KDM4C promotes mouse hippocampal neural stem cell proliferation through modulating ApoE expression.

KDM4C is implicated in the regulation of cell proliferation, differentiation, and maintenance in various stem cell types. However, its function in neural stem cells (NSCs) remains poorly understood. Therefore, this study aims to investigate the role and regulatory mechanism of KDM4C in NSCs. Primary hippocampal NSCs were isolated from neonatal mice, and both in vivo and in vitro lentivirus-mediated overexpression of KDM4C were induced in these hippocampal NSCs. Staining results revealed a significant increase in BrdU- and Ki-67-positive cells, along with an elevated number of cells in S phases due to KDM4C overexpression. Subsequently, RNA-seq was employed to analyze gene expression changes following KDM4C upregulation. GO enrichment analysis, KEGG analysis, and GSEA highlighted KDM4C-regulated genes associated with development, cell cycle, and neurogenesis. Protein-protein interaction analysis uncovered that ApoE protein interacts with several genes (top 10 upregulated and downregulated) regulated by KDM4C. Notably, knocking down ApoE mitigated the proliferative effect induced by KDM4C overexpression in NSCs. Our study demonstrates that KDM4C overexpression significantly upregulates ApoE expression, ultimately promoting proliferation in mouse hippocampal NSCs. These findings provide valuable insights into the molecular mechanisms governing neurodevelopment, with potential implications for therapeutic strategies in neurological disorders.

UTX inhibition suppresses proliferation and promotes apoptosis in patient-derived glioblastoma stem cells by modulating periostin expression.

Glioblastoma stem cells (GSCs) exert a crucial influence on glioblastoma (GBM) development, progression, resistance to therapy, and recurrence, making them an attractive target for drug discovery. UTX, a histone H3K27 demethylase, participates in regulating multiple cancer types. However, its functional role in GSCs remains insufficiently explored. This study aims to investigate the role and regulatory mechanism of UTX on GSCs. Analysis of TCGA data revealed heightened UTX expression in glioma, inversely correlating with overall survival. Inhibiting UTX suppressed GBM cell growth and induced apoptosis. Subsequently, we cultured primary GSCs from three patients, observing that UTX inhibition suppressed cell proliferation and induced apoptosis. RNA-seq was performed to analyze the gene expression changes after silencing UTX in GSCs. The results indicated that UTX-mediated genes were strongly correlated with GBM progression and regulatory tumor microenvironment. The transwell co-cultured experiment showed that silencing UTX in the transwell chamber GSCs inhibited the well plate cell proliferation. Protein-protein interaction analysis revealed that periostin (POSTN) played a role in the UTX-mediated transcriptional regulatory network. Replenishing POSTN reversed the effects of UTX inhibition on GSC proliferation and apoptosis. Our study demonstrated that UTX inhibition hindered POSTN expression by enhancing the H3K27me2/3 level, eventually resulting in inhibiting proliferation and promoting apoptosis of patient-derived GSCs. Our findings may provide a novel and effective strategy for the treatment of GBM.

CCN3/NOV Regulates Proliferation and Neuronal Differentiation in Mouse Hippocampal Neural Stem Cells via the Activation of the Notch/PTEN/AKT Pathway.

