Peripheral blood lymphocytes from patients with ...

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Paraskevi Moutsatsou a, James N. Tsoporis a,* , Vasileios Salpeas , Ekaterini Bei , Basel ...... Donato R, Sorci G, Riuzzi F, Arcuri C, Bianchi R, Brozzi F, et al.
DOI 10.1515/cclm-2013-0978      Clin Chem Lab Med 2014; aop

Paraskevi Moutsatsoua, James N. Tsoporisa,*, Vasileios Salpeas, Ekaterini Bei, Basel Alevizos, Chrysoula Anagnostara, Shehla Izhar, Gerald Proteau, Emmanouil Rizos, Erifili Hatziagelaki, Ioannis K. Toumpoulis, Ioannis K. Rizos and Thomas G. Parker

Peripheral blood lymphocytes from patients with bipolar disorder demonstrate apoptosis and differential regulation of advanced glycation end products and S100B Abstract Background: This study addresses the expression of the glycosylated proteins known as advanced glycation end products (AGEs), the calcium binding protein S100B and the apoptotic parameters cytochome c and caspase-3 activity in peripheral lymphocyte cytosolic extracts from a sample of bipolar disorder (BD) patients and healthy (control) subjects. Methods: Cross-sectional study of 35 patients with a clinical diagnosis of bipolar disease (10 euthymic, 12 depressed, 13 manic) and 10 healthy control subjects. Lymphocytes were used as a surrogate model in BD diagnosis and treatment. AGEs and S100B in lymphocyte cell extracts were measured by commercially available enzyme-linked immunosorbent assay. Results: AGEs were lower in all BD patients compared to healthy subjects. Depressed patients had approximately two-fold higher S100B levels compared to healthy subjects. Manic and depressed BD patients had increased superoxide dismutase mRNA levels. Apoptosis as measured by BAX/Bcl2 ratio, cytochrome c release, caspase-3 activity was increased in manic and depressed patients compared to healthy subjects. In the depressed patients, S100B levels correlated with cytochrome c release. Conclusions: In conclusion, our study shows decreased AGEs and increased S100B levels and caspase downstream apoptosis in peripheral lymphocytes of BD patients that may underlie disease etiopathogenesis. Keywords: advanced glycation end products; apoptosis; bipolar disorder; lymphocytes; S100B.

a

Paraskevi Moutsatsou and James N. Tsoporis contributed equally to this work.

*Corresponding author: James N. Tsoporis, Division of Cardiology, Department of Medicine, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Toronto, Ontario, Canada, Phone: +1 416-864-6060X77625, Fax: +1 416-864-6034, E-mail: [email protected] Paraskevi Moutsatsou and Ekaterini Bei: Laboratory of Biological Chemistry, Medical School, University of Athens, Athens, Greece Vasileios Salpeas: Laboratory of Biological Chemistry, Medical School, University of Athens, Athens, Greece; and 2nd Academic Department of Cardiology, Attikon University Hospital, University of Athens Medical School, Athens, Greece Basel Alevizos and Chrysoula Anagnostara: Department of Psychiatry, Medical School, Attikon University Hospital, University of Athens, Athens, Greece Shehla Izhar, Gerald Proteau and Thomas G. Parker: Division of Cardiology, Department of Medicine, Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, University of Toronto, Toronto, Ontario, Canada Emmanouil Rizos: Department of Psychiatry, Medical School, Eginition Hospital, University of Athens, Athens, Greece Erifili Hatziagelaki: 2nd Department of Internal Medicine-Propaedeutic, Research Institute and Diabetes Centre, Attikon University Hospital, University of Athens Medical School, Athens, Greece Ioannis K. Toumpoulis: Department of Cardiac Surgery, Attikon University Hospital, University of Athens, Athens, Greece Ioannis K. Rizos: 2nd Academic Department of Cardiology, Attikon University Hospital, University of Athens Medical School, Athens, Greece

