The Effect Of Pin1on Toxicity Of Mutant Huntingtin And Its Underlying Mechanism

Huntington’s disease (HD) is a hereditary neurodegenerative disease due to theexpansion of CAG repeats expansion in the first exon of HD gene that encodeshuntingtin (Htt). The pathology of HD is characterized by loss of striatal and corticalneurons selective. The mutant Htt encoded by HD gene can produce cytotoxicitythrough a variety of ways and form aggregates by misfolding in brain neuroncytoplasmic and nuclear. The aggregated-Htt is more toxic than non-aggregated-Htt orsoluble Htt. Therefore, it is great significance to clear the mutant Htt and reduce theformation of aggregates for reducing the onset and preventing the development of HD.Peptidyl-prolyl cis-trans isomerse (Pin1) is a conserved enzyme in the parvulinfamily. Pin1can specifically binds phosphorylated ser/Thr-Pro protein motifs andcatalyzes the cis/Thr-Pro isomerization of the substrate proteins, regulate theirbiological functions from cell cycle progression to signal transduction events. Theneurodegeneration process often associated with Pinl downregulated and dysfunction.Pin1gene knockout mice showed age-dependent neurodegeneration. Pin1is closelyrelated to the occurrence and the development of Alzheimer’s disease; the supplementof exogenous Pinl can restore the biological activity of the microtubule-associatedprotein (tau), reduce neuronal death witn Pinl lacked. In the disease of Caymancerebellar ataxia, Pin1can produce neuroprotective by inhibiting the pathogenic proteinBNIP-H binding with glutamine. These results suggest that Pin1may beneuroprotective against neurodegeneration. However, it has not been reported thatwhether Pin1has the same effects to mutant Htt in the neurodegenerative disease ofHD. In this study, by using the cellular and molecular biology methods we stud ied theeffect of Pin1to mutant huntingtin-induced toxicity, and discussed its possiblemechanisms. The results showed that overexpression of Pin1in the mouseneuroblastoma cells (N2a cells) can inhibit mutant Htt cytotoxicity and promotedegradation of mutant Htt, enhance the stability of the E3ubiquitin ligase C-terminus ofheat shock protein70-interacting protein (CHIP) and the activity of the ubiquitin-proteasome system (UPS).Pin1inhibits cytotoxicity of mutant Htt. To ensure the relationshipof the Pin1activity and the toxicity of mutant Htt, we detected the effect of theover-expression of Pin1on the cell viability in stable normal or mutant Htt N2a cells.The results of MTT assay showed that the over-expression of Pin1didn’t affect the cellviability of20Q cells. However, in the160Q cells, the over-expression of Pin1couldincrease the cell viability. Then we applied caspase-3active immunoblot analysis to testthe cytotoxicity effect of mutant Htt by expressing different level Pin1. In lowconcentration range, with different concentration of Pin1transfected to160Q-Htt cells,western-blot and Biodot results show that with the Pin1concentration increasedgradually, Caspase-3reduce gradually. The results demonstrated that Pin1hadprotection against mutant Htt cytotoxicity in mutant Htt-expressing cells.Pin1reduces both aggregated and soluble mutant Htt in N2acells expressing mutant huntingtin. To identify wether Pin1could reducethe mutant Htt, under fluorescence microscope we observed that Pin1overexpressioncould reduce both aggregated and soluble mutant Htt, and it was reversible by Pin1silence. The western-blot result showed that both aggregated and soluble Htt wasreduced by Pin1overexpression. It was confirmed by Western-blot and filter-trapimmunoblot analysis that Pin1decreased mutant Htt aggregates in a dose dependent.At the same time, RT-PCR results showed that Pin1overexpression did not alter theexpression of mutant Htt. Thus Pin1could promote both aggregated and soluble Httdegradation.Pin1reduces mutant Htt through the ubiquitin-proteasomesystem by promoting the stability of the CHIP. Because CHIP has notonly the role of the molecular chaperones to correct misfolded protein but also the roleof the E3ubiquitin ligase and can promote degradable activity of UPS substrate by promoting ubiquitination. To clarify whether Pin1could promote mutant Httdegradation through CHIP, different concentration of Pin1was transfected to160Q-Httcells, Western-blot results showed that with the Pin1concentration increasing gradually,CHIP protein expressed increase gradually, and at the same time, RT-PCR resultsshowed that Pin1overexpression did not alter the expression of CHIP. Bycycloheximide inhibition assay, we found that expression of Pin1can promote CHIPstability in N2a cells. Therefore, we believe that Pin1promots the stability CHIP.(Cycloheximide is a compound that has the inhibitory effects on the protein synthesisprocess in eukaryotes. It hinders the process of translation by interfering with thetranslocation step in the process of protein synthesis. It is often used to inhibit theprotein synthesis in vitro eukaryotic cells on the biopharmaceutics study, and in themolecular biology can be used to determine the half-life of the protein (enzyme). Itoften use cycloheximide handling cells and then use the Western-blot experiments toshow how the target protein changed by the time.)To further identify that Pin1can reduce mutant Htt by CHIP, we use CHIP-SiRNAto silence CHIP in160Q-Htt cells. We found that after CHIP silenced, aggregated andsoluble Htt were increased significantly, and compared silence CHIP over-expressingof Pin1with silence control over-expressing of Pin1, we found that mutant Httincreased significantly. These Results indicated that Pin1degradated mutant Htt byCHIP.To assess whether Pin1promote mutation Htt degradation via the ubiquitin-proteasome system, the same cycloheximide inhibition assay was applied toobserved the ubiquitination of total protein. The resuts show that Pin1promoteubiquitination of total protein.Taken together, Pin1could stabilize CHIP, increase ubiquitination of mutant Htt byCHIP and then promote degradation of mutant Htt through UPS, which leads to theinhibition of cytotoxicity of mutant Htt.