1. Ubiquitin Proteasome System in Parkinson's Disease:
7th June 2012,
ISCOMS, Groningen
Ubiquitin Proteasome System in Parkinson's Disease:
a keeper or a witness?
Martins-Branco D, Esteves AR, Santos D, Arduino DM, Swerdlow RH, Januario C, Oliveira CR, Cardoso SM

2. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Parkinson’s Disease (PD) In
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Mitochondria
Apoptosis
Fusion
Fission
Mitochondria
Apoptosis
Fusion
Fission
Autophagy
Macro
CMA
UPS
Ubiquitin
Proteasome
20S
26S PD
Major Symptoms
Bradykinesia
Tremor at rest
Rigidity
Postural instability
Protein Quality Control System
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

3. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Our Hypothesis
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Mitochondria
Apoptosis
Fusion
Fission
Mitochondria
Apoptosis
Fusion
Fission
Evidence exists supporting the notion that oxidative stress and impaired mitochondrial function may trigger the etiopathogenesis of the disorder.
Thus, in this work we propose to focus on the role of UPS as a protein quality control system and evaluate how mitochondrial dysfunction potentiates aSN aggregation through direct study of proteasome activity and ubiquitin-dependent aSN clearance.
Autophagy
Macro
CMA
UPS
Ubiquitin
Proteasome
20S
26S PD
Protein Quality Control System
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

4. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
PD models
Peripheral Blood Mononuclear Cells (PBMC)
SH-SY5Y ndufa2 KD cells
PD Cybrids
*Treated with Lactacystin
*Treated with Lactacystin
*Lactacystin concentration used (2µM) did not affect viability in both cell-line models. N=3.
Object of study and techniques
UPS
Protein Aggregation
Mitochondria
Proteasome enzymatic activity
aSN oligomers formation
ETC CX I activity
NADH-ubiquinone oxiredutase assay (Esteves et al., 2008)
Fluorimetric proteasomal activity analysis (Domingues et al., 2008)
Immunoblotting (Esteves et al. 2010), Immunoprecipitation and Dot Blot
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

5. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Mitochondrial function in PD cellular models
Mitochondria
Apoptosis
Fusion
Fission
As previously shown by our group, ETC CXI activity is reduced in
platelets of PD patients and in PD Cybrids (Esteves, et al., 2008).
We further characterized SH-SY5Y ndufa2 KD cells and observed that ETC CXI activity was reduced (p=0.0071) as compared to wild-type SH-SY5Y cells.
This mitochondrial impairment was due to the decrease in
ndufa2 gene expression in SH-SY5Y ndufa2 KD cells (p=0.0256)
NADH-ubiquinone oxiredutase assay (Esteves et al., 2008)
Immunoblotting (Esteves et al. 2010)
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

6. SH-SY5Y ndufa2KD cells (in vitro model)
UPS
Protein Aggregation

7. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
SH-SY5Y ndufa2KD cells: UPS
Despite ndufa2 KD cells have a deficient ETC, ATP-dependent chymotrypsin-like activity was found unchanged and ATP-independent chymotrypsin-like activity chiefly increased in ndufa2 KD cells (p=0.0207).
Fluorimetric proteasomal activity analysis (Domingues et al., 2008)
UPS
Ubiquitin
Proteasome
20S
26S
However, levels of total protein ubiquitination and ubiquitin monomer were increased.
Lactacystin induced an increase in the accumulation of both ubiquitinated species and ubiquitin monomer
Immunoblotting (Esteves et al. 2010)
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

8. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
SH-SY5Y ndufa2KD cells: aSN oligomerization
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
We observed that aSN oligomerization increased in SH-SY5Y ndufa2 KD when compared to respective parental cell-line (p=0.0007).
aSN oligomerization increased with lactacystin treatment.
Immunoblotting (Esteves et al. 2010)
In ndufa2 KD cells we observed an increase in the levels of ubiquitinated aSN as compared to parental cells (p=0.0157). Interestingly, lactacystin potentiated the accumulation of ubiquinated aSN in parental cells (p=0.0325), but failed to do so in ndufa2 KD cells, which indicates a previous impairment of UPS.
Immunoprecipitation
Molecular Mechanisms of Disease Group
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Martins-Branco et al.

