Sirtuin-2 Protects Neural Cells from Oxidative Stress and Is Elevated in Neurodegeneration

Sirtuins are highly conserved lysine deacetylases involved in ageing, energy production, and lifespan extension. The mammalian SIRT2 has been implicated in Parkinson's disease (PD) where studies suggest SIRT2 promotes neurodegeneration. We therefore evaluated the effects of SIRT2 manipulation in toxin treated SH-SY5Y cells and determined the expression and activity of SIRT2 in postmortem brain tissue from patients with PD. SH-SY5Y viability in response to oxidative stress induced by diquat or rotenone was measured following SIRT2 overexpression or inhibition of deacetylase activity, along with α-synuclein aggregation. SIRT2 in human tissues was evaluated using Western blotting, immunohistochemistry, and fluorometric activity assays. In SH-SY5Y cells, elevated SIRT2 protected cells from rotenone or diquat induced cell death and enzymatic inhibition of SIRT2 enhanced cell death. SIRT2 protection was mediated, in part, through elevated SOD2 expression. SIRT2 reduced the formation of α-synuclein aggregates but showed minimal colocalisation with α-synuclein. In postmortem PD brain tissue, SIRT2 activity was elevated compared to controls but also elevated in other neurodegenerative disorders. Results from both in vitro work and brain tissue suggest that SIRT2 is necessary for protection against oxidative stress and higher SIRT2 activity in PD brain may be a compensatory mechanism to combat neuronal stress.


Efficiency of SIRT2 transfection
Western blot analysis of diquat treated SH-SY5Y cells showed a significant increase in SIRT2 levels in SIRT2 transfected cells with a 3-3.5 fold increase in vehicle (0.2% PBS) treated cells compared to the empty vector transfected cells (p<0.001 compared to both empty vector and empty vector+AGK2, Supplementary Figure 1). Similar increase was seen in 10μM diquat treated cells, interestingly a small increase of 12% in SIRT2 levels was observed in SIRT2 transfected cells compared to SIRT2 transfected cells treated with AGK2 (p<0.01). Similar to diquat treated cells, the levels of SIRT2 increased by 2.5-2.75 fold in 20μM rotenone treated cells and by 4-4.5 fold in 0.5μM rotenone treated cells (p<0.001) compared to empty vector transfected cells (Supplementary Figure 1). Note: The endogenous SIRT2 level remained undetected in SH-SY5Y cells possibly due to low level of SIRT2 in SH-SY5Y cells and over-expression of SIRT2 only resulted in expression of Isoform 3 of SIRT2 (41kDa).

Supplementary Figure 1 Transfection efficiency of SIRT2 in toxin treated SH-SY5Y cells.
SIRT2 was over-expressed in SH-SY5Y cells and control cells were transfected with empty vector following which one set of cells was treated with toxin alone and another with SIRT2 inhibitor AGK2 and toxin. Cells were harvested and the samples were probed for SIRT2 expression; a-d show the representative blot of SIRT2 and GAPDH in cells treated with a) 20μM diquat, b) 10μM diquat, c) 20μM rotenone and d) 0.5μM rotenone. e-h represent the data are presented as fold-untreated (+SD) in cells treated with a) 20μM diquat, b) 10μM diquat, c) 20μM rotenone and d) 0.5μM rotenone. Data presented are from three independent assays (n=3). *p<0.05 when compared to 0.2% PBS/DMSO, one-way ANOVA (Bonferroni corrected), ###p<0.001and ##p<0.01 when compared to empty vector treatment, ~~~p<0.001 and ~p<0.05 when compared to empty vector + AGK2 treatment and $$$p<0.001 and $$p<0.01 when compared to SIRT2 overexpressing cells, two-way ANOVA (Bonferronicorrected).

Efficiency of SIRT2 inhibition
In order to test the efficiency of SIRT2 inhibition by AGK2, levels of acetylated α-tubulin (K40), was measured in the samples. As shown in Supplementary Figure 2, the levels of acetylated α-tubulin were elevated by 1.2-1.6 fold in cells co-incubated with AGK2 and diquat (p<0.001 in empty vector + AGK2 cells and p<0.01 in SIRT2+AGK2 cells) compared to empty vector transfected cells. The levels of acetylated α-tubulin were reduced by 50% in SIRT2 transfected cells (p<0.01; Supplementary Figure 2) compared to empty vector transfected cells.
These findings suggest that AGK2 is a potent SIRT2 inhibitor. The levels of acetylated αtubulin were significantly higher in AGK2 treated cells in both rotenone treatments (20μM or 0.5μM rotenone: p<0.001) and the levels were reduced in SIRT2 transfected cells (20μM or 0.5μM rotenone: p<0.001; Supplementary Figure 2).

