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Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder
European Neuropsychopharmacology (]]]]) ], ]]]–]]]
www.elsevier.com/locate/euroneuro
Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder Marcio Gerhardt Soeiro-de-Souzaa, Antonio L. Teixeirab, Elvis C. Mateob, Marcus V. Zanettic,d, Flavia G. Rodriguesb, Vanessa J. de Paulac, Julia F. Bezerrac, Ricardo A. Morenoa, Wagner F. Gattazc,d, Rodrigo Machado-Vieirac,d,e,n a
Mood Disorders Program (GRUDA), Institute and Department of Psychiatry, University of Sao Paulo, Brazil Laboratory of Immunopharmacology, IBC, Federal University of Minas Gerais, Brazil c Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil d Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, Brazil e ETPB, Mood and Anxiety Disorders Program, National Institute of Mental Health, National Institutes of Health, and Department of Health and Human Services, Bethesda, MD, United States b
Received 1 November 2013; received in revised form 12 March 2014; accepted 19 March 2014
KEYWORDS
Abstract
Bipolar disorder; Telomerase; Lithium; Treatment; Depression; Epigenetics
Telomeres are DNA-protein complexes that cap linear DNA strands, protecting DNA from damage. Recently, shorten telomeres length has been reported in bipolar disorder (BD) and depression. The enzyme telomerase regulates telomeres' length, which has been associated with cellular viability; however it is not clear how telomerase may be involved in the pathophysiology and therapeutics of BD. In the present study, leukocyte telomerase activity was assessed in 28 medication-free BD depressed individuals (DSM-IV-TR criteria) at baseline and after 6 weeks of lithium therapy (n =21) also matching with 23 healthy controls. There was no difference between telomerase activity in subjects with BD depression (before or after lithium) and controls. Improvement of depressive symptoms was negatively associated with telomerase activity after 6 weeks of lithium therapy. This is the first study describing telomerase activity in BD research. Overall, telomerase activity seems not directly involved in the pathophysiology of short-term BD. Lithium's antidepressant effects may involve regulation at telomerase activity. Further studies with larger samples and long-term illness are also warranted. Published by Elsevier B.V.
n
Corresponding author at: Laboratory of Neuroscience (LIM27), Department and Institute of Psychiatry, University of Sao Paulo, Brazil. E-mail address: [email protected] (R. Machado-Vieira).
http://dx.doi.org/10.1016/j.euroneuro.2014.03.005 0924-977X/Published by Elsevier B.V.
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
2
M.G. Soeiro-de-Souza et al.
1.
Introduction
Bipolar disorder (BD) is a serious and chronic psychiatric disorder, with high rates of comorbidities related to “accelerated aging” (Sodhi et al., 2012). Recently, leukocyte telomeres were suggested as a novel locus linking mood disorders and accelerated aging (Everson-Rose and Lewis, 2005; Simon et al., 2006; Penninx et al., 1998; Martinsson et al., 2013; Wassertheil-Smoller et al., 2004; Kinser and Lyon, 2013; Sodhi et al., 2012; Wolkowitz et al., 2010, 2011b). Telomere dysfunction has been associated with multiple environmental and biological stressors (EversonRose and Lewis, 2005; Epel et al., 2010; Penninx et al., 1998; Heidinger et al., 2012; Wassertheil-Smoller et al., 2004; Zglinicki, 2002; Sodhi et al., 2012; Epel et al., 2004; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013) and eventually can result in programmed cell death (apoptosis) (Wong and Collins, 2003). Few studies on peripheral blood mononuclear cells (PBMCs) suggest altered telomere function in mood disorders. In depression and BD, shorter mean telomere length has been reported (Everson-Rose and Lewis, 2005; Simon et al., 2006; Penninx et al., 1998; Hartmann et al., 2010; Wassertheil-Smoller et al., 2004; Hoen et al., 2011; Sodhi et al., 2012; Wikgren et al., 2012; Rizzo et al., 2013). The mechanism that leads to telomere shortening in mood disorders is not clear; also it is not known how mood stabilizers influence telomerase length activity. Telomeres are DNA nucleoprotein complexes at the end of chromosomes, which protect DNA from deterioration and fusion with other chromosomes (Everson-Rose and Lewis, 2005; Penninx et al., 1998; Blackburn, 2001; WassertheilSmoller et al., 2004; Sodhi et al., 2012). Telomere length and telomerase activity are two distinct processes. Telomere length declines with cell replication, and is replenished by its enzyme telomerase, which adds six-base DNA repeats onto the telomeric ends of chromosomes (Simon et al., 2006; Feng et al., 1995; Martinsson et al., 2013; Blackburn, 2001; Kinser and Lyon, 2013; Wolkowitz et al., 2010, 2011b). In addition, telomerase activity regulates