RhoGDI3 at the trans-Golgi network participates in NLRP3 inflammasome activation, VSMC phenotypic modulation, and neointima formation.

BACKGROUND AND AIMS: The pathological roles and mechanisms of Rho-specific guanine nucleotide dissociation inhibitor 3 (RhoGDI3) in vascular smooth muscle cell (VSMC) phenotypic modulation and neointima formation are currently unknown. This study aimed to investigate how RhoGDI3 regulates the Nod-like receptor protein 3 (NLRP3) inflammasome in platelet-derived growth factor-BB (PDGF-BB)-induced neointima formation. METHODS: For in vitro assays, human aortic VSMCs (HA-VSMCs) were transfected with pcDNA3.1-GDI3 and RhoGDI3 siRNA to overexpress and knockdown RhoGDI3, respectively. HA-VSMCs were also treated with an NLRP3 inhibitor (CY-09) or agonist (NSS). Protein transcription and expression, cell proliferation and migration, Golgi morphology, and protein binding and colocalization were measured. For the in vivo assays, balloon injury (BI) rats were injected with recombinant adenovirus carrying RhoGDI3 shRNA. Carotid arterial morphology, protein expression and colocalization, and activation of the NLRP3 inflammasome were measured. RESULTS: PDGF-BB treatment induced transcription and expression of RhoGDI3 through PDGF receptor αß (PDGFRαß) rather than PDGFRαα or PDGFRßß in HA-VSMCs. RhoGDI3 suppression blocked PDGF-BB-induced VSMC phenotypic transformation. In contrast, RhoGDI3 overexpression further promoted PDGF-BB-induced VSMC dedifferentiation. The in vivo results also confirmed that RhoGDI3 expressed in VSMCs participated in neointima formation and muscle fiber and collagen deposition caused by balloon injury. In addition, PDGF-BB increased binding of RhoGDI3 to NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) at the trans-Golgi membrane, which depended on the normal Golgi network. However, recruitment of NLRP3 and ASC to the trans-Golgi network after PDGF-BB treatment was independent of RhoGDI3. Moreover, RhoGDI3 knockdown significantly inhibited ASC expression and NLRP3 inflammasome assembly and activation and reduced NLRP3 protein stability in PDGF-BB-treated HA-VSMCs. Inhibiting NLRP3 effectively prevented PDGF-BB-induced VSMC phenotypic modulation, and an NLRP3 agonist reversed the decline in VSMC phenotypic transformation caused by RhoGDI3 knockdown. Furthermore, RhoGDI3 suppression reduced the protein levels and assembly of NLRP3 and ASC, and the activation of the NLRP3 inflammasome in VSMCs in a rat balloon injury model. CONCLUSIONS: The results of this study reveal a novel mechanism through which RhoGDI3 regulates VSMC phenotypic modulation and neointima formation by activating the NLRP3 inflammasome.

LncRNA KCNQ10T1 shuttled by bone marrow mesenchymal stem cell-derived exosome inhibits sepsis via regulation of miR-154-3p/RNF19A axis.

This study aims to discuss the role of exosomes KCNQ10T1 derived from bone marrow mesenchymal stem cells (BMMSCs) in sepsis and to further investigate its potential molecular mechanisms. Exosomes extracted from BMMSCs are identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blot. Fluorescence labeling is applied to detect the internalization of exosomes in receptors. The proliferation ability, migration ability, and invasion ability of HUVECs are determined by CCK-8, EdU, wound healing, and Transwell. The levels of inflammatory cytokines in sepsis cells are quantitatively detected by ELISA. Kaplan-Meier survival curve is used to describe the overall survival. RT-qPCR is used to detect mRNA expression of related genes. Bioinformatics analysis is performed to search the downstream target of KCNQ1OT1 and miR-154-3p and the interaction is verified by luciferase reporter assay. Exosomes derived from BMMSCs alleviated the toxicity in sepsis cell models and animal models. In mice with septic cell models, exosomal KCNQ10T1 was down-regulated and associated with lower survival. Overexpression of KCNQ10T1 inhibited the proliferation and metastasis of LPS-induced HUVECs. Further research illustrated that miR-154-3p was the downstream target gene of KCNQ1OT1 and RNF19A was the downstream target gene of miR-154-3p. Importantly, functional research findings indicated that KCNQ1OT1 regulated sepsis progression by targeting miR-154-3p/RNF19A axis. Our study demonstrates that the exosomal KCNQ1OT1 suppresses sepsis via mediating miR-154-3p/RNF19A, which provides a latent target for sepsis treatment.

