MEF2D And Degradation By Chaperone-mediated Autophagy In Parkinson’s Disease

Background and aim:Parkinson’s disease (PD) is the most common neurodegenerative disorder affectingmovement. The disease is estimated to have a prevalence rate of1%at age65,affectingmillions of people worldwide. PD is characterized by the degenerative loss of pigmenteddopaminergic neurons in the substantial nigra pars compacta (SNc) of the brain. Theincidence of the disease is dependents on the increased of age. Its main clinical symptomsare myotonia, r motor retardation, esting tremor, and attitude disturbance and so on. Someof them also have the damage of congnitive function which seriously affects the quality oflife and working. Our current understanding of how these etiological factors of PDcontribute to loss of DA neurons is still incomplete. Nosetiology shows that it is thoughtto be related to environmental toxins, gene mutation, genetic factors, oxidative stress,immune system abnormalities, iron ion aggregation and neuronal excitotoxicity. With the intensive aging of the population, represented by the PD central nervous degenerativedisease has become one of the major lethal diseases only after cardiovascular or CerebralVascular diseases, malignant tumor and stroke, causing serious burden to the patients andthe whole society. Therefore, to explore the pathogenesis of the nervous systemdegenerative disease and to find the best treatment strategies have become the current hottopic in the field of neuroscience research.Autophagy refers to the process by which lysosomes degrade intracellular constituents,including both organelles and soluble proteins. It can be further classified into three types,namely macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)，depending on how lysosomes receive the cargo. Three forms of autophagy dispose bothsoluble proteins and bulky organelles. Proper function of autophagic process is veryimportant for cellular homeostasis and defects in this pathway have been identified to playa role in a growing list of human disorders, including cancer, ageing, infectious diseases,heart disease, and neurological diseases. Since the long-lived neuronal cells arepermanently postmitotic, they are thought to be particularly vulnerable to the disruption ofautophagy. Indeed, dysregulation of autophagy has been implicated both in chronicneurodegenerative diseases such as Alzheimer’s disease, PD, Huntington disease, as wellas amyotrophic lateral sclerosis, and in acute neuronal damages such as ischemia andtrauma. Deletion of genes required for macroautophagy in the central nervous system isshown to cause degenerative loss of neurons. Unlike macroautophagy and microautophagythat employ vesicles to deliver substrates, CMA targets specific proteins via chaperoneprotein hsc70, which subsequently delivers substrates to lysosomes through membranereceptor LAMP-2A.Although the precise reasons for the selective loss of SNc DA neurons are not entirelyclear, exposure to neurotoxins and the ensuring oxidative stress are considered to be one ofthe important factors either triggering or facilitating the pathogenic process. Transcriptionfactor MEF2D is known to promote neuronal survival in several model systems and hasbeen shown recently to be a substrate of CMA in DA neurons. Dysfunction of CMA hasbeen implicated in the pathogenesis of some forms of familial Parkinson’s disease (PD). CMA is important in maintaining the normal turnover of neuronal survival factor myocyteenhancer factor2D (MEF2D) in dopaminergic (DA) neurons. But whether neurotoxinsmay regulate CMA-MEF2D in neurons remains unknown. What is the specific molecularmechanism? Here we will discuss all these issues.Method:We chose to test this in a mouse midbrain dopaminergic progenitor cell line SN4741because it has been widely used to study the neuronal stress response induced by PDrelated toxins. So we use it to be the Parkinson model in vitro. PD C57BL/6mice modelswere induced by unilateral(the left SNc) stereotaxic intra-striatal6-OHDA injections. Wealso took the brain tissues of PD patients for the vivo study.6-OHDA is a neurotoxin usedto model PD in rodent and to selectively kill dopaminergic and noradrenergic neurons inexperimental models of PD both in vivo and in vitro. Both of the two PD models weretreated with6-OHDA. To measure the survival of DA cell and activity of MEF2D, weused MTT and Luciferase reporter gene assay. Then we observed the modification ofMEF2D which was affected by oxidative stress through two ways of Protein oxidationdetection, DNP and BIAM. After treated with several inhibitors of autophagy, wediscussed the relationship between neurotoxins and CMA with western blot andImmunoprecipitation. We isolated the purified lysosomes from SN cell and then testedBinding and uptake by lysosomes to see alters of activity of CMA. Meanwhile, weexamined whether6-OHDA deregulated CMA in SN4741cells by real-time PCR andImmunofluorescence. We also used the PD animal model and brain tissues of PD patientsas vivo study to see the oxidized changes of MEF2D through the Protein oxidationdetection and2D Gel Electrophoresis.Result:1．