Search

P53 is a tumour suppressor protein thought to be primarily involved in cancer biology, but recent evidence suggests it may also coordinate novel functions in the CNS, including mediation of pathways underlying neurodegenerative disease. In microglia, the resident immune cells of the brain, p53 activity can promote an activation-induced pro-inflammatory phenotype Jayadev et al. (2011) [1], as well as neurodegeneration Davenport et al. (2010) [2]. Synapse degeneration is one of the earliest pathological events in many chronic neurodegenerative diseases Conforti et al. (2007) and Clare et al. (2010) [3,4] and may be influenced by early microglial responses. Here we examined synaptic properties of neurons following modulation of p53 activity in rat microglia exposed to inflammatory stimuli. A significant reduction in the expression of the neuronal synaptic markers synaptophysin and drebrin, occurred following microglial activation and was seen prior to any visible signs of neuronal cell death, including neuronal cleaved caspase-3 activation. This synaptic marker loss together with microglial secretion of the inflammatory cytokines tumour necrosis factor α (TNF-α) and interleukin 1-β (IL-1β) was abolished by the removal of microglia or inhibition of microglial p53 activation. These results suggest that transcriptional-dependent p53 activities in microglia may drive a non-cell autonomous process of synaptic degeneration in neurons during neuroinflammatory degenerative diseases., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: Microglia, the immune effector cells of the CNS and the signaling molecule Wnt, both play critical roles in neurodevelopment and neurological disease. Here we describe the inducible release of exosomes from primary cultured rat microglia following treatment with recombinant carrier-free Wnt3a., Results: Wnt3a was internalised into microglia, being detectable in early endosomes, and secreted in exosomes through a GSK3-independent mechanism. Electron microscopy demonstrated that exosomes were elliptical, electron-dense (100 nm) vesicles that coalesced with time in vitro. In contrast to microglia, primary cortical neurons released exosomes constitutively and the quantity of exosomes released was not altered by Wnt3a treatment. The proteomic profile of the microglial-derived exosomes was characterised using liquid chromatography-tandem mass spectrometry (LC/MS/MS) and the vesicles were found to be associated with proteins involved in cellular architecture, metabolism, protein synthesis and protein degradation including β-actin, glyceraldehyde-3-phosphate dehydrogenase, ribosomal subunits and ubiquitin (45 proteins in total). Unlike lipopolysaccharide, Wnt3a did not induce a neurotoxic, pro-inflammatory phenotype in primary microglia., Conclusion: These findings reveal a novel mechanism through which Wnt3a signals in microglia resulting in the release of exosomes loaded with proteinaceous cargo.

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.

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.

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.

Chromogranin A (CgA) is associated with microglial activation cascades implicated in neurodegeneration in Alzheimer's, Pick's and Parkinson's diseases. In primary rat microglia, CgA-mediated inducible nitric oxide (iNOS) expression, nitric oxide (NO) production, mitochondrial depolarisation and apoptosis were inhibited by PP2 (Src kinase inhibitor). CgA-mediated iNOS expression and NO production were also inhibited by U0126 (MEK inhibitor), but mitochondrial depolarisation and apoptosis were not. PP2 inhibited ERK phosphorylation; therefore, Src mediates CgA-induced ERK phosphorylation leading to iNOS expression and NO production. Glutamate release induced by CgA was independent of both pathways. These findings provide insights into the way microglia are activated by CgA and the microglial signalling mechanisms associated with neurological disorders such as Alzheimer's disease.

Microglial activation is implicated in the neurotoxicity of neurodegenerative diseases. Raised intracerebral levels of albumin are associated with the pathology of Alzheimer's disease, multiple sclerosis, and stroke where blood-brain barrier damage is evident. We report here that treatment of primary cultured microglia and the N9 microglial cell line with pure albumin, or albumin in which fatty acids and immunoglobulins remain attached (fraction V), induced a rise in intracellular calcium. This rise in intracellular calcium was mediated via Src tyrosine kinase and phospholipase C. The albumin-induced calcium response was coupled to microglial proliferation, which was prevented by BAPTA, U73122 or PP2 but not mimicked by thapsigargin. In contrast, peritoneal macrophages were resistant to albumin- or fraction V-induced calcium responses and proliferation, whilst primary cultured astrocytes or the TSA-3 astrocyte cell line were responsive to fraction V albumin but not pure albumin. Furthermore, cerebellar granule neurones did not respond to albumin. These data suggest that albumin may play a role in microglial activation in pathological situations involving blood-brain barrier impairment, and that the specific responses of microglia to albumin allow a distinction to be made between the signalling responses of microglia, blood-borne macrophages, astrocytes and neurones.