SIRT1 activating compounds reduce oxidative stress and prevent cell death in neuronal cells

Abstract

Activation of SIRT1, an NAD+-dependent deacetylase, prevents retinal ganglion cell (RGC) loss in optic neuritis, an inflammatory demyelinating optic nerve disease. While SIRT1 deacetylates numerous protein targets, downstream mechanisms of SIRT1 activation mediating this neuroprotective effect are unknown. SIRT1 increases mitochondrial function and reduces oxidative stress in muscle and other cells, and oxidative stress occurs in neuronal degeneration. We examined whether SIRT1 activators reduce oxidative stress and promote mitochondrial function in neuronal cells. Oxidative stress, marked by reactive oxygen species (ROS) accumulation, was induced in RGC-5 cells by serum deprivation, or addition of doxorubicin or hydrogen peroxide, and resulted in significant cell loss. SIRT1 activators resveratrol (RSV) and SRTAW04 reduced ROS levels and promoted cell survival in RGC-5 cells as well as primary RGC cultures. Effects were blocked by SIRT1 siRNA. SIRT1 activators also increased expression of succinate dehydrogenase (SDH), a mitochondrial enzyme, and promoted deacetylation of PGC-1α, a co-enzyme involved in mitochondrial function. Results show SIRT1 activators prevent cell loss by reducing oxidative stress and promoting mitochondrial function in a neuronal cell line. Results suggest SIRT1 activators can mediate neuroprotective effects during optic neuritis by these mechanisms, and they have the potential to preserve neurons in other neurodegenerative diseases that involve oxidative stress.

Keywords: SIRT1; mitochondria; neuroprotection; optic neuropathy; oxidative stress; resveratrol.

Figure 1

ROS accumulate in the optic nerve during EAE. Eight-week-old female SJL mice were immunized with proteolipid protein and were sacrificed 11 days later. Optic nerves of EAE and control mice were isolated and stained with MitoSOX Red. (A) Cross-sections show high levels of MitoSOX Red staining, a marker of superoxide anion, throughout the parenchyma of EAE optic nerves, with significantly less staining in control optic nerves. One representative nerve from a control mouse, and one EAE optic nerve, is shown at 10× original magnification (top) and 40× original magnification (bottom). (B) The average intensity of MitoSOX staining is significantly higher in EAE optic nerves as compared to control optic nerves (^*p < 0.05).

Figure 2

Cell viability and MitoSOX staining in cultured RGC-5 cells in response to stressors. (A) RGC-5 cells were plated in serum-containing medium for 16 h and then stressed by serum starvation of RGC-5 cells for the next 48 h. A significant decrease in numbers of viable cells, counted by trypan blue exclusion, (^*p < 0.05) occurs by 24 h after serum removal. (B) There is an associated increase in the number of MitoSOX Red staining cells in serum-deprived cultures as compared to serum-containing cultures. (C) RGC-5 cells were plated in serum-containing medium for 16 h and then treated with 1 μM doxorubicin for 24 h. Cell viability was assessed by trypan blue exclusion. Doxorubicin induces a significant decrease (^***p < 0.001) in RGC-5 cell number compared to control cultures, beginning within 6 h of incubation. (D) Increased superoxide staining is observed in doxorubicin treated RGC-5 cultures. (E) RGC-5 cells were plated in serum-containing medium for 16 h, then treated with 500 μM H₂O₂ for 24 h. Cell viability was assessed using PrestoBlue™ Cell Viability Reagent. A significant decrease in cell viability (^*p < 0.05; ^***p < 0.001) occurs after H₂O₂ treatment. (F) H₂O₂ induces an increase in MitoSOX staining.

Figure 3

H₂O₂-induced loss of neuronal differentiated RGC-5 cells attenuated by SIRT1 activators. (A) RGC-5 cells were plated in serum-containing medium for 16 h and then differentiated into neurite-sprouting neuronal cells by addition of 1 μM staurosporine for 6 h. Phase-contrast photographs demonstrate morphologic changes, including presence of neurites, in differentiated cells. (B) Twenty-four hours treatment of differentiated RGC-5 cells with varying doses of H₂O₂ shows a significant dose dependent decrease in cell viability (^*p < 0.05 for 300 μM H₂O₂ vs. control; ^***p < 0.001 for 500 μM H₂O₂ vs. control; ^***p < 0.001 for 700 μM H₂O₂ vs. control) as measured by PrestoBlue Cell Viability Reagent. (C) Pretreatment with 0.25 μM RSV or 5 μM of SRTAW04 beginning 30 min before H₂O₂ treatment, and continuing through 24 h exposure to 500 μM H₂O₂, shows a significant attenuation of RGC-5 death as compared to cells exposed to H₂O₂ alone (^*p < 0.05; ^**p < 0.01). The ability of RSV and SRTAW04 to attenuate RGC-5 cell death is blocked by addition of SIRT1 inhibitor NAM (200 μM). NAM alone does not alter cell viability.

