N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse.

OBJECTIVE: Dopaminergic neuronal death in Parkinson's disease (PD) is accompanied by oxidative stress and preceded by glutathione depletion. The development of disease-modifying therapies for PD has been hindered by a paucity of animal models that mimic these features and demonstrate an age-related progression. The EAAC1(-/-) mouse may be useful in this regard, because EAAC1(-/-) mouse neurons have impaired neuronal cysteine uptake, resulting in reduced neuronal glutathione content and chronic oxidative stress. Here we aimed to (1) characterize the age-related changes in nigral dopaminergic neurons in the EAAC1(-/-) mouse, and (2) use the EAAC1(-/-) mouse to evaluate N-acetylcysteine, a membrane-permeable cysteine pro-drug, as a potential disease-modifying intervention for PD. METHODS: Wild-type mice, EAAC1(-/-) mice, and EAAC1(-/-) mice chronically treated with N-acetylcysteine were evaluated at serial time points for evidence of oxidative stress, dopaminergic cell death, and motor abnormalities. RESULTS: EAAC1(-/-) mice showed age-dependent loss of dopaminergic neurons in the substantia nigra pars compacta, with more than 40% of these neurons lost by age 12 months. This neuronal loss was accompanied by increased nitrotyrosine formation, nitrosylated α-synuclein, and microglial activation. These changes were substantially reduced in mice that received N-acetylcysteine. INTERPRETATION: These findings suggest that the EAAC1(-/-) mouse may be a useful model of the chronic neuronal oxidative stress that occurs in PD. The salutary effects of N-acetylcysteine in this mouse model provide an impetus for clinical evaluation of glutathione repletion in PD.

Pro-inflammatory response and adverse drug reactions: mechanisms of action of ximelagatran on chemokine and cytokine activation in a monocyte in vitro model.

There is a lack of suitable human in vitro systems which can predict drug hepatotoxicity that in many cases involves inflammatory responses mediated by macrophages. In the present investigation we used an in vitro model based on human THP-1 cells to evaluate the inflammatory cytokine/chemokine activation properties of ximelagatran, a drug previously shown to cause elevation of liver transferases in a subset of patients. Treatment of the cells with ximelagatran caused an intracellular accumulation of the metabolites hydroxymelagatran and melagatran. A decreased viability and increased release of the pro-inflammatory cytokines and chemokines IL-8, VEGF and MCP-1 was seen. Ximelagatran exposure caused activation of ERK1/2 and JNK as evident from determination of the phosphorylation status. In accordance, the release of IL-8 was attenuated by inhibitors of the ERK- and JNK-pathways. It is concluded that human monocytes might constitute a valuable additional in vitro model for monitoring the basis for cytotoxic action of drugs.

Glucocorticoid treatment induces expression of small heat shock proteins in human satellite cell populations: consequences for a desmin-related myopathy involving the R120G alpha B-crystallin mutation.

A missense mutation (R120G) of the molecular chaperone alpha B-crystallin has recently been linked to a familial form of desmin-related myopathy characterized by intrasarcoplasmic aggregates of desmin. It was previously demonstrated that the mutant R120G had a defective chaperone-like function. However, the cellular and physiopathological consequences of R120G mutant expression in human muscle cells are as yet unclear. Thus, we developed a cellular model for the study of this R120G alpha B-crystallin-related desmin-related myopathy. We demonstrate that dexamethasone enhances alpha B-crystallin and HSP27 expression in normal and desmin-related myopathy-derived muscle cells. In the undifferentiated desmin-related myopathy satellite cell population no intracytoplasmic aggregates were observed. However, in differentiated satellite cells derived from a related myopathy patient, we observed an enhanced plasma membrane localization of alpha B-crystallin following glucocorticoid. We also observed that the protective effect against stress of alpha B-crystallin is altered upon glucocorticoid-induced small heat shock protein expression for the desmin-related myopathy-derived cells.

Increased sensitivity for troglitazone-induced cytotoxicity using a human in vitro co-culture model.

Drug-induced hepatotoxicity is a major reason for withdrawal of drugs from development as well as from the market. A major problem predicting hepatotoxicity is the lack of suitable predictive in vitro system. Drug-induced hepatotoxicity is usually associated with the recruitment of immune cells to the liver accelerating an inflammatory response often initiated by activation of the Kupffer cells. In order to evaluate whether the introduction of inflammatory cells could increase the sensitivity for drug-induced cytotoxicity we developed an in vitro co-culture system based on two human cell lines; a hepatoma (Huh-7) and monocytic (THP-1) cell line. As model drugs we chose two peroxisome proliferator activated receptor gamma (PPAR gamma) agonists, the hepatotoxic troglitazone and the non-hepatotoxic rosiglitazone. In the co-cultures, troglitazone caused an enhanced cytotoxicity as compared to single cultures of either cell line, whereas little cytotoxicity was seen after treatment with rosiglitazone. Troglitazone treatment increased gene expression of pro-inflammatory mediators and stress-related genes in both cell types, which in general was more pronounced in co-cultures than in single cell cultures. Based on these results we suggest that co-cultures of human hepatoma cells and monocytes might provide an important in vitro system for better prediction of cytotoxicity mediated by potential hepatotoxins.

