Ubiquitin proteasome system in Parkinson's disease: a keeper or a witness?

Abstract

Objective: The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models.

Method: We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations.

Results: We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients.

Interpretation: aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.

Figure 1. aSN aggregation in PD cellular models

(A) Densitometry analysis of triton-soluble aSN oligomers in SH-SY5Y ndufa2 KD cells and representative WB. N=3, ***p<0.001. (B) Densitometry analysis of triton-soluble aSN oligomers in PD Cybrids and representative WB. N=3, *p<0.05 (C) Densitometry analysis of triton-soluble aSN oligomers in PD patients PBMC and representative WB.

Figure 2. UPS function in SH-SY5Y ndufa2 KD cells

(A) Proteasome 26S and 20S chymotryosin-like activity. N=4, *p<0.05. (B) Densitometry analysis of total ubiquitinated protein content. N=3, p=0,0534 (C) Densitometry analysis of ubiquitin monomer. N=3, *p<0.05. (D) Representative WB of ubiquitinated proteins in SH-SY5Y control and ndufa2 KD cell lines under basal and lactacystin-treated conditions.

Figure 3. UPS function in PD Cybrids

(A) Proteasome 26S and 20S chymotryosin-like; trypsin-like and PGPH activities. N=4, *P<0.05. (B) Densitometry analysis of total ubiquitinated protein content. N=3, **p<0.01. (C) Representative WB of ubiquitinated proteins in CT and PD cybrid cell lines under basal and lactacystin-treated conditions.

Figure 4. UPS function in PBMC of LOPD and EOPD patients

(A) Proteasome 26S and 20S chymotrypsin-like activity. *p<0,05 (B) Densitometry analysis of total ubiquitinated protein content and representative WB. (C) Ubiquitination influence on proteasome activity in LOPD patients: 26S chymotrypsin-like activity do not correlated with ubiquitinated protein content. N=13, Pearson r=0.2840, p=0.3470, r2=0.08066; 20S chymotrypsin-like activity has a significant positive correlation with ubiquitinated protein content. N=13, Pearson r=0.6486, *P<0,05, r2=0,4207.

Figure 5. Ubiquitinated aSN in PD cell-line models

(A) Densitometry analysis of the ratio between ubiquitinated aSN and aSN after aSN IP in SH-SY5Y ndufa2 KD; and representative WB. SH-SY5Y ndufa2 KD cells show an increased amount of ubiquitinated aSN compared to parental cell-line. Lactacystin induced an increase in aSN ubiquitination in parental cells. N=2 *p<0.05. (B) Densitometry analysis of ratio between ubiquitinated aSN and aSN after aSN IP in PD Cybrids and representative WB. PD cybrids show an increase in the amount of ubiquitinated aSN compared to CT cybrids. Lactacystin promote the accumulation of ubiquitinated aSN in CT cells. N=2, p<0.05. p<0,05. (C) aSN oligomers levels have a positive correlation with total ubiquitination in PBMC of PD patients. N=11, Pearson r=0.6924, *p<0.05, r2=0.04795.

Figure 6. aSN quantification in the plasma of PD patients

Densitometry analysis of aSN levels in the plasma of PD patients and representative dot blot. There is an increase in the amount of aSN in the plasma of PD patients, which is significative in EOPD. N=4-10. *P<0,05.

Figure 7. Correlation between Age and Chymotrypsin-like activity in PBMC

(A) In control individuals 26S chymotrypsin-like activity has a significant negative correlation with age in control indivuduals. N=8, Pearson r=−0,754, *p<0,05, r2=0,5686 and 20S chymotrypsin-like activity isnegatively correlated with age. N=8, Pearson r=−0,468, p=0,2422, r2=0,219 (B) In LOPD patients 26S chymotrypsin-like activity is negatively correlated with age. N=14, Pearson r=−0,5275, p=0,0526, r2=0,2783; 20S chymotrypsin-like activity has a significant negative correlation with age. N=14, Pearson r = −0,6864, **p<0,01, r2=0,4712 (C) In EOPD patients 26S chymotrypsin-like activity is negatively correlated with age. N=4, Pearson r=−0,7365, p=0,2635, r2=0,5424 and 20S chymotrypsin-like activity is positively correlated with age. N=6, Pearson r=0,5648, p=0,2429, r2=0,3190.

Figure 8. Correlations studies between demographic characteristics and Ubiquitination or aSN oligomers

(A) In LOPD patients ubiquitination has a significantly negative correlation with age. N=13, Pearson r=−0,6748, *p<0,05, r2=0,4553; aSN has a very low and weak positive correlation with age. N=13, Pearson r=0,2511, p=0,4079, r2=0,06306 (B) In LOPD patients ubiquitination has a positive correlation with duration of disease. N=10, Pearson r=0,6058, p=0,0634, r2=0,3670; aSN has a very low and weak positive correlation with duration of the disease. N=10, Pearson r=0,2214, p=0,5387, r2=0,04902 (C) In LOPD patients aSN/ubiquitin ratio has a positive correlation with age. N=13, Pearson r=0,736, **p<0,01, r2=0,5417 (D) In LOPD patients aSN/ubiquitin ratio has a very weak negative correlation with duration of disease. N =10, Pearson r=0,4997, p=0,1414, r2=0,2497.