The Role Of Autophagic Degradation Of Disease-related Proteins In ALS And Intervention Study Of Trehalose In ALS Transgenic Mice

Part I Intervention study on trehalose in NSC-34cells transfected withALS-related proteinsObjective: We assessed whether autophagy activated by trehalose is ableto accelerate ALS related proteins degradation in NSC-34cells and whethertrehalose could reduce the insults of those toxic proteins on cells. This studycould provide in vitro experimental evidence to explore the effect ofautophagy manipulation on the ALS pathology.Methods: NSC34cells transient-transfected with WT TDP-43, Q331KTDP-43, M337V TDP-43, TDP-25, TDP-35were treated with trehalose, withconcentrations varying from0to100mM. Levels of endogenous LC3-II, P62and ALS related proteins were detected by immunoblotting. To verify thatwhether the protein degradation effect of trehalose depends on the autophagypathway,3-MA and wortmannin, two specific autophagy inhibitor, were usedto inhibit autophagosome formation. To further investigate inducedautophagosome formation by trehalose, bafilomycin A1, which blocksautophagosome-lysosome fusion, was used. Activated caspase-3, cytosoliccytochrome c and ROS generation after trehalose treatment were also detectedvia immunoblotting or flow cytometry.Results: We observed that trehalose at the concentration of50and100mM significantly accelerated the clearance of Q331K TDP-43, M337VTDP-43(P<0.05), but trehalose at any concentration had no effect on WTTDP-43degradation(P>0.05). Similar to the reduction of TDP-43, a decreasein P62levels was also observed(P<0.05). A prominent increase occurred in thelevels of LC3-II at concentrations of50mM and100mM, indicating thattrehalose can induce the autophagy pathway in NSC34cells transfected withboth mutant TDP-43and its C-terminal fragments in a dose dependent manner (P<0.05). Cotreatmeant with wortmannin or3-MA blocked the increase in thelevel of LC3-II and the degradation of TDP-25(P<0.05). Trehalosesignificantly increased LC3-II levels in the presence of bafilomycin A1,compared with bafilomycin A1alone (P<0.05). Trehalose reduced the amountof activated caspase-3and cytosolic cytochrome c in cells transfected withEGFP-TDP-25. Trehalose treatment resulted in a decrease of ROS generationin cells transfected with TDP-43c-terminal fragments compared with control.Conclusion:1Trehalose could induce autophagy in the NSC-34cells in adose-dependent manner.2Trehalose activates autophagy beyond autophagosome-lysosomefusion.3Trehalose could accelerate the degradation of Q331K TDP-43, M337VTDP-43protein as well as TDP-25and TDP-35(two truncated fragment ofTDP-43), but there was no effect on the degradation of WT TDP-43.4Trehalose protects cells against mitochondria-dependent apoptoticinsults.Part II: Intervention study on trehalose on autophagic degradation ofmutant SOD1and glial activation in transgenic ALS G93A-SOD1miceObjective: For further confirmation of the effect of trehalose on theALS mice model, we treated SOD1G93A mice with2%trehalose from60days of age to assess the autophagy pathway and levels of mutant SOD1. Thisstudy could provide in vivo experimental evidence for further survival study.Methods: Transgenic human SOD1-G93A mice and their non-transgeniclittermates were used for this study. We treated transgenic SOD1-G93A miceand their non-transgenic littermates by spontaneous oral administrationstarting at60days of age with2%(w/v) trehalose. We identified theSOD1-G93A transgenic mice by PCR with the primer sequence andidentification methods provided by Jackson Laboratory, and observed thephenotype of transgenic SOD1-G93A mice. The levels of LC3-II, p62andmutant SOD1was detected by immunoblotting. Microglial and astrocytic activation were also assessed using immunoblotting and immunostaining.Results: The levels of LC3-II in the spinal cords of the transgenicSOD1-G93A and non-transgenic mice were significantly increased after onemonth of treatment, while P62and human SOD1decreased (P<0.05). Thelevels of CD11b and GFAP protein were reduced by trehalose treatment(P<0.05). Both astrogliosis and microgliosis in the spinal cord sections