1 Rapamycin Protects Against Rotenone-induced Apoptosis Through Autophagy Induction Tianhong Pan, MD, PhD; Wenjie Xie, MD; Pawan Rawal,MDJoseph Jankovic, MD; Weidong Le, MD, PhDDepartment of Neurology, Baylor College of Medicine, Houston, TXAAN, Seattle, 04/30/2009
2 This Study Was Supported by DISCLOSUREThis Study Was Supported byDiana Helis Henry Medical Research Foundation Carolyn Weiss Law Seed Funding National Parkinson Foundation grant to the Baylor College of Medicine Center of Excellence
4 Pathogenesis of Neurodegeneration in Parkinson’s Disease Genetic factors(DJ-1, PINK1, LRRK2, etc)Environmental toxinAgingMitochondrial DysfunctionMutations in -synucleinATPFree radicalsMutation inparkin, UCHL1 -synuclein aggregationMutation in ATP13A2UPS dysfunctionALP dysfunctionAccumulation of Aggregated/Misfolded ProteinsUPS = Ubiquitin Proteasome SystemALP = Autophagy Lysosome PathwayDopaminergic neuron death
5 Protein Degradation Routes Ubiquitin-proteasome system (UPS)ProteinDegradationAutophagy-lysosome pathway (ALP)
6 ALP in mammalian cells Macroautophagy CMA (Autophagy) Microautophagy Various SignalsCMACytosolic proteineg. -synucleinBafilomycin A1 (Baf1)LysosomeCytosolic Chaperon(hsc70)3-methyladenine (3MA)EnzymesmTORCytosolic protein-molecular chaperone complexAutophagosome fuses with lysosomeMisfolded/aggregated proteins or cell componentsLamp2aAmino and fatty acids are released into cytoplasmaAutophagosome/MitophahgosomeTwo concentric membranes engulf cell components or aggregated proteins to be degradedAutophagolysosomeAutophagic Vacuoles (AVs)MicroautophagyMitophagyPan et al. Brain (2008), 131,ALP = Autophagy Lysosome Pathway
7 Autophagy (Macroautophagy) A process of bulk degradation ofLong-lived, stable proteinsEntire organelles (eg. mitochondria)Aggregated proteins that fail to be degraded by UPS
8 HYPOTHESISAutophagy enhancement may prevent accumulation of aggregated/misfolded proteins and of damaged mitochondria, postulated to be two major pathogenic mechanisms of neurodegeneration associated with PD.
9 OBJECTIVETo explore the potential neuroprotective effects of autophagy enhancement on neurotoxin-induced injury and its possible mechanisms
10 REAGENTS Rapamycin Rotenone FDA-approved antibiotic and immuno- suppressantEnhances autophagy via inhibition ofmammalian target of rapamycin (mTOR), a negative regulator of autophagyRotenoneAn inhibitor of mitochondrial complex I,used as a model for neurotoxin-inducedneurodegeneration in PD
11 METHODSThe human neuroblastoma SH-SY5Y cells were treated with rapamycin at various concentrations for different time durationsThe cells were exposed to rotenone with/without rapamycin pretreatment on both small interference RNA of Atg5 (Atg5 siRNA)-transfected cells, in which the autophagy was suppressed, and non-transfected cells.After specific treatment, the cells were either harvested for protein isolation for Elisa assay or immunoblotting assay, or were fixed for immunostaining assay and electron microscopy analysis.
19 CONCLUSION 1Rapamycin exerts a neuroprotective role by interfering with pro-apoptotic insults via enhanced clearance of misfolded/aggregated proteins and/or of dysfunctional mitochondria through autophagy enhancement.
20 Anti-apoptosis via Autophagy Enhancement by Rapamycin
21 CONCLUSION 2Autophagy enhancers, such as rapamycin, may be considered potential therapeutic agents for the treatment of PD.
22 Therapeutic Targets for PD Misfolded/Aggregated ProteinsInjured Mitochondria
23 Novel Therapeutic Strategy for PD AutophagyEnhancement
24 AcknowledgementParkinson’s Disease Center and Movement Disorder Clinic:Joseph Jankovic, MDParkinson’s Disease Research Lab:Weidong Le, MD, PhDPawan Rawal, MDYunchen Wu, MD, PhDWenjie Xie, MDInstitutional Core Grant #CA16672 High Resolution Electron Microscopy facility, UTMDACCKenneth Dunner
25 Parkinson’s Disease Center and Movement Disorders Clinic