Inhibition of Ribosomal RNA Processing 15 Homolog (RRP15) Suppressed Tumor Growth, Invasion and Epithelial to Mesenchymal Transition (EMT) of Colon Cancer.

Although ribosomal RNA processing 15 Homolog (RRP15) has been implicated in the occurrence of various cancers and is considered a potential target for cancer treatment, its significance in colon cancer (CC) is unclear. Thus, this present study aims to determine RRP15 expression and biological function in CC. The results demonstrated a strong expression of RRP15 in CC compared to normal colon specimens, which was correlated with poorer overall survival (OS) and disease-free survival (DFS) of the patients. Among the nine investigated CC cell lines, RRP15 demonstrated the highest and lowest expression in HCT15 and HCT116 cells, respectively. In vitro assays demonstrated that the knockdown of RRP15 inhibited the growth, colony-forming ability and invasive ability of the CC cells whereas its overexpression enhanced the above oncogenic function. Moreover, subcutaneous tumors in nude mice showed that RRP15 knockdown inhibited the CC growth while its overexpression enhanced their growth. Additionally, the knockdown of RRP15 inhibited the epithelial-mesenchymal transition (EMT), whereas overexpression of RRP15 promoted the EMT process in CC. Collectively, inhibition of RRP15 suppressed tumor growth, invasion and EMT of CC, and might be considered a promising therapeutic target for treating CC.

MiR-130a-3p regulates neural stem cell differentiation in vitro by targeting Acsl4.

In the adult mammalian brain, neural stem cells (NSCs) are the precursor cells of neurons that contribute to nervous system development, regeneration, and repair. MicroRNAs (miRNAs) are small non-coding RNAs that regulate cell fate determination and differentiation by negatively regulating gene expression. Here, we identified a post-transcriptional mechanism, centred around miR-130a-3p that regulated NSC differentiation. Importantly, overexpressing miR-130a-3p promoted NSC differentiation into neurons, whereas inhibiting miR-130a-3p function reduced the number of neurons. Then, the quantitative PCR, Western blot and dual-luciferase reporter assays showed that miR-130a-3p negatively regulated acyl-CoA synthetase long-chain family member 4 (Acsl4) expression. Additionally, inhibition of Acsl4 promoted NSC differentiation into neurons, whereas silencing miR-130a-3p partially suppressed the neuronal differentiation induced by inhibiting Acsl4. Furthermore, overexpressing miR-130a-3p or inhibiting Acsl4 increased the levels of p-AKT, p-GSK-3ß and PI3K. In conclusion, our results suggested that miR-130a-3p targeted Acsl4 to promote neuronal differentiation of NSCs via regulating the Akt/PI3K pathway. These findings may help to develop strategies for stem cell-mediated treatment for central nervous system diseases.

circRNA Acbd6 promotes neural stem cell differentiation into cholinergic neurons via the miR-320-5p-Osbpl2 axis.

Neural stem cells (NSCs) persist in the dentate gyrus of the hippocampus into adulthood and are essential for both neurogenesis and neural circuit integration. Exosomes have also been shown to play vital roles in regulating biological processes of receptor cells as a medium for cell-to-cell communication signaling molecules. The precise molecular mechanisms of exosome-mediated signaling, however, remain largely unknown. Here, we found that exosomes produced by denervated hippocampi following fimbria-fornix transection could promote the differentiation of hippocampal neural precursor cells into cholinergic neurons in coculture with NSCs. Furthermore, we found that 14 circular RNAs (circRNAs) were upregulated in hippocampal exosomes after fimbria-fornix transection using high-throughput RNA-Seq technology. We further characterized the function and mechanism by which the upregulated circRNA Acbd6 (acyl-CoA-binding domain-containing 6) promoted the differentiation of NSCs into cholinergic neurons using RT-quantitative PCR, Western blot, ELISA, flow cytometry, immunohistochemistry, and immunofluorescence assay. By luciferase reporter assay, we demonstrated that circAcbd6 functioned as an endogenous miR-320-5p sponge to inhibit miR-320-5p activity, resulting in increased oxysterol-binding protein-related protein 2 expression with subsequent facilitation of NSC differentiation. Taken together, our results suggest that circAcbd6 promotes differentiation of NSCs into cholinergic neurons via miR-320-5p/oxysterol-binding protein-related protein 2 axis, which contribute important insights to our understanding of how circRNAs regulate neurogenesis.

miR-103-3p targets Ndel1 to regulate neural stem cell proliferation and differentiation.