Figure 4

SRTAW04 and RSV attenuate ROS in RGC-5 cells. RGC-5 cells were grown for 24 h with or without (control) 500 μM H₂O₂, and treated continuously with 0.25 μM RSV or 5 μM SRTAW04 where indicated, beginning 30 min before addition of H₂O₂. Cells were stained with MitoSOX Red to assess ROS accumulation. (A) Significant attenuation of MitoSOX staining is shown in representative cultures treated with RSV and SRTAW04 compared to untreated H₂O₂-containing cultures for both non-differentiated (top) and differentiated (bottom) RGC-5 cells. (B) The average intensity of MitoSOX staining is significantly higher in H₂O₂ cultures as compared to control cultures (^*p < 0.05), and compared to RSV (^*p < 0.05) and SRTAW04 (^*p < 0.05) treated cultures of differentiated RGC-5 cells.

Figure 5

SRTAW04 and RSV attenuate H₂O₂ induced loss of mitochondrial potential. RGC-5 cells were plated in serum-containing medium for 16 h and then differentiated into neuronal cells by addition of 1 μM staurosporine for 6 h. Differentiated RGC-5 cells were maintained for 24 h with or without (control) 500 μM H₂O₂, and treated with 0.25 μM RSV or 5 μM SRTAW04 where indicated, beginning 30 min before addition of H₂O₂. JC-1 and TMRM stains were used to assess mitochondrial membrane potential. (A) For comparison, a representative field containing similar cell numbers was photographed from each culture (despite overall lower cell numbers in cultures containing H₂O₂ without SIRT1 activators). Green JC-1 staining (top row) labeling all mitochondria, including those with low membrane potential, demonstrates similar mitochondrial numbers in each culture. Red JC-1 staining (second row) labeling of normal mitochondria shows significant loss of membrane potential in cells exposed to H₂O₂, and this is reversed by addition of RSV or SRTAW04. Merged images of green and red JC-1 staining (third row) show a high green to red ratio in cells exposed to H₂O₂, with a lower ratio following addition of RSV or SRTAW04. Red TMRM staining (bottom row) of normal mitochondria shows similar loss of membrane potential in cells exposed to H₂O₂ that is reversed by addition of RSV or SRTAW04. (B) The average ratio of green to red staining of JC-1 is significantly higher in H₂O₂ cultures as compared to control cultures (^*p < 0.05), and compared to RSV (^*p < 0.05) and SRTAW04 (^*p < 0.05) treated cultures. (C) The average intensity of TMRM staining is significantly lower in H₂O₂ cultures as compared to control cultures (^***p < 0.001), and compared to RSV (^*p < 0.05) and SRTAW04 (^*p < 0.05) treated cultures.

Figure 6

RSV and SRTAW04 protection of RGC-5 cells is SIRT1-dependent. Staurosporine-differentiated RGC-5 cells were transfected with SIRT1 siRNA (A) or a control siRNA (B) and cultured with or without 500 μM H₂O₂, and with or without RSV (0.25 and 3 μM) or SRTAW04 (2.5 and 5 μM) as indicated. SIRT1 siRNA blocked the ability of RSV and SRTAW04 to prevent loss of RGC-5 cells (^***p < 0.001 vs. controls). This effect is specific to SIRT1, as RSV and SRTAW04 do prevent H₂O₂-induced RGC-5 loss in cells transfected with control siRNA (^*p < 0.05). (C) Expression of SIRT1 was measured by Western blot of protein extracts from RSV-treated RGC-5 cultures not transfected with siRNA. Twenty-four hours after initiation of treatment, H₂O₂ shows a significant decrease of SIRT1 protein expression (^***p < 0.001), whereas treatment with RSV significantly attenuates this decrease (^**p < 0.01).

Figure 7

RSV treatment increases markers of mitochondrial function in stressed RGC-5 cells. Staurosporine-differentiated RGC-5 cells were cultured with or without 500 μM H₂O₂, and with or without 0.25 μM RSV for 24 h. (A) Western blot analysis shows a significant decrease in SDH expression (^**p < 0.01) during H₂O₂ treatment which is attenuated by treatment with RSV (^*p < 0.05). (B) Western blot analysis shows similar effects on SOD2 expression. The significant decrease (^**p < 0.01) during H₂O₂ treatment, compared to controls, is not found in cells treated with RSV (^*p < 0.05). (C) Protein extracts were immunoprecipitated with anti-PGC-1α antibodies, blotted, and hybridized with anti-PGC-1α and anti-acetylated lysine antibodies to assess the acetylation state of PGC-1α. H₂O₂ treatment significantly increases the proportion of acetylated PGC-1α (^*p < 0.05) compared to controls, and RSV and SRTAW04 treatment each prevent this acetylation (^*p < 0.05).

Figure 8

SIRT1 activators prevent loss of primary RGCs. Retinal cells were dissociated from neonatal mice and plated overnight prior to transfection with siRNA for 24 h and then treated 24 h with 500 μM H₂O₂ with or without 3 μM RSV. Cells were stained with antibodies to Brn3a to detect RGCs within mixed retinal cell cultures, and numbers of Brn3a positive cells were counted. (A) Brn3a staining demonstrates the presence of RGCs with neuronal morphology in these primary cultures. Original magnification × 20 (left) and × 63 (right). (B) H₂O₂ induces a significant decrease in the number of RGCs (^***p < 0.001) which is attenuated by treatment with RSV (^**p < 0.01) in the presence of control siRNA. SIRT1 siRNA transfection blocks the ability of RSV to attenuate RGC loss, as cell numbers remain significantly reduced compared to controls (^***p < 0.001).