The self-renewal and multipotent potentials in neural stem cells (NSCs) maintain the normal physiological functions of central nervous system (CNS). The abnormal differentiation of NSCs would lead to CNS disorders. However, the mechanisms of how NSCs differentiate into astrocytes, oligodendrocytes (OLs) and neurons are still unclear, which is mainly due to the complexity of differentiation processes and the limitation of the cell separation method. In this study, we modeled the dynamics of neural cell interactions in a systemic approach by mining the high-throughput genomic and proteomic data, and identified 8615 genes that are involved in various biological processes and functions with significant changes during the differentiation processes. A total of 1559 genes are specifically expressed in neural cells, in which 242 genes are NSC specific, 215 are astrocyte specific, 551 are OL specific, and 563 are neuron specific. In addition, we proposed 57 transcriptional regulators specifically expressed in NSCs may play essential roles in the development courses. These findings provide more comprehensive analysis for better understanding the endogenous mechanisms of NSC fate determination.
Animals ; Astrocytes ; cytology ; metabolism ; Cell Differentiation ; genetics ; Gene Expression Profiling ; Gene Regulatory Networks ; Mice ; Neural Stem Cells ; cytology ; metabolism ; Oligodendroglia ; cytology ; metabolism ; Protein Interaction Mapping

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP cells mainly generated NeuN neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP cells at the young adult stage is significantly different from that at the adult stage.
Animals ; Astrocytes ; cytology ; metabolism ; Brain ; cytology ; growth & development ; metabolism ; Calbindins ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Mice ; Mice, Transgenic ; Nerve Tissue Proteins ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Neurons ; cytology ; metabolism ; Nuclear Proteins ; metabolism

Animals ; Astrocytes ; cytology ; Cell Differentiation ; Cellular Reprogramming ; Induced Pluripotent Stem Cells ; cytology ; Kruppel-Like Transcription Factors ; genetics ; metabolism ; Mice ; Neurons ; cytology ; Octamer Transcription Factor-3 ; genetics ; metabolism

The blood-brain barrier (BBB) is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein 1 (DMP1)-proteoglycan (PG), which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMP1-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 (S89) in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine (S89G), which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that regulated BBB formation, but also assigned a new function to DMP1-PG.
Animals ; Astrocytes ; cytology ; metabolism ; Blood-Brain Barrier ; cytology ; metabolism ; Cells, Cultured ; Extracellular Matrix Proteins ; genetics ; metabolism ; Female ; Glycosylation ; Male ; Mice ; Proteoglycans ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1 (+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 microg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained. IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P< 0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.
Astrocytes/cytology ; Astrocytes/*metabolism ; Cell Line, Transformed ; Cells, Cultured ; Galanin/*biosynthesis ; Galanin/genetics ; Genetic Vectors ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Transfection

The regulation of astroglia on synaptic plasticity in the CA1 region of rat hippocampus was examined. Rats were divided into three groups: the newly born (< 24 h), the juvenile (28-30 days) and the adult groups (90 - 100 days), with each group having 20 animals. The CA1 region of rat hippocampus was immunohistochemically and electron-microscopically examined, respectively, for the growth of astroglia and the ultrastructure of synapses. The high performance liquid chromatography was employed to determine the cholesterol content of rat hippocampus. In the newly-born rats, a large number of neurons were noted in the hippocampal CA1 region of the newly-born rats, and few astroglia and no synaptic structure were observed. In the juvenile group, a few astroglias and some immature synapses were found, which were less than those in adult rats (P < 0.01). The cholesterol content was 2.92 +/- 0.03 mg/g, 11.20 +/- 3.41 mg/g and 12.91 +/- 1.25 mg/g for newly born, the juvenile and the adult groups, respectively, with the differences among them being statistically significant (P < 0.01). Our study suggests that the astrocytes may play an important role in the synaptic formation and functional maturity of hippocampal neurons, which may be related to the secretion of cholesterol from astrocytes.
Age Factors ; Animals, Newborn ; Astrocytes/cytology ; Astrocytes/metabolism ; Astrocytes/*physiology ; CA1 Region, Hippocampal/*physiology ; CA1 Region, Hippocampal/*ultrastructure ; Cell Communication/physiology ; Cholesterol/metabolism ; Neuronal Plasticity/*physiology ; Random Allocation ; Rats, Wistar ; Synapses/*physiology ; Synapses/ultrastructure

OBJECTIVETo study the effects of deltamethrin (DM) on cell survival rate and intracellular Ca(2+) ([Ca(2+)]i) concentration in primary cultured astrocytes of rat.

METHODSThe cell survival rate was measured by Typan Blue assay; the intracellular [Ca(2+)]i concentration was determined by the fluorescent Ca(2+) indicator Fura-2/AM.

RESULTSThe survival rate of astrocytes was decreased to 91.9% after astrocytes were incubated with 1 x 10(-5) mol/L DM for 72 h (P < 0.05). The cell survival rates were 89.0%, 84.8%, 81.2% and 79.2% respectively when astrocytes were administered with 1 x 10(-4) mol/L DM for 4, 12, 24 and 72 h, which were remarkably lower than control groups (P < 0.01). Comparing with controls and before DM treatment, sharp increases in [Ca(2+)]i concentration [(451.4 +/- 42.3), (536.9 +/- 47.5) and (870.9 +/- 100.5) nmol/L respectively] were observed when astrocytes were incubated with 1 x 10(-7), 1 x 10(-6) and 1 x 10(-5) mol/L DM for 5 minutes (P < 0.01). After astrocytes were treated with 1 x 10(-8), 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) mol/L DM for 15 minutes, the [Ca(2+)]i concentrations were decreased to (124.3 +/- 6.0), (131.3 +/- 19.1), (118.9 +/- 1.4), (136.6 +/- 3.8) nmol/L respectively, which were significantly different from those of controls and before treatment. And this situation was almost keeping stable to 30 min.

