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Caloric restriction rescues yeast cells from alpha-synuclein toxicity through autophagic control of proteostasis

Sampaio-Marques, Belém, Pereira, Hélder, Santos, Ana R., Teixeira, Alexandra, Ludovico, Paula·2018·Aging

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Figure 1 Caloric restriction abrogates α-synuclein (SNCA)-induced toxicity by upregulating ubiquitin-proteasome system activity. (A) Chronological lifespan (CLS) and SNCA levels of stationary wild type cells harbouring the vector control or expressing the human SNCA grown under regular (2% glucose) or CR (0.5% glucose) conditions. (B) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (E) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (F) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. (G) RPN4 and (H) RPN5 mRNA relative expression levels. Three reference genes (ACT1-actin, PDA1-alpha subunit of pyruvate dehydrogenase and TDH2-isoform 2 of glyceraldehyde-3-phosphate dehydrogenase) were used as internal standards and for the normalization of mRNA expression levels. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under regular or CR conditions expressing vector control or SNCA. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).
Figure 1 Caloric restriction abrogates α-synuclein (SNCA)-induced toxicity by upregulating ubiquitin-proteasome system activity. (A) Chronological lifespan (CLS) and SNCA levels of stationary wild type cells harbouring the vector control or expressing the human SNCA grown under regular (2% glucose) or CR (0.5% glucose) conditions. (B) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (E) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (F) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. (G) RPN4 and (H) RPN5 mRNA relative expression levels. Three reference genes (ACT1-actin, PDA1-alpha subunit of pyruvate dehydrogenase and TDH2-isoform 2 of glyceraldehyde-3-phosphate dehydrogenase) were used as internal standards and for the normalization of mRNA expression levels. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under regular or CR conditions expressing vector control or SNCA. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).CC BY · PubMed Central
Figure 2 Autophagy inhibition leads to upregulation of the ubiquitin-proteasome system activity in α-synuclein (SNCA)-expressing cells under caloric restriction. (A) Chronological lifespan (CLS) and SNCA levels of SNCA-expressing stationary wild type cells, under caloric restriction (CR, 0.5% glucose) conditions, in the presence or absence of chloroquine (CQ), an inhibitor of autophagy. (B) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (*p≤0.05, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of CQ. (C) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (D) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (E) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (F) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (G) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (H) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. Statistical significance represented in (C), (D), (F) and (H) was determined by Student's t-test (*p≤0.05, ***p≤0.001, ****p≤0.0001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).
Figure 2 Autophagy inhibition leads to upregulation of the ubiquitin-proteasome system activity in α-synuclein (SNCA)-expressing cells under caloric restriction. (A) Chronological lifespan (CLS) and SNCA levels of SNCA-expressing stationary wild type cells, under caloric restriction (CR, 0.5% glucose) conditions, in the presence or absence of chloroquine (CQ), an inhibitor of autophagy. (B) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (*p≤0.05, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of CQ. (C) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (D) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. (E) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (F) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. (G) Ubiquitination profile determined by Western blotting using an anti-mono and polyubiquitination antibody. (H) Graphical representation of the intensity of total UB/Pgk1 obtained by densitometric analysis. Statistical significance represented in (C), (D), (F) and (H) was determined by Student's t-test (*p≤0.05, ***p≤0.001, ****p≤0.0001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).CC BY · PubMed Central
Figure 3 Pharmacological inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction but has no major impact on autophagy. (A) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001) between cells grown under CR (0.5% glucose) conditions expressing vector control or SNCA in the presence or absence of bortezomib (Bort). (B) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. Statistical significance represented in (B) and (D) was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of Bort. (E) Chronological lifespan (CLS) and SNCA levels of pdr5Δ cells expressing SNCA grown under CR conditions, in the presence or absence of Bort. (F) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (***p≤0.001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of caloric restricted cells expressing vector control (G) or SNCA (H) in the absence or presence of Bort. Blots represented in (G) are from the same gel, as in (H). (I) Densitometric analysis of the ratio between the free GFP versus the total GFP. Bands were quantified by Quantity One software. Significance of the data was determined by two-way ANOVA (**p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).
