SIRT3 deacetylated and increased citrate synthase activity in PD model

https://doi.org/10.1016/j.bbrc.2017.01.163Get rights and content

Abstract

SIRT3 have been found to be neuroprotective in many neurological diseases, but its detail mechanism is only partially understood. In this study, MPP+ was used to treat SH-SY5Y cells as the cellular model of PD to test the role of SIRT3 and the mechanism may be involved in. We focused on the changes and relationship between SIRT3 and the key mitochondrial enzymes citrate synthase (CS) and isocitrate dehydrogenase 2 (IDH2). We found MPP+ decreased SIRT3 expression. And our results showed that the enzymatic activities of CS and IDH2 were significantly reduced in MPP+ treatment cells, while protein acetylation of CS and IDH2 increased. However overexpressed-SIRT3 partially reversed at least, the decline of CS activity and the increase of CS protein acetylation. IDH2 did not showed the same changes. The study suggested that SIRT3 deacetylated and activated CS activity. Hence, we conclude that SIRT3 exhibits neuroprotection via deacetylating and increasing mitochondrial enzyme activities.

Introduction

Parkinson's disease (PD) is the second most common neurodegenerative disorder, as well as the most common movement disorder associated with the elderly [1]. PD, affects more than 1% of the population over the age of 65 years and more than 4% of the population by the age of 85 years [2]. So far there are no effective treatments or preventive therapies [3]. Therefore, it's of great importance to study the underlying mechanisms of dopaminergic (DA) neuronal degeneration and develop the relevant neuroprotective/therapeutic strategies. There are various factors involved in PD pathogenesis, of which mitochondrial dysfunction is the most remarkable one. It is generally accepted that DA neurons turn sensitive to mitochondrial dysfunction particularly in energy status. Neuronal damage has been closely connected with the reduction of intracellular adenosine triphosphate (ATP) level in the brain of PD patients, and in models of PD [4], [5]. Given that mitochondrial-based oxidative damage is a major postulated mechanism for PD pathogenesis [6], many investigators are focusing on mitochondrial enzymes.

Sirtuins, NAD+-dependent protein deacetylases, participates in systemic metabolic regulation, of which SIRT3, SIRT4, and SIRT5 are mitochondrial localized [3]. As the most principal one, SIRT3 is expected to participate in PD progression [3], but how it works still has been unknown. Recent findings suggest that SIRT3 deacetylates and activates mitochondrial enzymes, of which involved in fatty acid β-oxidation, amino acid metabolism, the electron transport chain, and antioxidant defenses [7].

Accumulating evidences have suggested that protein acetylation, as a major post-translational modification, is recognized as a major regulatory mechanism in mitochondrial function and neuronal health [8]. Our previous study found that MPP+ treatment decreased the expression of SIRT1 of DA neuron, which weakened the deacetylation to H3K14 and strengthened the combination of H3K14 with HIF1α promoter, and then increased the transcription activity of HIF1α [9].

Citrate synthase (CS), isocitrate dehydrogenase 2 (IDH2) and pyruvatede hydrogenase (PDH) are the gatekeeper enzymes of the citric acid cycle (TCA), which occur in the mitochondrial matrix and generate high-energy product for oxidative phosphorylation. Impaired CS activity could thus impact mitochondrial function and exacerbate age-related hearing loss [10]. Johnson et al. found that a missense mutation of CS is responsible for the ahl4-related hearing loss of A/J strain mice [10]. Other researchers found that PDH α1 subunit can be deacetylated by SIRT3 in vitro and in vivo, altering the activity of PDH as well as tumor cell metabolism [11]. Whether CS, IDH2 and PDH are participated in PD or associate with SIRT3 are unclear. The aim of this study is to examine the effects of SIRT3 on PD pathogenesis and uncover the molecular mechanisms involved.

Section snippets

Cell culture and treatments

SH-SY5Y cells were maintained in Dulbecco's modified eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (GIBCO, Gaithersburg, MD, USA) and grown in a CO2 incubator maintained at atmospheric oxygen levels and 5% CO2. MPP+ (Sigma–Aldrich, St. Louis, MO, USA) stocks were dissolved in phosphate buffered saline (PBS) at a stock concentration at 125 mM which was stored at −20 °C. MPP+ was further diluted in serum free DMEM to achieve the final concentrations.

CCK8 cell viability assay

Cell viability was

MPP+ stimulated the proliferation inhibition, apoptosis, cell cycle arresting and mitochondrial dysfunction

MPP+ has been vastly used in all sorts of cell damage and PD cell models. In this study, to evaluate the neurotoxicity of MPP+ in SH-SY5Y cells, we examined the cell inhibition rate by CCK-8 method. We treated cells with MPP+ at various concentrations (0, 0.5, 1, 2.5, 5 mM)for 24 h. Compared to the control group, cells treated with MPP+ at a concentration of 2.5 mM achieved an approximate 50% increase in cell inhibition rate. So, the most suitable concentration is 2.5 mM. We used the indicated

Discussion

Sirtuins, including SIRT1-7, widely exists in various organisms from bacteria to humans [12]. SIRT1 is located in the nucleus, it regulates adaptive changes in gene expression in response to bioenergetic challenges [13]. While SIRT3 are mitochondrial localized, where it plays a critical role in regulating mitochondrial metabolic pathways [13]. Mitochondria are energy supply station of cells and contain many metabolic pathways. So the regulation of SIRT3 will affect not only the function of

Conflict of interest statement

The authors report no conflicts of interest with this study.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China [grant numbers 81371410, 81171205], and the Biomedical Multidisciplinary Program of Shanghai Jiao Tong University [grant number YG2014MS31 to Yun-Cheng Wu], This work was supported by grant from Shanghai Natural Science Foundation (No. 16ZR1434000) to Te Liu.

References (25)

Cited by (54)

  • Acetylation of citrate synthase inhibits Bombyx mori nucleopolyhedrovirus propagation by affecting energy metabolism

    2022, Microbial Pathogenesis
    Citation Excerpt :

    In Escherichia coli, multiple lysine sites were found to be acetylated in CS, and the acetylation of lysine 295 significantly reduced the enzyme activity [18]. In mammalian cells, the acetylation of many mitochondrial metabolic enzymes is regulated by the deacetylase Sirt3 [19], and overexpression of Sirt3 can significantly down-regulate the acetylation level of CS, as well as partially up-regulate the catalytic activity [20]. These data suggest that acetylation of CS is essential for enzyme activity.

  • Mitochondrial sirtuins, metabolism, and aging

    2022, Journal of Genetics and Genomics
    Citation Excerpt :

    Importantly, Sirt3 expression changes during the course of Alzheimer's disease in humans (Weir et al., 2012; Yin et al., 2018). In addition, SIRT3 is proposed to function in a protective manner in Parkinson's disease (PD) pathogenesis (Cui et al., 2017; Shi et al., 2017). However, more recent work proposes that SIRT3 can play an unappreciated deleterious role in disease development.

View all citing articles on Scopus
View full text