Neural stem cells (NSCs) persist in the subgranular zone (SGZ) throughout the lifespan and hold immense potential for the repair and regeneration of the central nervous system, including hippocampal-related diseases. Several studies have demonstrated that cellular communication network protein 3 (CCN3) regulates multiple types of stem cells. However, the role of CCN3 in NSCs remains unknown. In this study, we identified CCN3 expression in mouse hippocampal NSCs and observed that supplementing CCN3 improved cell viability in a concentration-dependent manner. Additionally, in vivo results showed that the injection of CCN3 in the dentate gyrus (DG) increased Ki-67- and SOX2-positive cells while decreasing neuron-specific class III beta-tubulin (Tuj1) and doublecortin (DCX)-positive cells. Consistently with the in vivo results, supplementing CCN3 in the medium increased the number of BrdU and Ki-67 cells and the proliferation index but decreased the number of Tuj1 and DCX cells. Conversely, both the in vivo and in vitro knockdown of the Ccn3 gene in NSCs had opposite effects. Further investigations revealed that CCN3 promoted cleaved Notch1 (NICD) expression, leading to the suppression of PTEN expression and eventual promotion of AKT activation. In contrast, Ccn3 knockdown inhibited the activation of the Notch/PTEN/AKT pathway. Finally, the effects of changes in CCN3 protein expression on NSC proliferation and differentiation were eliminated by FLI-06 (a Notch inhibitor) and VO-OH (a PTEN inhibitor). Our findings imply that while promoting proliferation, CCN3 inhibits the neuronal differentiation of mouse hippocampal NSCs and that the Notch/PTEN/AKT pathway may be a potential intracellular target of CCN3. Our findings may help develop strategies to enhance the intrinsic potential for brain regeneration after injuries, particularly stem cell treatment for hippocampal-related diseases.

Activation of metabotropic glutamate receptor 4 regulates proliferation and neural differentiation in neural stem/progenitor cells of the rat subventricular zone and increases phosphatase and tensin homolog protein expression.

Neural stem/progenitor cells (NSPCs) persist in the mammalian subventricular zone throughout life, where they can be activated in response to physiological and pathophysiological stimuli. A recent study indicates metabotropic glutamate receptor 4 (mGluR4) is involved in regulating NSPCs behaviors. Therefore, defining mGluR4 function in NSPCs is necessary for determining novel strategies to enhance the intrinsic potential for brain regeneration after injuries. In this study, mGluR4 was functionally expressed in SVZ-derived NSPCs from male Sprague-Dawley rats, in which the cyclic adenosine monophosphate concentration was reduced after treatment with the mGluR4-specific agonist VU0155041. Additionally, lateral ventricle injection of VU0155041 significantly decreased 5-bromo-2'-deoxyuridine (BrdU)+ and Ki67+ cells, while increased Doublecortin (DCX)/BrdU double-positive cells in SVZ. In cultured NSPCs, mGluR4 activation decreased the ratio of BrdU+ cells, G2/M-phase cells, and inhibited Cyclin D1 expression, whereas it increased neuron-specific class III ß-tubulin (Tuj1) expression and the number of Tuj1, DCX, and PSA-NCAM-positive cells. However, pharmacological blocking mGluR4 with the antagonist MSOP or knockdown of mGluR4 abolished the effects of VU0155041 on NSPCs proliferation and neuronal differentiation. Further investigation demonstrated that VU0155041 treatment down-regulated AKT phosphorylation and up-regulated expression of the phosphatase and tensin homolog protein (PTEN) in NSPCs culture. Moreover VU0155041-induced proliferating inhibition and neuronal differentiating amplification in NSPCs were significantly hampered by VO-OHpic, a PTEN inhibitor. We conclude that activation of mGluR4 in SVZ-derived NSPCs suppresses proliferation and enhances their neuronal differentiation, and regulation of PTEN may be involved as a potential intracellular target of mGluR4 signal. Cover Image for this issue: https://doi.org/10.1111/jnc.15052.

Attenuation of Acute Intracerebral Hemorrhage-Induced Microglial Activation and Neuronal Death Mediated by the Blockade of Metabotropic Glutamate Receptor 5 In Vivo.