CONCLUSIONThe cell survival rate was decreased and the [Ca(2+)]i concentration was temporarily increased when astrocytes were treated with DM.

Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Cell Survival ; drug effects ; Cells, Cultured ; Insecticides ; toxicity ; Nitriles ; Pyrethrins ; toxicity ; Rats

OBJECTIVETo investigate the effect of propofol on the expression of thrombospondin-1 (THBS-1) mRNA and protein in purified newborn rat cortical astrocytes in vitro.

METHODSAstrocytes were isolated from newborn rat cortex and grown in culture before exposure to propofol at 3, 10, 30, 100 or 300 µmol/L for 6 h, 12, or 24 h. The mRNA level of THBS-1 was detected by RT-PCR, and the protein level of THBS-1 was detected by immunofluorescence cytochemistry and Western blotting.

RESULTSPropofol exposure caused significantly upregulated THBS-1 level in cultured astrocytes (P<0.05) to a level about 1.3 times higher than that in control cells. The mRNA and protein levels of THBS-1 in cultured rat cortical astrocytes were upregulated by exposures to 10, 30 and 100 µmol/L propofol (P<0.01). High expression of THBS-1 mRNA and protein was detected in the cells with exposures for different durations (P<0.05), especially in the 12 h group (P<0.01).

CONCLUSIONPropofol at clinically relevant concentrations can modulate the level of THBS-1 secreted by astrocytes of rat cerebral cortex in vitro.

Animals ; Astrocytes ; drug effects ; metabolism ; Cells, Cultured ; Cerebral Cortex ; cytology ; Propofol ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Thrombospondin 1 ; metabolism

OBJECTIVETo study the effects of resveratrol on amino acid release in cultured astrocytes with stretch injury and explore its possible mechanism for central nervous system protection.

METHODSCultured rat astrocytes were treated with different concentrations of resveratrol for 12 h and the subsequent changes in Glu and Gln release and lactate dehydrogenase(LDH) leakage were examined after stretch injury.

RESULTSHigh-performance liquid chromatography showed that stretch injury increased Glu release from the astrocytes (P<0.05), and at the concentration of 1 micromol/L, resveratrol further increased Glu release (P<0.05), whereas at 100 micromol/L, resveratrol obviously inhibit Glu release (P<0.05). LDH leakage increased significantly after stretch injury of the astrocytes (P<0.05), and resveratrol acted to further increase LDH leakage at 1 micromol/L but inhibited LDH leakage at 100 micromol/L (P<0.05).

CONCLUSIONResveratrol can inhibit the release of Glu and LDH leakage from rat astrocytes to protect the cells from stretch injury.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; metabolism ; Cells, Cultured ; Glutamine ; metabolism ; Glutathione ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Neuroprotective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology ; Stress, Mechanical

OBJECTIVE: To observe the internalization of beta-amyloid protein (Abeta) in primary cultured neurons and effect of astrocyte on it. METHODS: The purified cortical neurons of mouse were cultured for 14 d, and were divided into a control group and an Abeta group. Each group was further divided into 3 subgroups. The neurons and 3 different concentration fluorescein or Abeta1-42-fluo were co-incubated for 24 h. The internalization of Abeta and the location of Abeta in subcellular structure were examined by the laser scanning confocal microscope combined with the image analysis method directly or after immunofluorescence staining. Neurons and astrocytes were co-cultured for 14 d. The cultured neurons and astrocytes were divided into a control group and a Abeta group. The cultures were treated with 200 nmol/L fluorescein or 200 nmol/L Abeta1-42-fluo for 24 h respectively. The effect of astrocyte on the internalization was analyzed by the above method. RESULTS: There were no fluorescent granules within neurons in every fluorescein group. The purified cortical neurons could internalize 100 nmol/L, or 200 nmol/L Abeta1-42-fluo in 24 h. The fluorescent granules of Abeta1-42 distributed within perikaryon and processes. The internalization was related to the concentration of Abeta. The part of Abeta was located in the lysosome of neurons indicated by immunofluorescence staining. Compared with the purified neurons, the neurons co-cultured with the astrocytes internalized Abeta increased in the internalization of Abeta. There was significant difference between the purified neurons and the co-cultured neurons with astrocytes (P<0.05). CONCLUSION Neurons could internalize the proper concentration of Abeta. Astrocyte might facilitate the internalization of Abeta in neurons.
Alzheimer Disease ; metabolism ; Amyloid beta-Peptides ; metabolism ; Amyloid beta-Protein Precursor ; metabolism ; Animals ; Animals, Newborn ; Astrocytes ; cytology ; Cells, Cultured ; Cerebral Cortex ; cytology ; metabolism ; Coculture Techniques ; Endocytosis ; genetics ; Mice ; Neurons ; cytology ; metabolism