tianhong pan, md, phd; wenjie xie, md; pawan rawal,md joseph jankovic, md; weidong le, md, phd...
TRANSCRIPT
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