Astrocytic mRNA Expression Research Articles

Oxidative stress in rat cortical neurons and astrocytes induced by paraquat in vitro.

Oxidative stress has been discussed as crucial mechanism of neuronal cell death in the adult brain. However, it was not clear until now whether neurons are more sensitive to oxidative stress than the other cells in the brain, e.g. astrocytes. Therefore both cell types were exposed to oxidative stress provoked by the redox-cycling compound paraquat. Cortical neurons were found to be more sensitive towards paraquat toxicity than astrocytes as shown by MTT and Neutral Red assay, two different cytotoxicity assays. Mitochondrial functions were determined by the mitochondrial membrane potential and intracellular ATP concentrations. Again cortical neurons were more severely impaired (by paraquat than astrocytes). The production of reactive oxygen species after paraquat exposure was much higher in cortical neurons than in astrocytes and correlated with a higher depletion of GSH (intracellular glutathion). Lipid peroxidation could be shown in astrocytes via the breakdown product malondialdehyde (MDA) whereas in cortical neurons 4-hydroxynonenal (4-HNE) was detected as this endpoint. If and how oxidative stress influences the antioxidant defense was determined via changes in the expression of antioxidant enzymes. Paraquat exposure lead to a 2-3 fold increase of catalase, MnSOD and CuZnSOD mRNA expression in astrocytes. In contrast to astrocytes, in cortical neurons catalase and MnSOD mRNA levels were only marginally elevated above 1.5-fold by treatment with paraquat. Expression levels of glutathione peroxidase (GPx) mRNA were the only one that were not changed in both cell types after paraquat exposure. It is concluded that the more marked increase in expression levels of antioxidant enzymes may render astrocytes more resistant to oxidative stress than neuronal cells.

Muramyl dipeptide potentiates cytokine-induced activation of inducible nitric oxide synthase in rat astrocytes

We investigated the influence of muramyl dipeptide (MDP), a cell wall component of Gram-positive bacteria, on cytokine-induced nitric oxide (NO) production in rat primary astrocytes. MDP alone did not induce NO release in astrocyte cultures. However, MDP increased astrocyte NO production and subsequent nitrite accumulation triggered by IFN-γ. IFN-γ-activated expression of mRNA for inducible NO synthase (iNOS) and iNOS transcription factor interferon regulatory factor-1 (IRF-1) was markedly enhanced in astrocytes treated with MDP. The potentiating effect of MDP on IFN-γ-induced NO production in astrocytes was completely blocked with protein tyrosine kinase (PTK) inhibitor genistein or mitogen activated protein kinase (MAPK) inhibitor PD98059. In contrast, protein kinase C (PKC) inhibitor calphostin C did not affect ability of MDP to augment IFN-γ-triggered astrocyte NO synthesis. These results suggest that MDP synergizes with IFN-γ in the induction of iNOS gene in astrocytes through mechanisms involving PTK and MAPK, but not PKC activation. Finally, MDP also augmented NO production and iNOS mRNA expression in astrocytes treated with IL-1β.

Regulation of beta-chemokine mRNA expression in adult rat astrocytes by lipopolysaccharide, proinflammatory and immunoregulatory cytokines.

Astrocytes constitute a part of the blood-brain barrier. Chemokine expression by astrocytes may contribute to leucocyte infiltration within the central nervous system (CNS) during inflammation. To investigate factor(s) regulating chemokine expression by astrocytes, we studied the induction of beta-chemokine mRNA expression in adult rat astrocytes. Astrocyte-derived monocyte chemoattractant protein- (MCP-1), RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta mRNA were induced by interferon-gamma (IFN-gamma). Tumour necrosis factor-alpha (TNF-alpha) induced MCP-1, RANTES and MIP-1beta mRNA expression, and lipopolysaccharide (LPS) induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression in astrocytes. LPS-induced MCP-1, MIP-1alpha and MIP-1beta mRNA expression by astrocytes was antagonized by transforming growth factor (TGF)-beta1 and interleukin (IL)-10. TGF-beta1 and IL-10 also down-regulated MCP-1 and RANTES mRNA expression induced by TNF-alpha. IL-10, but not TGF-beta1, inhibited MIP-1beta mRNA expression induced by TNF-alpha. The results of this in vitro study suggest that beta-chemokine mRNA expression by adult rat astrocytes can be induced by LPS or proinflammatory cytokines, while regulatory cytokines, such as TGF-beta1 and IL-10, down-regulate astrocyte-derived beta-family chemokine mRNA expression induced by LPS, IFN-gamma and TNF-alpha. Further study of CNS chemokines will enhance our understanding of leucocyte recruitment to the CNS and suggest therapeutic strategies for neurological disorders.