Introduction Bipolar disorder (BD), a severe neuropsychiatric disease [1], has been associated with increased oxidative stress. Reactive oxygen species are elevated in the brain and in lymphocytes of BD patients [2, 3]. BD patients are characterized by alterations in brain glucose metabolism [4] and energy metabolism disturbances including elevations in lactate and a shift in energy redox state from oxidative

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2      Moutsatsou et al.: Peripheral blood lymphocytes from patients with bipolar disorder demonstrate apoptosis

phosphorylation towards glycolysis [5, 6]. In BD, accelerated oxidative stress and deregulation of glucose metabolism may cause cells to accumulate the receptor for advanced glycation end product (RAGE) ligands AGEs and the calcium binding protein S100B. AGEs are pathologically modified proteins formed by non-enzymatic glycation and oxidation of proteins and/or lipids, and contribute to protein misfolding and aggregation normally during aging, and at an accelerated rate in diabetes [7]. AGEs have been identified in a variety cells, including lymphocytes, and mediate multiple pathogenic effects including apoptosis intracellularly, and extracellularly in part through interaction with RAGE [8]. S100B is expressed in astrocytes, oligodendrocytes and schwann cells and by interacting with intracellular target proteins, indirectly controls growth, apoptosis, and morphology of cells [9, 10]. S100B is also secreted from cells and interacts with RAGE resulting in concentration-dependent trophic or apoptotic responses. In brain, S100B stimulates neurite outgrowth and apoptosis at nanomolar and micromolar concentrations, respectively [9]. The interaction between S100B and RAGE expressed on neurons induces the expression of the receptor itself and proinflammatory cytokines implicated in the psychopathology of various psychiatric diseases including depression and schizophrenia [11, 12]. Recent studies have reported elevated serum levels of S100B in patients with schizophrenia from activated, damaged or dysfunctional astrocytes from these patients [11–13]. Polymorphisms in RAGE have also been linked with schizophrenia [14, 15]. Interestingly, the secreted form of RAGE, sRAGE that lacks the transmembrane domain, and competes with cell-bound RAGE by binding the same ligands, has been reported to regulate the detrimental effects of S100B observed in patients with schizophrenia [13]. Although astrocyte S100B release represents the major source of elevated serum S100B levels [16], extracranial sources [17, 18] such as lymphocytes cannot be ruled out [18, 19]. S100B is localized in the cytoplasm of lymphocytes [18, 20] and comprise 2%–4% of the lymphocyte population displaying a CD3+CD8+ T cell phenotype [18]. Lymphocytes also secrete S100B, suggesting crosstalk between adaptive and innate immune systems mediated by S100B [18]. Lymphocyte S100B and AGEs have not been addressed in BD. The expression of these RAGE ligands in peripheral lymphocytes may be important with regard to the study of its mechanism of action in BD and may have a predictive value for treatment response. A number of studies have reported altered apoptotic factors and their mediated responses in BD. Changes include DNA damage in peripheral blood [21], increased

pro-apoptotic serum activity [22], mitochondrial dysfunction [23] and altered apoptotic factors in postmortem brain of BD patients [24]. These studies implicate the involvement of cell death in the pathophysiology of BD. A limitation of most of these studies is non-availability of medical diagnosis, and lack of information on whether the patients were in the manic or depressive phase at the time of death. The present study was designed to evaluate the expression of AGEs and S100B in conjunction with markers of apoptosis (caspase 3 activity, cytochrome c release and the apoptotic gene ratio of proapototic BAX/antiapototic Bcl2) and markers of oxidative stress (transcript levels of the antioxidant enzymes superoxide dismutase (SOD), catalase and HSP70) in lymphocytes of control subjects and BD patients categorized into euthymic, manic and depressed. As in our previous studies [25, 26], to define intracellular signaling pathways that may play a role in the pathophysiology of BD, we used lymphocytes as our model system, since alterations in BD described at the peripheral blood cell level may reflect the altered state of the central nervous system [27, 28].