Figure 3 Pharmacological inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction but has no major impact on autophagy. (A) Chymotrypsin- and trypsin like activities. The assay was normalized to the total protein amount. Significance was determined by two-way ANOVA (*p≤0.05, **p≤0.01, ***p≤0.001) between cells grown under CR (0.5% glucose) conditions expressing vector control or SNCA in the presence or absence of bortezomib (Bort). (B) RPN4 mRNA relative expression levels as described in the legend of Figure 1. (C) UPS activity measured by monitoring the ubiquitin/proteasome-dependent proteolysis of the short-lived protein UBG76V-GFP. GFP was detected by Western blotting using a GFP-specific antibody. (D) Graphical representation of GFP/Pgk1 obtained by densitometric analysis. Statistical significance represented in (B) and (D) was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001) comparing caloric restricted vector control or SNCA-expressing cells in the presence or absence of Bort. (E) Chronological lifespan (CLS) and SNCA levels of pdr5Δ cells expressing SNCA grown under CR conditions, in the presence or absence of Bort. (F) Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS. Significance was determined by two-way ANOVA (***p≤0.001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of caloric restricted cells expressing vector control (G) or SNCA (H) in the absence or presence of Bort. Blots represented in (G) are from the same gel, as in (H). (I) Densitometric analysis of the ratio between the free GFP versus the total GFP. Bands were quantified by Quantity One software. Significance of the data was determined by two-way ANOVA (**p≤0.01, ***p≤0.001, ****p≤0.0001) between cells grown under CR conditions expressing vector control or SNCA in the presence or absence of Bort. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).CC BY · PubMed Central
Figure 4 Genetic inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction conditions but has no major impact on autophagy. Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS of caloric restricted vector control and SNCA expressing wild type cells compared with RPN4 (A) and UMP1 (B) deleted cells. (C) Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of wild type, rpn4Δ and ump1Δ caloric restricted cells expressing vector control or SNCA. Blots from same strain were run in the same gel. Densitometric analysis of the ratio between the free GFP versus the total GFP of rpn4Δ (D) and ump1Δ (E) cells. Data from wild type cells is repeated in (D) and (E) for easier interpretation. Bands were quantified by Quantity One software. (F) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type and cue5Δ cells expressing the vector control or SNCA in normal (2% glucose) or CR (0.5% glucose) growth conditions. (G) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type cells expressing the vector control or SNCA in the absence or presence of chloroquine (CQ). Significance was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) comparing wild type with each mutant strain in the same conditions. For (G) the comparison was done between vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).
Figure 4 Genetic inhibition of the ubiquitin-proteasome system activity decreases lifespan of α-synuclein (SNCA)-expressing cells grown under caloric restriction conditions but has no major impact on autophagy. Mean (50% survival) and maximum (10% survival) lifespans determined from curve fitting of the survival data from CLS of caloric restricted vector control and SNCA expressing wild type cells compared with RPN4 (A) and UMP1 (B) deleted cells. (C) Autophagy flux assessed by the GFP-Atg8 processing assay (immunoblotting analysis with antibody against GFP) of wild type, rpn4Δ and ump1Δ caloric restricted cells expressing vector control or SNCA. Blots from same strain were run in the same gel. Densitometric analysis of the ratio between the free GFP versus the total GFP of rpn4Δ (D) and ump1Δ (E) cells. Data from wild type cells is repeated in (D) and (E) for easier interpretation. Bands were quantified by Quantity One software. (F) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type and cue5Δ cells expressing the vector control or SNCA in normal (2% glucose) or CR (0.5% glucose) growth conditions. (G) Mean and maximum lifespans determined from curve fitting of the survival data from CLS of wild type cells expressing the vector control or SNCA in the absence or presence of chloroquine (CQ). Significance was determined by Student's t-test (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001) comparing wild type with each mutant strain in the same conditions. For (G) the comparison was done between vector control or SNCA-expressing cells in the presence or absence of CQ. Data represents mean ± SEM of at least three biological independent replicas. The error bars represent the standard error of the mean (SEM).CC BY · PubMed Central

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Metadata

Authors
Sampaio-Marques, B., Pereira, H., Santos, A. R., Teixeira, A., & Ludovico, P.
Affiliations
Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, Braga, Portugal
Venue
Aging
Published
2018-12-07
Volume
10
Issue
12
Pages
3821-3833
ISSN
1945-4589
Cited by
14
References
7
Language
en

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