Fig. 8: Cis- and trans-RSV have opposite effects on ARH3-mediated chromatin de-ADP-ribosylation and ‘trapped’-PARP1 mediates the neurotoxic effects of trans-RSV.
a Cis-RSV removes auto-PARylated PARP1 from chromatin and trans-RSV ‘traps’ PARP1 onto the chromatin. Representative immunoblots and quantification from chromatin fraction of cortical neurons (DIV 9) depicting PARP1 and PAR, ARH3, TyrRS after treatment with cis- and trans-RSV (50 µM) for 1 hr. b ARH3 levels are not affected in the hippocampal tissues of AD patients. Representative immunoblots and quantification for ARH3 using anti-ARH3 antibody in the hippocampal region of AD patients (n = 5) with age and sex-matched controls (n = 5). c TyrRS interacts with ARH3. Immunoprecipitated (IP) ARH3 from cortical neurons (DIV 9) was immunoblotted (IB) using anti-TyrRS and anti-ARH3 antibodies to detect the interaction of TyrRS with ARH3. Total TyrRS and ARH3 were detected by WB in the whole cell lysate (WCL). d siRNA knockdown of PARP1 rescues trans-RSV-mediated neurotoxicity. Rat cortical neurons (DIV 7) were transfected with siRNA against PARP1 (siRNAPARP1) or control siRNA (75 nM) and then treated with cis-RSV (50 µM) or trans-RSV (50 µM) for 72 hr. Neuronal viability was assessed and quantified using an MTT assay. The knockdown was verified using immunoblot and quantified using specific antibodies for PARP1. e trans-RSV inhibits nucleoside incorporation in a DNA fiber assay. Cortical neurons (DIV 9/10) were treated with cis-or trans-RSV (50 μM) for 8 hr followed by a 30 min pulse labeling using 50 µM of nucleoside analog, CldU (5-chloro-2′-deoxyuridine). DNA fiber assay was performed according to the published protocol followed by immunostaining for single-stranded (ss) DNA (red) and CldU (green). Representative images (scale bar, 100 µm) showing the incorporation of CldU into DNA during the repair process was assessed using ImageJ by counting the number of CldU positive DNA fibers for a total of 300 fibers per condition. All data represent mean ± SEM with statistical significance calculated using two-tailed Student’s unpaired t-test.