of90-day-old ALS mice treated with trehalose were less pronounced than thoseof the untreated ones (P<0.05).Conclusion:1Trehalose enhanced the autophagy pathway in transgenic SOD1-G93A2Trehalose accelerated the degradation of mutant SOD1in the spinalcord of transgenic SOD1-G93A.3Trehalose inhibited microglial and astrocytic activation.Part III: Intervention study of trehalose on disease progression and motorfunction in transgenic SOD1-G93A mice.Objective: To test whether trehalose has a beneficial effect on ALS,survival study was in turn conducted with litter-mate matched femaleSOD1-G93A transgenic mice (n=15/group) in investigator-blinded trials. Theeffect of trehalose on bodyweight, disease onset, progression, motor functionwere accessed during the trial. The levels of mutant human SOD1, LC3-II andP62at endstage were detected using immunoblotting. Microglial andastrocytic activation were also assessed using immunoblotting andimmunostaining. Anti-NeuN staining of the lumbar spinal cord section wasperformed to examine the effect of trehalose on motor neuron loss in thespinal cords.Results: In both the transgenic and non transgenic mice the weight ofthe treatment group remained slightly lower than that of their correspondingcontrol group throughout the duration of the study. This difference washowever statistically insignificant indicating that trehalose has little effect onbodyweight of ALS mice. Treatment with trehalose delayed the onset ofdisease by nearly4days (ctrl:94.43±1.53days, trehalose:98.71±1.46days, P=0.039<0.05). The time which took the treatment group to reach score2wasdelayed by6days as compared to the control group.(ctrl:111.57±2.22days，trehalose117.93±2.43days，P=0.048<0.05).The time to reach score3and endstage, were similarly delayed in the treatment group by5days and7daysrespectively （time to reach score2ctrl:126.0±2.84days, trehalose:131.5±2.6days; survival ctrl:130.93±2.81days,trehalose:138.21±3.317days).However, these two differences were not statistically significant (P=0.228and0.091respectively), indicating that trehalose had an effect in the earlystages of the disease, delaying onset and the time to reach score3, but wasineffective in the later progressions of ALS. At early stages, control miceperformed less well on rotarod assessment than trehalose treated mice. It wasonly on the98th day that the difference between the two groups wassignificant (P=0.048<0.05).In later stages, however, the performance of thetransgenic mice on the rotarod showed no distinguishable difference with thecontrol mice. The difference between performance of the treatment andcontrol group on the hanging wire were more marked in the hanging wireassessment. Statistically significant differences were observed on the98th,102th and112th day (P=0.031,0.028and0.029, respectively). Trehalosefailed to decrease mutant SOD1and P62levels in mice at endstage, both ofwhich remained constant, while at the same time raising levels of LC3-II.Numbers of motor neurons were similar between the two groups, indicatingthat trehalose could not significantly preserve the loss of motor neuron. Thelevels of CD11b and GFAP were reduced by trehalose treatment (P<0.05).Both astrogliosis and microgliosis in the spinal cord sections of ALS mice atendstage treated with trehalose were less pronounced than those of theuntreated ones (P<0.05).Conclusion:1Trehalose delayed the onset of the disease by nearly4days as well asdelaying the time to reach neurological score2by nearly6days. But it failedto delay the time to reach neurological score3and death.2Trehalose alleviated the impairment of motor performance at earlier stages of the disease，the beneficial effects of trehalose on motor behaviorrecoveries and the reduction of mutant SOD1protein were lost in later stages.3Trehalose increased the number of autophagosome but failed todecrease mutant SOD1protein in transgenic SOD1-G93A mice at endstage.4Trehalose had significant inhibitive effect on astrocytosis andmicroglial activation in transgenic SOD1-G93A mice at endstage.5Trehalose could not significantly preserve the loss of motor neuron intransgenic SOD1-G93A mice at endstage.