transcription of growth factors in adverse conditions (Epel et al., 2010, 2004; Sung et al., 2005; Heidinger et al., 2012; Calado and Young, 2009; Zglinicki, 2002; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013). Hippocampal telomerase was shown to modulate depressive-like behaviors (Wong and Collins, 2003; EversonRose and Lewis, 2005; Zhou et al., 2011; Blackburn, 2001; Penninx et al., 1998; Wassertheil-Smoller et al., 2004; Sodhi et al., 2012). Recently, an increase in telomerase activity in MDD was described, also associated with depression severity and predicting response to sertraline (Everson-Rose and Lewis, 2005; Feng et al., 1995; Simon et al., 2006; Wolkowitz et al., 2012; Penninx et al., 1998; Blackburn, 2001; Hartmann et al., 2010; Wassertheil-Smoller et al., 2004; Hoen et al., 2011; Sodhi et al., 2012; Wikgren et al., 2012; Rizzo et al., 2013). In BD, studies on telomere dysfunctions were limited to investigation on telomere length in leukocytes, which does not necessary reflect telomerase activity. These studies report reduced telomere length in BD (Simon et al., 2006; Elvsåshagen et al., 2011; Rizzo et al., 2013). Also, a high number of short telomeres were found in bipolar II disorder
(BD-II), associated with lifetime number of depressive episodes (Epel et al., 2010, 2004; Elvsåshagen et al., 2011; Heidinger et al., 2012; Zglinicki, 2002; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013). Telomerase activity may reduce the impact of oxidative stress, conferring resistance to physical and chemical stressors (Sung et al., 2005; Rubio et al., 2004; Calado and Young, 2009). Although lithium has been widely used as an antidepressant in BD (Yatham et al., 2013; Machado-Vieira et al., 2009), its long term use was associated with longer telomeres (Martinsson et al., 2013). To date, no study on telomerase activity has been performed in BD. Also, lithium seems to regulate telomere length in BD but its association with telomerase activity is not known. Thus, we aimed to investigate the leukocyte telomerase activity in bipolar depression before and after a six-week trial with lithium, also matching with a healthy control group.
2.
Experimental procedures
Subjects were evaluated between August 2010 and June 2012 at the Institute of Psychiatry, University of Sao Paulo, Brazil. Twenty-eight patients, 21 (70%) women, age 28.5(75.3) years with bipolar I disorder (BD-I) (39%) or BD-II (61%), current episode depressive, as diagnosed by Structured Clinical Interview for Axis I DSM-IV-TR Disorders (SCID) entered the study. Patients had a score greater than or equal to 18 on the 21-item Hamilton Depression Scale (HDRS) and Young Mania Rating Scale baseline score lower than 7 (except for 2 patients). Also, before the treatment was started 24 (85%) patients were drug-free for at least 6 months. At day one, patients were given lithium at a dosage of 450 mg/day, and subsequent dosage adjustment was allowed at a flexible manner, according to clinical response and serum levels. Mean serum lithium level at the endpoint was 0.48 mEq/L (mean dose: 728.5 mg7118.9). Seven patients dropped out the study or had adjunctive medications and thus were not included. All patients evaluated here were in lithium monotherapy (six used hypnotics as needed for a maximum of 5 days). In order to avoid other potential confounding factors, patients had no severe medical illness, were free of comorbid substance abuse or dependence, and had no axis I comorbidity. Twenty-three age-matched healthy controls were evaluated (10 women; age 27.176.6 years). Controls were excluded if they had lifetime history of any axis I psychiatric disorder (by SCID-I), or any first-degree relative with a mental disorder. The local institutional ethics committee approved the study and all patients provided written consent before they enter in the study. Clinical response was defined as a decrease of 50% or more in the Hamilton Depression Rating Scale (HDRS) at endpoint and remission as HDRSo8 at endpoint.
2.1.
Procedure
Psychometric assessments were made at baseline, on week 1, week 2, week 4, and week 6 (endpoint). Assessment of symptoms was performed with the HDRS. The Young Mania Rating Scale (YMRS) was used to evaluate potential switch to mania. Blood samples of patients were collected for this objective at baseline (before treatment) and at endpoint (week 6), while healthy controls had only one-point sample collection.
2.2.
PBMC collection
Blood was collected in EDTA tubes between 8:00 a.m. and 10:00 a.m. PBMCs were isolated using ficoll-hypaque gradient centrifugation
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
Telomerase in Bipolar Disorder
3
(GE Healthcare Biosciences) according to the manufacturer's specifications. Samples were washed three times in phosphate buffered saline (PBS) and stored in aliquots at 80 1C.
2.3. Photometric enzyme immunoassay for detection of telomerase activity, utilizing the Telomeric Repeat Amplification Protocol – TRAP (Telo TAGGG Telomerase PCR ELISA kit) Telomerase activity was determined using the Telo TAGGG Telomerase PCR ELISA Kit (Roche, Mannheim, Germany). The assay is divided into three steps: extraction; elongation/amplification and hybridization/detection by ELISA. 1) Extraction: Samples were thawed and centrifuged at 3000g for 10 min at 4 1C, to eliminate the DMSO. The cells were washed twice in phosphate buffer solution (PBS) and resuspended in 400 ml of PBS and then the cells were counted using a hemocytometer, Neubauer. Cells were resuspended in 200 ml lysis reagent, pre-cooled on ice by retropipetting at least 3 times and incubated on ice for 30 min. The lysate was centrifuged at 16,000g for 20 min at 4 1C. The supernatant (175 ml) was transferred to a fresh tube. 2) Elongation/amplification: 25 ml of reaction mixture was transferred into a tube for PCR amplification and later 3 ml of cells extract was added (samples and controls); finally sterile water was added to make up the reaction mixture to a final volume of 50 ml. Tubes were transferred to a thermal cycler (GeneAmpPCR System 9600, Perkin-Elmer) using the following protocol: primer elongation (25 min at 25 1C for 1 cycle), telomerase inactivation (5 min at 94 1C for 1 cycle), amplification: denaturation–annealing–polymerization (30 s at 94 1C, 30 s at 50 1C, and 90 s at 72 1C, respectively for 30 cycles), 10 min at 72 1C for 1 cycle. 3) Hybridization/detection by ELISA: Per sample 20 ml of denaturating reagent was transferred into a tube carefully, and then 5 ml of the amplification product was added and incubated at 25 1C for 10 min. Later, 225 ml hybridization buffer was added per tube and mixed thoroughly by vortexing briefly. Next, the reaction follows the manufacturer's protocol. Finally, the absorbance of the samples was measured at 450 nm using a microplate (ELISA) reader (with a reference wavelength of approximately 690 nm) within 30 min after addition of the stop reagent (A450 nm units/ 10,000 cells).
2.4.
Statistical analysis
Kolmogorov–Smirnov test was used to check if the sample distribution was normal. Between-group comparison of the demographic variables was done by Wilcoxon–Mann–Whitney test for continuous variables, and Chi square test for categorical variables. Withingroup comparisons were carried out with repeated measures ANOVA model for controlling age, gender body mass index and tobacco use. Correlations between variables were assessed by Pearson's correlation coefficient. All tests were two-tailed with α=0.05.
Table 1
3. 3.1.
Results Demographics
Demographic and clinical data are summarized in Table 1. The mean lifetime duration of BD was 3.171.5 years. The BD and control subjects did not significantly differ in age, body mass index and current tobacco use. Mean HDRS baseline was 22.573.7, while mean endpoint HDRS was 9.377.2. Mean YMRS baseline was 5.675.4, while mean endpoint YMRS was 3.470.6.
3.2.
Telomerase activity: BD vs. controls
Telomerase activity did not differ between controls and BD subjects in unmedicated bipolar depression (df=49; P=0.64) (Figure 1). Neither baseline telomerase activity (P=0.31) nor endpoint telomerase activity (0.48) differs between BD types I and II. Moreover, treatment response rates did not differ between BD types I and II (P= 0.07). Also, telomerase activity did not decrease after six weeks of lithium treatment (BD posttreatment mean=0.054270.003; P=0.34)
3.3. Telomerase activity and depression symptoms (HDRS) Telomerase activity showed no association with symptoms severity at baseline (r = 0.38; P = 0.85) or HDRS scores at endpoint (P = 0.07). However, a negative association between improvement of depressive symptoms after lithium treatment and endpoint telomerase activity was observed (r = 0.56; P= 0.007) (Figure 2). When considering response criteria (16 responders out of 21 subjects at endpoint), there was no significant change on telomerase activity, as well when considering the remission criteria (data not shown). Serum levels of lithium neither correlate with telomerase activity at endpoint (r= 0.29; P =0.18) nor with the change on telomerase activity during the follow-up period (week 6 – baseline) (r= 0.13; P =0.5).
4.
Discussion
To the best of our knowledge, this is the first study to investigate telomerase activity in BD. Here we evaluated subjects with BD during depressive episodes and treated with lithium for 6 weeks. We report a significant negative association between lithium's antidepressant effects and
Sociodemographic data.
Variable
Controls (n = 23)
Bipolar disorder (n= 28)
P
Age Gender (female/male) Body mass index (kg/m2) Use tabacco currently (yes/no) Disease duration (months)
27.1776.6 10/13 25.7274.3 6/17
28.575.3 21/7 28.6475.6 8/20 37.94718.5
0.42 0.02 0.14 0.96
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
4
M.G. Soeiro-de-Souza et al.
Figure 1
Mean telomerase activity before and after 6 week lithium treatment in BD (Po0.05) and matched with healthy controls.
Figure 2
Posttreatment telomerase activity correlation with HDRS change (week 6 – baseline).
