1. 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

2. 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

3. BACKGROUND

4. 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

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

6. 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,
ALP = Autophagy Lysosome Pathway

7. 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

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

10. 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

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 Rapamycin’s 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/Aggregated
Proteins
Injured Mitochondria

23. Novel Therapeutic Strategy for PD
Autophagy
Enhancement

24. 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

25. Parkinson’s Disease Center and Movement Disorders Clinic