Supplementary Figure 2 AGK2 treatment and its effect on expression of α-tubulin in toxin treated SH-SY5Y cells.
SIRT2 was over-expressed in SH-SY5Y cells and control cells were transfected with empty vector following which one set of cells was treated with toxin alone and another with SIRT2 inhibitor AGK2 and toxin. Cells were harvested and the samples were probed for α-tubulin expression; a and c show the representative blot of α-tubulin, acetylated α-tubulin and GAPDH in cells treated with a) 20μM diquat or 10μM diquat and c) 20μM rotenone or 0.5μM rotenone. The graphs represent the data are presented as fold-untreated (+SD) in cells treated with b) 20μM diquat or 10μM diquat, d) 20μM rotenone or 0.5μM rotenone. Data presented are from three independent assays (n=3). *p<0.05 when compared to 0.2% PBS/DMSO, one-way ANOVA (Bonferroni corrected), ###p<0.001and ##p<0.01 when compared to empty vector treatment, ~~~p<0.001 and ~p<0.05 when compared to empty vector + AGK2 treatment and $$$p<0.001 and $$p<0.01 when compared to SIRT2 overexpressing cells, two-way ANOVA (Bonferroni-corrected).

Cellular location of SIRT2 under oxidative stress Supplementary Figure 3: SIRT2 localisation following oxidative stress.
Cells were treated with vehicle or with 10µM or 20µM diquat, or 20µM rotenone and stained to show localisation of SIRT2 within cells. Under basal conditions, SIRT2 showed a predominantly cytoplasmic localisation but following oxidative stress showed additional translocation to the nucleus. The efficiency of AGK2 as a potent SIRT2 inhibitor was analysed by treating the recombinant SIRT2 with different doses of AGK2 ranging from 1µM -60µM. As shown in Figure 3, treatment of recombinant SIRT2 with AGK2 showed diminished activity of SIRT2 with AGK2

Supplementary Figure 4: Activity of AGK2 against Recombinant Sirtuins.
Recombinant SIRT1, SIRT2 or SIRT3 was used in the activity assay at 100ng/well and at a dose range of 1µM-60µM for AGK2 in order to define the inhibitory characteristics of AGK2 as a specific SIRT2 inhibitor. AGK2 showed highly selective inhibition of SIRT2 up to 60µM.
The efficiency of the substrate Ac-RHKK(Ac)-AMC to measure SIRT activity was also analysed using recombinant SIRT1, SIRT2 and SIRT3. As shown in Figure 4, the substrate showed comparable deacetylase activity with the three recombinant SIRTs and no significant statistical difference was observed in activity of SIRT1, SIRT2 or SIRT3 (Supplementary Recombinant SIRTs were used for the activity assay at 100ng/well to measure the deacetylase activity of SIRT1-SIRT3 towards the substrate. Recombinant sirtuins showed similar specific activity using the Ac-RHKK(Ac)-AMC substrate.

Cellular distribution of SIRT2 in the human brain
The location of SIRT2 was determined in different regions of the hippocampus-CA1, CA2, CA3 and CA4. In controls, SIRT2 was localised in the nucleus in CA1 neurones ( Figure 6a) and CA2 neurones ( Figure 6b) and was localised in both the nucleus and cytoplasm in CA3 neurones ( Figure 6c) and CA4 neurones (Figure 6d), whereas in PD SIRT2 was present in the cytoplasm and occasionally present in the nucleus in CA1-4 ( Figure 6 a-d). As in PD, in PDD cases, SIRT2 was localised both in the nucleus and cytoplasm in CA1 and CA2 but was prominently in the cytoplasm in CA3 and CA4 (Figure 6 a-d). In the DLB and AD, SIRT2 was present in the cytoplasm in CA1 (Figure 6a) neurones and in other CA subfields occasional nuclear localisation was observed but it was pre-dominantly present in the cytoplasm ( Figure   6 b-d). Determination of SIRT2 sub-cellular localisation of SIRT2 in the cerebellum did not show any significant difference in disease groups and controls. In general, nuclear staining of Purkinje cells and granular neurones was observed in all groups (Figure 6e). SIRT2 did not show a staining pattern that could be associated to Lewy bodies. These results suggest that there was no significant effect of disease conditions on the location of SIRT2 within the cell.  Table 3 Summary table presenting localisation of SIRT2 in different brain regions of PD, PDD, DLB, AD and control groups.