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P. K. Mukherjee, N. G. Bazan; Upregulation of Proinflammatory Genes (COX-2 and B-94) Under the Influence of Mutant Glutamine Expansion Inhibited by NPD1 and PEDF/DHA in Human Retinal Pigment Epithelial Cells (HRPE). Invest. Ophthalmol. Vis. Sci. 2010;51(13):3714.
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The mutation of the ataxin-1 protein causes polyglutamine diseases due to glutamine expansions on the polypeptide chain. Docosahexaenoic acid (DHA) and pigment epithelium-derived growth factor (PEDF) promote neuroprotection through neuroprotectin D1 (NPD1) synthesis and may foster survival in stroke and neurodegenerative diseases. Ataxin-1 mutation interacts with its normal cellular complex, but the function of the complex is repressed due to the mutation. The function of the complex is to up-regulate and inhibit certain gene expression. The purpose of this study is to investigate the up-regulation of proinflammatory genes cyclooxygenase-2 (COX-2) and B-94 under the influence of mutant glutamine expansion and the effect of NPD1 and PEDF with DHA in human retinal pigment epithelial (HRPE) cells.
HRPE cells, grown overnight, were cotransfected with 82Q polyglutamine expansions and COX-2 promoter (830) luciferase constructs by Fugene-6 according to the manufacturer’s protocol (Roche). A promoterless β-galactosidase construct was used to cotransfect as transfection control. Transfected cells were harvested, cell extracts were made, and luciferase assays were performed using luciferin as substrate. Western blot analysis of COX-2 and B-94 was performed using respective antibodies.
Our results indicated that a transfected ataxin-1 mutant gene with 82 polyglutamine expansions in human retinal pigment epithelial (HRPE) cells induce apoptosis in cell involving the upregulation of (COX-2) and B-94. These upregulations were inhibited by NPD1 as well as PEDF with DHA. Western blot analysis indicated that the ataxin-1 mutant induced proinflammatory COX-2 and proapoptotic B-94 proteins, and NDP1 and PEDF with DHA were able to inhibit the induction of these proteins in HRPE cells.
The expression of proinflammatory/proapoptotic proteins COX-2 and B-94 were reduced when the HRPE cells transfected with ataxin-1 mutant were exposed to NPD1 or a mixture of DHA/PEDF. These neuroprotective agents enhance survival of the HRPE cells when polyglutamine diseases are induced in HRPE cells. These results demonstrate that survival signaling is mediated by NPD1 in an experimental model of neurodegeneration. NPD1 may be useful in therapeutic strategies for treating neuronal diseases like age-related macular degeneration (AMD) and stroke.
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