The regulation of adult neural stem cells (NSCs) is critical for lifelong neurogenesis. MicroRNAs (miRNAs) are a type of small, endogenous RNAs that regulate gene expression post-transcriptionally and influence signaling networks responsible for several cellular processes. In this study, miR-103-3p was transfected into neural stem cells derived from embryonic hippocampal neural stem cells. The results showed that miR-103-3p suppressed neural stem cell proliferation and differentiation, and promoted apoptosis. In addition, miR-103-3p negatively regulated NudE neurodevelopment protein 1-like 1 (Ndel1) expression by binding to the 3' untranslated region of Ndel1. Transduction of neural stem cells with a lentiviral vector overexpressing Ndel1 significantly increased cell proliferation and differentiation, decreased neural stem cell apoptosis, and decreased protein expression levels of Wnt3a, ß-catenin, phosphor-GSK-3ß, LEF1, c-myc, c-Jun, and cyclin D1, all members of the Wnt/ß-catenin signaling pathway. These findings suggest that Ndel1 is a novel miR-103-3p target and that miR-103-3p acts by suppressing neural stem cell proliferation and promoting apoptosis and differentiation. This study was approved by the Animal Ethics Committee of Nantong University, China (approval No. 20200826-003) on August 26, 2020.

MiR-674-5p Suppresses the Proliferation and Migration of Glioma Cells by Targeting Cul4b.

Glioma multiforme (GBM) is the most common malignant primary brain tumors. Despite the considerable advances in GBM treatment, it is still one of the most lethal forms of brain tumor. New clinical biomarkers and therapeutic targets are immediately required. MicroRNAs (miRNAs) are a class of small, evolutionarily conserved noncoding RNAs and have emerged as the key regulators of many cancers. Here in this study, we showed that miR-674-5p was probably an important regulator of glioma cell growth. After the transfection with miR-674-5p mimic or inhibitor, we found that the expression level of miR-674-5p was negatively related with cell proliferation and migration in C6 cells. Based on the prediction of the target genes of miR-674-5p on the website, we chose Cullin 4B (Cul4b), a gene upregulated in GBM, and proved that it was a target of miR-674-5p. In addition, we explored the role of miR-674-5p in glioma growth in vivo. Taken together, the present study indicated that miR-674-5p suppressed glioma cell proliferation and migration by targeting Cul4b.

miR-33-3p Regulates PC12 Cell Proliferation and Differentiation In Vitro by Targeting Slc29a1.

MicroRNA-33-3p (miR-33-3p) has been widely investigated for its roles in lipid metabolism and mitochondrial function; however, there are few studies on miR-33-3p in the context of neurological diseases. In this study, we investigated the functional role of miR-33-3p in rat pheochromocytoma PC12 cells. A miR-33-3p mimic was transduced into PC12 cells, and its effects on proliferation, apoptosis, and differentiation were studied using the MTS assay, EdU labeling, flow cytometry, qRT-PCR, western blot, ELISA, and immunofluorescence. We found that miR-33-3p significantly suppressed PC12 cell proliferation, but had no effect on apoptosis. Furthermore, miR-33-3p promoted the differentiation of PC12 cells into Tuj1-positive and choline acetyltransferase-positive neuron-like cells. Mechanistically, miR-33-3p repressed the expression of Slc29a1 in PC12 cells. Importantly, knocking down Slc29a1 in PC12 cells inhibited proliferation and induced differentiation into neuron-like cells. In conclusion, this study showed that miR-33-3p regulated Slc29a1, which in turn controlled the proliferation and differentiation of PC12 cells. Thus, we hypothesize that the miR-33-3p/Slc29a1 axis could be a promising therapeutic target for recovering neurons and the cholinergic nervous system.

