated the effects of metabotropic glutamate receptor 5 (mGluR5) on NPC proliferation under hypoxia, the expression of cyclin D1, and the activation of the mitogen‐activated protein kinases (MAPKs) signaling pathway in cell culture. The results showed that hypoxia induced mGluR5 expression on NPCs in vitro. Under hypoxia, the mGluR5 agonists DHPG and CHPG significantly increased NPC proliferation in cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division, and expression of cyclin D1, with decreasing cell death. The mGluR5 siRNA and antagonist MPEP decreased the NPC proliferation and expression of cyclin D1, with increasing cell death. Phosphorylated JNK and ERK increased with the proliferation of NPCs after DHPG and CHPG treatment under hypoxia, while p‐p38 level decreased. These results demonstrate that the expression of mGluR5 was upregulated during the proliferation of rat NPCs stimulated by hypoxia in vitro. The activation of the ERK and JNK signaling pathway and the expression of cyclin D1 were increased in this process. These finding suggest the involvement of mGluR5 in rat NPC proliferation and provide a target molecule in neural repair after ischemia/hypoxia injury of CNS.

Developmental distribution pattern of metabotropic glutamate receptor 5 in prenatal human hippocampus

ObjectiveMetabotropic glutamate receptor 5 (mGluR5) is concentrated in zones of active neurogenesis in the prenatal and postnatal rodent brain and plays an important role in the regulation of neurogenesis. However, little is known about mGluR5 in the prenatal human brain. Here, we aimed to explore the expression pattern and cellular distribution of mGluR5 in human fetal hippocampus.MethodsThirty-four human fetuses were divided into four groups according to gestational age: 9–11, 14–16, 22–24 and 32–36 weeks. The hippocampus was dissected out and prepared. The protein and mRNA expression of mGluR5 were evaluated by Western blot and immunohistochemistry or real-time PCR. The cellular distribution of mGluR5 was observed with double-labeling immunofluorescence.ResultsBoth mGluR5 mRNA and protein were detected in the prenatal human hippocampus by real-time PCR and immunoblotting, and the expression levels increased gradually over time. The immunohistochemistry results were consistent with immunoblotting and showed that mGluR5 immunoreactivity was mainly present in the inner marginal zone (IMZ), hippocampal plate (HP) and ventricular zone (VZ). The double-labeling immunofluorescence showed that mGluR5 was present in neural stem cells (nestin-positive), neuroblasts (DCX-positive) and mature neurons (NeuN-positive), but not in typical astrocytes (GFAP-positive). The cells co-expressing mGluR5 and nestin were mainly located in the IMZ, HP and subplate at 11 weeks, all layers at 16 weeks, and CA1 at 24 weeks. As development proceeded, the number of mGluR5/nestin double-positive cells decreased gradually so that there were only a handful of double-labeled cells at 32 weeks. However, mGluR5/DCX double-positive cells were only found in the HP, IZ and IMZ at 11 weeks.ConclusionThe pattern of mGluR5 expression by neural stem/progenitor cells, neuroblasts and neurons provides important anatomical evidence for the role of mGluR5 in the regulation of human hippocampal development.

Hypoxia-regulated neurotrophin-3 expression by multicopy hypoxia response elements reduces apoptosis in PC12 cells

We have previously reported that 5xa0copies of the hypoxia response element (HRE) can conditionally regulate brain-derived neurotrophic factor gene expression under hypoxic/ischemic conditions in mice. In the present study, we investigated the controlled expression of neurotrophin-3 (NT-3) by HRE under hypoxic conditions and determined the protective effects of conditionally expressed NT-3 on hypoxia-induced apoptosis in PC12 cells. Five copies of the HRE (5HRE) and the simian virusxa040 minimal promoter (SV40mp) were employed to construct a cassette, and transfer of therapeutic gene, NT-3, into PC12 cells was achieved using a retroviral vector. Our results showed that the retroviral vector, pLNC-5HRE-NT3, was successfully constructed and transfected into PC12 cells. Compared with normal conditions, in which NT-3 was expressed at low levels, the expression of NT-3 significantly increased under hypoxic conditions in 5HRE-NT3 transgenic PC12 cells (P<0.05). By contrast, in NT-3 transgenic PC12 cells without HRE, we found no significant difference in NT-3 expression between the normoxic and hypoxic groups. The conditional adjustment of NT-3 expression by 5HRE significantly reduced apoptosis induced by hypoxia in 5HRE-NT3 transgenic PC12 cells (P<0.05) but not in 5HRE-enhanced green fluorescent protein (EGFP) transgenic PC12 cells and PC12 cells without gene transfer. In addition, the hypoxia-induced upregulation of both p38 and caspase-3 activities was suppressed in 5HRE-NT3 transgenic PC12 cells under hypoxic conditions (P<0.05). Taken together, these results demonstrate that 5HRE-SV40mp regulates NT-3 gene expression in response to hypoxia in PC12 cells. The data presented in this study may prove useful in future gene therapy studies for the treatment of ischemic diseases.

mGluR5 promotes the differentiation of rat neural progenitor cells into cholinergic neurons and activation of extracellular signal-related protein kinases.

