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Author's personal copy Journal of Affective Disorders 151 (2013) 667–672
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Research report
Identification of altered dipeptidyl-peptidase activities as potential biomarkers for unipolar depression Jingti Deng a, Jonathan R. Lamb a, Astrid P. Mckeown a, Sam Miller a, Pierandrea Muglia b, Paul C. Guest c, Sabine Bahn c, Enrico H. Domenici b, Hassan Rahmoune a,c,n a
Clinical Pharmacology Unit, GlaxoSmithKline, Addenbrooke's Hospital, Cambridge CB2 2GG, UK Medicines Research Centre, GlaxoSmithKline, Via Fleming 4, 37134 Verona, Italy c Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, Cambridge University, Tennis Court Road, Cambridge CB2 1QT, UK b
art ic l e i nf o
a b s t r a c t
Article history: Received 4 December 2012 Accepted 23 July 2013 Available online 31 July 2013
Background: Changes in circulatory aminopeptidases [dipeptidyl-peptidase-IV (DPP-IV), Prolyl-oligopeptidase (POP) and Leucine aminopeptidase (LAP)] activities have been found to be associated with psychiatric illnesses and inflammatory diseases. Methods: The discriminatory indices of aminopeptidases activities were assessed by enzymatic assays in plasma samples from 240 unipolar depression (UD) patients and 264 matched controls. In addition the relationship between soluble and cellular DPP-IV activity was determined in plasma and blood cells from healthy subjects. Results: Greater than 95% of the plasma DPP-IV activity could be blocked by inhibitors, demonstrating the specificity of the assay. Also, DPP-IV protein and activity levels were strongly correlated. In contrast, only 50% of the membrane-bound activity in blood cells was inhibited, which suggested that other similar peptidases may be present in these cells. UD patients had decreased plasma levels of DPP-IV and POP activities compared to healthy controls with a concomitant increase in LAP activity. Finally, testing of the LAP/DPP-IV ratio resulted in good discrimination of UD patients from controls with an area under the curve—receiver operating characteristic of 0.70. Limitations: Further biological validation studies using different cohorts are warranted. Conclusions: The finding that plasma DPP-IV activity was decreased and LAP activity was increased in UD patients suggests the potential value for testing the levels of these enzymes for improved classification of patients. In addition, the changes in these enzymes, suggests that the proteolytic maturation of their proneuropeptide and prohormone subtrates may also be affected in UD, resulting in altered production of the associated bioactive peptides. Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
Keywords: Unipolar depression Biomarkers Plasma Peripheral blood mononuclear cells Aminopeptidase
1. Introduction The aminopeptidase family of proteases is involved in the proteolytic processing of precursor proteins to produce biologically active neuropeptides and hormones. These include dipeptidyl-peptidase (DPP)-IV (Boonacker and Van Noorden, 2003), prolyl-oligopeptidase (POP) (Garcia-Horsman et al., 2007) and leucine aminopeptidase (LAP) (Matsui et al., 2006). DPP-IV selectively removes amino-terminal dipeptides from precursor proteins containing proline or alanine in the second position
n Corresponding author at: Cambridge Centre for Neuropsychiatric Research, Department of Chemical Engineering and Biotechnology, Cambridge University, Tennis Court Road, Cambridge CB2 1QT, UK. Tel.: +44 1223 334 160; fax: +44 12 2333 4162. E-mail addresses:
[email protected],
[email protected] (H. Rahmoune).