The activation of microglia in response to intracerebral hemorrhagic stroke is one of the principal components of the progression of this disease. It results in the formation of pro-inflammatory cytokines that lead to neuronal death, a structural deterioration that, in turn interferes with functional recovery. Metabotropic glutamate receptor 5 (mGluR5) is highly expressed in reactive microglia and is involved in the pathological processes of brain disorders, but its role in intracerebral hemorrhage (ICH) remains unknown. We hypothesized that mGluR5 regulates microglial activation and ICH maintenance. In this study, collagenase-induced ICH mice received a single intraperitoneal injection of the mGluR5 antagonist-, MTEP, or vehicle 2 h after injury. We found that acute ICH upregulated mGluR5 and microglial activation. mGluR5 was highly localized in reactive microglia in the peri-hematomal cortex and striatum on days 3 and 7 post-ICH. The MTEP-mediated pharmacological inhibition of mGluR5 in vivo resulted in the substantial attenuation of acute microglial activation and IL-6, and TNF-α release. We also showed that the blockade of mGluR5 markedly reduced cell apoptosis, and neurodegeneration and markedly elevated neuroprotection. Furthermore, the MTEP-mediated inhibition of mGluR5 significantly reduced the lesion volume and improved functional recovery. Taken together, our results demonstrate that ICH injury enhances mGluR5 expression in the acute and subacute stages and that mGluR5 is highly localized in reactive microglia. The blockade of mGluR5 reduces ICH-induced acute microglial activation, provides neuroprotection and promotes neurofunctional recovery after ICH. The inhibition of mGluR5 may be a relevant therapeutic target for intracerebral hemorrhagic stroke.

Activation of type 4 metabotropic glutamate receptor promotes cell apoptosis and inhibits proliferation in bladder cancer.

Bladder cancer, the second most common genitourinary malignancy, severely endangers the human health. Rising evidence suggests that metabotropic glutamate receptors (mGluRs) are involve in tumor progression. In this study, we observed that metabotropic glutamate receptor 4 (mGluR4) was functionally expressed in normal and cancerous bladder cells and its expression was positively correlated with high bladder cancer grading. We further confirmed that the activation of mGluR4 by VU0155041, an mGluR4-specific agonist, decreased cyclic adenosine monophosphate (cAMP) concentration and cell viability, promoted apoptosis and inhibited proliferation in bladder cancer cells, whereas MSOP (group III mGluR antagonist) or mGluR4 knockdown eliminated the effects of mGluR4 activity. Western blotting revealed the decreased cyclin D1 expression, increased procaspase-8/9/3 cleavage, and unbalanced Bcl-2/Bax expression in bladder cancer cell lines after mGluR4 activation, and likewise MSOP and mGluR4 knockdown abrogated the actions of mGluR4 activity. In vivo study showed that mGluR4 activation significantly inhibited tumor growth of bladder cancer via suppressing proliferation and promoting apoptosis. Furthermore, upregulation of phosphatase and tensin homolog (PTEN) and inhibition of Akt phosphorylation were also observed after mGluR4 activation. Similar with VU0155041, the Akt-specific inhibitor markedly promoted apoptosis and inhibited proliferation. Nevertheless, the PTEN-specific inhibitor significantly abolished the mGluR4 activation-induced cell apoptosis and proliferative inhibition in bladder cancer cell lines. These results indicate that mGluR4 can regulate the switch between survival and death via the cAMP/PTEN/AKT signaling pathway in bladder cancer cells. Our findings suggest that mGluR4 has diagnostic and prognostic potential for bladder cancer, and the development of mGluR4 agonist may be a promising strategy for bladder cancer treatment.

Rational selection of antibacterial drugs and postoperative nursing for gynecologic and obstetric surgery patients.

Infection after gynecologic surgery is very common and frequent. If the control is not good, it will lead to serious consequences. Therefore, it is necessary to use antibiotics in the period of obstetrics and gynecology. This study will explore the use of antimicrobial agents in gynecologic and obstetric surgery, thus standardizing the use of antibiotics in the process of obstetrics and gynecology. Through the analysis of the use of antibacterials, we can see that the highest utilization rate of 5 kinds of antibacterial drugs followed by Cefaclor Sustained Release Tablets (65.7%), metronidazole (32.5%), cefathiamidine (26.8%), enoxacin (22.5%) and cefoperazone tazobactam sodium (11.8%). At the same time, the hospital should improve the consciousness of rational drug use and strengthen the administration of antibacterials in the operative period of obstetrics and gynecology. The application of antibiotics in the operative period of the department of obstetrics and gynecology can improve the current situation of its irrational use. Nursing work must take strict aseptic operation to prevent cross infection. At the same time, we should strengthen the observation of the effect of medication, monitor the body temperature and blood pressure, and identify the side effects of drugs.

