Survivin mutant protects differentiated dopaminergic SK-N-SH cells against oxidative stress

Abstract

Oxidative stress is due to an imbalance of antioxidant/pro-oxidant homeostasis and is associated with the progression of several neurological diseases, including Parkinson's and Alzheimer's disease and amyotrophic lateral sclerosis. Furthermore, oxidative stress is responsible for the neuronal loss and dysfunction associated with disease pathogenesis. Survivin is a member of the inhibitors of the apoptosis (IAP) family of proteins, but its neuroprotective effects have not been studied. Here, we demonstrate that SurR9-C84A, a survivin mutant, has neuroprotective effects against H₂O₂-induced neurotoxicity. Our results show that H₂O₂ toxicity is associated with an increase in cell death, mitochondrial membrane depolarisation, and the expression of cyclin D1 and caspases 9 and 3. In addition, pre-treatment with SurR9-C84A reduces cell death by decreasing both the level of mitochondrial depolarisation and the expression of cyclin D1 and caspases 9 and 3. We further show that SurR9-C84A increases the antioxidant activity of GSH-peroxidase and catalase, and effectively counteracts oxidant activity following exposure to H₂O₂. These results suggest for the first time that SurR9-C84A is a promising treatment to protect neuronal cells against H₂O₂-induced neurotoxicity.

Figure 1. SurR9-C84A attenuate H₂O₂ induced cell death.

SK-N-SH cells were differentiated using 20 µM retinoic acid for 10 days and ddifferentiated media were replaced with growth media. (A) Differentiated cells were treated with different concentration of SurR9-C84A for 24 hr and cell viability was determined using MTT assay. (B) Differentiated SK-N-SH cells were treated with different concentration of H₂O₂ and cell viability was determined using MTT assay. (C, D) Differentiated SK-N-SH cells were pre-treated with 75 µg/ml of SurR9-C84A or ascorbic acid for 24 hr followed by treatment with 300 µM of H₂O₂ for 24 hr. The cell viability and toxicity were determined using (C) MTT and (D) LDH assays, respectively. Data are representative of at least three independent experiments and expressed as mean±SEM; *P<0.05, **P<0.01.

Figure 2. SurR9-C84A prevents H₂O₂ induced apoptosis and necrosis in differentiated SK-N-SH cells.

SK-N-SH cells were differentiated with 20 µM RA for 10 days. Differentiation media were replaced with growth media and cells were treated with (A–C) 300 µM H₂O₂, (D–E) Control, (G–I) 75 µg/ml SurR9-C84A +300 µM H₂O₂ and (J–L) 75 µg/ml ascorbic acid +300 µM H₂O₂ for 24 hr. (M) graph shows the percentage of apoptotic and necrotic cells. Neuronal cells were stained with TUNEL/Propidium iodine double staining and analysed using confocal microscopy as described in materials and methods. The values are presented as the percentage of total number of cells and shown as mean±SEM of three independent experiments. At least 100 cells were counted in each treatment. Bar is 10 µm.

Figure 3. SurR9-C84A prevents mitochondrial depolarization.

SK-N-SH cells were differentiated with 20 µM retinoic acid for 10 days. Differentiated media were replaced with growth media and cells were pre-treated with 75 µg/ml of SurR9-C84A or ascorbic acid for 24 hr followed by treatment with 300 µM of H₂O₂ for 24 hr. At the end of incubation mitochondrial membrane depolarization was qualified and quantified with MitoLight Mitochondrial kit using both techniques of (A) confocal microscopy and (B) spectrofluorometery (see material and method). Green fluorescence (detection of monomers) indicates the presence of depolarized mitochondria (apoptotic cells). Red fluorescence (J-aggregates) indicates the functional and polarized mitochondria. Values are presented as a percentage of increase in mitochondrial depolarization. Data are representative of at least three independent experiments and expressed as mean±SEM; *P<0.05, **P<0.01.

Figure 4. SurR9-C84A pre-treatment prevents expression of neuronal cell death markers such as cyclin D1, caspase 9 and 3.

SK-N-SH cells were differentiated for 10 days with 20 µM RA. After the differentiation period the media was replaced with cell growth media and cells were pre-treated with 75 µg/ml SurR9-C84A or 75 µg/ml ascorbic acid for 24 hr followed by treatment with 300 µM H₂O₂ for 24 hr. (A) cell lysate was prepared and western blot analysis was performed to study the expression of cyclin D1. The loading of each lane was normalised to the level of β-Actin. (B) ELISA assay was performed for cleavage of caspase9 and 3 as described in materials and methods. SurR9-C84A protects the nucleus damage. Cells were pre-treated with 75 µg/ml SurR9-C84A or ascorbic acid for 24 hr followed by treatment with 300 µM H₂O₂ for 24 hr and stained with PI. Arrows show the damage nuclei. (C) Percentage of cells with abnormal nuclei.