Inducible nitric oxide synthase is key to peroxynitrite-mediated, LPS-induced protein radical formation in murine microglial BV2 cells

Abstract

Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer’s disease, multiple sclerosis, and Parkinson’s disease. Though LPS-induced microglial activation in models of Parkinson’s disease (PD) is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation is not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor) and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, are involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-kB inhibitor PDTC and the P38 MAPK inhibitor SB202190 were used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.

Sulforaphane Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity by Activating the Keap1-Nrf2-ARE Pathway and Inhibiting the MAPKs and NF-κB Pathways

“…The activated microglia produce neurotrophic or neurotoxic mediators in neurodegenerative lesions [6,32,40,41]. Microglial activation has been correlated with the severity of HD by a clinical and positron emission tomography study [42].…”

“…Microglial activation has been correlated with the severity of HD by a clinical and positron emission tomography study [42]. Therefore, regulating microglial activation may be an attractive therapeutic strategy for various neurological diseases, including HD [6,40,41]. SFN attenuates lesion volume after spinal cord injury [17] and the neuropathic pain after spinal nerve transaction [35] by inhibiting microglial activation and proinflammatory cytokine expression.…”

Mitochondrial dysfunction and cell death in neurodegenerative diseases through nitroxidative stress

“…For instance, large amounts of NO seems to be important in causing ischemic brain injury (Garcia-Bonilla et al, 2014) and many other neuronal diseases (Butterfield et al, 2007, 2014). Direct evidence for the involvement of iNOS in neuronal diseases was provided by the experimental results of using iNOS -knockout mice, a specific inhibitor of iNOS 1400W (Kumar et al, 2014), or an anti-sense oligonucleotide specific to iNOS (Maggio et al, 2012). For instance, iNOS -knockout mice were resistant to behavioral and synaptic deficits or cerebral plaque formation and premature mortality in animal models of AD (Medeiros et al, 2007; Nathan et al, 2005).…”

Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

“…LPS-activated microglia are known to express iNOS and release NO (Kumar et al, 2014;Wen et al, 2011). We therefore tested whether blocking iNOS with the specific inhibitor N-(3-(aminomethyl)benzyl)acetamidine dihydrochloride (1400 W) prevented uptake of live PC12 cells, and found that it partially inhibited this phagocytosis (Fig.…”