Impaired DNA Damage Response By Overexpressed The Amyotrophic Lateral Sclerosis-related Protein Poly-PR

Aim:The GGGGCC hexanucleotide repeat expansion in the non-coding region of C9orf72 gene is a major contributor to the pathogenesis of amyotrophic lateral sclerosis(ALS).The GGGGCC repeat expansions can be translated into five dipeptide repeat proteins(DPRs).It has been previously reported that arginine rich poly-PR,localized in the nucleolus,was strikingly toxic to the cells.However,the mechanism implicated in the toxicity of poly-PR remains unclear.Therefore,we designed related experiments to explore the mechanisms underlying cell damage caused by Poly-PR.Methods:We used the constructed GFP-PR30(PR with 30 repeats)plasmid,GFP-PR30 lentivirus and FITC-PR20 peptides to treat HEK-293T cells,U2OS cells,SH-SY5Y cells and mouse primary cortical neurons.Immunofluorescence and Western blot were used to determine the expression and localization of poly-PR and DNA damage repair related proteins.Lactate dehydrogenase(LDH)release assay was used to detect the toxicity of poly-PR.The comet assay was used to measure the degree of DNA damage in primary neurons and U2OS cells caused by poly-PR.Immunoprecipitation was used to detect the co-binding of the protein PARP1/PAR and poly-PR.Hydrogen peroxide(H2O2)was administrated to U2OS cells to induce cell DNA damage,and the cellular localization of PAR was detected by immunofluorescence.U2OS cells were irradiated with a laser confocal microscope to detect the effect of poly-PR on sarcoma fusion protein(FUS)recruitment at DNA damage site.The key process of DNA damage caused by poly-PR was verified by using PAR hydrolase inhibitor(PARGi).The degree of DNA damage in the motor cortex in GFP-PR28 transgenic mice was detected using immunohistochemistry staining.Results:We found that poly-PR was localized in the nucleoli.Poly-PR caused DNA damage and cell death in U2OS cells and primary cortical neurons.Poly-PR was co-localized with PARP1 and PAR.Poly-PR inhibited the damaged DNA-dependent PARP1 activation and PAR redistribution in the cytoplasm,which ultimately inhibited the recruitment of FUS.The inhibition of this repair pathway led to the death of neurons.PARGi significantly inhibited the axon break of mouse cortical neurons caused by poly-PR.In addition,wide range of DNA damage response,that is indicated by γH2AX foci stainging,was determined in the motor cortex of GFP-PR28 transgenic mice.Conclusion:Poly-PR causes DNA damage in neurons,and impairs the DNA damage repair response by inhibiting the activity of PARP1,reducing the PAR entry into the nucleus,and thus eventually causing apoptosis.PAR hydrolase inhibitor(PARGi)can effectively block cell death caused by poly-PR.