(Lambeir et al., 2003) and is widely distributed (Mentlein, 1999). It is present as both membrane-bound and circulating forms with indistinguishable protease activity (Drucker, 2003). LAP catalyses removal of leucine residues from the amino-terminus of proteins although its biological role is still not well understood. However, many studies have indicated that it plays a role in proteolytic maturation of proproteins involved in various central and peripheral processes (Lambeir et al., 2003). POP removes amino-terminal peptides from small proteins by cleavage on the carboxy-terminal side of proline resides and is highly expressed in the brain (GarciaHorsman et al., 2007). It is also involved in the processing of bioactive peptides which are involved in regulation of mood and behaviour. The aminopeptidases have been linked to the pathophysiology of various diseases including neuropsychiatric disorders and metabolic conditions such as type II diabetes mellitus (Drucker,
0165-0327/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jad.2013.07.015
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J. Deng et al. / Journal of Affective Disorders 151 (2013) 667–672
2003). Although UD is a severe mental disease affecting primarily the brain, it is becoming more apparent that the whole body is involved (Brandt et al., 2007; Hildebrandt et al., 2000). Studies over the last two decades have shown that many patients with UD have inflammatory, hormonal and metabolic abnormalities, similar to those seen in cardiovascular diseases and diabetes (Hildebrandt et al., 2000). DPP-IV has been proposed as a diagnostic or prognostic marker for various cancers and neuropsychiatric disorders. For example, previous studies have found that changes in aminopeptidase activity may be involved in neuropsychiatric conditions such as unipolar depression (UD) (Maes et al., 1997). Also, DPP-IV is used currently as a therapeutic target for diabetes as a means of increasing insulin release from pancreatic β-cells (Ahren and Schmitz, 2004). DPP-IV is also known to be involved in immune system regulation through effects on T cell activation and chemotaxis (Ohnuma et al., 2008), and it appears to be negatively associated with inflammation (Busso et al., 2005; Tanaka et al., 1994). Such studies also indicate a link between inflammation and depressive symptoms. Lower circulatory peptidase activity has also been proposed as a predictor for depressive symptoms following interferon-alpha (IFN-α) immunotherapy (Maes and Bonaccorso, 2004). Also, treatment of high-risk melanoma patients with IFN-α resulted in a decrease of POP serum activity and this was associated with increased symptoms of depression (Van Gool et al., 2004). Neuropsychiatric conditions such as UD and anorexia nervosa have been associated with decreased serum DPP-IV levels (Hildebrandt et al., 1999). Conversely, serum POP activity has been found to be increased in bipolar disorder, mania and schizophrenia (Maes et al., 1994, 1995, 1996) and previous reports have also suggested its potential use as a prognostic (Tarrago et al., 2005) and/or diagnostic biomarker (Maes et al., 1996). This is interesting since POP catalyses the proteolytic production of hormones and neuropeptides (Polgar, 2002) that are known to be involved in regulation of mood disorders. In this study, we have analysed plasma from a large cohort of UD patients and matched controls in attempt to characterise changes in the activities of DPP-IV, LAP and POP activity. It was of particular importance to determine whether any differences in these enzymatic activities could be used as a means of classifying UD patients compared to controls.
2. Methods
Table 1 Study population demographic data. UD Subjects Gender: Female—number (%) Male—number (%) Age: mean (+/ SD) (years) BMI: mean (+/ SD) (kg/m2)
240 164 76 53.2 25.9
Control
(68.3) (31.7) (14.2) (4.0)
264 171 93 48.9 24.2
(64.8) (35.2) (14.1) (3.4)
Patients were included in the study if they were diagnosed with recurrent UD of moderate or severe intensity according to the Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV or the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10). Most of the UD subjects were euthymic at the time of sampling. Controls were screened for the presence of anxiety and mood disorders using the Composite International Diagnostic Screener (Wittchen et al., 1998) and healthy participants with mental disorders or co-morbiditie (as described above) were also excluded from the present study. Blood was collected by venous puncture into EDTA-containing tubes and then centrifuged at 1000 g for 10 min at 4 1C to pellet the cellular material. The resulting supernatants (plasma) were collected and aliquoted into Eppendorf tubes and frozen immediately at 80 1C. 2.2. Preparation of peripheral blood mononuclear cells To determine plasma and cellular DPP-IV activities, a proof of conduct study was conducted whereby 50–100 mL of fresh blood were obtained from matched healthy volunteers (n ¼14) who had been screened for medical disorders such as diabetes, autoimmune disease or recent infections. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation at 400 g for 30 min at 25 1C on an endotoxin-free Histopaque-1077 cushion. PBMCs were collected from the blood/Histopaque-1077 interface and treated with a cell lysis solution (Promega Corporation, Madison, WI 53711-5399, USA). Next, the cells were washed be brief centrifugation in 50 mL of phosphate buffered saline, the pellets were gently re-suspended and the cells counted using a Beckman Z-Coulter counter (Beckman Coulter, High Wycombe, UK). The study protocol and informed consent forms were reviewed and approved by Cambridge local research ethics committee.
2.1. Study population
2.3. Measurement of aminopeptidase activities
The full subject population was 1022 UD patients and 1000 controls from the Max Planck Institute and affiliated hospitals in Munich, Germany as part of a biomarkers study sponsored by GlaxoSmithkline. Blood samples were collected between 2002 and 2003 with informed written consent by all participants in accordance with Good Clinical Practise guidelines and the declaration of Helsinki, as modified by the 48th World Medical Association. To reduce the heterogeneity of patients and controls as described by Domenici et al. (2010), a total of 240 UD patients and 264 age and gender matched controls were selected for the present functional biomarkers study. Briefly, Exclusion criteria were body mass index (BMI) less than 18.5 4 BMI4 40 kg/m2 and the presence of comorbidities such as multiple sclerosis, all cancers, Parkinson's disease, rheumatoid arthritis, inflammatory bowel disorders, psoriasis, emphysema, chronic bronchitis, hayfever, diabetes and cardiovascular diseases (Table 1). Subjects who smoked more than twenty cigarettes per day were also excluded. All other potential confounders were included as covariates in the analyses.