Life-Long Dietary Restriction Attenuates Age-Related Increases in Hippocampal Glial Fibrillary Acidic Protein mRNA

The expression of astrocyte-specific glial fibrillary acidic protein increases after experimental lesions and is elevated throughout the brain in aged rodents and primates. Clusterin (ApoJ) expression increases in astrocytes and microglia after lesions, but changes during aging have not been reported. Dietary restriction (DR) delays the onset and progression of many age-related physiological deficits in rodents. This study showed that the age-related increase in glial fibrillary acidic protein mRNA in the hippocampus was attenuated in 24-month-old male Fischer 344 rats subjected to a 50% DR beginning at 6 weeks of age. ApoJ mRNA expression in astrocytes was unchanged by DR. These results demonstrate that DR can delay neurodegeneration in aged rats as assessed by a marker of reactive astrogliosis.

Regulatory sequences for the transcription of the laminin B2 gene in astrocytes

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types in order to understand the transcriptional regulation of laminin B2 gene in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed different patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U251 and rat C6 glioma cells, a greater number of nuclear proteins ranging from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet active in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in both astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5′-flanking regions responsible for basal levels of B2 laminin promoter activity revealed a silencer-like segment between −830 and −224 which reduced promoter activity. Deletion analysis further revealed that B2 laminin promoter possesses a highly active short promoter (−94 to +106) and basal transcriptional activity resides within −61 to +106. DNase 1 footprinting, gel-shift competition assays and site-directed mutagenesis of a highly active short promoter revealed that this region contained binding sites for cell-type nuclear factors. The shortest construct containing only residues −21 to +106 was inactive in HepG2 and U251 glioma cells. In primary astrocytes, however, this construct showed a high level of transcriptional activity. Deletion of 47 bp (+59 to +106) in 5′-UTR completely blocked promoter activity in astrocytes confirming that this downstream region is important for transcriptional activity in primary astrocytes. Together, these results suggest that astrocytes may utilize mutually exclusive transcription factors and regulatory sequences, in addition to common factors in the control of the laminin B2 promoter.

Regulation of Clusterin Secretion and mRNA Expression in Astrocytes by Cytokines

Clusterin is an authentic Sertoli cell secretory product initially identified in the ram and rat testis. Subsequent studies have shown that this protein is present in almost all organs and in multiple species. Its mRNA increases in the brain undergoing degeneration as a result of infection, brain injury, and other pathological conditions such as Alzheimer's disease. However, its site(s) of synthesis and modulator(s) in the brain are not known. The objectives of this study were to determine if astrocytes could synthesize and secrete clusterin in vitro and to investigate the effects of various cytokines on the secretion and the mRNA expression of clusterin in the primary cultures of astrocytes. Astrocytes were isolated from cerebral cortices of neonatal rats and enriched to a purity of greater than 95% as judged by immunocytochemical staining using antibody against glial fibrillary acidic protein (GFAP), a specific marker of astrocytes. Using immunoprecipitation techniques, we have demonstrated that astrocytes actively synthesize and Secrete clusterin in vitro. Immunocytochemical staining using a monospecific antibody against clusterin showed that this protein is localized in the entire cytoplasm and the processes of astrocytes. Treatment of astrocytes with either interleukin-1β, or interleukin-2, induced a significant increase in the production and the mRNA levels of clusterin, whereas other cytokines including interleukin-3, interleukin-6, and interferon-γ had no apparent effect. The results of this study suggest that clusterin may be a marker to study the immune response in the brain.