Hepatitis B virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and M2-like macrophages.

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.

Ameliorative effects of tannic acid on carbon tetrachloride-induced liver fibrosis in vivo and in vitro.

We investigated the ameliorative effects and potential mechanisms of tannic acid (TA) in carbon tetrachloride (CCl4)-intoxicated mice and hepatic stellate cells (HSCs). Liver fibrosis was observed in CCl4 (800 ml/kg)-induced mice, and high viability was observed in CCl4 (10 mM)-intoxicated HSCs. Pre-treatment of mice with TA (25 or 50 g/kg/day) significantly ameliorated hepatic morphology and coefficient values and reduced the activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), the concentrations of malondialdehyde (MDA) and serum levels of endothelin-1 (ET-1). In addition, TA increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and endothelial nitric oxide synthase (eNOS) and the serum level of NO. Moreover, TA reduced the expression of angiotensin II receptor-1 (ATR-1), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), caspase-3, c-fos, c-jun, the ratio of Bax/bcl-2, tissue inhibitor of metalloproteinase-1 (TIMP-1) and TA increased matrix metal proteinase-9 (MMP-9), matrix metalloproteinase-1 (MMP-1). Furthermore, TA (0.01 µM, 0.1 µM or 1 µM) decreased the TIMP-1/MMP-1 ratio and reduced the viability of HSCs. These results indicated that TA exerts significant liver-protective effects in mice with CCl4-induced liver fibrosis. The potential mechanism may rely on the inhibition of collagen accumulation, oxidative stress, inflammation and apoptosis.

Pathological functions of interleukin-22 in chronic liver inflammation and fibrosis with hepatitis B virus infection by promoting T helper 17 cell recruitment.

UNLABELLED: It is well established that interleukin (IL)-22 has hepatoprotective and antifibrotic functions in acute liver injury models; however, its function in patients with liver fibrosis and liver cirrhosis (LC) remains obscure. In the current study, we demonstrated that expression of numerous IL-22 pathway-associated genes was significantly up-regulated in hepatitis B virus (HBV)-infected liver tissues, compared to normal controls, through microarray analysis. In agreement with these findings, liver-infiltrating IL-22(+) cells were largely increased in HBV-infected patients with LC, compared to those without LC or healthy subjects, and were positively associated with liver fibrosis staging scores. Immunohistochemistry and flow cytometric analyses revealed that IL-22 was produced by multiple intrahepatic immune cells and, preferentially, by T-helper (Th) 17 cells in LC patients. In an HBV transgenic (Tg) mouse model of T-cell-mediated chronic liver inflammation and fibrosis, blockade of IL-22 attenuated hepatic expression of chemokine (C-X-C motif) ligand 10 and chemokine (C-C motif) ligand 20 (CCL20) and subsequently reduced Th17 recruitment and liver inflammation and fibrosis progression. In vitro treatment with IL-22 stimulated hepatic stellate cells (HSCs) to secrete several chemokines and subsequently promoted Th17 cell chemotaxis. Blocking C-X-C chemokine receptor type 3 or CCL20 reduced Th17 cell chemotaxis by IL-22-treated HSCs. CONCLUSIONS: IL-22 plays a pathological role in exacerbating chronic liver inflammation and fibrosis by recruiting hepatic Th17 cells in HBV-infected patients and HBV Tg mice.

Folate Deficiency and/or the Genetic Variant Mthfr677C >T Can Drive Hepatic Fibrosis or Steatosis in Mice, in a Sex-Specific Manner.

