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. 2017 Jun 2;18(1):46.
doi: 10.1186/s12868-017-0364-1.

SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease

Preeti Singh  1   2 , Peter S Hanson  1 , Christopher M Morris  3   4
Affiliations

SIRT1 ameliorates oxidative stress induced neural cell death and is down-regulated in Parkinson's disease

Preeti Singh et al. BMC Neurosci. .

Abstract

Background: Sirtuins (SIRTs) are NAD+ dependent lysine deacetylases which are conserved from bacteria to humans and have been associated with longevity and lifespan extension. SIRT1, the best studied mammalian SIRT is involved in many physiological and pathological processes and changes in SIRT1 have been implicated in neurodegenerative disorders, with SIRT1 having a suggested protective role in Parkinson's disease. In this study, we determined the effect of SIRT1 on cell survival and α-synuclein aggregate formation in SH-SY5Y cells following oxidative stress.

Results: Over-expression of SIRT1 protected SH-SY5Y cells from toxin induced cell death and the protection conferred by SIRT1 was partially independent of its deacetylase activity, which was associated with the repression of NF-кB and cPARP expression. SIRT1 reduced the formation of α-synuclein aggregates but showed minimal co-localisation with α-synuclein. In post-mortem brain tissue obtained from patients with Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies and Alzheimer's disease, the activity of SIRT1 was observed to be down-regulated.

Conclusions: These findings suggests a negative effect of oxidative stress in neurodegenerative disorders and possibly explain the reduced activity of SIRT1 in neurodegenerative disorders. Our study shows that SIRT1 is a pro-survival protein that is downregulated under cellular stress.

Keywords: Alpha-synuclein; Cell survival; Oxidative stress; Parkinson’s disease; SIRT1.

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Figures

Fig. 1
Fig. 1
Effect of SIRT1 and its deacetylase activity on cell viability of toxin treated SH-SY5Y cells. SIRT1WT and SIRT1H363Y were over-expressed in SH-SY5Y cells and control cells were transfected with empty pLenti CMV vector following which cells were treated with diquat (20 or 10 μM) or rotenone (20 or 0.5 μM) for 20 h and viability was measured by reduction of Alamar Blue. Data are presented as mean % control ± SD from three independent assays (n = 3). ***p < 0.001 and **p < 0.01 when compared to 0.2% vehicle (PBS/DMSO), one-way ANOVA (Bonferroni corrected), ###p < 0.001 and ##p < 0.01 when compared to empty vector treatment and ~~~p < 0.001 when compared to SIRT1WT, two-way ANOVA (Bonferroni corrected)
Fig. 2
Fig. 2
Expression of NF-κB in toxin treated SH-SY5Y. SIRT1WT and SIRT1H363Y were over-expressed in SH-SY5Y cells and control cells were transfected with empty vector following which cells were treated with diquat (a 20 or b 10 μM) or rotenone (c 20 or d 0.5 μM) for 20 h. Cells were harvested and the samples were probed for NF-κB. Data are presented as fold-untreated (+SD) from three independent assays (n = 3) with comparison to GAPDH as a housekeeping control protein. ***p < 0.001 when compared to 0.2% PBS, one-way ANOVA (Bonferroni corrected), ###p < 0.001 when compared to empty vector treatment, ~~~p < 0.001, ~~p < 0.01 and ~p < 0.05 when compared to SIRT1WT cells, two-way ANOVA (Bonferroni corrected). Images are representative blot of NF-κB and GAPDH
Fig. 3
Fig. 3
Expression of cleaved PARP-1 in toxin treated SH-SY5Y. SIRT1WT and SIRT1H363Y were over-expressed in SH-SY5Y cells and control cells were transfected with empty vector following which cells were treated with diquat (a 20 or b 10 μM) or rotenone (c 20 or d 0.5 μM) for 24 h. Cells were harvested and the samples were probed for cPARP-1. Data are presented as fold-untreated (+SD) from three independent assays (n = 3) with comparison to GAPDH as a housekeeping control protein. ***p < 0.001 and **p < 0.01 when compared to 0.2% PBS, one-way ANOVA (Bonferroni corrected), ###p < 0.001 when compared to empty vector treatment, ~p < 0.05 when compared to SIRT1WT cells, two-way ANOVA (Bonferroni corrected). Images are representative blot of cPARP1 and GAPDH
Fig. 4
Fig. 4
Localisation of SIRT1 and phospho-α-synuclein in toxin treated SH-SY5Y cells. Cellular distribution of SIRT1 and phospho-α-synuclein was determined using fluorescent immunocytochemistry. Images show α-synuclein immunostaining, SIRT1 immunostaining and all staining merged including DAPI in a 20 μM diquat and b 20 μM rotenone treated cells. Scale bars—white scale bar 50 μM and red scale bar 20 μM; magnification: ×40
Fig. 5
Fig. 5
Phospho-α-synuclein aggregate formation in toxin treated SH-SY5Y cells. SIRT1WT and SIRT1H363Y overexpressing SH-SY5Y cells were treated with toxin (20 or 10 μM diquat or 20 or 0.5 μM rotenone) and 0.2% PBS or DMSO; cells transfected with empty vector were used as a control. Cells were immunostained with phospho-α-synuclein. Images were captured through GFP filter under ×63 magnification. The captured images represent phospho-α-synuclein staining and the bar graphs represent the aggregate quantification in diquat or rotenone treated cells. Each bar represents % phospho-α-synuclein aggregates (±SD) from three independent assays (n = 3). ***p < 0.001, **p < 0.01 and *p < 0.05 when compared to 0.2% vehicle, one-way ANOVA (Bonferroni corrected), ###p < 0.001 and ##p < 0.01 when compared to control cells, ~~~p < 0.001 and ~p < 0.05 when compared to SIRT1WT overexpressing cells, two-way ANOVA (Bonferroni corrected). Scale bar 20 μM
Fig. 6
Fig. 6
Expression of SIRT1 protein in post mortem brain in Parkinson’s Disease. The levels of SIRT1 were determined in different regions of Parkinson’s disease (PD) patients and compared to a control-cohort. SIRT1 band intensity was normalised with GAPDH as a housekeeping protein. Data are presented as fold change (±SD) with respect to control from three independent replicates of SIRT1/GAPDH. ***p < 0.001, **p < 0.01 and *p < 0.05 (t test). Images are representative blots of SIRT1 and GAPDH. M denotes molecular weight marker lane
Fig. 7
Fig. 7
Expression of SIRT1 protein in post mortem brain of Parkinson’s disease with dementia. The levels of SIRT1 were determined in different regions of Parkinson’s disease with dementia patients and compared to a control cohort. SIRT1 band intensity was normalised with GAPDH as a housekeeping protein. Data are presented as fold change (±SD) with respect to control from three independent replicates. **p < 0.01 (t test). Images are representative blots of SIRT1 and GAPDH. M denotes molecular weight marker lane
Fig. 8
Fig. 8
Total sirtuin activity and specific SIRT1 activities in frontal and temporal cortex in neurodegeneration. Total SIRT and SIRT1 activities were measured using a fluorometric enzymatic activity assay with specific SIRT1 inhibition in the frontal and temporal cortices of PD, PDD, DLB and AD patients and were compared to cohort-control group. ***p < 0.001, **p < 0.01 and *p < 0.05 when compared to control, one-way ANOVA (Bonferroni corrected)
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