Differential expression of alpha 1-antichymotrypsin in the aged human brain.

The localization of alpha 1-antichymotrypsin (ACT) mRNA and ACT immunoreactivity (ACT-IR) were examined in 12 brains obtained at post-mortem from elderly patients, four of whom had Alzheimer's disease. A biotinylated oligonucleotide probe was used for in situ hybridization histochemistry and the relationship between the expression of both ACT mRNA and ACT-IR and the extent of beta protein or tau deposition were investigated. The extent of beta-plaques, tau-tangles, and ACT-IR were rated from (-) to (++). In brains without plaques and tangles, there were no detectable ACT mRNA signals in the gray matter, and those in the white matter were weak; in these brains, ACT-IR was generally weak. The brains with beta-plaques but no tangles showed weak ACT mRNA expression in astrocytes of both the gray and white matter; they also showed weak ACT-IR in the astrocytes. In the brains from patients with Alzheimer's disease with both plaques and tangles, ACT mRNA was expressed intensely in a majority of the astrocytes in the white and gray matter. Some senile plaques-associated astrocytes expressed ACT mRNA and ACT-IR was strong in the white matter astrocytes. ACT-IR and ACT mRNA expression in astrocytes was correlated with the extent of beta and tau depositions.

DEMONSTRATION OF SERINE PROTEASE INHIBITORS IN THE ALZHEIMER-TYPE DEMENTIA BRAINS

I examined the localization of α1-antichymotrypsin (ACT) mRNA and ACT immunoreactivity (ACT-IR) in 12 brains obtained from elderly autopsy cases, including 4 of Alzheimer-type dementia. I investigated the relationship between the ACTmRNA and ACT-IR and the extent of β or tau deposits. In the brains without plaques and tangles, there were no detectable ACTmRNA signals in the gray matter, and those in the white matter were weak. In these brains, ACT-IR was generally weak. The brains with fl-plaques but no tangles showed weak ACT mRNA expression in the astrocytes in both the gray and white matter. They also showed weak ACT-IR in the astrocytes. In the Alzheimer brains with both plaques and tangles, ACTmRNA was expressed intensely in a majority of the astrocytes in the white and gray matter. Some senile plaque-associated astrocytes expressed ACTmRNA. ACT-IR was abundant in the white matter astrocytes. Among protease inhibitors and proteases, only ACT was associated with β-plaques. ACT-IR and ACT mRNA expression in astrocytes was well-correlated with the extent of β and tau deposits. It can be assumed that ACT plays an important role in amyloidogenesis.

Induction and regulation of class II major histocompatibility complex mRNA expression in astrocytes by interferon-γ and tumor necrosis factor-α

Astrocytes can function as antigen-presenting cells (APC) upon expression of class II major histocompatibility complex (MHC) antigens, which are induced by interferon-γ (IFN-γ). Previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (TNF-α) enhances IFN-γ-mediated class II antigen expression on astrocytes. We have now investigated the effect of IFN-γ and TNF-α on class II MHC mRNA expression in astrocytes using Northern blot analysis. Astrocytes do not constitutively express mRNA for class II MHC. Kinetic analysis of class II MHC mRNA expression in IFN-γ-treated cells demonstrated an 8 h time lag, which was followed by an increase over the next 16 h. Optimal expression of class II mRNA was detected after a 24 h incubation with IFN-γ. This level of expression was further enhanced by the simultaneous addition of IFN-γ and TNF-α to the astrocytes, while TNF-α alone had no effect on class II mRNA expression. TNF-α does not act by increasing the stability of IFN-γ-induced class II mRNA, indicating its action is not at that specific level of post-transcriptional control. Furthermore, astrocyte class II mRNA expression was inhibited when cycloheximide (CHX) was added together with IFN-γ or IFN-γ/TNF-α, and when CHX was added up to 4 h after treatment with IFN-γ or IFN-γ/TNF-α. These results indicate that astrocyte class II mRNA expression is mediated by newly synthesized proteins induced by IFN-γ and/or IFN-γ/TNF-α. The expression of class II antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses.