The Role Of Mitophagy And Protective Effect Of Diallyl Trisulfide On Cellular Model Of Amyotrophic Lateral Sclerosis

Background: Amyotrophic lateral sclerosis (ALS), a fatalneurodegenerative disease, is characterized by muscle atrophy, spasticity andweakness, and leads to death within three to five years of diagnosis. Recently,TAR DNA binding protein of43kDa (TDP-43) was identified as the majorprotein constituent of ubiquitinated inclusions in brains of ALS andfrontotemporal lobar degeneration. TDP-43is encoded by the gene TARDBPon human chromosome1. It may be involved in specific pre-mRNA splicingand transcription, mRNA stability and the biosynthesis of microRNAs. Up tonow, over40dominant mutations in the TDP-43gene have been linked tosporadic and familial ALS, which provided strong evidence for a direct linkbetween TDP-43dysfunction and neurodegeneration. TDP-43C-terminalfragments are recovered in the sarkosyl-insoluble fraction of affected brainregions of ALS patients, it has been hypothesized that they serve to seedTDP-43aggregation and play an important role in disease pathogenesis. It iswidely accepted that mitochondria is a converging point of multiplepathological pathways in ALS. A large number of researches indicated thatmitochondrial dysfunction participated in the degeneration of ALS patients,animal models for mutant SOD1-linked ALS, in which the accumulation ofmutants or wild type (WT) SOD1in mitochondria represents a critical point.Correspondingly, TDP-43-expressing models also showed mitochondrialdysfunction, indicated by accumulation of reactive oxygen species (ROS), lossof mitochondrial membrane potential (Ψm), and impairment of therespiratory chain. Mitophagy plays an important role in the quality control ofmitochondria, which has been implicated in Parkinson’s disease andAlzheimer’s disease, but very little is known about their role in ALS.Diallyl trisulfide (DATS), one of the major organosulfur compounds in garlic oil，has received considerable attention because of its anticancer effect.It can not only ofer protection against chemically induced cancer in animalmodels by modulation of carcinogen metabolism, but also suppress growth ofcancer cells in vitro and in vivo by inhibition of cancer cell proliferation andcell cycle arrest, induction of apoptosis, histone modification, and suppressionof angiogenesis. The other known health benefits of DATS includeantithrombotic effects, improvement of the immune function, lowering ofblood glucose level, radioprotection, and protection against microbialinfections. In addition, its neuroprotective effects were demonstrated bystudies from our laboratory using organotypic spinal cord culture andtransgenic mice model of ALS. However, that was not confirmed in cellularmodel.Part Ⅰ: The effects of full-length TDP-43and its C-terminal fragmentson mitochondrial function and morphologyObjective: To investigate whether full-length TDP-43and its C-terminalfragments impair mitochondrial function and morphology.Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first determined the expression levelsby western blots. We next observed the subcellular localization of TDP-43byconfocal microscopy, and further investigated the effects of human TDP-43onmitochondrial function, indicators of mitochondrial activity such asmitochondrial membrane potential and levels of cellular ROS were determined.Finally we examined morphological changes of mitochondria in transfectedcells by confocal microscopy.Results:(1) Human TDP-43plasmids were sucessfully expressed inNSC34cells. Western blot data showed the expression levels of full-lengthTDP-43were very similar to endogenous TDP-43, but the expression levels ofTDP-25and TDP-35were approximately50%of that endogenous TDP-43.(2) In untransfected cells, TDP-43was localized to the nucleus. In cells transfected with full-length TDP-43, WT and mutant TDP-43predominantlyexpressed in the nucleus, but TDP-43signals were also detected in thecytoplasm. Compared with full-length TDP-43, C-terminal fragments weremore likely to form compact cytoplasmic and nuclear inclusions.(3) Quantification by flow cytometry showed that both WT and mutantTDP-43could lead to decrease in mitochondrial membrane potential.Compared with control cells, full-length TDP-43and its C-terminal fragmentssignificantly increased ROS production.(4) We also investigated morphological changes of mitochondria byconfocal microscopy, and clustered and uneven distributed mitochondria weremore common in full-length and truncated TDP-43transfection cells.Conclusion: Compared with control cells, full-length TDP-43and itsC-terminal fragments significantly decreased mitochondrial membranepotential levels, increased ROS production, and disturbed mitochondrianormal distribution.Part Ⅱ: The effects of full-length TDP-43and its C-terminal fragmentson mitophagy in NSC34cellsObjective: To investigate whether full-length TDP-43and its C-terminalfragments activate mitophagy.Methods: NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35, and the cells wereharvested after48h of transfection. We first confirmed the purity