Dataset Information

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

ABSTRACT:

Objective

Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.

Methods

Experimental cerebral ischemia was induced, in vitro with oxygen-glucose deprivation (OGD), or in vivo via bilateral common carotid artery occlusion (BCCAO). Genetic knock-down in vitro via siRNA, or in vivo P2Y12 transgenic mice (P2Y12-/- or P2Y12+/-), or in vivo treatment with clopidogrel, were used to manipulate the receptor. Neuron death, microglial activation, and microglial migration were assessed.

Results

The addition of microglia to neuron-astrocyte cultures increases neurotoxicity following OGD, which is mitigated by microglial P2Y12 deficiency (P<0.05). Wildtype microglia form clusters around these neurons following injury, which is also prevented in P2Y12 deficient microglia (P<0.01). P2Y12 knock-out microglia migrated less than WT controls in response to OGD-conditioned neuronal supernatant. P2Y12 (+/-) or clopidogrel treated mice subjected to global cerebral ischemia suffered less neuronal injury (P<0.01, P<0.001) compared to wild-type littermates or placebo treated controls. There were also fewer microglia surrounding areas of injury, and less activation of the pro-inflammatory transcription factor, nuclear factor Kappa B (NFkB).

Interpretation

P2Y12 participates in ischemia related inflammation by mediating microglial migration and potentiation of neurotoxicity. These data also suggest an additional anti-inflammatory, neuroprotective benefit of clopidogrel.

SUBMITTER: Webster CM

PROVIDER: S-EPMC3733797 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Publications

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

Webster Corey M CM Hokari Masaaki M McManus April A Tang Xian Nan XN Ma Hualong H Kacimi Rachid R Yenari Midori A MA

PloS one 20130805 8

<h4>Objective</h4>Microglia are among the first immune cells to respond to ischemic insults. Triggering of this inflammatory response may involve the microglial purinergic GPCR, P2Y12, activation via extracellular release of nucleotides from injured cells. It is also the inhibitory target of the widely used antiplatelet drug, clopidogrel. Thus, inhibiting this GPCR in microglia should inhibit microglial mediated neurotoxicity following ischemic brain injury.<h4>Methods</h4>Experimental cerebral ...[more]

PMID: 23940669

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Project description:BackgroundNeuroinflammation and immune responses occurring minutes to hours after stroke are associated with brain injury after acute ischemic stroke (AIS). PPARγ coactivator-1α (PGC-1α), as a master coregulator of gene expression in mitochondrial biogenesis, was found to be transiently upregulated in microglia after AIS. However, the role of microglial PGC-1α in poststroke immune modulation remains unknown.MethodsPGC-1α expression in microglia from human and mouse brain samples following ischemic stroke was first determined. Subsequently, we employed transgenic mice with microglia-specific overexpression of PGC-1α for middle cerebral artery occlusion (MCAO). The morphology and gene expression profile of microglia with PGC-1α overexpression were evaluated. Downstream inflammatory cytokine production and NLRP3 activation were also determined. ChIP-Seq analysis was performed to detect PGC-1α-binding sites in microglia. Autophagic and mitophagic activity was further monitored by immunofluorescence staining. Unc-51-like autophagy activating kinase 1 (ULK1) expression was evaluated under the PGC-1α interaction with ERRα. Finally, pharmacological inhibition and genomic knockdown of ULK1 were performed to estimate the role of ULK1 in mediating mitophagic activity after ischemic stroke.ResultsPGC-1α expression was shortly increased after ischemic stroke, not only in human brain samples but also in mouse brain samples. Microglia-specific PGC-1α overexpressing mice exhibited significantly decreased neurologic deficits after ischemic injury, with reduced NLRP3 activation and proinflammatory cytokine production. ChIP-Seq analysis and KEGG pathway analysis revealed that mitophagy was significantly enhanced. PGC-1α significantly promoted autophagic flux and induced autolysosome formation. More specifically, the autophagic clearance of mitochondria was enhanced by PGC-1α regulation, indicating the important role of mitophagy. Pharmacological inhibition or knockdown of ULK1 expression impaired autophagic/mitophagic activity, thus abolishing the neuroprotective effects of PGC-1α.ConclusionsMechanistically, in AIS, PGC-1α promotes autophagy and mitophagy through ULK1 and reduces NLRP3 activation. Our findings indicate that microglial PGC-1α may be a promising therapeutic target for AIS.

Project description:Microglia are the resident phagocytes of the central nervous system, and microglial activation is considered to play an important role in the pathogenesis of neurodegenerative diseases. Recent studies with single-cell RNA analysis of CNS cells in Alzheimer's disease and diverse other neurodegenerative conditions revealed that the transition from homeostatic microglia to disease-associated microglia was defined by changes of gene expression levels, including down-regulation of the P2Y12 receptor gene (P2Y12R). However, it is yet to be clarified in Alzheimer's disease brains whether and when this down-regulation occurs in response to amyloid-β and tau depositions, which are core pathological processes in the disease etiology. To further evaluate the significance of P2Y12 receptor alterations in the neurodegenerative pathway of Alzheimer's disease and allied disorders, we generated an anti-P2Y12 receptor antibody and examined P2Y12 receptor expressions in the brains of humans and model mice bearing amyloid-β and tau pathologies. We observed that the brains of both Alzheimer's disease and non-Alzheimer's disease tauopathy patients and tauopathy model mice (rTg4510 and PS19 mouse lines) displayed declined microglial P2Y12 receptor levels in regions enriched with tau inclusions, despite an increase in the total microglial population. Notably, diminution of microglial immunoreactivity with P2Y12 receptor was noticeable prior to massive accumulations of phosphorylated tau aggregates and neurodegeneration in rTg4510 mouse brains, despite a progressive increase of total microglial population. On the other hand, Iba1-positive microglia encompassing compact and dense-cored amyloid-β plaques expressed P2Y12 receptor at varying levels in amyloid precursor protein (APP) mouse models (APP23 and AppNL-F/NL-F mice). By contrast, neuritic plaques in Alzheimer's disease brains were associated with P2Y12 receptor-negative microglia. These data suggest that the down-regulation of microglia P2Y12 receptor, which is characteristic of disease-associated microglia, is intimately associated with tau rather than amyloid-β pathologies from an early stage and could be a sensitive index for neuroinflammatory responses to Alzheimer's disease-related neurodegenerative processes.

Dataset Information

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

Objective

Methods

Results

Interpretation

Publications

Microglial P2Y12 deficiency/inhibition protects against brain ischemia.

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