resting telomerase activity in drug-free BD patients, suggesting a possible role of telomerase in the therapeutics of BD. Regarding the pathophysiology of BD, no difference on telomerase activity was observed between patients and controls. Telomerase activity has been associated with depression in preclinical models and is activated in response to specific types of brain damage, such as ischemic injury. This enzyme modulates depressive-like behaviors, possibly involving neurogenesis (Zhou et al., 2011). Moreover, chronic and acute stress models showed to upregulate telomerase expression (Beery et al., 2012; Epel et al., 2010). In humans, Wolkowitz et al. (2012) reported an increase in telomerase activity in unipolar depression subjects compared to controls, also directly associated with the severity of depressive episode. Moreover, the same study found an increased telomerase activity after treatment with sertraline (8 weeks) in those with lower pre-treatment telomerase activity (Wolkowitz et al., 2012). The potential role of leukocyte telomerase activity in antidepressant response has never been described in BD. Here, lower telomerase activity was associated with superior improvement of depressive
symptoms with lithium. Lithium is a first line agent approved to treat bipolar depression in monotherapy (Yatham et al., 2013). This finding may represent a decreased cellular stress associated with clinical improvement, since acute stress exposure has been linked to increased telomerase activity (Epel et al., 2010). BD has been associated with oxidative stress and inflammation, and both can alter the telomere function (Wolkowitz et al., 2011a; Zglinicki, 2002). The association between telomerase activity and improvement of depressive symptoms with lithium might be also associated with apoptosis regulation proteins. However, we did not find a significant difference on telomerase activity in subjects during a bipolar depression episode compared to that of healthy controls, not supporting a role for this enzyme in the pathophysiology of the illness. The main strengths of the present investigation include the inclusion of mostly drug-naïve unmedicated subjects of young age and short duration of illness. Also, patients underwent similar treatment with lithium for the same period (6-week) for depressive episodes and the study also included a well characterized control group with healthy subjects. Limitations
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
Telomerase in Bipolar Disorder include a relative small sample size, the open-label nature of the trial and lack of assessment of potential lifestyle variables. Moreover, both telomere length and telomerase activity may better represent the current cellular senescence status. In addition, these measures may vary between different PBMC subsets. Overall, this is the first study describing telomerase activity in BD and its potential association with clinical improvement with lithium treatment. The association between lower telomerase activity and improvement of depressive symptomatology after lithium treatment in BD might reflect a novel target for antidepressant response in BD. Future studies in this promising area are warranted.
Role of funding source This study was sponsored by Sao Paulo Research Foundation (Fapesp, Brazil, Grant 2009-14891).
Contributors All authors contributed for this manuscript.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments This study was sponsored by Sao Paulo Research Foundation (Fapesp, Brazil, Grant 2009-14891). The Laboratory of Neuroscience (LIM27) is supported by the Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS).
References Beery, A.K., et al., 2012. Chronic stress elevates telomerase activity in rats. Biol. Lett. 8 (6), 1063–1066. Blackburn, E.H., 2001. Switching and signaling at the telomere. Cell 106 (6), 661–673. Calado, R.T., Young, N.S., 2009. Telomere diseases. N. Engl. J. Med. 361 (24), 2353–2365. Elvsåshagen, T., et al., 2011. The load of short telomeres is increased and associated with lifetime number of depressive episodes in bipolar II disorder. J. Affect. Disord. 135 (1-3), 43–50. Epel, E.S., et al., 2004. Accelerated telomere shortening in response to life stress. Proc.Natl. Acad. Sci. USA 101 (49), 17312–17315. Epel, E.S., et al., 2010. Dynamics of telomerase activity in response to acute psychological stress. Brain Behav. Immun. 24 (4), 531–539. Everson-Rose, S.A., Lewis, T.T., 2005. Psychosocial factors and cardiovascular diseases. Ann.Rev. Public Health 26, 469–500. Feng, J., et al., 1995. The RNA component of human telomerase. Science 269 (5228), 1236–1241 (New York, N.Y.). Hartmann, N., et al., 2010. Telomere length of patients with major depression is shortened but independent from therapy and severity of the disease. Depress. Anxiety 27 (12), 1111–1116. Heidinger, B.J., et al., 2012. Telomere length in early life predicts lifespan. Proc.Natl. Acad. Sci. USA 109 (5), 1743–1748.
