Nitric Oxide Dysregulation in Platelets from Patients with Advanced Huntington Disease

et al. (2014) Nitric Oxide Dysregulation in Platelets from Patients with Advanced Huntington Disease. PLoS ONE 9(2): e89745. doi:10.1371/journal.pone.0089745 Nitric Oxide Dysregulation in Platelets from Patients with Advanced Huntington Disease Albino Carrizzo 0 Alba Di Pardo 0 Vittorio Maglione 0 Antonio Damato 0 Enrico Amico 0 Luigi Formisano 0 Carmine Vecchione 0 Ferdinando Squitieri 0 Cristoforo Scavone, Universidade de Sao Paulo, Brazil 0 1 IRCCS Neuromed , Pozzilli (IS) , Italy , 2 Department of Science and Technology, University of Sannio , Benevento , Italy , 3 Department of Medicine and Surgery, University of Salerno , Salerno , Italy Nitric oxide (NO) is a biologically active inorganic molecule involved in the regulation of many physiological processes, such as control of blood flow, platelet adhesion, endocrine function, neurotransmission and neuromodulation. In the present study, for the first time, we investigated the modulation of NO signaling in platelets of HD patients. We recruited 55 patients with manifest HD and 28 gender- and age-matched healthy controls. Our data demonstrated that NO-mediated vasorelaxation, when evoked by supernatant from insulin-stimulated HD platelets, gradually worsens along disease course. The defective vasorelaxation seems to stem from a faulty release of NO from platelets of HD patients and, it is associated with impairment of eNOS phosphorylation (Ser1177) and activity. This study provides important insights about NO metabolism in HD and raises the hypothesis that the decrease of NO in platelets of HD individuals could be a good tool for monitoring advanced stages of the disease. - . These authors contributed equally to this work. Huntington disease (HD), a dominantly transmitted neurodegenerative disorder, is characterized by the progressive striatal and cortical neurodegeneration associated with motor, cognitive and behavioral disturbances [1]. The disease-causing mutation is an expansion of a CAG trinucleotide repeat (.36 repeats) encoding a polyglutamine (polyQ) stretch in N-terminal region of huntingtin (Htt), a ubiquitous protein whose function is still unclear. Elongated polyQ stretch endows mutant Htt (mHtt) with toxic properties and results in the development of a broad array of cell dysfunctions [2]. Although the disease has traditionally been described as a disorder purely of the brain, emerging evidence indicates that abnormalities outside the central nervous system (CNS) are commonly found in HD [3]. However, whether a correlation exists between central pathology and peripheral defects is still poorly understood. Mutant Htt has been widely described to be expressed either in central or in peripheral tissues and to exert its toxic effect in both neuronal and non-neuronal cells with similar mechanisms [4]. Previous studies highlighted the ability of mHtt to interfere with a number of molecular mechanisms in peripheral tissues, which have been suggested to virtually reflect central dysfunctions in HD and potentially useful to better understand some genetic and biochemical aspects of the disease [5,6]. Among all the several dysfunctions, the dysregulation of nitric oxide (NO)/NO synthase (NOS) pathway is suggested to potentially represent a critical contributor to HD pathology [7]. NO, known as an important signaling molecule, normally acts in many tissues and regulates a diverse range of physiological and cellular processes such as control of blood flow, platelet adhesion, neurotrasmission and neuromodulation [8]. Production of NO is mediated by different NOS isoforms in both CNS and peripheral tissues [9]. Human platelets, that normally express the endothelial form of NOS (eNOS), whose activity is regulated by phosphorylation at different Serine/Threonine residues [10], represent an important source of peripheral NO. Under pathological condition, production of NO can be either protective or toxic, depending on the stage of the disease, the isoforms of NOS involved and, the initial pathological event [7]. To date, many are the studies that described NO as a potential key mediator of neurodegeneration [11,12]. NO dysfunction in CNS has been previously demonstrated to be involved in different processes leading to progressive striatal damage and to abnormal cerebral blood flow (CBF) in both HD experimental models and patients [7,13]. However, there is no actual evidence proving NO abnormalities in peripheral blood cells. In this study, we carried out experiments on platelets, which represent a validated peripheral model for testing potential impairment of NO regulation, obtained from patients with manifest HD. Our data highlighted defective NO metabolism in the advanced stages of HD and, for the first time, reveled a possible stage-dependent dysregulation of peripheral eNOS signaling in the disease. Subjects A total of 55 HD patients (11 stage I, 17 stage II, 16 stage III and 11 stage IV), and 28 gender- and age-matched healthy controls were recruited in this study. Control group was divided in two subgroups consisting of a younger control group with a mean age of 43.867.4 and an older control group with a mean age of 59.763.4 in order to match them with early (stage I and II) and late (stage III and IV) HD patients, respectively. Classification of control subjects in young and old groups has been performed in a manner similar to that described previously [14]. Subjects demographic, clinical and genetic characteristics of both controls and HD patients are reported in Table 1. All the subjects with suspect of cardiovascular, psychiatric or neurodegenerative disorders other than HD, were excluded from this study. Most patients were taking benzodiazepines; some of the patients in stage III-IV were receiving low doses of atypical neuroleptics (olanzapine, 2.510 mg; risperidone, 13mg or tetrabenazine, 12.5 25 mg). None of the patients were taking medication for any cardiovascular diseases. Clinical examinations were conducted using the Unified Huntingtons Disease Rating Scale (UHDRS) to measure motor, cognitive, behavioral and general function (Huntington Study Group, 1996) [15]. The disease stage was calculated according to the Total Functional Capacity (TFC) score [16]. Ethics Statement All HD patients revealed a CAG repeat expansion mutation, and all of them, as well as control individuals, were requested to sign an informed consent before study recruitment. All human experiments were performed in accordance with the Declaration of Helsinki and after approval from the local Ethical Committee of Istituto Neurologico Mediterraneo IRCCS Neuromed [17]. Platelet isolation Twenty-five millilitres of blood were collected into acid citrate dextrose (ACD: 85 mmol/L sodium citrate, 65 mmol/L citric acid, and 125 mmol/L dextrose; 2,5 ml ACD:25 ml of blood) and platelet-rich plasma was obtained by centrifugation at 130 g for 20 minutes. The resultant platelet-rich plasma (PRP) was used as source of platelets. P (...truncated)