NADPH oxidase is the primary source of superoxide induced by NMDA receptor activation.

Neuronal NMDA receptor (NMDAR) activation leads to the formation of superoxide, which normally acts in cell signaling. With extensive NMDAR activation, the resulting superoxide production leads to neuronal death. It is widely held that NMDA-induced superoxide production originates from the mitochondria, but definitive evidence for this is lacking. We evaluated the role of the cytoplasmic enzyme NADPH oxidase in NMDA-induced superoxide production. Neurons in culture and in mouse hippocampus responded to NMDA with a rapid increase in superoxide production, followed by neuronal death. These events were blocked by the NADPH oxidase inhibitor apocynin and in neurons lacking the p47(phox) subunit, which is required for NADPH oxidase assembly. Superoxide production was also blocked by inhibiting the hexose monophosphate shunt, which regenerates the NADPH substrate, and by inhibiting protein kinase C zeta, which activates the NADPH oxidase complex. These findings identify NADPH oxidase as the primary source of NMDA-induced superoxide production.

Glutamate activates c-fos in glial cells via a novel mechanism involving the glutamate receptor subtype mGlu5 and the transcriptional repressor DREAM.

Activation of c-fos in brain is related to coupling of neuronal activity to gene expression, but also to pathological conditions such as seizures or excitotoxicity-induced cell death. Glutamate activates c-fos in neurons through the calcium-dependent phosphorylation of CREB by ERK and/or CaMKIV kinase pathways downstream NMDA-receptors. In glial cells, however, the activation of c-fos by glutamate is poorly understood. Because glial cells actively modulate neuronal excitability and the brain's response to injury, we studied the mechanisms by which glutamate activates c-fos in rat cortical glial cells. Glutamate potently induced c-fos mRNA in a calcium-dependent manner, as demonstrated by using the calcium chelator BAPTA-AM. Glutamate-induced c-fos mRNA expression was not sensitive to inhibitors of ERK, p38(MAPK), or CaMK pathways, indicating that glial c-fos is activated by a distinct mechanism. Thapsigargin abolished the glutamate effect on c-fos mRNA, indicating ER calcium mobilization. Additionally, glutamate induction of c-fos mRNA was sensitive to the mGluR5 antagonist MPEP but not the NMDA-R antagonist MK-801. In luciferase reporter assays, DRE, which actively represses c-fos by binding the calcium-binding transcriptional repressor DREAM, was activated by glutamate, whereas SRE and CRE were not. Finally, glutamate caused the nuclear export of DREAM in astrocytes, and transfection of astrocytes with a mutant variant of DREAM that constitutively binds DNA inhibited glutamate-induced c-Fos expression. These findings are in sharp contrast to the mechanism described in neurons and suggest a novel pathway activated by glutamate in glial cells that employs mGluR5, ER calcium, and the derepression of c-fos at the DRE.

Activation of c-fos by lipopolysaccharide in glial cells via p38 mitogen-activated protein kinase-dependent activation of serum or cyclic AMP/calcium response element.

Pathological conditions such as ischaemic stroke and inflammatory disorders cause c-fos activation in the brain. This activation contributes to the initiation of the brain's inflammatory response, orchestrated by activated glial cells. The inflammatory signalling cascades leading to c-fos activation in glial cells are not well characterized. Thus, we have attempted a detailed analysis of the cis-acting elements, transcription factors and upstream kinase pathways involved in the activation of c-fos by lipopolysaccharide (LPS) in primary rat cortical glial cells. We found that (1) LPS-induced c-fos mRNA levels were sensitive to p38 mitogen-activated protein kinase (MAPK) inhibitors but not to mitogen-activated/extracellular signal-regulated kinase (ERK) or calcium-calmodulin-dependent kinase inhibitors, (2) LPS activated both serum response element (SRE) and cyclic AMP/calcium response element (CRE)-driven luciferase reporters in transient transfection assays, (3) LPS induced the phosphorylation of Elk1 CRE-binding protein (CREB)/activated transcription factor-1 (ATF-1) and the activation of GAL4-Elk1 and GAL4-CREB chimeric proteins, and (4) mutation of both SRE and CRE elements was necessary and sufficient to completely abolish LPS induction of a rat c-fos proximal promoter-luciferase reporter. Thus, c-fos activation by LPS in glial cells occurs via the SRE or CRE in an independent manner, and involves the Elk1 or CREB/ATF-1 transcription factors. Elk1-mediated transactivation was dependent on p38 MAPK, suggesting a crucial role of these factors in mediating inflammatory responses in the CNS.