SCOPE: Disturbances in one-carbon metabolism contribute to nonalcoholic fatty liver disease (NAFLD) which encompasses steatosis, steatohepatitis, fibrosis, and cirrhosis. The goal is to examine impact of folate deficiency and the Mthfr677C >T variant on NAFLD. METHODS AND RESULTS: This study uses the new Mthfr677C >T mouse model for the human MTHFR677C >T variant. Mthfr677CC and Mthfr677TT mice were fed control diet (CD) or folate-deficient (FD) diets for 4 months. FD and Mthfr677TT alter choline/methyl metabolites in liver and/or plasma (decreased S-adenosylmethionine (SAM):S-adenosylhomocysteine (SAH) ratio, methyltetrahydrofolate, and betaine; increased homocysteine [Hcy]). FD, with contribution from Mthfr677TT, provokes fibrosis in males. Studies of normal livers reveal alterations in plasma markers and gene expression that suggest an underlying predisposition to fibrosis induced by FD and/or Mthfr677TT in males. These changes are absent or reverse in females, consistent with the sex disparity of fibrosis. Sex-based differences in methylation potential, betaine, sphingomyelin, and trimethylamine-N-oxide (TMAO) levels may prevent fibrogenesis in females. In contrast, Mthfr677TT alters choline metabolism, dysregulates expression of lipid metabolism genes, and promotes steatosis in females. CONCLUSION: This study suggests that folate deficiency predisposes males to fibrosis, which is exacerbated by Mthfr677TT, whereas Mthfr677TT predisposes females to steatosis, and reveal novel contributory mechanisms for these NAFLD-related disorders.

R-Loop Defines Neural Stem/Progenitor Cells During Mouse Neurodevelopment.

Neural stem/progenitor cells (NSPCs) are present in the mammalian brain throughout life and are involved in neurodevelopment and central nervous system repair. Although typical epigenetic signatures, including DNA methylation, histone modifications, and microRNAs, play a pivotal role in regulation of NSPCs, several of the epigenetic regulatory mechanisms of NSPCs remain unclear. Thus, defining a novel epigenetic feature of NSPCs is crucial for developing stem cell therapy to address neurologic disorders caused by injury. In this study, we aimed to define the R-loop, a three-stranded nucleic acid structure, as an epigenetic characteristic of NSPCs during neurodevelopment. Our results demonstrated that R-loop levels change dynamically throughout neurodevelopment. Cells with high levels of R-loops consistently decreased and were enriched in the area of neurogenesis. Additionally, these cells costained with SOX2 during neurodevelopment. Furthermore, these cells with high R-loop levels expressed Ki-67 and exhibited a high degree of overlap with the transcriptional activation markers, H3K4me3, ser5, and H3K27ac. These findings suggest that R-loops may serve as an epigenetic feature for transcriptional activation in NSPCs, indicating their role in gene expression regulation and neurogenesis.

Prevalence and prevalence trends of transfusion transmissible infections among blood donors at four Chinese regional blood centers between 2000 and 2010.

BACKGROUND: In China, high prevalence of HBV and HCV parallels with the growing epidemic of syphilis and HIV in the general population poses a great threat to blood safety. This study investigated the prevalence of serologic markers for transfusion transmissible infections (TTIs) among four Chinese blood centers. METHODS: We examined whole blood donations collected from January 2000 through December 2010 at four Chinese blood centers. Post-donation testing of TTIs (HIV, HBV, HCV and syphilis) were conducted using two different enzyme-linked immunosorbent assay kits for each seromarker. The prevalence of serologic markers for TTIs (%) was calculated and additional analysis was conducted to examine donor characteristics associated with positive TTIs serology. RESULTS: Of the 4,366,283 donations, 60% were from first-time donors and 40% were from repeated donors. The overall prevalence of HIV, HBsAg, HCV and syphilis was 0.08%, 0.86%, 0.51% and 0.47%, respectively. The prevalence profile of TTIs varied among different blood centers and appeared at relatively high levels. Overall, the prevalence of HBsAg and HCV demonstrated a decline trend among four blood centers, while the prevalence of HIV and syphilis displayed three different trends: constantly steady, continually increasing and declining among different centers. CONCLUSIONS: This study reflects the risk of TTIs has been greatly reduced in China, but blood transfusion remains an ongoing risk factor for the spread of blood-borne infections, and further work and improvements are needed to strengthen both safety and availability of blood in China.

Periostin attenuates oxygen and glucose deprivation-induced death of mouse neural stem cells via inhibition of p38 MAPK activation.

Promoting neural stem cells (NSCs) survival in the harsh niche is essential to cell replacement therapy for various central nervous system diseases. As an integral component of the extracellular matrix, Periostin (POSTN) has been shown to protect various cell types from hypoxia-ischemia damage. This study aimed to investigate the neuroprotective effects of POSTN on NSCs injury induced by oxygen and glucose deprivation (OGD). Under challenge with OGD, cell viability significantly decreased in cultured mouse NSCs, and supplement POSTN rescued cell viability in a concentration-dependent manner, as shown by CCK-8. TUNEL and propidium iodide/Hoechst staining showed that POSTN pretreatment protected NSCs against OGD-induced apoptosis. Western blot assay demonstrated that POSTN pretreatment inhibited cleavage of caspase-3 and restored the balance of Bcl-2/Bax. And pretreatment with cilengitide (an inhibitor of POSTN receptors) abolished the protective effect of POSTN. Further investigation demonstrated that supplement POSTN inhibited phosphorylation of p38 in a concentration-dependent manner. Moreover, the neuroprotective effect of POSTN was hampered by anisomycin, an activator of p38. We conclude that POSTN pretreatment in cultured mouse NSCs mitigated OGD-induced cell death, and inhibition of the p38 MAPK pathway might be one of the underlying mechanisms. Our findings may provide a novel strategy for enhancing both endogenous and exogenous NSCs survival after ischemia and hypoxia injury.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Sex-Specific Gene Expression in Brain of Young Mice and Embryos.

Food fortification and increased vitamin intake have led to higher folic acid (FA) consumption by many pregnant women. We showed that FA-supplemented diet in pregnant mice (fivefold higher FA than the recommended level (5xFASD)) led to hyperactivity-like behavior and memory impairment in pups. Disturbed choline/methyl metabolism and altered placental gene expression were identified. The aim of this study was to examine the impact of 5xFASD on the brain at two developmental stages, postnatal day (P) 30 and embryonic day (E) 17.5. Female C57BL/6 mice were fed a control diet or 5xFASD for 1 month before mating. Diets were maintained throughout the pregnancy and lactation until P30 or during pregnancy until E17.5. The 5xFASD led to sex-specific transcription changes in a P30 cerebral cortex and E17.5 cerebrum, with microarrays showing a total of 1003 and 623 changes, respectively. Enhanced mRNA degradation was observed in E17.5 cerebrum. Expression changes of genes involved in neurotransmission, neuronal growth and development, and angiogenesis were verified by qRT-PCR; 12 and 15 genes were verified at P30 and E17.5, respectively. Hippocampal collagen staining suggested decreased vessel density in FASD male embryos. This study provides insight into the mechanisms of neurobehavioral alterations and highlights potential deleterious consequences of moderate folate oversupplementation during pregnancy.

ISL1 Promotes Human Glioblastoma-Derived Stem Cells' Self-Renewal by Activation of Sonic Hedgehog/GLI1 Function.