9. PD Cybrids (ex vivo model)
UPS
Protein Aggregation

10. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
PD Cybrids: UPS
We found a decrease in ATP-dependent trypsin-like activity in PD cybrids (p=0.0438). Interestingly, in our ex vivo model, we could not observe an up-regulation of ATP-independent proteasomal function, like previously described for ndufa2 KD cells.
Fluorimetric proteasomal activity analysis (Domingues et al., 2008)
UPS
Ubiquitin
Proteasome
20S
26S
We observed an increase in protein ubiquitination levels (p=0.0044), as it was previously showed by our group (Esteves, et al., 2010).
Lactacystin treatment promoted a non significant accumulation of ubiquitinated species.
Immunoblotting (Esteves et al. 2010)
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

11. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
PD Cybrids: aSN oligomerization
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
As it was previously shown by our group (Esteves, et al., 2010), there was an increased aSN oligomerization in PD cybrids (p=0.0485).
aSN oligomerization increase with lactacystin treatment.
Immunoblotting (Esteves et al. 2010)
Levels of ubiquitinated aSN were increased in PD Cybrids (p=0.0044). Moreover, similarly to what we observed in ndufa2 KD cells, lactacystin was able to promote the accumulation of ubiquitinated aSN only in CT cells (p=0.0277). Again, PD cybrids with dysfunctional mitochondria also have an impaired UPS that may precede aSN ubiquitination, since lactacystin failed to potentiate the build-up of these aSN species.
Immunoprecipitation
Molecular Mechanisms of Disease Group
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Martins-Branco et al.

12. PD Patients’ PBMC (in vivo model)
UPS
Protein Aggregation

13. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
PD patients’ PBMC cells: UPS
Interestingly, we detected an increase of proteasome activities in younger groups, in both CT and PD cells, versus older individuals’, being this effect greater for ATP-dependent chymotrypsin-like activity.
Fluorimetric proteasomal activity analysis (Domingues et al., 2008)
Ubiquitination levels have a significant positive correlation with 20S chymotrypsin-like activity in LOPD group (p=0,0165)
UPS
Ubiquitin
Proteasome
20S
26S
Regardless of great variability, we can see an increase in the mean of ubiquitination levels in both disease groups when compared with their counterparts, similarly to what we observed in our other PD models.
There were also increased protein ubiquitination levels in younger individuals group
Immunoblotting (Esteves et al. 2010)
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

14. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
PD patients’ PBMC cells: aSN oligomerization
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
In PBMC of PD patients we can just observe a tendency to an increased aSN oligomerization, probably due to high variability between human samples.
Immunoblotting (Esteves et al. 2010)
In PBMC of PD patients we evaluated the interplay between UPS and aSN accumulation by determining a statistically significant positive correlation between aSN and total ubiquitin content (p=0,0182)
Immunoprecipitation
Since we observed in PBMC of PD patients a tendency for aSN accumulation we quantified the levels of aSN in the plasma. We observed an increase in aSN secretion in the both groups, although we only obtained statistical significance in the EOPD group compared to the respective age-matched control group (p=0,0111)
Dot Blot
Molecular Mechanisms of Disease Group
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15. Correlation studies in PBMC PD model
UPS
Protein Aggregation

16. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Correlation studies in PBMC PD model
UPS
Protein Aggregation
Due to high variability observed in the previous results with PBMC model, some correlation studies were performed in order to better understand the influence of some demographic characteristics of patients’ population
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

17. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Correlation studies in PBMC PD model
We observed a negative correlation between age of individuals and 26S and 20S chymotrypsin-like activities, in both control individuals (p=0.0307)
and LOPD patients (p=0.0067).
Concerning to ubiquitination levels, there is a negative correlation between age and ubiquitination levels in LOPD group of patients (p=0,0114).
aSN/ubiquitin ratio is not correlated with duration of disease but is positively correlated with age, with an exponential nonlinear fit (p=0,0041).
Interestingly, ubiquitination has a positive correlation tendency with duration of disease.
UPS
Ubiquitin
Proteasome
20S
26S
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

18. Molecular Mechanisms of Disease Group
Introduction
Materials & Methods
Results
Discussion
Molecular Mechanisms of Disease Group
Neurology Department
Martins-Branco et al.

19. Final Remarks Highlights
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Protein Aggregation
Alpha-sinuclein
Lewy Bodies
Mitochondria dysfunction trigger Ubiquitin-Protesome System impairment in PD models
PD models show increased total protein ubiquitination and alpha- synuclein levels
Ubiquitinated aSN is increased, particularly in the in vitro and ex vivo models
Proteasome inhibition fail to increase ubiquitinated aSN in these cell-line models
ATP-independent proteasomal activity is chiefly increased the in vitro PD model
Mitochondria
Apoptosis
Fusion
Fission
Mitochondria
Apoptosis
Fusion
Fission
Autophagy
Macro
CMA
UPS
Ubiquitin
Proteasome
20S
26S PD
Protein Quality Control System

20. Acknowledgments Molecular Mechanisms of Disease Group
Mitochondrial dysfunction in the pathogenesis of Alzheimer´s and Parkinson’s diseases
Sandra M. Cardoso (PhD)
Ana Raquel Esteves (PhD)
Daniela M. Arduíno (PhD student)
Diana F F Silva (PhD student)
Daniel Santos (PhD student)
Diogo Martins Branco (Master student)
Catarina R. Oliveira (Consultant CNC)
Neurology C Department: Cristina Januário
Fradique Moreira
Russel H. Swerdlow
GAPI
FMUC