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

Molecular Mechanisms of Disease Group Introduction Materials & Methods Results Discussion Parkinson’s Disease (PD) In http://en.wikipedia.org/wiki/Parkinson's_disease 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.

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.

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.

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.

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

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.

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 Neurology Department Martins-Branco et al.

PD Cybrids (ex vivo model) UPS Protein Aggregation

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.

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 Neurology Department Martins-Branco et al.

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

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.

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 Neurology Department Martins-Branco et al.

Correlation studies in PBMC PD model UPS Protein Aggregation

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.

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.

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

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

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