ofmitochondrial isolation, obtained from the homogenization method, bytransmission electron microscope. Then we examined the protein level ofhuman and endogenous TDP-43in the mitochondria and cytoplasm bywestern blot, observed distributive characters of TDP-43and mitochondria byconfocal microscopy, and determined LC3B and P62expression levels inmitochondrial and cytosol fractions.Results:(1) We first confirmed the purity of mitochondrial isolation,obtained from the homogenization-centrifugation method, by transmission electron microscope; the micrograph showed that mitochondria isolated bythis technique preserved morphological integrity very well，and the purity ofmitochondria maintained in90%above. Western blot data showed that actin(a cytosol maker) was undetectable in mitochondrial fraction, and VDAC1(amitochondrial maker) was also undetectable in cytosol fraction.(2) We examined TDP-43in the mitochondrial fractions by western blotsand confocal microscopy. Our data showed that WT and mutant TDP-43could be present and localized in the mitochondria, and the expression level ofWT TDP-43in mitochondrial fraction was very similar to mutant TDP-43.(3) We further evaluated mitophagy in purified mitochondria bymeasuring the levels of LC3-II and p62. expression of WT and mutantTDP-43led to sequestration of endogenous nuclear TDP-43after48htransfection. Furthermore, in cells transfected with WT or mutant TDP-43, thelevel of LC3-II markedly increased and p62decreased in the mitochondrialfractions. In addition, VDAC1, decreased in cells expressed TDP-25andTDP-35, compared with control. Moreover, in the mitochondrial fractions,TDP-25and TDP-35clearly up-regulated the levels of LC3-II but reduced theexpression of p62.Conclusion: Mitochondria isolated by homogenization-centrifugationmethod preserved morphological integrity very well and high purity, and aportion of exogenous TDP-43were localized in the mitochondria. WT, mutantTDP-43and its C-terminal fragments all induced LC3-II but reduced p62inthe mitochondrial fractions, indicating that mitophagy was activated at thesubcellular level.Part Ⅲ: The protective effect of Diallyl trisulfide on cellular model ofamyotrophic lateral sclerosisObjective: To investigate the protective effect and mechanism Diallyltrisulfide on cellular model of amyotrophic lateral sclerosisMethods: NSC34cells were exposed to different concentrations andtimes of DATS, and cell viability was measured by the CCK-8assay. NSC34cells were treated with appropriate DATS for indicated time, and we examined LC3B level by western blot. In addition, cells expressing pEGFP-LC3weretreated with DATS for indicated concentration and time, and the accumulationof pEGFP-LC3in response to treatment were analyzed by fluorescencemicroscopy. We also investiged the effect of DATS on human TDP-43intransfected cells by western blots，and determined which means DATS causedthe clearance of TDP-43using the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. We finally examined Nrf2in the nucleus andantioxidase levels after the treatment of DATS in transfected cells.Results:(1) There was no marked difference in the degree of toxicityfrom0to10μM for24h. In contrast, cell viability measured by the CCK-8assay was significantly decreased from20to100μM in NSC34cells in adose-dependent manner. Next the cells were treated with10μM DATS atincreasing times and cell viability was analyzed. The CCK-8leveldramatically decreased at48h after the addition of DATS.(2) NSC34cells were exposed to DATS from0to10μM for24h, andwestern blotting analysis revealed a steadily increasing quantity of theLC3-II form. In addition, time-course analysis showed that24h of exposure toDATS stimulated the greatest amount LC3-II protein. Furthermore, we alsotreated the pEGFP-LC3transfected cells with DATS and found that there wereincreased numbers of punctate pEGFP-LC3dots in the treated cells indose-dependent manner.(3) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. Treatment with10μMDATS for24h resulted in decrease in human TDP-43protein levels, both inWT and mutants transfected cells by western blots. Furthermore, we treatedTDP-25transfected cells with the autophagy inhibitor Baf A1and theproteasome inhibitor MG132. These inhibitor data revealed that TDP-25levels were not decreased by24h in cells treated with DATS and Baf A1compared to DATS alone. In virtually, they were decreased after exposure ofDATS and MG132, indicating that DATS promotes clearance of misfolded proteins by induction of autophagy.(4) NSC34cells were transiently transfected with different humanTDP-43plasmids, including WT, Q331K, M337V TDP-43and twoC-terminal fragments termed as TDP-25and TDP-35. After treatment with10μM DATS for24h, western blot data showed the treatment of DATSsignificantly induced nuclear translocation of Nrf2, increased antioxidaseHO-1and NQO1erxpression.Conclusion: In TDP-43-associated ALS cellular models, low-doseDATS could up-regulate autophagy flux, cause the clearance of exogenousTDP-43by autophagy, activate Nrf2/ARE pathway, enhance the expression ofHO-1and NQO-1, and confer its robust neuroprotection.