Regulatory Effect And Underlying Mechanisms Of Autophagy On Age-Related Sensitivity To Parkinson’s Disease Onset

Part I Autophagy stress is involved in the MPTP-induced toxicityto dopaminergic neurons in substantia nigraObjective To investigate the changes of autophagic activity in C57BL mice afterMPTP treatment and to explore the possible role of autophagy in Parkinson’s diseasepathogenesis.Methods C57/BL mice were intraperitoneally injected with MPTP·HCl (14mg/kg free base in saline） four times a day at2-h intervals. And rapamycin were givenonce daily on two days before and on the same day as MPTP injection. Rolling bar testsand pole climbing tests were performed at the7thday after MPTP exposure. Tyrosinehydroxylase (TH）-positive neurons and terminals in substantia nigra pars compacta(SNpc） and striatum were detected with immunostaining. Western blotting was used toexamine the protein levels of TH and α-synuclein. Transmission Electron Microscope(TEM） was used to examine autophagosome formation. Furthermore, autophagy fluxwas evaluated by detecting the protein level of LC3-II and P62.Results It was found that parkinsonian mice had significant shorter rolling bartest latency and longer pole-climbing time after MPTP treatment for7days. Autophagyinducer rapamycine rescured the movement impairment in parkinsonian mice. Therewas a significant decrease of SNc and striatum TH positive cells in parkinsonian mice.MPTP exposure also dramatically decreased the protein level of TH and increased theprotein level of α-synuclein in SNpc, while rapamycine pretreatment relieved the DAneuron loss caused by MPTP. TEM observations showed significant morphologicchanges in parkinsonian mice, as indicated by mitochondrial swelling andautophagosomes accumulation in SNc neurons, after MPTP and rapamycin treatment. LC3, the marker protein of autophagy, was also found to be significantly increased inparkinsonian mice and rapamycin pretreatment further enhanced it. In addition, P62, theautophagy flux marker, was also increased in parkinsonian mice. However, this wasreduced by rapamycin pretreatment.Conclusion Autophagy stress,caused by MPTP,contributed to the pathogenesisof PD.Autophagy inducer rapamycin increased the autophagic degradation of α-synuclein by facilitating autophagic flux and thus showed a neuroprotective effect onDA neurons. Part II Autophagy is involved in age-related sensitivity toparkinsonian pathogenesisObjective To explore the involvement of autophagy in high sensitivity toMPTP-caused neurotoxicity in aged C57BL mice.Methods MPTP-induced parkinsonian model were developed in different aged(3and20months old） male C57BL mice. Rolling bar tests and pole climbing testswere performed on the1st,4th,7th day after MPTP exposure. Tyrosine hydroxylase(TH）-positive neurons and fibers in substantia nigra pars compacta (SNpc） andstriatum were detected with immunostaining. Transmission Electron Microscope(TEM）was used to examine autophagosome. Western blotting was used to examine theprotein levels of TH, α-synuclein, LC3-II, P62and HDAC6as well.Results Compared with3-month-old PD mice,16-month-old PD mice showedsignificant shorter pole-climbing time after MPTP treatment for7days. But there’s nostatistical difference in the rolling bar test latency between these two groups.Age-associated changes in16-month-old parkinsonian mice included the reduction inthe densities of TH positive cells/fibers in SNc and striatum, lower TH proteinexpression, andα-synuclein accumulation as well. Under TEM, we observed increasedlipofuscin and swollen mitochondria in16-month-old parkinsonian mice. In16-month-old mice treated with saline, the protein levels of LC3-II and P62declinedwhile P62protein increased with age. Although MPTP treatment increased the protein levels of LC3-II, P62and HDAC6in16-month-old mice group, there’s no statisticalsignificance. Compared with3-months-old PD mice,16-months-old PD mice showedlower protein expressions of LC3-II and HDAC6.Conclusion Ageing aggravated the DA neurons injury caused by MPTP.16-month-old mice showed a high sensitivity to MPTP neurotoxicity. Autophagy in SNcdeclines with age. In3-month-old mice, autophagy stress mediated by HDAC6weakened the cytotoxicity of MPTP to a certain degree. While in16-month-old mice,autophagy decompensation may be responsible for the higher sensitivity to MPTPneurotoxicity in aged mice. Part III The underlying mechanisms of autophagy in age-relatedsensitivity to parkinsonian pathogenesisObjective To evaluate the neuroprotective effects of autophagy-targeted drugsrapamycin or treholose and determine the underlying mechanisms of autophagicregulation in age-related sensitivity to neurotoxicity to dopaminergic neurons.Methods MPTP-induced parkinsonian model were developed in different aged(3and20months old） male C57BL mice. The groups were dealt with rapamycinvehicle, high, medium and low dosages of rapamycin and treholose. Rolling bar testswere performed on the7thday after MPTP exposure and TEM was used to the examineautophagosome. Western blotting was used to examine the protein levels of TH,α-synuclein, LC3-II, P62, HDAC6and LAMP1.Results After treatment with aapamycin at different doses,3-months-old and16-month-old parkinsonian mice both presented improved behavior in movement. In3-month-old group, the effects of rapamycin are inversely proportional to its dosage.Specifically, both high and moderate dosage of rapamycin promoted the formation ofautophagosome in3-and16-month-old parkinsonian mice. Rapamycin activatedautophagic flux and up-regulated HDAC6protein expression in16-month-old and theseeffects were proportional to rapamycin dosage. Although treatment with high dose ofrapamycin led to the increases of LC3-II and HDAC6, autophagic flux remained incompleted. Notably, all doses of rapamycin up-regulated the protein expression ofLAMP1in both3-and16months-old group. It was noted that the increase in3-month-old parkinsonian mice was inversely proportional to the rapamycin dose.Treholose promoted α-synuclein degradation by enhancing autophagic flux, which thensynergistically produced neuroprotective effects in3-month-old parkinsonian mice. But,the same dosage of rapamycin did not showed any effect in16months-old parkinsonian mice.Conclusion Rapamycin and treholose increased the autophagic degradation ofα-synuclein by enhancing autophagic flux in3-month-old PD mice and thus exertedneuroprotection on DA neurons. Owing to the low dosage group of rapamycinsynergistically increased the high level LAMP1protein expression, the low dosagegroup of rapamycin showed the strongest neuroprotective effect.16-month-old micemay need higher doses of autophagy targeted-drugs to activate the autophagic flux dueto the low autophagic activity in compensation in aged mice.