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G. Hoppe, J. Sears; Downregulation of Major Non-Histone Nuclear Protein Hmgb1 Hypersensitizes Neurons to UV-Light-Induced DNA Damage. Invest. Ophthalmol. Vis. Sci. 2007;48(13):618.
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To investigate a neuroprotective role of Hmgb1, an abundant chromatin protein involved in DNA transcription, synthesis and repair. Since in contrast to other tissues, expression of Hmgb1 in retinal photoreceptors undergoes significant daily changes, we hypothesized that protection of photoreceptor DNA from light-induced damage is one of the crucial functions of Hmgb1 in retina.
Immunohistochemistry was performed on eyes of Long-Evans rats entrained to cyclic light (12h light:12h dark), or cultured neuronal cell lines B35 and SK-N-SH using polyclonal antibody for Hmgb1 and monoclonal antibodies recognizing DNA lesions, e.g., cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydroguanine (8oxoG). shRNA constructs (5 for human and 5 for mouse Hmgb1) pre-cloned into lentiviral expression vectors by the RNAi Consortium were utilized to generate viral particles capable of RNA interference-mediated silencing.
Rhythmic expression of Hmgb1 in rat retinal photoreceptors coincided with light phase, but could also be induced by light during dark phase after a 6-hr lag period. Levels of CPDs characteristic for direct UV-induced DNA damage were increased in light-exposed retina. Light-dependent elevation of CPDs was found only in photoreceptor nuclei, but not in other retinal neurons. Hmgb1 can be efficiently downregulated by RNAi in cultured rat and human neuronal cell lines, B35 and SK-N-SH respectively. SK-N-SH cells exhibited variable expression levels of Hmgb1, and the rates of CPD formation in UV-challenged cells inversely correlated with levels of nuclear Hmgb1. Thus, Hmgb1 appears to protect cultured neurons from direct DNA damage by UV. However, indirect DNA damage due to oxidative modification exemplified by 8oxoG was insensitive to the Hmgb1 content.
Rhythmic diurnal expression of Hmgb1 is entrained by light. Suppression of Hmgb1 protein levels increases susceptibility of cultured neurons to DNA damage by UV light. Correlation between light-induced DNA lesions and levels of Hmgb1 in photoreceptor nuclei suggest a unique genoprotective and therefore neuroprotective role for Hmgb1 in retina.
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