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

This Study Was Supported by 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

BACKGROUND

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  -synuclein aggregation Mutation in ATP13A2 UPS dysfunction ALP dysfunction Accumulation of Aggregated/ Misfolded Proteins UPS = Ubiquitin Proteasome System ALP = Autophagy Lysosome Pathway Dopaminergic neuron death

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

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

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

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.

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

REAGENTS Rapamycin Rotenone 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

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.

RESULTS

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

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

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

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

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

Injured Mitochondria Cleared via Autophagy

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.

Anti-apoptosis via Autophagy Enhancement by Rapamycin

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

Therapeutic Targets for PD Misfolded/Aggregated Proteins Injured Mitochondria

Novel Therapeutic Strategy for PD Autophagy Enhancement

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

Parkinson’s Disease Center and Movement Disorders Clinic