5 Hoen, P.W., et al., 2011. Depression and leukocyte telomere length in patients with coronary heart disease: data from the heart and soul study. Psychosom. Med. 73 (7), 541–547. Ilmonen, P., Kotrschal, A., Penn, D.J., 2008. Telomere attrition due to infection. PLoS One 3 (5), e2143. Kinser, P.A., Lyon, D.E., 2013. Major depressive disorder and measures of cellular aging: an integrative review. Nurs. Res. Pract. 2013, 469070. Kotrschal, A., Ilmonen, P., Penn, D.J., 2007. Stress impacts telomere dynamics. Biol. Lett. 3 (2), 128–130. Machado-Vieira, R., et al., 2009. The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. Bipolar Disord. 11 (Suppl 2), 92–109. Martinsson, L., et al., 2013. Long-term lithium treatment in bipolar disorder is associated with longer leukocyte telomeres. Transl. Psychiatry 3, e261. Penninx, B.W., et al., 1998. Chronically depressed mood and cancer risk in older persons. J. Natl. Cancer Inst. 90 (24), 1888–1893. Price, L.H., et al., 2013. Telomeres and early-life stress: an overview. Biol. Psychiatry 73 (1), 15–23. Rizzo, L.B., et al., 2013. Immunosenescence is associated with human cytomegalovirus and shortened telomeres in type I bipolar disorder. Bipolar Disord. 15 (8), 832–838. Rubio, M.A., Davalos, A.R., Campisi, J., 2004. Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress. Exp. Cell Res. 298 (1), 17–27. Shlush, L.I., et al., 2011. Telomere elongation followed by telomere length reduction, in leukocytes from divers exposed to intense oxidative stress–implications for tissue and organismal aging. Mech. Ageing Dev. 132 (3), 123–130. Simon, N.M., et al., 2006. Telomere shortening and mood disorders: preliminary support for a chronic stress model of accelerated aging. Biol. Psychiatry 60 (5), 432–435. Sodhi, S.K., et al., 2012. Evidence for accelerated vascular aging in bipolar disorder. J. Psychosom. Res. 73 (3), 175–179. Sung, Y.H., et al., 2005. The pleiotropy of telomerase against cell death. Mol. Cells 19 (3), 303–309. Wassertheil-Smoller, S., et al., 2004. Depression and cardiovascular sequelae in postmenopausal women. The Women's Health Initiative (WHI). Arch. Intern. Med. 164 (3), 289–298. Wikgren, M., et al., 2012. Short telomeres in depression and the general population are associated with a hypocortisolemic state. Biol. Psychiatry 71 (4), 294–300. Wolkowitz, O.M., et al., 2010. Depression gets old fast: do stress and depression accelerate cell aging? Depress. Anxiety 27 (4), 327–338. Wolkowitz, O.M., et al., 2012. Resting leukocyte telomerase activity is elevated in major depression and predicts treatment response. Mol. Psychiatry 17 (2), 164–172. Wolkowitz, O.M., Mellon, S.H., et al., 2011a. Leukocyte telomere length in major depression: correlations with chronicity, inflammation and oxidative stress – preliminary findings. PLoS One 6 (3), e17837. Wolkowitz, O.M., Reus, V.I., Mellon, S.H., 2011b. Of sound mind and body: depression, disease, and accelerated aging. Dialogues Clin. Neurosci. 13 (1), 25–39. Wong, J.M.Y., Collins, K., 2003. Telomere maintenance and disease. Lancet 362 (9388), 983–988. Yatham, L.N., et al., 2013. Canadian Network for Mood and Anxiety Treatments (CANMAT) and International Society for Bipolar Disorders (ISBD) collaborative update of CANMAT guidelines for the management of patients with bipolar disorder: update 2013. Bipolar Disord. 15 (1), 1–44. Zglinicki, von, T., 2002. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27 (7), 339–344. Zhou, Q.-G., et al., 2011. Hippocampal telomerase is involved in the modulation of depressive behaviors. J. Neurosci. 31 (34), 12258–12269.
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
www.elsevier.com/locate/euroneuro
Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder Marcio Gerhardt Soeiro-de-Souzaa, Antonio L. Teixeirab, Elvis C. Mateob, Marcus V. Zanettic,d, Flavia G. Rodriguesb, Vanessa J. de Paulac, Julia F. Bezerrac, Ricardo A. Morenoa, Wagner F. Gattazc,d, Rodrigo Machado-Vieirac,d,e,n a
Mood Disorders Program (GRUDA), Institute and Department of Psychiatry, University of Sao Paulo, Brazil Laboratory of Immunopharmacology, IBC, Federal University of Minas Gerais, Brazil c Laboratory of Neuroscience, LIM-27, Institute and Department of Psychiatry, University of Sao Paulo, Brazil d Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of São Paulo, Brazil e ETPB, Mood and Anxiety Disorders Program, National Institute of Mental Health, National Institutes of Health, and Department of Health and Human Services, Bethesda, MD, United States b
Received 1 November 2013; received in revised form 12 March 2014; accepted 19 March 2014
KEYWORDS
Abstract
Bipolar disorder; Telomerase; Lithium; Treatment; Depression; Epigenetics
Telomeres are DNA-protein complexes that cap linear DNA strands, protecting DNA from damage. Recently, shorten telomeres length has been reported in bipolar disorder (BD) and depression. The enzyme telomerase regulates telomeres' length, which has been associated with cellular viability; however it is not clear how telomerase may be involved in the pathophysiology and therapeutics of BD. In the present study, leukocyte telomerase activity was assessed in 28 medication-free BD depressed individuals (DSM-IV-TR criteria) at baseline and after 6 weeks of lithium therapy (n =21) also matching with 23 healthy controls. There was no difference between telomerase activity in subjects with BD depression (before or after lithium) and controls. Improvement of depressive symptoms was negatively associated with telomerase activity after 6 weeks of lithium therapy. This is the first study describing telomerase activity in BD research. Overall, telomerase activity seems not directly involved in the pathophysiology of short-term BD. Lithium's antidepressant effects may involve regulation at telomerase activity. Further studies with larger samples and long-term illness are also warranted. Published by Elsevier B.V.
n
Corresponding author at: Laboratory of Neuroscience (LIM27), Department and Institute of Psychiatry, University of Sao Paulo, Brazil. E-mail address: [email protected] (R. Machado-Vieira).
http://dx.doi.org/10.1016/j.euroneuro.2014.03.005 0924-977X/Published by Elsevier B.V.
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
2
M.G. Soeiro-de-Souza et al.
1.