CircHECTD1 Regulates Cell Proliferation and Migration by the miR-320-5p/SLC2A1 Axis in Glioblastoma Multiform.

Glioblastoma multiform (GBM) is the most common and malignant primary brain cancer in adults, and thus, novel potential therapeutic targets for diagnosis and treatment are urgently needed. Circular RNAs (circRNAs) are a class of widespread and diverse endogenous RNAs that have been suggested as potential critical mediators during progression of various tumors. In this study, we investigated the involvement of circHECTD1 in GBM progression. CircHECTD1 Lentivirus, miR-320-5p mimic, and SLC2A1 Lentivirus were transduced into cancer cells independently or together. circHECTD1, miR-320-5p, and SLC2A1 level were detected by qRT-PCR. Western blot and qRT-PCR were applied to measure the expression of SLC2A1, CyclinD1, CDK2, and PCNA. Flow cytometry, EdU, colony formation, Transwell and wound-healing assays were conducted to assess cell proliferation and migration. Luciferase reporter assays were performed to determine the effect of miR-320-5p on circHECTD1 or SLC2A1. Xenograft experiments were implemented to evaluate tumor growth in vivo. CircHECTD1 expression led to the promotion of proliferation and migration of GBM cells. In addition, circHECTD1 acted as a ceRNA to interact with miR-320-5p, which targeted the solute carrier family 2 member 1 (SLC2A1). In vivo experiments also revealed that circHECTD1 promoted tumor growth. Collectively, our findings showed that the circHECTD1-miR-320-5p-SLC2A1 regulatory pathway promoted the progression of GBM, suggesting that circHECTD1 may be a therapeutic target for GBM.

Expression and function of Ndel1 during the differentiation of neural stem cells induced by hippocampal exosomesticle.

BACKGROUND: In the brain of adult mammals, neural stem cells persist in the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus, which are specialized niches with proliferative capacity. Most neural stem cells are in a quiescent state, but in response to extrinsic stimuli, they can exit from quiescence and become reactivated to produce new neurons, so neural stem cells are considered to be a potential source for cell replacement therapy of many nervous system diseases. We characterized the expression of Ndel1 during the differentiation of neural stem cells induced by hippocampus exosomes, and assessed the effect of Ndel1 on neural stem cells differentiation. METHODS: Hippocampal exosomes were isolated and extracted, and co-cultured exosomes with neural stem cells. Western blot, flow cytometry, and immunofluorescence analyses were used to analyze expression of neuronal markers. Further, utilizing high-throughput RNA sequencing technology, we found that nudE neurodevelopment protein 1-like 1 was significantly upregulated in exosomes derived from denervated hippocampus, and then characterized its mechanism and function during neural stem cells differentiation by qRT-PCR, western blot, flow cytometry, and immunofluorescence analyses. RESULTS: Our results revealed that exosomes of denervated hippocampus promoted the differentiation of neural stem cells into neuron. Hence, we identified that nudE neurodevelopment protein 1-like 1 was significantly upregulated and highly expressed in the nervous system. In addition, we found that miR-107-3p may regulate neural stem cell differentiation by targeting Ndel1. CONCLUSIONS: Our results revealed that deafferentation of the hippocampal exosomes co-cultured with neural stem cells could promote them to differentiate into neurons. Hence, we found that miR-107-3p may regulate neural stem cells differentiation by targeting Ndel1. Importantly, Ndel1 enhanced spatial learning and hippocampal neurogenesis in rats after fimbria fornix transection in vivo. These findings set the stage for a better understanding of neurogenesis, a process that 1 day may inspire new treatments for central nervous system diseases.

GAS5 which is regulated by Lhx8 promotes the recovery of learning and memory in rats with cholinergic nerve injury.

