Qian Jiao

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.

Neurotrophin-3 gene transduction of mouse neural stem cells promotes proliferation and neuronal differentiation in organotypic hippocampal slice cultures

Summary Background The transplantation of neural stem cells (NSCs) has been accepted as a promising therapeutic strategy for central nervous system disorders. However, the beneficial effect of NSC transplantation upon functional recovery is limited due to the unfavorable microenvironment (niche) at the site of trauma or degenerative disease in the brain. Combination of transplantation of NSCs with neurotrophins may overcome the hurdles of impaired cell survival and neuronal differentiation. Material/Methods In the current study, the neurotrophin-3 (NT-3) gene was transduced into cultured mouse embryonic cortical NSCs via an AAV vector (NSC-NT-3). The effect of NT-3 over-expression on cell proliferation and differentiation in NSCs was observed by immunohistochemistry, cell culture and organotypic hippocampal slice cultures. Results The characteristics of self-renewal and multiple differentiation of NSCs were well-preserved. Cells in the NSC-NT-3 group proliferated faster and differentiated into more β-tubulin III-positive neurons compared to the control group in vitro. Furthermore, cells in the NSC-NT-3 group survived in a significantly higher percentage and undertook neuronal differentiation preferably in organotypic hippocampal slice cultures. Conclusions Our results suggest that the transduction of NT-3 into NSCs could effectively promote NSCs survival, proliferation, and neuronal differentiation in vitro without change of the stemness of NSCs. This work also offers evidence to better understand the safety and efficiency of combined treatment with NT-3 and NSCs for the central nervous system disorders.

The Mental Retardation-Associated Protein srGAP3 Regulates Survival, Proliferation, and Differentiation of Rat Embryonic Neural Stem/Progenitor Cells

The mental retardation-associated protein, srGAP3 is highly expressed in neurogenic sites. It is thought to regulate the key aspects of neuronal development and functions. Little is known about the interaction between srGAP3 and immature neural stem cells/neural progenitor cells (NSCs/NPCs). In the current study, the expression of srGAP3 in NSCs/NPCs was detected. Then, survival, proliferation, differentiation, and morphological alteration of NSCs/NPCs were assessed after a lentivirus-mediated knockdown of srGAP3. The results showed that srGAP3 is highly expressed in NSCs/NPCs both in vitro and in vivo. After knockdown of srGAP3 (LV3-srGAP3 infection), viability and proliferation of NSCs/NPCs dramatically decreased, approximately 85% displayed a similar morphology with type I cells that have no or only few indistinguishable processes. After 7 days culture in a differentiation medium, 62.5%±8.3% of cells in the srGAP3 knockdown group were nestin-positive and 24.8%±5.8% of them were β-tubulin III-positive, which are significantly higher (30.2%±9.9% and 14.6%±2.7%) than in the control group (LV3-NC infection). In addition, cells in the knockdown group had significantly fewer, but longer processes. Our results demonstrate that srGAP3 knockdown negatively regulates NSCs/NPCs survival, proliferation, differentiation, and morphological alteration, particularly, process formation. Taken together, our results provide strong evidence that srGAP3 is involved in the regulation of biological behavior and the morphological features in rat NSCs/NPCs in vitro.

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.

Spatial relationship between NSCs/NPCs and microvessels in rat brain along prenatal and postnatal development

Neurogenesis and angiogenesis are two parallel processes that occur in brain development and repair, and so share some molecular signals. In order to better understand the interaction between the genesis of neural cells and vessels during brain development, the density of microvessels and the number of nestin positive neural stem/neural progenitor cells (NSCs/NPCs) around microvasculature in various brain regions was quantified. Results showed that the density of microvessels remained at a relative low level during embryonic development and dramatically increased after postnatal day 3 (P3), especially in subventricular zone. The number of nestin positive NSCs/NPCs per microvessel in neurogenic brain regions continually increased with fetal brain development and then gradually dropped down during postnatal development. The highest density of NSCs/NPCs appeared at postnatal day 1 (P1) and dramatically decreased after P3. Similar pattern was observed in striatum. In the olfactory bulb, the cerebral cortex and cerebellum, the dramatic decrease of NSCs/NPCs density appeared after P7, especially in the cerebral cortex. Our results demonstrated that anatomically, the spatial relationship between NSCs/NPCs and microvessels changed during brain development. The alteration patterns in neurogenic brain regions differed from non‐neurogenic brain regions.

Low concentration of serum helps to maintain the characteristics of NSCs/NPCs on alkali-treated PHBHHx film in vitro.

