Altered mitochondrial acetylation profiles in a kainic acid model of temporal lobe epilepsy

Abstract

Impaired bioenergetics and oxidative damage in the mitochondria are implicated in the etiology of temporal lobe epilepsy, and hyperacetylation of mitochondrial proteins has recently emerged as a critical negative regulator of mitochondrial functions. However, the roles of mitochondrial acetylation and activity of the primary mitochondrial deacetylase, SIRT3, have not been explored in acquired epilepsy. We investigated changes in mitochondrial acetylation and SIRT3 activity in the development of chronic epilepsy in the kainic acid rat model of TLE. Hippocampal measurements were made at 48 h, 1 week and 12 weeks corresponding to the acute, latent and chronic stages of epileptogenesis. Assessment of hippocampal bioenergetics demonstrated a ≥ 27% decrease in the ATP/ADP ratio at all phases of epileptogenesis (p < 0.05), whereas cellular NAD+ levels were decreased by ≥ 41% in the acute and latent time points (p < 0.05), but not in chronically epileptic rats. In spontaneously epileptic rats, we found decreased protein expression of SIRT3 and a 60% increase in global mitochondrial acetylation, as well as enhanced acetylation of the known SIRT3 substrates MnSOD, Ndufa9 of Complex I and IDH2 (all p < 0.05), suggesting SIRT3 dysfunction in chronic epilepsy. Mass spectrometry-based acetylomics investigation of hippocampal mitochondria demonstrated a 79% increase in unique acetylated proteins from rats in the chronic phase vs. controls. Pathway analysis identified numerous mitochondrial bioenergetic pathways affected by mitochondrial acetylation. These results suggest SIRT3 dysfunction and aberrant protein acetylation may contribute to mitochondrial dysfunction in chronic epilepsy.

Keywords: Acetylation; Epilepsy; Mass spectrometry; Mitochondria; Proteomics; SIRT3.

Figure 1

Impaired hippocampal bioenergetics during epileptogenesis. (A) ATP/ADP ratio and (B) nicotinamide adenine dinucleotide (NAD+) in the hippocampus during epileptogenesis. Values are mean ± SEM (n = 3-18 per group). *, p < 0.05 vs Control.

Figure 2

Reduced SIRT3 protein expression and activity, as assessed by global levels of mitochondrial acetylation, in hippocampus at chronic time point of epileptogenesis. Hippocampal (A) SIRT3 protein expression and (B) global levels of acetylated proteins in the mitochondria during epileptogenesis. SIRT3 expression normalized to same loading control for both Control and Kainic acid-treated at each time point (acute: GAPDH, latent: GAPDH, chronic: actin). Data are normalized to Control mean value. Representative blot images below. Values are mean ± SEM (n = 3-14 per group). *, p < 0.05 vs Control.

Figure 3

Decreased Nampt protein expression during acute and chronic phases and no change in SIRT1 protein expression at all time points of epileptogenesis in hippocampus. Hippocampal nuclear-cytoplasmic (A) Nampt and (B) SIRT1 protein expression during epileptogenesis. Nampt expression normalized to total protein load for both Control and Kainic acid (KA)-treated at all time points. SIRT1 expression normalized to same loading control for both Control and KA-treated at each time point (acute: GAPDH, latent: GAPDH, chronic: actin). Data are normalized to Control mean value. Representative blot images below. Values are mean ± SEM (n = 3-14 per group). *, p < 0.05 vs Control.

Figure 4

Increased hippocampal acetylation of SIRT3 protein substrates in chronic time point. Enhanced levels of (A) acetylated manganese superoxide dismutase (Ac-MnSOD) lysine 122 (K122), (B) acetylated NADH Dehydrogenase (Ubiquinone) 1 Alpha Subcomplex 9 (Ac-Ndufa9) to total Ndufa9 and (C) acetylated isocitrate dehydrogenase 2 (Ac-IDH2) in hippocampus of spontaneously epileptic rats at chronic time point. Ac-MnSOD and ac-IDH2 expression normalized to total protein load for both Control and Kainic acid (KA)-treated. Ndufa9 expression normalized to VDAC for both Control and KA-treated. Data are normalized to Control mean value. Representative blot images below. Values are mean ± SEM (n = 3-11 per group). *, p < 0.05 vs Control.

Figure 5

Mitochondrial acetylomics analysis in a model of KA induced epileptogenesis. Enriched mitochondrial samples were subjected to acetyl-immunopurification followed by nHPLC-MS/MS analysis. A significant number of unique features were identified in the KA samples. In total, 333 proteins were identified and include 227 which overlap between the samples, while 38 were unique to control samples alone and 68 were unique to KA treatment. This analysis identified 490 and 543 acetyl peptides in control and KA samples out of 1179 and 1270 total peptides, respectively.