CART Peptide Is a Potential Endogenous Antioxidant and Preferentially Localized in Mitochondria

Reddy PH (2012) CART Peptide Is a Potential Endogenous Antioxidant and Preferentially Localized in Mitochondria. PLoS ONE 7(1): e29343. doi:10.1371/journal.pone.0029343 CART Peptide Is a Potential Endogenous Antioxidant and Preferentially Localized in Mitochondria Peizhong Mao 0 Charles K. Meshul 0 Philippe Thuillier 0 Natalie R. S. Goldberg 0 P. Hemachandra 0 Reddy 0 Stephen D. Ginsberg, Nathan Kline Institute and New York University School of Medicine, United States of America 0 1 The Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University , Beaverton, Oregon , United States of America, 2 The Department of Public Health & Preventive Medicine, and the Knight Cancer Institute, Oregon Health & Science University , Portland, Oregon , United States of America, 3 The Research Services, Portland VA Medical Center , Portland, Oregon , United States of America, 4 Departments of Behavioral Neuroscience and Pathology, Oregon Health & Science University , Portland, Oregon , United States of America, 5 The Department of Physiology and Pharmacology, Oregon Health & Science University , Portland, Oregon , United States of America The multifunctional neuropeptide Cocaine and Amphetamine Regulated Transcript (CART) is secreted from hypothalamus, pituitary, adrenal gland and pancreas. It also can be found in circulatory system. This feature suggests a general role for CART in different cells. In the present study, we demonstrate that CART protects mitochondrial DNA (mtDNA), cellular proteins and lipids against the oxidative action of hydrogen peroxide, a widely used oxidant. Using cis-parinaric acid as a sensitive reporting probe for peroxidation in membranes, and a lipid-soluble azo initiator of peroxyl radicals, 2,29-Azobis(2,4dimethylvaleronitrile) we found that CART is an antioxidant. Furthermore, we found that CART localized to mitochondria in cultured cells and mouse brain neuronal cells. More importantly, pretreatment with CART by systemic injection protects against a mouse oxidative stress model, which mimics the main features of Parkinson's disease. Given the unique molecular structure and biological features of CART, we conclude that CART is an antioxidant peptide (or antioxidant hormone). We further propose that it may have strong therapeutic properties for human diseases in which oxidative stress is strongly involved such as Parkinson's disease. - Funding: These studies were supported by the American Heart Association (Beginning Grant-in-Aid 0565527Z) to PM, the Department of Veterns Affairs Merit Review Program to CK, and the National Institutes of Health to PT (CA112083-01) and to PHR (AG028072, AG026051), and to ONPRC (RR000163). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The neuroendocrine system plays a primary role in the regulation of major physiological processes such as development, growth, metabolism, reproduction, and adaptation to the environment via peptide and steroid hormones, neurotransmitters and their receptors [14]. The neuropeptide, cocaine- and amphetamineregulated transcript (CART) gene was first cloned in 1995 [5]. CART has been implicated in a variety of physiological processes, including food intake, reward and endocrine regulations [6,7]. CART knockout (KO) mice have been found to display some agingassociated symptoms, including increased body weight. In humans, a mutation in the CART gene has been found in an obese family [8,9]. Interestingly, a recent report showed that CART levels within cerebrospinal fluid were significantly reduced by 30% in DLB (dementia with Lewy bodies) patients compared to normal controls as well as to Alzheimers disease patients [10]. The presence of Lewy bodies is considered to be the neuropathologic hallmark of Parkinsons disease (PD), one of the most common neurodegenerative diseases caused by degeneration of dopaminergic neurons in substantia nigra (SN) [11]. In the center nervous system CART is widely expressed in different tissues and regions, including cortex, midbrain and spinal cord [1214]. Increasing studies have shown putative anatomical and functional network between the CART-containing neurons and the mesolimbic dopaminergic system [9,1518]. The network connecting the SN, nucleus accumbens (NA), and dorsal striatum as well as ventral tegmental area (VTA) is mainly involved in dopaminergic motor pathways, suggesting that CART has the capacity to modulate mesolimbic dopamine, which could have implications for the treatment not only of psychostimulant abuse but also for the treatment of other disorders with midbrain dopamine involvement, such as PD. Furthermore, CART is highly expressed in the mammalian and human hypothalamus, pituitary and adrenal gland (HPA axis), as well as in the circulatory system [7,12,19] (Figure S1). This feature indicates a possible role for CART in stress and natural homeostasis, including the cellular defense system. Interestingly CART expression is regulated by stress in a regionally and time specific manner, and CART is also regulated by corticosteroid actions in the brain, including the hippocampus [9,20]. Although much is known about CART in terms of its structure, expression and function, little is known about CART receptors and its other interaction proteins, which has hampered basic studies and clinical investigations [7,21]. Recently using yeast two-hybrid screening, for the first time we identified the mitochondrial protein succinate dehydrogenase, subunit B (SDHB) as the first CART binding partner [22]. SDHB is a critical enzyme for both the Krebs cycle and the mitochondrial electron transport chain (ETC), where it is known as complex II. Furthermore, we found that CART could stimulate the activities of SDH and complex II under basal condition and ischemic condition (oxygen-glucose-deprivation, OGD) and preserve ATP levels after OGD in rat primary neurons [22]. Notably a recent report showed that only one SDHB gene mutation caused severe clinical phenotypes in a young patient including multiple system disorders, such as paraganglioma, type 2 diabetes, ischemic stroke, renal failure and congestive heart failure [23]. This further highlights the potential importance of SDHB and CART in pathways compromised in inherited genetic diseases. In addition, a significant common pathological feature of these disorders is oxidative damage. However whether CART has an antioxidant activity is unknown. Thus, in this report we investigated the hypothesis that CART plays a protective role in the oxidative stress using in vitro and in vivo systems. Interestingly, our data demonstrate that CART peptide is a strong antioxidant and it may be a potential therapeutic candidate for Parkinsons disease. TAT-EGFP-CART protein is an effective CART molecule In order to increase CA (...truncated)