The role of mitochondrial NAD+-dependent SIRT3 deacetylase during chronic pain

Millions of people worldwide suffer from chronic pain as a result of damage to or dysfunction of the nervous system under various disease conditions. Neuropathic pain is a debilitating form of treatment-resistant chronic pain and responds poorly to the clinically available therapies. Neuropathic pain is characterized by symptoms including hyperalgesia, allodynia, paresthesia and spontaneous pain. Symptoms of neuropathic pain can be attributed to a variety of alterations in pain-related gene expression and modification in primary afferent or spinal cord neurons. Recent studies from animal models have led to understanding of its pathobiology which includes complex interrelated pathways leading to peripheral and central neuronal sensitization. Considerable evidence implicates superoxide (SO) and peroxynitrite (PN) in the development of chronic pain, the transition of acute to chronic pain, as well as opiate-induced hyperalgesia and antinociceptive tolerance. PN seems to be a key toxic mediator for target-based therapeutic strategies. Numerous studies demonstrate that pharmacologic inhibition of SO and PN can prevent and reverse the characteristic pathologies associated with inflammatory pain, neuropathic pain, and morphine-induced hyperalgesia and tolerance. Alterations in glutamatergic neurotransmission and neuroinflammation, as well as modulation of ion channels underlie the development of central sensitization (a putative pathophysiologic state underlying persistent and chronic pain) associated with acute and chronic inflammatory and noninflammatory neuropathic pain. Glutamate-mediated activation of the NMDA receptor is fundamental in acute hyperalgesia, chronic neuropathic pain states and opiate tolerance. Overt activation of this receptor leads to free radicals formation linked to several diseases associated with glutamate-mediated neurotoxicity. More recently mitochondrial dysfunction has been postulated to represent an important component of neuropathic pain states. Reducing the accompanying mitochondrial SO levels lessens hyperalgesia. These results indicates that downstream mitochondrial Ca+ uptake and consequent production of free radicals is necessary for synaptic plasticity underlying chronic pain. Furthermore, this enhanced ROS production alters the mitochondrial genome and proteome through a number of mechanisms, including the accumulation of the lipid peroxidation product 4-hydroxynonenal(4-HNE) in pathologic quantities near lipid-rich cellular membranes. The cellular impact of 4-HNE generation is not fully understood, however, at basal levels, it has been implicated in intracellular signalling, and in excess, it is know to interfere with proteomic activity via Michael type and Schiff base adduct formation with Cys amino acid residues of proteins. Cys-rich regions are present in plasma membrane receptors and ion channels, including the NMDA receptors. Thiol-containing proteins that function in redox signaling and physiological regulation are susceptible to two-electron oxidation by lipid hydroperoxides, aldehydes, quinones and disulfides. Aberrant generation of nonradical oxidants at rates comparable to normal oxidation may be sufficient to disrupt function. Sirtuins, the class III histone deacetylases (HDACs), are widely distributed (there are seven mammalian enzymes SIRT1-SIRT7) and have been shown to regulate a variety of physiopathological processes, such as inflammation, cellular senescence, differentiation, metabolism and cell cycle regulation. Sirtuins catalize the deacetylation of the -amino group of lysine residues of histones and non-histone proteins and are involved in regulating transcriptional activity and protein function such as nuclear factor (NF)-kB, p53, peroxisome proliferator-activated receptor(PPAR)-and endothelial nitric oxide synthase. Only three mitochondrial sirtuins have been identified, SIRT3-5. On the basis of knockout studies, SIRT3 was determined to be the major mitochondrial protein deacetylase. Targets of SIRT3 deacetylation are widespread and are implicated in a host of cellular pathways, including oxidative phosphorylation, fatty acid metabolism, oxidative stress response, and alcohol metabolism. It is therefore likely that alterations in protein acetylation due to metabolic and oxidative stress may negatively impact these pathways, disrupting normal mitochondrial processes. Given the sequence homology among the sirtuin family of proteins, it is likely that SIRT3 is also susceptible to protein carbonylation as it has been shown in a model of alcoholic liver disease. Naturally occurring dietary polyphenols, such as resveratrol, curcumin, quercetin and catechins have antioxidant and anti-inflammatory properties and have also been shown to activate sirtuins directly or indirectly in a variety of models. Therefore, activation of SIRT3 by polyphenols could be beneficial in therapeutic intervention of chronic pain and for the modulation of hyperalgesia obsersed during opioid tolerance. A better understanding of molecular and cellular mechanisms underlying the pathogenesis of the “pain state disease” is crucial for the development of effective therapeutic strategies to treat chronic neuropathic pain and to maintain opiate efficacy during chronic dosing without engendering tolerance or unacceptable side effects. In this study, we investigate the involvement of SIRT3 modulation by free radicals products in preventing experimental neuropathic pain induced by the loose ligation of the sciatic nerve of the rat (CCI). We, also, evaluate the role of SIRT3 modulation during the opioid induced hyperalgesic response and the protective effects of a natural antioxidant, the bergamot polyphenolic fraction (BPF) in both experimental model of chronic pain. In rats, CCI induced a thermal and mechanical hyperalgesia that last up to 21 days post injury. These events were significantly reduced by chronic administration of a natural antioxidant, BPF, compared with the well-known, clinically used, pregabalin. Furthermore, the development of CCI induced pain seems to be correlated with a significant malonyldialdheide (MDA) and hydroxynonenal (HNE, a lipid peroxidation product) accumulation which resulted in mitochondrial proteins carbonylation of SIRT3 lysine residues; consequently, SIRT3 carbonylation led to inactivation of its biological functions. BPF pre-administration was able to reduce the CCI-induced HNE production and SIRT3 carbonylation restoring SIRT3 biological activity. These findings demonstrate, for the first time, that deactivation of sirtuins is involved in neuropathic pain and that activation of SIRT3 by antioxidants is beneficial during oxidative stress induced hyperalgesia and allodynia. In particular, we have demonstrated a close relation between oxidative stress, hyperalgesia, allodynia and sirtuins inactivation and that sirtuins protection by a natural antioxidant, BPF, may represent a new target in therapeutic intervention for the management of pain suffering patients. In addition to these data, we aim to prove the involvement of SIRT3 modulation by free radicals in an other model of pain where the role of ROS have been well studied as the chronic morphine induced hyperlagesia and tolerance. When morphine has been administred for over 5 days loose its therapeutic efficacy and we could measured the paradoxic hyperalgesic response in the treated animals. Also in this experimental condition, the BPF was able to reinstate the therapeutic efficacy of opioid reducing the concomitant ROS formation. Firthermore, we show that in this condition SIRT3 undergo nitration of the tyr residues and looses its capability of deacetylate the mithochondria. The results of our study will support the hypothesis that free radicals post-translational modulation of key proteins involved in the regulation of protein acetylation is an important pathway used by superoxide/peroxynitrite to mediate the development and maintenance of chronic pain. Prevention of post-translational modulation such as nitration and HNE-adduct formation and hyperalgesic response by antioxidants indicates the importance of the development of new therapeutical approach. All together, the generated evidence will support the redox basis of chronic pain hypothesis. There is considerable need to elucidate the nitration and the thiol redox signaling and identify critical sites where interventions could protect against disruption of function and/or restore normal signaling. The newly identified potential pathway will be crucial for developing new therapeutic strategies for prevention and treatment of pain instaurance with reduction of socioeconomic costs and amelioration of the patients quality of life. Keywords: Chronic Neuropathic pain, CCI, Sirtuins, SIRT3, Oxidative stress, Antioxidants, Bergamot Polyphenolic Fraction (BPF).