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

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

BACKGROUND

ATP Free radicals

-synuclein aggregation

Dopaminergic neuron death

Environmental toxin

Genetic factors(DJ-1, PINK1, LRRK2, etc)

Aging

Mutation in ATP13A2Mutation in

parkin, UCHL1

Mutations in -synuclein

UPS = Ubiquitin Proteasome System

Mitochondrial Dysfunction

Accumulation of Aggregated/ Misfolded Proteins

Pathogenesis of Neurodegeneration in Parkinson’s Disease

UPS dysfunction ALP dysfunction

ALP = Autophagy Lysosome Pathway

Ubiquitin-proteasome system

(UPS)

Protein Degradation Routes

Autophagy-lysosome pathway

(ALP)

Protein Degradation

Two concentric membranes engulf cell components or aggregated proteins to be

degraded

Autophagosome/Mitophahgosome Autophagolysosome

Autophagic Vacuoles (AVs)

Misfolded/aggregated proteins or cell

componentsAmino 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 (Autophagy)

CMA

Lamp2a

Cytosolic protein-molecular chaperone complex

ALP in mammalian cells

Pan et al. Brain (2008), 131, 1969-1978

ALP = Autophagy Lysosome Pathway

mTOR

Various Signals

Mitophagy

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

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.

To explore the potential neuroprotective

effects of autophagy enhancement on

neurotoxin-induced injury and its

possible mechanisms

OBJECTIVE

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

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

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

Acknowledgement

Parkinson’s Disease Center and Movement Disorders Clinic