Materials and methods Patient characteristics The work described in this article has been carried out in accordance with the code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The study received ethical approval from the Medical Faculty Ethics Committee and written informed consent was obtained from all research participants. We designed a cross-sectional study of lymphocyte AGEs and S100B in a clinical series of different bipolar disorders. The population included 10 healthy (control) subjects (4 males, 6 females, mean age ± SEM, 41.8 ± 3.6 years) and 35 bipolar patients (14 males, 21 females, 45.6 ± 4.0 years) (Table 1). The age of control subjects ranged from 24 to 55 years and the age of bipolar patients ranged from 21 to 69 years. Patients which met diagnostic inclusion criteria for bipolar disorder I or II were assessed based upon the consensus of more than two psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders IV (DSM-IV) [29], 21-item Hamilton Depression Rating Scale [30] or the Young Mania Rating Scale [31]. Depressive disorders due to medical or neurological conditions were exclusion criteria. Patients with co-morbid psychiatric disorders were also excluded. Bipolar patients on stable psychopharmacological treatment regimen for up to 6 weeks were recruited from outpatients of the Department of Psychiatry. The patients were on combined therapy with a mood stabilizer, an antidepressant and an antipsychotic agent (Table 1). Ten control subjects were recruited from the University staff and current medical problems and positive history of psychiatric and neurological disorders were exclusion criteria. Additionally, the exclusion criteria included history of current substance abuse or dependence in the preceding 6 months as defined by DSM-IV.

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Moutsatsou et al.: Peripheral blood lymphocytes from patients with bipolar disorder demonstrate apoptosis      3

Table 1 Demographic and clinical data of healthy subjects and patients with bipolar disorder.  

n   n (BDI/BDII)   Mean age, years   Male Gender, n (%)   Current smokers   HDRS (score-range)   YMRS (score-range)   Drug treatment AD, %   Aps, %   MS, %  

Healthy  subjects 

Bipolar subjects Euthymic 

Depressed 

Manic 

p-Value

10    41.8 ± 3.6  4 (40)  5 (50)     

10  9/1  47.7 ± 4.8  3 (30)  4 (40)  < 7  < 6 

12  10/2  45.7 ± 3.5  5 (41.7)  5 (41.7)  > 17–34   

13  13  43.3 ± 3.8  6 (46.2)  5 (38.5)    > 17–31 

0.878 0.568 0.951

     

50  70  100 

75  50  67 

0  92  62 

Includes subjects with BD and healthy donors. AD, antidepressants; AP, antipsychotics; BD, bipolar disorder; BDI, bipolar disorder type I; BDII, bipolar disorder type II; HDRS, Hamilton Depression Rating Scale; MS, mood stabilizers; YMRS, Young Mania Rating Scale. Bipolar patients: 1 drug free (depressed). Neither patients nor controls had alimentary restrictions or evidence of clinical malnutrition. The healthy control group was free of any medication at least 1 month prior to blood sampling. Current smoking was defined as smoking on more than one of the 30  days preceding blood sampling. The study received ethical approval from the Medical Faculty ethics committee.

Isolation of lymphocytes and subcellular fractionation Peripheral blood mononuclear cells were collected by venipuncture and lympocytes were isolated by centrifugation over a Ficoll-Paque Plus gradient, according to the manufacturer’s instructions (Amersham Biosciences, Pittsburgh, PA, USA). Cytosolic extracts were prepared from lymphocytes as described [32].

Determination of AGEs and S100B Ten micrograms of protein of lymphocyte cytosolic extract was used to perform each enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions [AGEs (Cell Biolabs, San Diego, CA, USA), S100B (ABNOVA, Taiwan), and RAGE (R&D Systems, Minneapolis MN, USA)]. The samples were analyzed blinded to the clinical status of the participants. The lowest level of S100B and RAGE that can be detected by this assay is 2.7 and 4.12 pg/mL, respectively, using a 50 μL sample size. The total AGEs detected by the kit included carboxy(methyl)lysine, pentosidine and other AGE structures. The lowest level of AGE that can be detected is 0.25 μg/mL. The intra- and inter-assay coefficients of variation values were