The Mechanisms Underlying Neurotoxicity Caused By Dipeptide Repeat Protein Poly-PR In C9orf72-related ALS/FTD

Aim:Dipeptide repeat protein poly-PR has been proved to have strong neurotoxicity in C9orf72-related amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).Moreover,DPR has cell-to-cell transmission characteristics similar to that of the mad cow disease protein(prion).However,the molecular mechanisms and transmission mechanisms underlying poly-PR contributed to neurodegeneration are unclear.We designed relevant experiments to explore the mechanisms underlying neuron death caused by poly-PR.Methods:The SH-SY5Y cells(a neuronal cell line)or mouse primary cortical neurons were treated with synthetic poly-PR(PR with 20 repeats,PR20)peptides or lentiviral vector expressed poly-PR(PR with 28 repeats,PR28).Inhibitors of endocytosis were used to explore the endocytosis pathways involved in poly-PR peptides uptake.Then Flow cytometry was used to quantify the fluorescence intensity of FITC to estimate the inhibitory effect of poly-PR peptides on transfer into neurons after the use of endocytosis inhibitors.Secondly,RNA interference(RNAi)was used to verify the endocytosis pathways involved in poly-PR peptides uptake.The neurotoxicity of poly-PR was detected by LDH release assay and Propidium iodide(PI)staining assay.The protein levels of ER stress marker CHOP,phosphorylated JNK and pro-apoptotic factors p53 and Bax after treatment with poly-PR were detected by Western blot.The expressions of ER stress markers and pro-apoptotic factors after treatment with poly-PR were detected by real-time quantitative polymerase chain reaction(real-time qPCR).Meanwhile,Immunofluorescence was used to detect the effect of poly-PR on the activation of pro-apoptotic factor p53 and cleaved-caspase-3.The LDH release experiment and PI staining assay were used to evaluate the protective effect of relevant inhibitors on blocking neurotoxicity caused by poly-PR,at the same time,Western blot was used to detect the expressions of ER stress marker CHOP and pro-apoptotic factors.Results:Chlorpromazine,an inhibitor of clathrin-mediated endocytosis,was found to significantly blocked poly-PR transfer into neurons.At the same time,using RNAi to knock clathrin heavy chain(CLTC)down can also block poly-PR transfer to cells,which is further suggested that poly-PR transferred into cells in a clathrin-dependent manner.We found that poly-PR transferred into neurons,causing ER stress,activation of JNK,and activation of pro-apoptotic factors p53 and Bax in SH-SY5Y cells and mouse primary cortical neurons.Activation of these pathways eventually leads to neuron death.To evaluate the effects of ploy-PR in a more disease-relevant context,we generated lentivirus vectors expressing PR with 28 repeats to infect primary neurons.The results also showed that the primary cortical neurons expressing PR28 caused ER stress and activation of pro-apoptotic factors.Using JNK inhibitor SP600125 can significantly inhibit the activation of JNK induced by poly-PR,so as to inhibit the activation of pro-apoptotic factors p53 and Bax.Chlorpromazine,an inhibitor of clathrin-mediated endocytosis,can significantly attenuate ER stress and suppress the activation of JNK and the upregulation of p53 and Bax caused by poly-PR treatment.Conclusion:Poly-PR transferred into neurons in a clathrin-dependent manner and induced cell death via ER stress and JNK-mediated pathway.An inhibitor of clathrin-mediated endocytosis chlorpromazine and a JNK inhibitor SP600125 blocked the neuron death caused by poly-PR.