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Sindhu Saraswathy, Narsing Rao; The induction of NLRC4 Inflammasome Activation in Photoreceptor mitochondrial oxidative stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1199.
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© ARVO (1962-2015); The Authors (2016-present)
Activation of the NLRC4 is known to activate inflammasome complex, primarily made up of caspase1, IL-1B and IL-18 which leads to a unique programmed cell death, pyroptosis. We recently developed a selective photoreceptor iNOS overexpressing transgenic animals with phenotype of robust photoreceptor mitochondrial oxidative stress. Using this model, we investigated effect of the oxidative stress on NLR-inflammasome activation and pyroptosis in the retina.
Retinas of 6 month old photoreceptor iNOS+/+ mice and wild type animals (C57BL/6) were subjected to PCR array to determine expression levels of NLRs. The retinas from similar groups of animals were processed for mtDNA isolation and oxidative stress mediated mtDNA lesions were quantified by qPCR. The mitochondrial and cytosolic DNA was also isolated from these retinas and Cox 1 gene levels were analyzed from the cytosolic DNA isolate using specific primers. The cytosolic extracts from both the iNOS+/+ and WT mice were subjected to imunoprecipitation studies to determine the interaction between NLRC4 and 8-OHdG (the marker for oxidized DNA).
There was significant upregulation of NLRC4, AIM2, ASC, activated caspase 1, IL1B and IL-18 . The downstream signaling molecules such as IL-12a, IL33, Ptgs2, Ripk2 and Txnip were also up regulated significantly. The mitochondrial DNA in the retina of iNOS+/+ mice had a strand break of 0.57 /10 kb with each mitochondrion containing 5 lesions compared to wild type (P<0.005). COX1 DNA levels were 6 fold higher in the retinal cytosol of iNOS+/+ mice. The cytosolic extract of iNOS+/+ mice showed increased NLRC4 levels compared to the WT controls. The imunoprecipitation studies showed interaction between NLRC4 and 8-OHdG in the iNOS+/+ mice whereas no interaction was observed in the WT control.
Retinas of iNOS+/+ mice with photoreceptor mt.oxidative stress showed mtDNA damage and escape of this DNA into the cytosol and interaction of the oxidized DNA with NLRC4. The upregulation of NLRC4 was in conjunction with display of activated caspase 1, IL-18 and IL-1b I and other signaling molecules involved in inflammasome activation. Such inflammasome activation is known to induce pyroptosis. Studies in this transgenic model reveal a novel mechanism of photoreceptor degeneration from mt.oxidative stress.
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