Transcript Tianhong Pan, MD, PhD - Baylor College of Medicine

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
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
Mutation in
parkin, UCHL1
ATP
Free radicals
 -synuclein aggregation
UPS dysfunction
Mutation in ATP13A2
ALP dysfunction
Accumulation of Aggregated/
Misfolded Proteins
UPS = Ubiquitin
Proteasome System
Dopaminergic neuron death
ALP = Autophagy
Lysosome Pathway
Protein Degradation Routes
Ubiquitin-proteasome system
Protein
Degradation
(UPS)
Autophagy-lysosome pathway
(ALP)
ALP in mammalian cells
Various
Signals
Macroautophagy
(Autophagy)
CMA
Bafilomycin A1
(Baf1)
Lysosome
mTOR
3-methyladenine
(3MA)
Enzymes
Cytosolic Chaperon
(hsc70)
Cytosolic protein-molecular
chaperone complex
Autophagosome
fuses with lysosome
Misfolded/aggregated
proteins or cell
components
Two concentric membranes
engulf cell components or
aggregated proteins to be
degraded
Cytosolic protein
eg. -synuclein
Lamp2a
Autophagosome/
Mitophahgosome
Amino and
fatty acids are
released into
cytoplasma
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

FDA-approved antibiotic and immunosuppressant
 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