Damage to the cholinergic system in central nervous system injuries such as traumatic brain injury (TBI) and neurodegenerative diseases leads to impaired learning and cognition. Neural stem cells (NSCs) have self-renewal capacity and multi-directional differentiation potential and considered the best source of cells for cell replacement therapy. However, how to promote the differentiation of NSCs into neurons is a major challenge in current research. Lhx8 has a specific effect on the development of the cholinergic nervous system, but its exact function is unclear. In this study, we found that Lhx8 could regulate the expression of Growth arrest-specific (GAS)5 which has been implicated in cancer but was less studied in the nervous system. Additionally, results from PCR, fluorescence in situ hybridization, and immunocytochemical analyses showed that GAS5 is mainly expressed in the cytoplasm of hippocampal neural stems cells and promotes their differentiation into neurons; the Morris water maze test demonstrated that GAS5 overexpression restored learning and memory in rats with cholinergic injury. These findings indicate that GAS5, which is regulated by Lhx8, improve brain function following cholinergic nerve injury.

MiR-210-3p Inhibits Proliferation and Migration of C6 Cells by Targeting Iscu.

Glioma is the most common primary brain tumor and the most malignant type of glioma is glioblastoma with the character of high mortality, high recurrence rate and poor prognosis. MicroRNAs act as an important component in glioma development and thus may be a potential target for the treatment of glioma. There were some researches indicated that miR-210-3p played a role in glioma development, but if it can inhibit glioma growth, as well as the underlying mechanism, is still uncertain. In the present study, we investigated the effects of miR-210-3p and its potential target gene Iscu on glioma (C6) cells proliferation and migration in vitro as well as the influence of miR-210-3p on glioma growth in vivo. The results showed that miR-210-3p inhibited the proliferation and migration of C6 cells by regulating the expression of its target gene Iscu in vitro. We also demonstrated that glioma growth was suppressed in immunodeficient mice when they were implanted with C6 cells overexpressing miR-210-3p. Our data indicated that miR-210-3p played an important role in the prevention of glioma growth by targeting Iscu and so miR-210-3p/Iscu axis might be a potential target for the treatment of glioma.

Gene expression changes induced by valproate in the process of rat hippocampal neural stem cells differentiation.

Valproate (VPA), an effective clinical approved anti-epileptic drug and mood stabilizer, has been believed to induce neuronal differentiation at the expense of inhibiting astrocytic and oligodendrocytic differentiation. Nevertheless, the involving mechanisms of it remain unclear yet. In the present study, we explored the global gene expression changes of fetus rat hippocampal neural stem cells following VPA treatment by high-throughput microarray. We obtained 874 significantly upregulated genes and 258 obviously downregulated genes (fold change > 2 and P < 0.05). Then, we performed gene ontology and pathway analyses of these differentially expressed genes and chose several genes associated with nervous system according to gene ontology analysis to conduct expression analysis to validate the reliability of the array results as well as reveal possible mechanisms of VPA. To get a better comprehension of the differentially regulated genes by VPA, we conducted protein-protein association analysis of these genes, which offered a source for further studies. In addition, we made the overlap between the VPA-downregulated genes and the predicted target genes of VPA-upregulated microRNAs (miRNAs), which were previously demonstrated. These overlapped genes may provide a source to find functional VPA/miRNA/mRNA axes during neuronal differentiation. This study first constructed a comprehensive potential downstream gene map of VPA in the process of neuronal differentiation.

Function and mechanism of long non-coding RNA Gm21284 in the development of hippocampal cholinergic neurons.

