1. α-Synuclein Binds Tom20 and
Inhibits Mitochondrial Protein
Import in Parkinson’s Disease
Paul J Barrett, PhD
Greenamyre Lab
Pittsburgh Institute for Neurodegenerative Diseases
University of Pittsburgh

2. Mitochondria Require Import of Proteins for Proper Function
+++
+++
Preprotein with
Mitochondrial targeting sequence (MTS)
+ + ++
Cleaved
MTS
Matrix
protein

3. α-Synuclein Interacts with Tom20 of the Import Machinery
Proximity Ligation Assays (PLA) Show Proximity Between αSyn and Tom20 in Rat Brain Tissue
Following Rotenone TreatmentPLA
αSyn/Tom20
PLA
αSyn/Tom22

4. α-Synuclein Levels Control the Interaction with Tom20
Knock Down (~30%) of αSyn Disrupts
the αSyn/Tom20 PLA Signal
Overexpression of αSyn Increases
the αSyn/Tom20 PLA Signal in the
Absence of Rotenone Treatment

5. Post-Translationally Modified α-Synuclein
Specifically Binds Tom20
PLA Experiments Show that Certain Post-Translational Modifications Promote the αSyn/Tom20 Interaction
In Vitro Binding Studies Show Specific Binding Between αSyn and Tom20
Monomer Oligomer
Kd=3.2uM
Dopamine-Modified
Kd=4.0uM
Phospho-Mimetic
Kd=4.6uM
Nitrate Modified

6. The α-Synuclein/Tom20 Interaction Inhibits Mitochondrial Import
αSyn Blocks the Import of Exogenous Mitochondrial Targeted GFP (mtGFP) in SH-SY5Y Cells
***, P<0.0001
Mimetic

7. The α-Synuclein/Tom20 Interaction Inhibits Mitochondrial Import
αSyn Blocks the Import of Endogenous Ndufs3 in HEK293 Cells
***, P<0.0001

8. The α-Synuclein/Tom20 Interaction Inhibits Mitochondrial Import
αSyn Blocks Import in Isolated Mitochondria
***, P<0.0001

9. Inhibition of Import Impairs Mitochondrial Function
Import Impairment Increases ROS Production
Import Impairment Decreases Respiration
***, P<0.0001
* * * * * *
*, P<0.001
Reduced
Oxidized
Vehicle Mono Oligo DA-Mod Phos-Mim Nit Mod
Tom20
SS/SH Ratio

10. Tom20 Overexpression Rescues Mitochondrial Import
Tom20 OE Rescues the Import of Endogenous Ndufs3 in HEK 293 Cells
***, P<0.0001 vs treatment
###, P<0.0001 vs vehicle
WT
Tom20 OE
WT
Tom20 OE
These results were mirrored using mtGFP in SH-SY5Y Cells
NDUFS3

11. Tom20 Overexpression Rescues Mitochondrial Import
Tom20 OE Rescues Import in Isolated Mitochondria
***, P<0.0001 vs treatment
###, P<0.0001 vs vehicle
WT
Tom20 OE

12. Tom20 Overexpression Rescues Mitochondrial Function
Tom20 OE Reduces ROS Production
***, P<0.0001 vs treatment
###, P<0.0001 vs vehicle
WT
Tom20 OE
Reduced
Oxidized
WT
Tom20 OE

13. Tom20 Overexpression Rescues Mitochondrial Function
Tom20 OE Restores Respiration to Basal Levels
Wild Type SH-SY5Y Tom20 OE SH-SY5Y

14. Import Impairment in Human Parkinson’s Disease
PLA Quantification
Ndufs3 Quantification
*,P<0.002
***,P<0.0001
ControlParkinson’s
TH PLA αSyn/Tom20 Ndufs3 Merge

15. Summary
PD Brain Tissue is αSyn/Tom20 PLA Positive
Indicating Potential Import Impairment in PD
Tom
22
Tom
40
Tom
40
5 6 7
Tom
20
PTM-α-synuclein
Tom
20
Tom
20
Certain PTM forms of αSyn Can Inhibit
Mitochondrial Protein Import
Tom20 Overexpression Can Rescue
Import and Function
• Loss of import leads to increased ROS
production, decreased respiration, and
decreased complex I activity
• Tom20 overexpression may be a viable
therapeutic for PD
ControlParkinson’s
TH
PLA
αSyn/Tom20 Ndufs3 Merge

16. Acknowledgments
• Greenamyre Lab
• Roberto Di Maio, PhD
• Eric Hoffman, PhD
• Xiaoping Hu
• Jennifer McCoy
• Alvetina Zharikov, PhD
• Anupom Borah, PhD
• Lauri Sanders, PhD
• Collaborators
• Dr. Teresa Hastings
• Caitlyn Barrett, PhD
• Dr. Edward Burton
• Dr. Charlene Chu
• Funding
• DSF Charitable Foundation
• Consolidated Anti-Aging
Foundation
• NIH
• US Department of Veteran’s
Affairs
• Blechman Foundation
• American Parkinson’s
Disease Association
Come see more at poster 6 in session 1!!

17. Overexpression of Mitochondrial-Targeting Sequence (MTS) Peptides Blocks the
αSyn/Tom20 Interaction and is Non-Toxic
MTS Transfection Before αSyn Treatment Blocks the αSyn/Tom20 Interaction
“Preventative”
MTS Transfection After αSyn Treatment Blocks the αSyn/Tom20 Interaction
“Therapeutic”
MTS Transfection Does Not Alter Ndufs3 Import or Mitochondrial Respiration

18. Tom20 Overexpression Does Not Change
Expression of Mitochondrial Proteins

19. Tom20 Overexpression Increases the Initial Rate of pOTC Import in
Isolated Mitochondria from SH-SY5Y Cells
Increasing Time, Constant Input (3uL) Increasing Input, Constant Time (5 Mins)
Results were validated in SH-SY5Y cells using mtGFP import

20. Asyn Fibrils Do Not Inhibit Import

21. DA-modified asyn reduces complex 1 activity
Import Impairment Decreases Complex 1 Activity
***, P<0.0001

22. Tom20 Overexpression Rescues Mitochondrial Import
Tom20 OE Rescues the Import of Exogenous Mitochondrial Targeted GFP (mtGFP) in SH-SY5Y Cells
***, P<0.0001 vs treatment
###, P<0.0001 vs vehicle
WT
Tom20 OE

23. Tom20/Tom22 PLA is Less in DA Neurons