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Tianhong Pan, MD, PhD; Wenjie Xie, MD; Pawan Rawal,MD Joseph Jankovic, MD; Weidong Le, MD, PhD

Rapamycin Protects Against Rotenone-induced Apoptosis Through Autophagy Induction . Tianhong Pan, MD, PhD; Wenjie Xie, MD; Pawan Rawal,MD Joseph Jankovic, MD; Weidong Le, MD, PhD Department of Neurology, Baylor College of Medicine, Houston, TX. AAN, Seattle, 04/30/2009. DISCLOSURE.

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Tianhong Pan, MD, PhD; Wenjie Xie, MD; Pawan Rawal,MD Joseph Jankovic, MD; Weidong Le, MD, PhD

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  1. Rapamycin Protects Against Rotenone-induced Apoptosis Through Autophagy Induction Tianhong Pan, MD, PhD; Wenjie Xie, MD; Pawan Rawal,MD Joseph Jankovic, MD; Weidong Le, MD, PhD Department of Neurology, Baylor College of Medicine, Houston, TX AAN, Seattle,04/30/2009

  2. DISCLOSURE This Study Was Supported by Diana Helis Henry Medical Research Foundation Carolyn Weiss Law Seed Funding National Parkinson Foundation grant to the Baylor College of Medicine Center of Excellence

  3. BACKGROUND

  4. Pathogenesis of Neurodegeneration in Parkinson’s Disease Genetic factors (DJ-1, PINK1,LRRK2, etc) Environmental toxin Aging Mitochondrial Dysfunction Mutations in -synuclein ATP Free radicals Mutation in parkin, UCHL1 Mutation in ATP13A2 -synuclein aggregation UPS dysfunction ALP dysfunction Accumulation of Aggregated/ Misfolded Proteins UPS = Ubiquitin Proteasome System ALP = Autophagy Lysosome Pathway Dopaminergic neuron death

  5. Protein Degradation Routes Ubiquitin-proteasome system (UPS) Protein Degradation Autophagy-lysosome pathway (ALP)

  6. ALP in mammalian cells Macroautophagy (Autophagy) Various Signals CMA Cytosolic protein eg. -synuclein Bafilomycin A1 (Baf1) Lysosome Cytosolic Chaperon (hsc70) Enzymes 3-methyladenine (3MA) mTOR Cytosolic protein-molecular chaperone complex Autophagosome fuses with lysosome Misfolded/aggregated proteins or cell components Amino and fatty acids are released into cytoplasma Lamp2a Autophagosome/ Mitophahgosome Autophagolysosome Two concentric membranes engulf cell components or aggregated proteins to be degraded Microautophagy Autophagic Vacuoles (AVs) Mitophagy Pan et al. Brain (2008), 131, 1969-1978 ALP = Autophagy Lysosome Pathway

  7. Autophagy (Macroautophagy) A process of bulk degradation of Long-lived, stable proteins Entire organelles (eg. mitochondria) Aggregated proteins that fail to be degraded by UPS

  8. HYPOTHESIS Autophagy 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. OBJECTIVE To explore the potential neuroprotective effects of autophagy enhancement on neurotoxin-induced injury and its possible mechanisms

  10. REAGENTS • Rapamycin • FDA-approved antibiotic and immuno- suppressant • Enhances autophagy via inhibition of mammalian target of rapamycin (mTOR), a negative regulator of autophagy • Rotenone • An inhibitor of mitochondrial complex I, used as a model for neurotoxin-induced neurodegeneration in PD

  11. METHODS • The human neuroblastoma SH-SY5Y cells were treated with rapamycin at various concentrations for different time durations • The 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.

  12. RESULTS

  13. Rapamycin Enhanced Autophagy in SH-SY5Y Cells LC3: Autophagy Marker ; Con = Control; Rapa = Rapamycin

  14. Rapamycin Protected Against Rotenone-Induced Apoptosis Con = control; Rapa = Rapamycin; Rot = Rotenone

  15. Autophagy Inhibition Blocked Rapamycin’s Neuroprotection Con = control; Rapa = Rapamycin; Rot = Rotenone

  16. Rapamycin Protected Mitochondrial Function Con = control; Rapa = Rapamycin; Rot = Rotenone

  17. Rapamycin Enhanced Degradation of Ubiquitinated Proteins Rapa = Rapamycin; Rot = Rotenone

  18. Injured Mitochondria Cleared via Autophagy

  19. CONCLUSION 1 Rapamycin 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 2 Autophagy enhancers, such as rapamycin, may be considered potential therapeutic agents for the treatment of PD.

  22. Therapeutic Targets for PD Misfolded/Aggregated Proteins Injured Mitochondria

  23. Novel Therapeutic Strategy for PD Autophagy Enhancement

  24. Acknowledgement Parkinson’s Disease Center and Movement Disorder Clinic: Joseph Jankovic, MD Parkinson’s Disease Research Lab: Weidong Le, MD, PhD Pawan Rawal, MD Yunchen Wu, MD, PhD Wenjie Xie, MD Institutional Core Grant #CA16672 High Resolution Electron Microscopy facility, UTMDACC Kenneth Dunner

  25. Parkinson’s Disease Center and Movement Disorders Clinic

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