Glioblastoma (GBM), the most aggressive primary heterogeneous primary brain tumor, is a glioma subtype that originates from the glial cells of the central nervous system. Glioblastoma stem cells (GSCs), situated at the top of the hierarchy, initiate and maintain the tumor and are largely accountable for GBM resistance to the mainstay treatment and recurrence. The LIM homeobox transcription factor islet 1 (ISL1) induces tumorigenicity in various tumors; however, its function in GSCs has been less reported. We aimed to generate GSCs from surgical specimens of human GBM and investigate the effect of ISL1 knockdown on GSCs. We established patient-derived GSCs, determined cancer stem cell marker expression, and immunostained GSCs to assess cell viability and apoptosis. We demonstrated that ISL1 deletion decreased the GSC viability and proliferation, and upregulated apoptosis. Moreover, we performed enzyme-linked immunosorbent assay and western blotting and found that ISL1 knockdown affected the expression of sonic hedgehog (SHH) and its downstream regulator GLI1, and further validated these results by supplementing the cells with recombinant SHH. Our results suggested that ISL1 played a critical role in regulating GBM growth and that an ISL1/SHH/GLI1 pathway was required for the maintenance of GBM progression and malignancy. The regulation of GSC growth through ISL1 might be a mechanism of interest for future therapeutic studies.

Activation of type 4 metabotropic glutamate receptor attenuates oxygen and glucose deprivation-induced apoptosis in human neural stem cells via inhibition of ASK1-p38 signaling pathway.

Hypoxic ischemic brain injury (HIBI) has been one of the most severe central nervous system (CNS) diseases with high fatality and disability rate. Neural stem cells (NSCs) persist in the mammalian brain throughout life and NSCs-associated therapies might be a promising strategy for the HIBI treatment. In this study, we identified that type 4 metabotropic glutamate receptor (mGluR4) was expressed in cultured human NSCs (hNSCs) isolated from the human fetus cortex and further established the oxygen and glucose deprivation (OGD) model in hNSCs to study the role of mGluR4 in hypoxic and ischemic injury. The results indicated that mGluR4 activation by using VU0155041 (mGluR4-specific agonist) markedly attenuated the OGD-induced alterations in TUNEL staining, apoptosis rate, cleavages of pro-caspase-8, -9, -3, and Bcl-2/Bax expression balance. Furthermore, mGluR4 activation inhibited the ASK1/p38 signaling pathway. Asiatic acid, as a p38 MAPK activator, is capable of abolishing the neuroprotective effect of mGluR4, while both NQDI-1 (ASK-1 inhibitor) and SB203580 (p38 MAPK inhibitor) exerted similar effects to VU0155041 in the OGD-induced hNSC damage. In conclusion, this study indicates that mGluR4 activation protects hNSCs against the OGD-induced cell death via inhibiting the ASK1-p38 pathway. Activation of mGluR4 might be a promising strategy for enhancing NSCs survival in hypoxic and ischemic injury.

Glypican 4 Regulates Aß Internalization in Neural Stem Cells Partly via Low-Density Lipoprotein Receptor-Related Protein 1.

Neural stem cell (NSC) damage has been reported in patients with Alzheimer's disease. Intracellular Aß plays a vital role in NSC damage. Heparan sulfate proteoglycans are potent mediators of Aß enrichment in the brain. We hypothesized the heparan sulfate proteoglycan glypican 4 (Gpc4) regulates Aß internalization by NSCs. We evaluated Gpc4 expression in NSCs from P0-P2 generations using immunofluorescence. Adenovirus and lentivirus were used to regulate Gpc4 expression in NSCs and APP/PS1 mice, respectively. Co-immunoprecipitation was used to determine the relationship between Gpc4, Aß, and low-density lipoprotein receptor-related protein 1 (LRP1). Intracellular Aß concentrations were detected using enzyme-linked immunosorbent assay and immunofluorescence. The role of Gpc4/LRP1 on toxic/physical Aß-induced effects was evaluated using the JC-1 kit, terminal deoxynucleotidyl transferase dUPT nick end labeling, and western blotting. Gpc4 was stably expressed in NSCs, neurons, and astrocytes. Gpc4 was upregulated by Aß in NSCs and regulated Aß internalization. Gpc4 attenuation reduced Aß uptake; Gpc4 overexpression increased Aß uptake. Gpc4 regulated Aß internalization through LRP1 and contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential and cell apoptosis, partly via LRP1. Therefore, Gpc4 is a key regulator of Aß enrichment in NSCs. Inhibiting Gpc4 rescued the Aß-induced toxic effect and attenuated the nontoxic Aß enrichment into intracellular toxic concentrations. Gpc4 contributed to Aß internalization and toxic/physical concentrations of Aß-induced mitochondrial membrane potential damage and cell apoptosis, partly via LRP1. These findings suggest a potential role of Gpc4 in treating Alzheimer's disease at an early stage, by targeting NSCs.