Introduction
Bipolar disorder (BD) is a serious and chronic psychiatric disorder, with high rates of comorbidities related to “accelerated aging” (Sodhi et al., 2012). Recently, leukocyte telomeres were suggested as a novel locus linking mood disorders and accelerated aging (Everson-Rose and Lewis, 2005; Simon et al., 2006; Penninx et al., 1998; Martinsson et al., 2013; Wassertheil-Smoller et al., 2004; Kinser and Lyon, 2013; Sodhi et al., 2012; Wolkowitz et al., 2010, 2011b). Telomere dysfunction has been associated with multiple environmental and biological stressors (EversonRose and Lewis, 2005; Epel et al., 2010; Penninx et al., 1998; Heidinger et al., 2012; Wassertheil-Smoller et al., 2004; Zglinicki, 2002; Sodhi et al., 2012; Epel et al., 2004; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013) and eventually can result in programmed cell death (apoptosis) (Wong and Collins, 2003). Few studies on peripheral blood mononuclear cells (PBMCs) suggest altered telomere function in mood disorders. In depression and BD, shorter mean telomere length has been reported (Everson-Rose and Lewis, 2005; Simon et al., 2006; Penninx et al., 1998; Hartmann et al., 2010; Wassertheil-Smoller et al., 2004; Hoen et al., 2011; Sodhi et al., 2012; Wikgren et al., 2012; Rizzo et al., 2013). The mechanism that leads to telomere shortening in mood disorders is not clear; also it is not known how mood stabilizers influence telomerase length activity. Telomeres are DNA nucleoprotein complexes at the end of chromosomes, which protect DNA from deterioration and fusion with other chromosomes (Everson-Rose and Lewis, 2005; Penninx et al., 1998; Blackburn, 2001; WassertheilSmoller et al., 2004; Sodhi et al., 2012). Telomere length and telomerase activity are two distinct processes. Telomere length declines with cell replication, and is replenished by its enzyme telomerase, which adds six-base DNA repeats onto the telomeric ends of chromosomes (Simon et al., 2006; Feng et al., 1995; Martinsson et al., 2013; Blackburn, 2001; Kinser and Lyon, 2013; Wolkowitz et al., 2010, 2011b). In addition, telomerase activity regulates transcription of growth factors in adverse conditions (Epel et al., 2010, 2004; Sung et al., 2005; Heidinger et al., 2012; Calado and Young, 2009; Zglinicki, 2002; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013). Hippocampal telomerase was shown to modulate depressive-like behaviors (Wong and Collins, 2003; EversonRose and Lewis, 2005; Zhou et al., 2011; Blackburn, 2001; Penninx et al., 1998; Wassertheil-Smoller et al., 2004; Sodhi et al., 2012). Recently, an increase in telomerase activity in MDD was described, also associated with depression severity and predicting response to sertraline (Everson-Rose and Lewis, 2005; Feng et al., 1995; Simon et al., 2006; Wolkowitz et al., 2012; Penninx et al., 1998; Blackburn, 2001; Hartmann et al., 2010; Wassertheil-Smoller et al., 2004; Hoen et al., 2011; Sodhi et al., 2012; Wikgren et al., 2012; Rizzo et al., 2013). In BD, studies on telomere dysfunctions were limited to investigation on telomere length in leukocytes, which does not necessary reflect telomerase activity. These studies report reduced telomere length in BD (Simon et al., 2006; Elvsåshagen et al., 2011; Rizzo et al., 2013). Also, a high number of short telomeres were found in bipolar II disorder
(BD-II), associated with lifetime number of depressive episodes (Epel et al., 2010, 2004; Elvsåshagen et al., 2011; Heidinger et al., 2012; Zglinicki, 2002; Ilmonen et al., 2008; Kotrschal et al., 2007; Shlush et al., 2011; Price et al., 2013). Telomerase activity may reduce the impact of oxidative stress, conferring resistance to physical and chemical stressors (Sung et al., 2005; Rubio et al., 2004; Calado and Young, 2009). Although lithium has been widely used as an antidepressant in BD (Yatham et al., 2013; Machado-Vieira et al., 2009), its long term use was associated with longer telomeres (Martinsson et al., 2013). To date, no study on telomerase activity has been performed in BD. Also, lithium seems to regulate telomere length in BD but its association with telomerase activity is not known. Thus, we aimed to investigate the leukocyte telomerase activity in bipolar depression before and after a six-week trial with lithium, also matching with a healthy control group.
2.
Experimental procedures
Subjects were evaluated between August 2010 and June 2012 at the Institute of Psychiatry, University of Sao Paulo, Brazil. Twenty-eight patients, 21 (70%) women, age 28.5(75.3) years with bipolar I disorder (BD-I) (39%) or BD-II (61%), current episode depressive, as diagnosed by Structured Clinical Interview for Axis I DSM-IV-TR Disorders (SCID) entered the study. Patients had a score greater than or equal to 18 on the 21-item Hamilton Depression Scale (HDRS) and Young Mania Rating Scale baseline score lower than 7 (except for 2 patients). Also, before the treatment was started 24 (85%) patients were drug-free for at least 6 months. At day one, patients were given lithium at a dosage of 450 mg/day, and subsequent dosage adjustment was allowed at a flexible manner, according to clinical response and serum levels. Mean serum lithium level at the endpoint was 0.48 mEq/L (mean dose: 728.5 mg7118.9). Seven patients dropped out the study or had adjunctive medications and thus were not included. All patients evaluated here were in lithium monotherapy (six used hypnotics as needed for a maximum of 5 days). In order to avoid other potential confounding factors, patients had no severe medical illness, were free of comorbid substance abuse or dependence, and had no axis I comorbidity. Twenty-three age-matched healthy controls were evaluated (10 women; age 27.176.6 years). Controls were excluded if they had lifetime history of any axis I psychiatric disorder (by SCID-I), or any first-degree relative with a mental disorder. The local institutional ethics committee approved the study and all patients provided written consent before they enter in the study. Clinical response was defined as a decrease of 50% or more in the Hamilton Depression Rating Scale (HDRS) at endpoint and remission as HDRSo8 at endpoint.