Metabotropic glutamate receptors (mGluRs) regulate neurogenesis in the mammalian central nervous system during development and throughout adulthood. However, the mechanisms remain unknown. The present study was aimed at investigating the effect of mGluR5 on the differentiation of rat neural progenitor cells (NPCs) into neurons as well as the underlying molecular mechanisms. NPCs were treated with mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), mGluR5 siRNA, and antagonist 6-methyl-2-(phenylethynyl) pyridine hydrochloride (MPEP), respectively. Three different subtypes of neurons (cholinergic, GABAergic, and dopaminergic neurons) were evaluated, and the activation of signaling pathways of mitogen-activated protein kinases was determined. Results showed that CHPG caused rat NPCs to differentiate into neurons, whereas mGluR5 siRNA and MPEP inhibited the cell differentiation. The proportion of cholinergic neurons increased with CHPG treatment and decreased after siRNA or MPEP treatment, whereas there were no significant changes in the proportions of GABAergic and dopaminergic neurons after treatment. The phosphorylated ERK1/2 levels increased after CHPG treatment and decreased after siRNA or MPEP treatment. In conclusion, our findings showed that mGluR5 caused rat NPCs to differentiate into cholinergic neurons by activating ERKs, suggesting that mGluR5 may play a significant role in the mechanism and treatment of degenerative diseases such as Alzheimer’s disease.

Retroviral vector-mediated hypoxia-regulated neurotrophin-3 gene transfer reduces apoptosis induced by hypoxia in PC12 cells

Background Gene therapy for ischemic diseases is a prospective strategy. However, excessive expression of therapeutic genes may produce undesired side effects. Recently, multiple copies hypoxia response elements (HRE) were developed to conditionally regulate gene expression under hypoxia. As a nerve growth factor, Neurotrophin3 (NT-3) possesses neural protect effects either in vitro or in vivo. To explore hypoxia-controlled NT-3 expression, we constructed a recombinant retrovirus vector with 5HRE and NT-3, and generated a gene transferred cell line PC12-5HRE-NT3 to determine effects of conditionally expressed NT-3 on apoptosis induced by hypoxia in PC12 cells.

mGluR5 regulated proliferation of neural stem cells after hypoxia with activation of MAPK signaling pathway

In this study, we investigated the effects of metabotropic glutamate receptor 5 (mGluR5) on NSC proliferation under hypoxia by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, diameter measurement of neurospheres, bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. The cell death of NSCs was evaluated by terminal dUTP nickend labeling (TUNEL) assay and Hoechst staining. The expression of cyclin D1 and the activation of mitogenactivated protein kinases (MAPKs) signaling pathway were analyzed by immunoblotting assay. Results The results showed that hypoxia promoted the mGluR5 expression on NSCs. Under hypoxia, mGluR5 agonist DHPG and CHPG significantly increased NSC proliferation in cell activity, diameter of neurospheres, bromodeoxyuridine (BrdU) incorporation and cell division, and expression of cyclin D1 with decreasing of cell death. mGluR5 siRNA and antagonist MPEP decreased the NSC proliferation and expression of cyclin D1 with increasing of cell death. Phosphorylated JNK and ERK increased with the proliferation of NSCs after mGluR5 agonist DHPG and CHPG treatment under hypoxia, while p-p38 level decreased. Conclusions These results demonstrated that the expression of mGluR5 was upregulated during the proliferation of NSCs stimulated by hypoxia in vitro. The activation of ERK and JNK signaling pathway and the expression of cyclin D1 were increased in the process. These finding suggesting the involvement of mGluR5 in NSC proliferation and providing a target molecule in neural repair after ischemia/hypoxia injury of CNS.

Self-renewal potential of NPCs decreased in vitro during human embryonic brain development with reduced activation of mitogen-activated protein kinases signaling.

Background Neural progenitor cells (NPCs) are multipotent and selfrenewing cells during development and throughout adulthood. A key question in NPCs transplantation therapies is which stage NPCs from human embryonic brain development are ideal donor. Methods In this study, we investigated the difference of survival, proliferation and apopto sis of NPCs from 12 w, 16 w and 20 w human embryonic brain, and the phosphorylation of mitogen-activated protein kinases (MAPKs) signaling molecules. The NPCs survival was evaluated by trypan blue staining. The NPCs proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, diameter measurement of neurospheres and cell cycle analysis. The cell death of NSCs was evaluated by Hoechst staining. The expression of phosphorylated extracellular signal regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 were analyzed by immunoblotting assay. Results The results showed that the survival of human NPCs gradually decreased with human embryonic brain development in vitro; the NPCs proliferation gradually decreased in cell activity, diameter of neurospheres and cell division with human embryonic brain development, and the NPCs apoptosis gradually increased. Phosphorylation of ERK1/2 gradually decreased with human embryonic brain development, however phosphorylation of p38 MAPK gradually increased, and there were no significant change in p-JNK2 level. Conclusions