Moderate Folic Acid Supplementation in Pregnant Mice Results in Altered Methyl Metabolism and in Sex-Specific Placental Transcription Changes.

SCOPE: Many pregnant women have higher folic acid (FA) intake due to food fortification and increased vitamin use. It is reported that diets containing five-fold higher FA than recommended for mice (5xFASD) during pregnancy resulted in methylenetetrahydrofolate reductase (MTHFR) deficiency and altered choline/methyl metabolism, with neurobehavioral abnormalities in newborns. The goal is to determine whether these changes have their origins in the placenta during embryonic development. METHODS AND RESULTS: Female mice are fed control diet or 5xFASD for a month before mating and maintained on these diets until embryonic day 17.5. 5xFASD led to pseudo-MTHFR deficiency in maternal liver and altered choline/methyl metabolites in maternal plasma (increased methyltetrahydrofolate and decreased betaine). Methylation potential (S-adenosylmethionine:S-adenosylhomocysteine ratio) and glycerophosphocholine are decreased in placenta and embryonic liver. Folic acid supplemented diet results in sex-specific transcriptome profiles in placenta, with validation of dietary expression changes of 29 genes involved in angiogenesis, receptor biology or neurodevelopment, and altered methylation of the serotonin receptor 2A gene. CONCLUSION: Moderate increases in folate intake during pregnancy result in placental metabolic and gene expression changes, particularly in angiogenesis, which may contribute to abnormal behavior in pups. These results are relevant for determining a safe upper limit for folate intake during pregnancy.

Activation of Type 4 Metabotropic Glutamate Receptor Regulates Proliferation and Neuronal Differentiation in a Cultured Rat Retinal Progenitor Cell Through the Suppression of the cAMP/PTEN/AKT Pathway.

Retinal progenitor cells (RPCs) remain in the eye throughout life and can be characterized by their ability for self-renewal as well as their specialization into different cell types. A recent study has suggested that metabotropic glutamate receptors (mGluRs) participate in the processes of multiple types of stem cells. Therefore, clarifying the functions of different subtypes of mGluRs in RPCs may provide a novel treatment strategy for regulating the proliferation and differentiation of endogenous RPCs after retinal degeneration. In this study, we observed that mGluR4 was functionally expressed in RPCs, with an effect on cell viability and intracellular cAMP concentration. The activation of mGluR4 by VU0155041 (VU, mGluR4 positive allosteric selective modulator) reduced the number of BrdU+/Pax6+ double-positive cells and Cyclin D1 expression levels while increasing the number of neuron-specific class III beta-tubulin (Tuj1)- and Doublecortin (DCX)-positive cells. The knockdown of mGluR4 by target-specific siRNA abolished the effects of VU on RPC proliferation and neuronal differentiation. Further investigation demonstrated that mGluR4 activation inhibited AKT phosphorylation and up-regulated PTEN protein expression. Moreover, the VU0155041-induced inhibition of proliferation and enhancement of neuronal differentiation in RPCs were significantly hampered by Forskolin (adenylyl cyclase activator) and VO-OHpic trihydrate (PTEN inhibitor). In contrast, the effect of LY294002 (a highly selective Akt inhibitor) on proliferation and differentiation was similar to that of VU. These results indicate that mGluR4 activation can suppress proliferation and promote the neural differentiation of cultured rat RPCs through the cAMP/PTEN/AKT pathway. Our research lays the foundation for further pharmacological work exploring a novel potential therapy for several retinal diseases.