6-OHDA induces oxidative modifications of MEF2D. Our data showed thatshort-term exposure to6-OHDA, a neurotoxin used to model PD in rodents, led to a clearreduction of MEF2D levels in the cytoplasm. While short exposure to6-OHDA did notchange nuclear MEF2D levels, prolonged treatment did reduce its levels in the nucleus. We then examined whether6-OHDA caused oxidative modifications of MEF2D inSN4741cells. Following a short treatment with6-OHDA, MEF2D wasimmunoprecipitated from isolated mitochondria, nuclear and cytoplasmic lysates ofSN4741and analyzed for carbonyl oxidation by Oxyblot. Exposure to6-OHDA causeda marked increase in the level of carbonyl oxidation of MEF2D in different cellularcompartments. The level of oxidized MEF2D was increased in a time-dependent mannerin cytosol. To corroborate this finding, we also tested whether MEF2D might be modifiedby other forms of oxidation. MEF2D has four cysteine residues within its sequence atpositions39,41,96and217. We examined the oxidation status of these cysteine residuesby a biotinylated iodoacetamide (BIAM) labeling method. Specifically, SN4741cellstreated with6-OHDA were lysed in a buffer containing BIAM, which does not react withoxidized cysteine residues. MEF2D was then isolated from the nuclear and cytoplasmiclysate by immunoprecipitation and labeled with BIAM. Our analysis indicated that6-OHDA greatly decreased the BIAM labeling of MEF2D protein, revealing increasedoxidation of cysteine residues. Different forms of natural vitamin E have disparatefunctions including antioxidition. We tested antioxidant vitamin E-Tocopherol andshowed that it protected MEF2D from6-OHDA induced reduction. Together, thesefindings demonstrate clearly that MEF2D is readily oxidized under oxidative stress.2.6-OHDA reduces the stability of MEF2D by CMA in DA SN4741cells. Ourstudies revealed that the level of MEF2D decreases as it is increasingly oxidativelymodified. These observations suggest that oxidative modifications may regulate MEF2Dstability. Since our previous studies have established MEF2D as a direct substrate ofCMA in DA neurons, we investigated whether neurotoxin-induced loss of MEF2D proteinwas mediated by a lysosomal pathway. We exposed SN4741cells to6-OHDA with orwithout NH4Cl. This analysis showed that6-OHDA reduced the level of MEF2D, andinhibition of lysosomal function by NH4Cl significantly blocked the6-OHDA-inducedloss of MEF2D. To show that6-OHDA reduces the level of MEF2D primarily byCMA-mediated degradation, we over-expressed a mutated form of MEF2D(MEF2D-N18) which has been shown to be resistant to degradation by CMA in SN4741 cells and then exposed the cells to6-OHDA. While6-OHDA reduced the levels of wildtype (wt) MEF2D, it failed to significantly alter the levels of MEF2D-N18. Furthermore,inhibition of macroautophagy had a smaller effect on the6-OHDA-induced loss ofMEF2D. Inhibition of the proteasome by MG132failed to attenuate6-OHDA-inducedloss of MEF2D. These findings suggest that proteasomes do not play a major role in6-OHDA-induced loss of MEF2D. Previous studies showed that the caspase pathwayplays a role in regulation of MEF2D stability. However, inhibition of caspase3activityby z-DEVD-fmk did not reverse the6-OHDA-induced MEF2D reduction. Together,these results support CMA as the primary mechanism by which6-OHDA promotesMEF2D degradation.3. Neurotoxin6-OHDA activates CMA in DA SN4741cells. We examined whether6-OHDA regulates CMA in SN4741cells by determining the level of the rate-limitingCMA regulator LAMP2A. We treated SN4741cells with6-OHDA and measured thelevels of LAMP2A mRNA by quantitative RT-PCR and protein by immunoblot. Thisanalysis showed that a short exposure to6-OHDA significantly increased the levels ofLAMP2A mRNA. Consistent with this,6-OHDA also caused an increase in the level ofLAMP2A protein as assessed by immunoblotting and immunocytochemistry. In addition,reducing LAMP2A levels by an anti-sense approach in SN4741cells significantlyprevented MEF2D degradation induced by6-OHDA. In contrast to LAMP2A, the level ofHsc70, another key CMA regulator, did not change significantly after6-OHDA