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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,

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Presentation on theme: "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,"— Presentation transcript:

1 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 Rapamycin Protects Against Rotenone-induced Apoptosis Through Autophagy Induction AAN, Seattle, 04/30/2009

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

3 BACKGROUND

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

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

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

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

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

10 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 REAGENTS

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 Rapamycins 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/AggregatedProteins Injured Mitochondria

23 Novel Therapeutic Strategy for PD Autophagy AutophagyEnhancement

24 Parkinsons Disease Center and Movement Disorder Clinic: Joseph Jankovic, MD Parkinsons 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 Acknowledgement

25 Parkinsons Disease Center and Movement Disorders Clinic


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