BACKGROUND: Increasing evidence has revealed that long non-coding RNAs (lncRNAs) play a pivotal role in the development of nervous system. Our previous studies have demonstrated that enhanced cholinergic neurogenesis occurs in the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) after cholinergic denervation, which is closely associated with the core transcription factor Lhx8. This study aimed to identify novel lncRNAs in a denervated hippocampal niche, which may affect cholinergic neurogenesis, and to explore the molecular mechanisms underlying cholinergic neurogenesis. METHODS: The gene expression profiles of the denervated hippocampus were examined by microarray analysis, and targeted lncRNAs were filtered using bioinformatics analysis. The lncRNA Gm21284 was predicted to be associated with Lhx8. RT-PCR and FISH were used to observe the expression and localization of Gm21284 in vitro and in vivo. The interaction between Gm21284 and Lhx8 and miR-30e-3P was verified using the luciferase reporter gene assay. Cell proliferation and differentiation was observed to reveal the effects of Gm21284 in cholinergic neurogenesis. RESULTS: Microarray analysis demonstrated 482 up-regulated and 135 down-regulated mRNAs, 125 up-regulated and 55 down-regulated lncRNAs, and 10 up-regulated and 3 down-regulated miRNAs in the denervated hippocampal niche. Overall, 32 lncRNAs were differentially expressed in the denervated hippocampal niche, which could interact with miR-30e-3p, miR-431, and miR-147. Among these 32 lncRNAs, Gm21284 and Adarb1 were identified after interleaving with lncRNAs in a co-expression network and WGCNA. Gm21284 was mainly located in the hippocampal DG. Furthermore, Gm21284-positive cells were considerably increased in the denervated hippocampus than in the normal side. EdU proliferation assay revealed that the proliferation of neural stem cells was repressed after the overexpression of Gm21284. Compared with the control group, the proportion of ChAT-positive cells increased at 7 days of differentiation of NSCs overexpressing Gm21284. CONCLUSION: Thus, Gm21284 functions as a competing endogenous RNA, which inhibits the proliferation of hippocampal NSCs and promotes their differentiation toward cholinergic neurons by inhibiting miR-30e-3P competitively.

Long non-coding RNA GAS5 promotes PC12 cells differentiation into Tuj1-positive neuron-like cells and induces cell cycle arrest.

Growth arrest-specific 5 (GAS5) is an anti-oncogene that has been extensively studied in tumors. However, research on GAS5 in the context of nervous system disease is rare at present. This study aimed to investigate the role of the long non-coding RNA GAS5 in rat pheochromocytoma cells (PC12 cells). GAS5-overexpressing lentivirus was transfected into PC12 cells, and expression levels of GAS5 and C-myc were detected by real-time PCR. Ratios of cells in S phase were detected by 5-ethynyl-2'-deoxyuridine. Immunohistochemical staining was used to detect the immunoreactivity of neuron microtubule markers Tuj1, doublecortin, and microtubule-associated protein 2. Apoptosis was detected by flow cytometry, while expression of acetylcholine in cells was detected by western blot assay. We found that GAS5 can promote PC12 cells to differentiate into Tuj1-positive neuron-like cells with longer processes. In addition, cell proliferation and cell cycle were significantly suppressed by GAS5, whereas it had no effect on apoptosis of PC12 cells. Our results indicate that GAS5 could increase the expression of choline acetyltransferase and acetylcholine release. Thus, we speculate that GAS5 is beneficial to the recovery of neurons and the cholinergic nervous system.

Effect of metastasis suppressor 1 on H1299 cells and its clinical significance in non-small cell lung cancer.

The present study aimed to investigate the effect of metastasis suppressor 1 (MTSS1) on the proliferation, migration and invasion of human H1299 non-small cell lung cancer cells and its clinical significance in non­small cell lung cancer. The target gene MTSS1-overexpressing lentivirus (LV-MTSS1) was transfected into H1299 cells and expression of MTSS1 was detected at the mRNA and protein levels. Cell Counting Kit-8, wound healing and Transwell assays revealed that the migration and invasion activities were significantly suppressed by MTSS1, but that it had no effect on cell proliferation. In addition, MTSS1 expression in tissue microarrays including samples from 223 cases of non-small cell lung cancer was tested by immunohistochemistry to explore the correlation between MTSS1 and clinicopathological characteristics and prognosis. MTSS1 suppressed H1299 cell migration and invasion, and its expression level can be used as a new independent factor for determining the prognosis of non-small cell lung cancer.

Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells.

Fimbria-fornix transection induces both exogenous and endogenous neural stem cells to differentiate into neurons in the hippocampus. This indicates that the denervated hippocampus provides an environment for neuronal differentiation of neural stem cells. However, the pathways and mechanisms in this process are still unclear. Seven days after fimbria fornix transection, our reverse transcription polymerase chain reaction, western blot assay, and enzyme linked immunosorbent assay results show a significant increase in ciliary neurotrophic factor mRNA and protein expression in the denervated hippocampus. Moreover, neural stem cells derived from hippocampi of fetal (embryonic day 17) Sprague-Dawley rats were treated with ciliary neurotrophic factor for 7 days, with an increased number of microtubule associated protein-2-positive cells and decreased number of glial fibrillary acidic protein-positive cells detected. Our results show that ciliary neurotrophic factor expression is up-regulated in the denervated hippocampus, which may promote neuronal differentiation of neural stem cells in the denervated hippocampus.

