Roles of microglial purinergic receptors in inflammatory conditions of the brain

Microglia, the resident immune cells of brain, mediate inflammatory responses leading to progressive neuronal damage in neurodegenerative diseases. Binding of ATP to purinergic receptors activates microglia thereby inducing cellular responses in inflamed brain cells. The two families of purinergic receptors, labelled P2XR (ionotropic) and P2YR (metabotropic) contribute to inflammatory responses in microglia. The first two parts of my study focused on the involvement and role of the ionotropic purinergic receptor, P2X₇R in mediating inflammatory responses such as secretion of pro-inflammatory factors in vitro and in vivo. The final part of my study concentrated on purinergic receptor-dependent intracellular Ca²⁺([Ca²⁺]i) mobilization and functional responses in human fetal microglia [i.e. foetal microglia]. A detailed in vivo study was carried out on the involvement of P2X₇R in mediating lipopolysaccharide (LPS)-induced inflammatory responses and neuronal damage in rat striatum. LPS-injected striatum exhibited a marked increase in the expression and production of P2X₇R compared with control (saline)-injected animals. Additionally, LPS injection upregulated a host of pro-inflammatory mediators and reduced neuronal viability. The P2X₇R antagonist, oxidized ATP (oxATP) was effective in attenuating expressions of all inflammatory mediators; most importantly oxATP was protective for striatal neurons. In vitro, I found LPS stimulation of cultured human microglia enhanced cellular expressions of inflammatory mediators and increased [Ca²⁺]i mobilization which were blocked with oxATP treatment. Overall, the results from this work indicate that P2X₇R plays a critical role in LPS-induced inflammatory responses including induction of neuronal damage. Subsequently a series of studies was designed to examine putative roles of P2X₇R in mediating inflammatory responses with relevance to the pathology typical of Alzheimer’s disease (AD). First, I found microglia isolated from AD brains expressed enhanced P2X₇R compared with microglia obtained from non-demented individuals. In a second study, human fetal microglia stimulated with Aβ₁₋₄₂ peptide exhibited markedly elevated levels of P2X₇R compared with untreated cells. Also, P2X₇R-mediated Ca²⁺ responses were increased with Aβ₁₋₄₂ pretreatment of cells relative to untreated cells. Finally, in vivo double immunostaining analysis showed considerable P2X₇R co-localized with microglia following injection of Aβ₁₋₄₂ into rat hippocampus. The overall results from this section of study show the involvement of P2X₇R in mediating microglial purinergic inflammatory responses in AD brain. We were also interested in the contribution of purinergic receptors other than P2X₇R in mediating inflammatory responses. I focused on cyclooxygenase-2 (COX-2) since this enzyme is highly elevated in inflamed brain and contributes to inflammation-induced cytotoxicity. In this research, we used a low concentration of ATP (100 μM) to eliminate contributions of P2X₇R since activation of this purinergic subtype receptor requires concentrations of ATP in excess of 1mM. In summary, we found that the block of P2XR (candidate P2X₄R) increased the duration of ATP-mediated [Ca²⁺]i responses and upregulated expression and production of COX-2. The prolonged response involved influx of Ca²⁺ through store-operated channels (SOC) and was suggested as a consequence of removal of cell depolarization by the block of P2XR. Inhibition of SOC was then shown to be effective in attenuating COX-2 expression in human microglia. These novel results link inhibition of P2XR, other than P2X₇R, with upregulation of COX-2 in human microglia with the link involving SOC-mediated Ca²⁺ influx. The overall findings from this study suggest that pharmacological manipulation of P2X₇R and other purinergic receptors could serve as a potential therapeutic intervention in modulating inflammatory responses in microglia.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat