ABSTRACT
Aberrant or chronic microglial activation is strongly implicated in neurodegeneration, where prolonged induction of classical inflammatory pathways may lead to a compromised blood-brain barrier (BBB) or vasculature, features of many neurodegenerative disorders and implicated in the observed cognitive decline. BBB disruption or vascular disease may expose the brain parenchyma to "foreign" plasma proteins which subsequently impact on neuronal network integrity through neurotoxicity, synaptic loss and the potentiation of microglial inflammation. Here we show that the blood coagulation factor fibrinogen (FG), implicated in the pathogenesis of dementias such as Alzheimer's disease (AD), induces an inflammatory microglial phenotype as identified through genetic microarray analysis of a microglial cell line, and proteome cytokine profiling of primary microglia. We also identify a FG-mediated induction of non-cell autonomous ER stress-associated neurotoxicity via a signaling pathway that can be blocked by pharmacological inhibition of microglial TNFα transcription or neuronal caspase-12 activity, supporting a disease relevant role for plasma components in neuronal dysfunction.
ABSTRACT
Microglial activation can lead to microglial apoptosis, which may serve to remove highly reactive and possibly neurotoxic microglia. However the loss of microglia may have consequences for future recovery, protection and repair. P53, a nuclear phosphoprotein transcription factor, is critical for activating the expression of genes involved in cell-cycle arrest and stress-induced apoptosis. In neurodegenerative diseases the expression of p53 is significantly increased in glial cells, and microglial numbers fall. Following activation with chromogranin A (100 nM), or beta-amyloid(25-35), (10 microM), microglia became apoptotic. Furthermore, p53 expression was enhanced, peaking at 4-6 h after exposure to activators. The p53 transcription inhibitor, pifithrin-alpha, (10 microM) significantly reduced the expression of p53 in microglia and significantly modulated the levels of microglial apoptosis induced by activation. Lithium chloride (5 mM), which can modulate p53-mediated pathways, also reduced p53 expression and reduced microglial apoptosis suggesting glycogen synthase kinase-3 plays a role. Regulating p53 pathways modulated microglial inducible nitric oxide synthase expression and tumour necrosis factor alpha secretion. Inhibiting p53 mediated microglial apoptosis prevented microglial neurotoxicity suggesting targeting of p53-mediated pathways in microglia may have therapeutic benefit in Alzheimer's disease.
Subject(s)
Amyloid beta-Peptides/pharmacology , Microglia/drug effects , Microglia/metabolism , Peptide Fragments/pharmacology , Signal Transduction/drug effects , Tumor Suppressor Protein p53/metabolism , Adjuvants, Immunologic/pharmacology , Animals , Animals, Newborn , Apoptosis/drug effects , Benzothiazoles/pharmacology , Cells, Cultured , Cerebellum/cytology , Chromogranin A/pharmacology , Drug Interactions , Enzyme-Linked Immunosorbent Assay/methods , Lithium Chloride/pharmacology , Neurons/drug effects , Neurons/metabolism , Polysaccharides/pharmacology , Rats , Rats, Wistar , Time Factors , Toluene/analogs & derivatives , Toluene/pharmacology , Transcription Factor CHOP/metabolism , Tumor Necrosis Factor-alpha/metabolism , Tumor Suppressor Protein p53/antagonists & inhibitorsABSTRACT
Chromogranin A (CgA), a neuroactive glycoprotein, is associated with microglial activation cascades implicated in neurodegeneration. Here we show that CgA-dependent inducible nitric oxide synthase (iNOS) expression and stress responses in microglia involved signalling via scavenger receptors (SR), since SR class-A (SR-A) ligands blocked iNOS expression, mitochondrial depolarisation, apoptosis and glutamate release. Furthermore, block of SR-A ameliorated CgA-induced microglial neurotoxicity. In contrast, block of CD36, or the receptor for advanced glycation end products (RAGE) did not prevent CgA-induced microglial activation and neurotoxicity. Thus, manipulation of specific scavenger receptor-coupled signalling pathways may provide avenues for therapeutic intervention in neurodegenerative diseases implicating microglial activation with chromogranin peptides.
Subject(s)
Chromogranin A/toxicity , Microglia/drug effects , Microglia/metabolism , Neurotoxins/toxicity , Receptors, Scavenger/metabolism , Signal Transduction/drug effects , Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid beta-Peptides/metabolism , Animals , Cell Line , Chromogranin A/chemistry , Molecular Sequence Data , Neurotoxins/chemistry , Plaque, Amyloid/drug effects , Plaque, Amyloid/metabolism , Rats , Rats, Wistar , Scavenger Receptors, Class A/metabolismABSTRACT
Microglial activation by blood-borne factors following blood-brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFalpha) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l-alpha-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood-brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.
Subject(s)
Cell Differentiation/drug effects , Macrophages, Peritoneal/drug effects , Microglia/drug effects , Serum Albumin/pharmacology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Animals, Newborn , Caspases/metabolism , Cell Death/drug effects , Cells, Cultured , Cerebellum/cytology , Culture Media, Conditioned/pharmacology , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Gene Expression Regulation/drug effects , Glutamic Acid/metabolism , Glutathione/metabolism , Lipopolysaccharides/pharmacology , Macrophages, Peritoneal/chemistry , Microglia/chemistry , Neurons/drug effects , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Polymyxin B/pharmacology , Rats , Rats, Wistar , Time FactorsABSTRACT
Microglia are present in an activated state in multiple sclerosis lesions. Incubation of primary cultured rat microglia with rat-brain derived myelin (0.1-1 microg/mL) for 24 h induced microglial activation; cells displayed enhanced ED1 staining, expression of inducible nitric oxide synthase, production and release of the cytokine tumour necrosis factor-alpha and glutamate release. Exposure of microglia to myelin induced the expression of neuronal caspases and ultimately neuronal death in cultured cerebellar granule cell neurons; neurotoxicity was directly because of microglial-derived soluble toxins. Co-incubation of microglia with agonists or antagonists of different metabotropic glutamate receptor (mGluR) subtypes ameliorated microglial neurotoxicity by inhibiting soluble neurotoxin production. Activation of microglial mGluR2 exacerbated myelin-evoked neurotoxicity whilst activation of mGluR3 was protective as was activation of group III mGluRs. These data show that myelin-induced microglial neurotoxicity can be prevented by regulation of mGluRs and suggest these receptors on microglia may be promising targets for therapeutic intervention in multiple sclerosis.