treatment.These findings suggest that6-OHDA may significantly enhance CMA activity viaLAMP2A in SN4741cells. To measure CMA activity more directly, we carried outlysosomal binding and uptake assays, the gold standard in determination of CMA activity.We treated SN4741cells with6-OHDA, prepared highly purified lysosomes free ofsignificant contamination of mitochondria and ER, and tested their binding capacity to aknown CMA substrate RNase A. This analysis indicated that6-OHDA significantlyincreased the amount of RNase A associated with lysosomes. Similarly,6-OHDA alsoincreased the amount of RNase A taken up by purified lysosomes. The increased uptakewas not due to non specific inhibitory effects of6-OHDA on either substrate or lysosomes since proteinase K was still able to efficiently degrade extra-lysosomal RNase A and thelevels of lysosomal luminal and member proteins cathepsin D and LAMP1remainedunchanged after6-OHDA treatment. Thus,6-OHDA activates CMA in dopaminergicSN4741cells.4. Oxidative modification of MEF2D facilitates its degradation by CMA. we tested thepossibility that oxidation of MEF2D facilitates its degradation by CMA. For this study,we treated SN4741cells with6-OHDA and blocked lysosomal activity simultaneouslywith NH4Cl. We then isolated MEF2D by immunoprecipitation and blotted precipitatedMEF2D for carbonyl oxidation by Oxyblot. Following6-OHDA treatment, inhibition oflysosomal function led to a significant accumulation of MEF2D with carbonyl oxidation.Similarly, reducing LAMP2A level by anti-sense LAMP2A also caused an accumulationof oxidized MEF2D. To test whether6-OHDA affects the interaction of MEF2D withHsc70, we over-expressed MEF2D in SN4741cells, treated cells with6-OHDA, incubatedcellular lysate with GST-Hsc70, and determined the amount of MEF2D bound toGST-Hsc70. Exposure to6-OHDA enhanced the interaction between MEF2D andGST-Hsc70in pull-down assay.6-OHDA also significantly increased the interactionbetween endogenous MEF2D and Hsc70in a co-immunoprecipitation assay. Consistentwith the above findings, we immunoprecipitated comparable levels of MEF2D fromcontrol and6-OHDA-treated lysates and showed that6-OHDA increased MEF2Doxidization. Incubation of these lysates with lysosomes purified from rat liver using alysosomal uptake assay showed that high levels of MEF2D oxidation correlated closelywith its increased uptake by lysosomes. These results strongly support that6-OHDA-induced oxidization of MEF2D significantly enhances its interaction with Hsc70and promotes its degradation by CMA.5. Oxidation of MEF2D is increased in brain tissues of a rodent PD model as wellas postmortem PD patient brain samples. we employed the6-OHDA-injection ofmice, a well-established rodent PD model. Following6-OHDA injection, weimmunoprecipitated MEF2D from brain lysates and blotted for carbonyl oxidation. Thisanalysis revealed robust oxidation of MEF2D in vivo, which paralleled a clear reduction in MEF2D DNA binding activity. Consistent with our cellular studies,6-OHDA alsocaused an increase in the level of LAMP2A but not Hsc70in mouse brain. Thiscorrelated with a decrease in MEF2D level in the SNc. To strengthen these findings, wetested the level of MEF2D carbonyl oxidation in postmortem PD brain tissues. We firstassessed the total level of MEF2D and showed that consistent with our previous report,MEF2D protein was increased in postmortem PD brains compared to controls. Weimmunoprecipitated MEF2D from control and PD postmortem brain lysates and blottedfor carbonyl formation. This analysis showed that the level of MEF2D carbonylformation in PD brains was significantly higher than that of controls. Consistent withincreased oxidative modifications, analysis by two-dimensional gel electrophoresisrevealed a clear shift in the pI of MEF2D that was immunoprecipitated from PDpostmortem brains and compared to controls. The ability of MEF2D in PD brain tissuesto bind DNA was markedly reduced. Overall, these studies indicate that MEF2D isoxidized in vivo in the context of PD.Conclusion:In this study we used morphology, molecular biology and immunology experimentmethods to confirm the exact molecular mechanism that neurotoxin6-OHDA modulatesthe important transcription factor MEF2D in DA neuron function from both in vivo and invitro at the first time. And we found that neurotoxin through two distinct mechanisms,oxidizing MEF2D and stimulating CMA activity, which collectively leads to accelerateddegradation of MEF2D. This regulatory mechanism may contribute to neurotoxin-andoxidative stress-induced toxicity in DA neurons and also provide new theoretical basis forclinical treatment of PD.