2.1.
Procedure
Psychometric assessments were made at baseline, on week 1, week 2, week 4, and week 6 (endpoint). Assessment of symptoms was performed with the HDRS. The Young Mania Rating Scale (YMRS) was used to evaluate potential switch to mania. Blood samples of patients were collected for this objective at baseline (before treatment) and at endpoint (week 6), while healthy controls had only one-point sample collection.
2.2.
PBMC collection
Blood was collected in EDTA tubes between 8:00 a.m. and 10:00 a.m. PBMCs were isolated using ficoll-hypaque gradient centrifugation
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
Telomerase in Bipolar Disorder
3
(GE Healthcare Biosciences) according to the manufacturer's specifications. Samples were washed three times in phosphate buffered saline (PBS) and stored in aliquots at 80 1C.
2.3. Photometric enzyme immunoassay for detection of telomerase activity, utilizing the Telomeric Repeat Amplification Protocol – TRAP (Telo TAGGG Telomerase PCR ELISA kit) Telomerase activity was determined using the Telo TAGGG Telomerase PCR ELISA Kit (Roche, Mannheim, Germany). The assay is divided into three steps: extraction; elongation/amplification and hybridization/detection by ELISA. 1) Extraction: Samples were thawed and centrifuged at 3000g for 10 min at 4 1C, to eliminate the DMSO. The cells were washed twice in phosphate buffer solution (PBS) and resuspended in 400 ml of PBS and then the cells were counted using a hemocytometer, Neubauer. Cells were resuspended in 200 ml lysis reagent, pre-cooled on ice by retropipetting at least 3 times and incubated on ice for 30 min. The lysate was centrifuged at 16,000g for 20 min at 4 1C. The supernatant (175 ml) was transferred to a fresh tube. 2) Elongation/amplification: 25 ml of reaction mixture was transferred into a tube for PCR amplification and later 3 ml of cells extract was added (samples and controls); finally sterile water was added to make up the reaction mixture to a final volume of 50 ml. Tubes were transferred to a thermal cycler (GeneAmpPCR System 9600, Perkin-Elmer) using the following protocol: primer elongation (25 min at 25 1C for 1 cycle), telomerase inactivation (5 min at 94 1C for 1 cycle), amplification: denaturation–annealing–polymerization (30 s at 94 1C, 30 s at 50 1C, and 90 s at 72 1C, respectively for 30 cycles), 10 min at 72 1C for 1 cycle. 3) Hybridization/detection by ELISA: Per sample 20 ml of denaturating reagent was transferred into a tube carefully, and then 5 ml of the amplification product was added and incubated at 25 1C for 10 min. Later, 225 ml hybridization buffer was added per tube and mixed thoroughly by vortexing briefly. Next, the reaction follows the manufacturer's protocol. Finally, the absorbance of the samples was measured at 450 nm using a microplate (ELISA) reader (with a reference wavelength of approximately 690 nm) within 30 min after addition of the stop reagent (A450 nm units/ 10,000 cells).
2.4.
Statistical analysis
Kolmogorov–Smirnov test was used to check if the sample distribution was normal. Between-group comparison of the demographic variables was done by Wilcoxon–Mann–Whitney test for continuous variables, and Chi square test for categorical variables. Withingroup comparisons were carried out with repeated measures ANOVA model for controlling age, gender body mass index and tobacco use. Correlations between variables were assessed by Pearson's correlation coefficient. All tests were two-tailed with α=0.05.
Table 1
3. 3.1.
Results Demographics
Demographic and clinical data are summarized in Table 1. The mean lifetime duration of BD was 3.171.5 years. The BD and control subjects did not significantly differ in age, body mass index and current tobacco use. Mean HDRS baseline was 22.573.7, while mean endpoint HDRS was 9.377.2. Mean YMRS baseline was 5.675.4, while mean endpoint YMRS was 3.470.6.
3.2.
Telomerase activity: BD vs. controls
Telomerase activity did not differ between controls and BD subjects in unmedicated bipolar depression (df=49; P=0.64) (Figure 1). Neither baseline telomerase activity (P=0.31) nor endpoint telomerase activity (0.48) differs between BD types I and II. Moreover, treatment response rates did not differ between BD types I and II (P= 0.07). Also, telomerase activity did not decrease after six weeks of lithium treatment (BD posttreatment mean=0.054270.003; P=0.34)
3.3. Telomerase activity and depression symptoms (HDRS) Telomerase activity showed no association with symptoms severity at baseline (r = 0.38; P = 0.85) or HDRS scores at endpoint (P = 0.07). However, a negative association between improvement of depressive symptoms after lithium treatment and endpoint telomerase activity was observed (r = 0.56; P= 0.007) (Figure 2). When considering response criteria (16 responders out of 21 subjects at endpoint), there was no significant change on telomerase activity, as well when considering the remission criteria (data not shown). Serum levels of lithium neither correlate with telomerase activity at endpoint (r= 0.29; P =0.18) nor with the change on telomerase activity during the follow-up period (week 6 – baseline) (r= 0.13; P =0.5).