Abstract Objective: Efforts have been made by tissue engineers to create a permissive environment for neural regeneration, and to enhance the efficiency of neural stem cell (NSC) transplantation. However, to acquire sufficient number of seed cells on the material appears to be the main obstacle to constructing functional transplantable NSC-biomaterial complexes. A culture system has been optimized in the current study to maintain the specific characteristics of NSCs/neural progenitor cells (NPCs) on the material and achieve sustaining increased multipotent seed cells. Methods: The PHBHHx film was selected as biomaterial and the surface was firstly modified with NaOH treatment. The NSCs/NPCs isolated from the cerebral cortex of rat embryos were cultured on the treated PHBHHx films in growth medium containing 1%, 5%, and 10% fetal bovine serum (FBS). Then the attachment, survival, proliferation, and differentiation of NSCs/NPCs were assessed. Results: NaOH treatment significantly increased the hydrophilicity of PHBHHx and enhanced NSCs/NPCs attachment. On the treated PHBHHx film, NSCs/NPCs survived well and actively proliferated in the medium containing 1% FBS. After 7–14 days in culture, approximately two-thirds of cells remained as nestin and Sox2 positive NSCs/NPCs. However, in the medium containing 5% and 10% FBS, NSCs/NPCs proliferation was reduced and differentiation, particularly glial differentiation was significantly promoted. Conclusion: Growth medium containing low concentration of FBS is favorable for maintaining the characteristics, in terms of self-renewal and multiple differentiation, of NSCs/NPCs on NaOH-treated PHBHHx films. This could be a useful method to construct functional transplantable NSCs/NPCs-biomaterial complex.

Cell-Cell Connection Enhances Proliferation and Neuronal Differentiation of Rat Embryonic Neural Stem/Progenitor Cells

Cell-cell interaction as one of the niche signals plays an important role in the balance of stem cell quiescence and proliferation or differentiation. In order to address the effect and the possible mechanisms of cell-cell connection on neural stem/progenitor cells (NSCs/NPCs) proliferation and differentiation, upon passaging, NSCs/NPCs were either dissociated into single cell as usual (named Group I) or mechanically triturated into a mixture of single cell and small cell clusters containing direct cell-cell connections (named Group II). Then the biological behaviors including proliferation and differentiation of NSCs/NPCs were observed. Moreover, the expression of gap junction channel, neurotrophic factors and the phosphorylation status of MAPK signals were compared to investigate the possible mechanisms. Our results showed that, in comparison to the counterparts in Group I, NSCs/NPCs in Group II survived well with preferable neuronal differentiation. In coincidence with this, the expression of connexin 45 (Cx45), as well as brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) in Group II were significantly higher than those in Group I. Phosphorylation of ERK1/2 and JNK2 were significantly upregulated in Group II too, while no change was found about p38. Furthermore, the differences of NSCs/NPCs biological behaviors between Group I and II completely disappeared when ERK and JNK phosphorylation were inhibited. These results indicated that cell-cell connection in Group II enhanced NSCs/NPCs survival, proliferation and neuronal differentiation through upregulating the expression of gap junction and neurotrophic factors. MAPK signals- ERK and JNK might contribute to the enhancement. Efforts for maintaining the direct cell-cell connection are worth making to provide more favorable niches for NSCs/NPCs survival, proliferation and neuronal differentiation.

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.

Dynamic expression of srGAP2 in cell nuclei and cytoplasm during the differentiation of rat neural stem cells in vitro.

Different SLIT-ROBO Rho GTPase-activating proteins (srGAPs) have different levels of expression and diverse functions during neural development. Although srGAP2 is expressed in developmental brain tissue, little is known about its influence on cellular development of the nervous system. In the current study, dynamic expression of endogenous srGAP2 during neural stem cell/progenitor cell (NSC/NPC) differentiation in vitro was investigated in order to elucidate the association between the dynamic expression of srGAP2 and neural development. srGAP2 was expressed in undifferentiated NSCs/NPCs, and differentiated neurons and astrocytes with distinct expression patterns. In conjunction with the differentiation of NSCs/NPCs in vitro, the number of srGAP2+ cells markedly reduced. The percentage of srGAP2+ cells in the population of nestin+ and β-tubulin III+ cells was significantly downregulated while in the population of glial fibrillary acidic protein-positive cells, almost all cells were srGAP2+. srGAP2 was predominantly expressed in the cell nucleus in all cell types. srGAP2 was also weakly expressed in the cytoplasm of nestin+ and β-tubulin III+ cells at 3 and 7 days in vitro. However levels were gradually downregulated during the process of differentiation and almost disappeared in β-tubulin III+ cells at 14 days. The results from the present study suggest that srGAP2 is involved in regulating NSC/NPC differentiation during neural development. The translocation of srGAP2 in the cytoplasm and cell nucleus in different cell types may function as a director in decisions regarding cell fate.