Fimbria-fornix (FF)-transected hippocampal extracts induce the activation of astrocytes in vitro.

Hippocampus is one of the neurogenesis areas in adult mammals, but the function of astrocytes in this area is still less known. In our previous study, the fimbria-fornix (FF)-transected hippocampal extracts promoted the proliferation and neuronal differentiation of radial glial cells in vitro. To explore the effects of hippocampal extracts on gliogenesis, the hippocampal astrocytes were treated by normal or ff-transected hippocampal extracts in vitro. The cells were immunostained by brain lipid-binding protein (BLBP), nestin, and SOX2 to assess their state of activation. The effects of astrocyte-conditioned medium on the neuronal differentiation of hippocampal neural stem cells (NSCs) were also investigated. After treatment of FF-transected hippocampal extracts, the number of BLBP, nestin, and Sox-positive cells were obviously more than the cells which treated by normal hippocampal extracts, these cells maintained a state of activation and the activated astrocyte-conditioned medium also promoted the differentiation of NSCs into more neurons. These findings suggest that the astrocytes can be activated by FF-transected hippocampal extracts and these activated cells also can promote the neuronal differentiation of hippocampal NSCs in vitro.

Transplantation of neural stem cells co-transfected with Nurr1 and Brn4 for treatment of Parkinsonian rats.

Neural stem cells (NSCs) tranplantation has great potential for the treatment of neurodegenerative disease such as Parkinson's disease (PD). However, the usage of NSCs is limited because the differentiation of NSCs into specific dopaminergic neurons has proven difficult. We have recently demonstrated that transgenic expression of Nurr1 could induce the differentiation of NSCs into tyrosine hydroxylase (TH) immunoreactive dopaminergic neurons, and forced co-expression of Nurr1 with Brn4 caused a dramatic increase in morphological and phenotypical maturity of these neurons. In this study, we investigated the effect of transplanted NSCs in PD model rats. The results showed that overexpression of Nurr1 promoted NSCs to differentiate into dopaminergic neurons in vivo, increased the level of dopamine (DA) neurotransmitter in the striatum, resulting in behavioral improvement of PD rats. Importantly, co-expression of Nurr1 and Brn4 in NSCs significantly increased the maturity and viability of dopaminergic neurons, further raised the DA amount in the striatum and reversed the behavioral deficit of the PD rats. Our findings provide a new potential and strategy for the use of NSCs in cell replacement therapy for PD.

In vitro differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs), derived from Wharton's jelly, into choline acetyltransferase (ChAT)-positive cells.

We isolated and expanded fibroblast-like cells from the Wharton's jelly of human umbilical cord successfully. Immunocytochemistry showed that they were positive for several markers of mesenchymal stem cells (CD73, CD90, and CD105) and integrin markers (CD29 and CD44), but negative for a hematopoietic cell maker (CD45) and an endothelial cell marker (CD31). Their differentiation into osteocytes and adipocytes under specific conditions indicated that they had multi-lineage differentiation potential. Therefore these results proved that the cells we obtained from Wharton's jelly were human umbilical cord mensenchymal stem cells (hUCMSCs). Using immunocytochemistry and Western blotting analysis, we found that after treatment with neuronal induction medium [NIM; consisting of brain-derived neurotrophic factor (BDNF) and low-serum media] for 14 days, hUCMSCs expressed a neuronal specific marker, microtubule associated protein 2 (MAP2), and extended neurite-like processes. After treatment with NIM, supplemented with hippocampal cholinergic neurostimulating peptide (HCNP) or rat denervated hippocampal extract [rDHE; derived from rat fimbria fornix (FF) transected hippocampus], hUCMSCs expressed choline acetytransferase (ChAT) and this action could be enhanced when cells were cultured with NIM, supplemented with HCNP and rDHE in combination. ELISA showed that these ChAT-positive cells could secrete acetylcholine (ACh). These findings indicate that hUCMSCs possess the potential of differentiation into functional ChAT-positive cells in vitro and provide a new candidate of cells for the cell transplantation to treat Alzheimer's disease (AD).