2.3.1. DPP-IV Plasma DPP-IV enzymatic activity was measured using a colorimetric assay in 96-well black plates (Corning Ltd., High Wycombe, UK) (Nagatsu et al., 1976). Plasma samples (μL) were incubated in 71 mM glycine/NaOH (pH 8.3) buffer containing 0.5 mg/mL Gly-Prop-nitroanilide substrate (Sigma) for 1 h at 37 1C. The released 4-nitroaniline chromogenic product was monitored at 405 nm in a Labsystems plate reader (Helsinki, Finland). Activity level was calculated by comparison of the readings to those of the p-nitroaniline (Immuno Kontact, Abingdon, Oxon, UK) standard. DPP-IV protein levels were measured using a DPP-IV enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (R&D systems, Abingdon, UK). Plasma and PBMC DPP-IV activities were measured in samples from healthy volunteers using the Merck & Co. DPP-IV inhibitor Sitagliptin-645825 (Thornberry and Weber, 2007) at concentrations ranging from 1.2 to 10 μM. Reactions were carried out for 20 min at room temperature, prior to addition of the DPP-IV substrate.
Author's personal copy J. Deng et al. / Journal of Affective Disorders 151 (2013) 667–672
2.3.2. POP Plasma POP activity was determined by a fluorimetric assay, as modified by Goossens et al. (1992) using the synthetic substrate Zglycyl-prolyl-4-methylcoumarinyl-7-amide (Bachem Feinchemikalien AG, Bubendorf, Switzerland). Samples containing plasma (10 μL) and assay buffer (90 μL; 50 mM Hepes, pH 7.5, 250 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol and 150 μM Z-glycyl-prolyl4-methylcoumarinyl-7-amide) were incubated at 37 1C for 1 h. The released 7-amino-4-methylcoumarin product was detected at 340 nm excitation and 465 nm emission using a Tecan plate reader (Crailsheim, Germany). Activity levels were calculated by comparison to readings of the MCA standard curve (Bachem Ltd., Saint Helens, UK). 2.3.3. LAP LAP activity was determined by colorimetric assay using a LeupNA substrate (Sigma). Samples containing plasma (5 μL) and substrate solution (95 μL; Tris–HCl, 50 mM, pH 7.2, 250 mM NaCL, 1 mg/mL Leu-pNA) were incubated at 37 1C for 60 min. Activity was calculated as described above for DPP-IV. All activities were expressed as international units/L plasma, which corresponds to the hydrolysis of 1 μmol substrate/min/L. All measures were performed in duplicate and corrected for
Table 2 Demographic data of UD and control subjects for assessment of DPP-IV protein levels. UD n Gender: Female—number (%) Male—number (%) Age: mean (+/ SD) (years) BMI: mean (+/ SD) (kg/m2)
Control
62 46 16 50.6 25.5
32 24 8 50.8 24.6
(74.2) (25.8) (11.1) (3.9)
(75.0) (25.0) (11.1) (4.4)
spontaneous substrate hydrolysis using blank wells without sample. Precision was evaluated by assessing the intra- and inter-assay coefficients of variation. 2.4. Statistics Aminopeptidase activities in UD patients and controls were compared using analysis of covariance (ANCOVA) on logtransformed values. This provided estimates of percentage differences between the groups, and values were adjusted for the co-variates of age, sex and BMI. Significant differences were determined by ANCOVA (po0.05). Pearson's correlation analysis was used to quantify the strength of the relationship between paired measurements. The accuracy of the measure values for distinguishing between UD patients and controls was evaluated using receiver operating characteristic (ROC) curve analysis. True positive rates (sensitivity) were plotted as a function of the false positive rate (100-Specificity) such that each point on the curve represented a sensitivity/specificity pair corresponding to a particular decision threshold (Metz, 1978; Zweig and Campbell, 1993).
3. Results 3.1. Study population and sample selection Aminopeptidases activities were assessed in plasma from UD patients (n ¼240) and control subjects (n ¼264) who were most closely matched for age and gender (Table 1). Another subset of 62 UD patients (n ¼31 UD with anxiety and n¼ 31 UD without anxiety symptoms) and 32 controls was selected to assess the correlation between plasma DPP-IV activity and protein levels. No differential aminopeptidases (DPP-IV, LAP and POP) activities were correlated with anxiety symptoms (Data not shown). Each
9.3% difference, p
