SIRT5 Deficiency Enhances Susceptibility to Kainate-Induced Seizures and Exacerbates Hippocampal Neurodegeneration not through Mitochondrial Antioxidant Enzyme SOD2

Abstract

Epilepsy is a common and serious neurological disorder characterized by occurrence of recurrent spontaneous seizures, and emerging evidences support the association of mitochondrial dysfunction with epilepsy. Sirtuin 5 (SIRT5), localized in mitochondrial matrix, has been considered as an important functional modulator of mitochondria that contributes to ageing and neurological diseases. Our data shows that SIRT5 deficiency strikingly increased mortality rate and severity of response to epileptic seizures, dramatically exacerbated hippocampal neuronal loss and degeneration in mice exposed to Kainate (KA), and triggered more severe reactive astrogliosis. We found that the expression of mitochondrial SIRT5 of injured hippocampus was relatively up-regulated, indicating its potential contribution to the comparably increased survival of these cells and its possible neuroprotective role. Unexpectedly, SIRT5 seems not to apparently alter the decline of antioxidant enzymes superoxide dismutase 2 (SOD2) and glutathione peroxidase (GPx) in hippocampus caused by KA exposure in our paradigm, which indicates the protective role of SIRT5 on seizures and cellular degeneration might through different regulatory mechanism that would be explored in the future. In the present study, we provided strong evidences for the first time to demonstrate the association between SIRT5 and epilepsy, which offers a new understanding of the roles of SIRT5 in mitochondrial functional regulation. The neuroprotection of SIRT5 in KA-induced epileptic seizure and neurodegeneration will improve our current knowledge of the nature of SIRT5 in central nervous system (CNS) and neurological diseases.

Keywords: SIRT5; SOD2; Sirtuin; kainate; mitochondria; oxidative stress; reactive astrogliosis; seizures.

Figure 1

Sirtuin 5 (SIRT5) deficiency increases mortality and seizure severity. Kaplan–Meier survival curves show statistically significant difference (P < 0.05) in survival between SIRT5^−/− and control mice over 24 h following kainate (KA) injection at the dosage of both 20 mg/kg (A) and 25 mg/kg (B). KA (20 mg/kg): SIRT5^−/− : n = 23, WT: n = 19; KA (25 mg/kg): SIRT5^−/− : n = 8, WT: n = 8. Survival was assessed using Kaplan–Meier analysis, and the statistically significant difference between two groups was determined with the log-rank test. (C) Seizure response over time in SIRT5^−/− (n = 19) and WT (n = 19) mice following KA injection (20 mg/kg). For each 20-min interval, the highest level of seizure activity was scored using seizure scale. With time, seizures were more severe in SIRT5^−/− mice. Data from seizure scoring were analyzed using two-way analysis of variance (ANOVA) for repeated measurements. (D) Integrated seizure severity of the cohort of mice. SIRT5^−/− : n = 23, WT: n = 19. The comparison was analyzed using unpaired Student’s t tests. Data in (C,D) are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., non-significant.

Figure 2

KA-induced hippocampal neuronal death and degeneration is dramatically reduced in SIRT5^−/− mice. All hippocampal slices were collected from WT and SIRT5^−/− mice 5 days after KA administration, and Nissl staining was performed for neuronal damage evaluation. (A) Representative hippocampus, CA1 and CA3 images from WT+Sal, SIRT5^−/−+Sal, WT+KA, SIRT5^−/−+KA animals. (B) Quantification was performed by counting the number of Nissl positive neurons (Sal groups: n = 4; KA groups: n = 6) in CA1 (B) and CA3 (C) regions. The data were carried out using two-way ANOVA followed by Bonferroni post hoc tests. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; n.s., non-significant; Sal, saline; KA, Kainate; WT, wild-type. Scale bar, 500 μm (A, top panel) and 100 μm (A, in hippocampal CA1 and CA3 regions).

Figure 3

SIRT5 deficiency exacerbates neurodegeneration in the hippocampal CA1 and CA3 regions after KA administration. (A) Representative sections of Fluoro-Jade C (FJc) staining in hippocampus of WT and SIRT5^−/− mice are shown 5 days after KA administration. Significantly increased FJc-positive neurons were detected in the CA1 and CA3 regions of SIRT5^−/− mice compared to WT controls. (B) Quantitative analysis of FJc fluorescence in hippocampal subregions 5 days after KA (n = 6 rats per group). The comparison was analyzed using unpaired Student’s t tests. Data are presented as mean ± SEM. *p < 0.05. Scale bar, 50 μm.

Figure 4

SIRT5 deficiency exacerbates reactive astrogliosis in the hippocampus of mice after KA administration. (A) Compared to nonreactive astrocytes in the control hippocampus, many astrocytes in the hippocampal CA1 and CA3 regions of KA administered mice were reactive and showed greater glial fibrillary acidic protein (GFAP) immunoreactivity, especially in SIRT5^−/− mice. Interestingly, the astrocyte reactivation in hippocampal dentate gyrus (DG) is not dramatically altered, comparable to that in the CA1 and CA3 regions. Quantification of the (B) total numbers of GFAP positive astrocytes and (C) the percentages of reactive astrocytes in different regions of whole hippocampus (n = 6 per group). The data were carried out using two-way ANOVA followed by Bonferroni post hoc tests. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01. n.s., non-significant. Scale bar, 500 μm (A, top panel) and 100 μm (A, in hippocampal CA1, CA3 and DG regions).

Figure 5

Expression level of SIRT5 in hippocampus in 24 h following KA administration. SIRT 5 expression levels were assessed by quantified with a quantitative real-time PCR (qPCR) and western blot. (A) SIRT 5 mRNA is dramatically upregulated in hippocampus of WT mice in 24 h after KA exposure. mRNA levels are expressed as a ratio to 18 S rRNA expression and normalized to baseline controls. Mean ± SEM, n = 4–5 per group. ***p < 0.001. (B) SIRT5 protein expression levels in 24 h after KA exposure. n = 4–5 per group. Although the total SIRT5 protein level was not notably altered (C), the ratio of SIRT5 protein to VDAC1, a mitochondrial marker, was significantly increased 24 h after KA exposure (D). The comparison was analyzed using unpaired Student’s t tests. Mean ± SEM, n = 4–5 per group. *p < 0.05.

Figure 6

SIRT5 deficiency does not affect the reduction of SOD2 and GPx proteins expression levels derived by KA exposure. The effect of SIRT5 deficiency on protein expression levels of SOD2 and GPx induced by KA exposure: representative Western blot image showing decreased protein levels of SOD2 (A) and GPx (C) in 24 h in the hippocampus of SIRT5^−/− and WT mice exposure to KA. Densitometry analysis of SOD2 and GPx are shown in (B,D). The data were carried out using two-way ANOVA followed by Bonferroni post hoc tests. Data are expressed as means ± SEM from n = 4–5 per group. *p < 0.05, **p < 0.01. n.s., non-significant. SOD2, superoxide dismutase 2; GPx, glutathione peroxidase.