UMI-77 Synergizes With TRAIL To Induce Apoptosis In Brain Glioma Cells And The Role Of PTPRU And DSTYK In The Nervous System

This thesis,divided into three parts,aims to explore the molecular mechanism underlying the pathogenesis of neurological disorders and provide novel therapeutic strategies for the treatment of these diseases.(1)UMI-77 synergizes with TRAIL to induce apoptosis in brain glioma cells.Malignant glioma is the most common and aggressive form of brain tumor.TRAIL(tumor necrosis factor-related apoptosis-inducing ligand)is a promising anticancer agent but is insufficient to induce apoptosis in gliomas.In this study,we showed that the small-molecule Mcl-1(myeloid cell leukemia-1)inhibitor UMI-77 sensitized glioma cells to TRAIL treatment.Combination of UMI-77 and TRAIL in glioma cells led to the activation of caspase-8 and Bid,cleavage of caspase-3 and poly-ADP ribose polymerase,the accumulation of tBid in the mitochondria and the release of cytochrome c into the cytosol,suggesting this combination induced mitochondrial outer membrane permeabilization and caspase-dependent apoptosis.UMI-77 untethered pro-apoptotic Bcl-2 proteins Bim and Bak from the sequestration of Mcl-1 and promoted the conformational activation of Bak.Small hairpin RNA of Bid reduced the cytotoxity of UMI-77 plus TRAIL as compared with control shRNA cells,suggesting that Bid is essential for the apoptosis induced by UMI-77 and TRAIL combination.Taken together,UMI-77 enhances TRAIL-induced apoptosis by unsequestering Bim and Bak,which provides a novel therapeutic strategy for the treatment of gliomas.(2)The role of PTPRU in midbrain dopaminergic neurons.PTPRU(protein tyrosine phosphatase receptor U)is a target gene of Parkinson's desease(PD)susceptibility protein Nurrl(the nuclear orphan receptor).PTPRU is involved in midbrain patterning and cell proliferation during early stages of development-However,whether PTPRU is required for the maintenance and survival of midbrain dopaminergic(mDA)neurons at late stages of development or in the adult midbrain remains unknown.In the present study,a floxed Ptpru mouse strain was generated and Ptpru was ablated by crossing with tyrosine hydroxylase(TH)-Cre mice which express Cre recombinase in postmitotic dopaminergic neurons.Loss of Ptpru resulted in a reduction in nuclear and somal size in both SNc(substantia nigra compact)and VTA(ventral tegmental area),which replicates the significant atrophy of SNc mDA neurons in PD patients.Reduced activity of Akt-mTORC1 signaling and phosphorylation levels of GSK3? following Ptpru ablation may be the mechanism underlying the reduced cell size.mDA neuronal markers in the midbrain of young adult Ptpru conditional knockout mice were maintained and their projections to striatum appeared undisturbed,suggesting that PTPRU is not required for the maintenance of mDA neuronal markers and nigrostriatal projection.Taken together,these results suggest a role of PTPRU in regulating the neuronal size of mDA neurons through modulating Akt-mTORC1 and GSK3? signalling.(3)The role of DSTYK in the nervous system and renal development.DSTYK(dual serine/threonine and tyrosine protein kinase)had been reported as a susceptibility gene for neurodegenerative desease SPG23(spastic paraplegia type 23).However,the function of DSTYK in the nervous system remains largely unknown.By using Dstyk knockout mice to analyze the consequences of Dstyk ablation,we found that loss of DSTYK resulted in impaired capabilities of learning and memory,but no significant brain developmental and morphological defects.This phenotype resembles symptoms of human SPG23.However,unlike SPG23 patients,Dstyk-/-mice showed no pigmentary abnormality of the fur and exhibited normal motor and balance capacities.Since clinical studies had also identified DSTYK as a causing gene of CAKUT(congenital anomalies of the kidney and urinary tract),we tried to determine whether DSTYK is involved in the renal development.Although the appearance of the urogenital system in Dstyk-/-mice appeared normal,the distribution of F-actin(fibrous actin)cytosk eleton in renal proximal convoluted tubules was obviously swelling and diffuse.To identify DSTYK binding partners,we generated a Flag-DSTYK knockin HEK 293T cell line with Flag tag sequence inserted to the N-terminus of endogenous DSTYK using CRISPR/Cas9 system.A total list of 67 proteins were identified by Co-immunoprecipitation/mass spectrometry that potentially interacts with DSTYK,mostly enzymes(39.0%),nucleic acid binding proteins(36.6%)and cytoskeletal proteins(14.6%).Profilin 1 and Profilin 2,small actin binding proteins regulating the architecture of F-actin cytoskeleton,were validated as DSTYK binding partners by Co-immunoprecipitation assay.These results demonstrate that DSTYK is involved in the process of learning and memory,and the regulation of F-actin cytoskeleton in the mouse renal proximal convoluted tubules.