4.
Discussion
To the best of our knowledge, this is the first study to investigate telomerase activity in BD. Here we evaluated subjects with BD during depressive episodes and treated with lithium for 6 weeks. We report a significant negative association between lithium's antidepressant effects and
Sociodemographic data.
Variable
Controls (n = 23)
Bipolar disorder (n= 28)
P
Age Gender (female/male) Body mass index (kg/m2) Use tabacco currently (yes/no) Disease duration (months)
27.1776.6 10/13 25.7274.3 6/17
28.575.3 21/7 28.6475.6 8/20 37.94718.5
0.42 0.02 0.14 0.96
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
4
M.G. Soeiro-de-Souza et al.
Figure 1
Mean telomerase activity before and after 6 week lithium treatment in BD (Po0.05) and matched with healthy controls.
Figure 2
Posttreatment telomerase activity correlation with HDRS change (week 6 – baseline).
resting telomerase activity in drug-free BD patients, suggesting a possible role of telomerase in the therapeutics of BD. Regarding the pathophysiology of BD, no difference on telomerase activity was observed between patients and controls. Telomerase activity has been associated with depression in preclinical models and is activated in response to specific types of brain damage, such as ischemic injury. This enzyme modulates depressive-like behaviors, possibly involving neurogenesis (Zhou et al., 2011). Moreover, chronic and acute stress models showed to upregulate telomerase expression (Beery et al., 2012; Epel et al., 2010). In humans, Wolkowitz et al. (2012) reported an increase in telomerase activity in unipolar depression subjects compared to controls, also directly associated with the severity of depressive episode. Moreover, the same study found an increased telomerase activity after treatment with sertraline (8 weeks) in those with lower pre-treatment telomerase activity (Wolkowitz et al., 2012). The potential role of leukocyte telomerase activity in antidepressant response has never been described in BD. Here, lower telomerase activity was associated with superior improvement of depressive
symptoms with lithium. Lithium is a first line agent approved to treat bipolar depression in monotherapy (Yatham et al., 2013). This finding may represent a decreased cellular stress associated with clinical improvement, since acute stress exposure has been linked to increased telomerase activity (Epel et al., 2010). BD has been associated with oxidative stress and inflammation, and both can alter the telomere function (Wolkowitz et al., 2011a; Zglinicki, 2002). The association between telomerase activity and improvement of depressive symptoms with lithium might be also associated with apoptosis regulation proteins. However, we did not find a significant difference on telomerase activity in subjects during a bipolar depression episode compared to that of healthy controls, not supporting a role for this enzyme in the pathophysiology of the illness. The main strengths of the present investigation include the inclusion of mostly drug-naïve unmedicated subjects of young age and short duration of illness. Also, patients underwent similar treatment with lithium for the same period (6-week) for depressive episodes and the study also included a well characterized control group with healthy subjects. Limitations
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005
Telomerase in Bipolar Disorder include a relative small sample size, the open-label nature of the trial and lack of assessment of potential lifestyle variables. Moreover, both telomere length and telomerase activity may better represent the current cellular senescence status. In addition, these measures may vary between different PBMC subsets. Overall, this is the first study describing telomerase activity in BD and its potential association with clinical improvement with lithium treatment. The association between lower telomerase activity and improvement of depressive symptomatology after lithium treatment in BD might reflect a novel target for antidepressant response in BD. Future studies in this promising area are warranted.
Role of funding source This study was sponsored by Sao Paulo Research Foundation (Fapesp, Brazil, Grant 2009-14891).
Contributors All authors contributed for this manuscript.
Conflict of interest The authors declare no conflict of interest.
Acknowledgments This study was sponsored by Sao Paulo Research Foundation (Fapesp, Brazil, Grant 2009-14891). The Laboratory of Neuroscience (LIM27) is supported by the Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS).
References Beery, A.K., et al., 2012. Chronic stress elevates telomerase activity in rats. Biol. Lett. 8 (6), 1063–1066. Blackburn, E.H., 2001. Switching and signaling at the telomere. Cell 106 (6), 661–673. Calado, R.T., Young, N.S., 2009. Telomere diseases. N. Engl. J. Med. 361 (24), 2353–2365. Elvsåshagen, T., et al., 2011. The load of short telomeres is increased and associated with lifetime number of depressive episodes in bipolar II disorder. J. Affect. Disord. 135 (1-3), 43–50. Epel, E.S., et al., 2004. Accelerated telomere shortening in response to life stress. Proc.Natl. Acad. Sci. USA 101 (49), 17312–17315. Epel, E.S., et al., 2010. Dynamics of telomerase activity in response to acute psychological stress. Brain Behav. Immun. 24 (4), 531–539. Everson-Rose, S.A., Lewis, T.T., 2005. Psychosocial factors and cardiovascular diseases. Ann.Rev. Public Health 26, 469–500. Feng, J., et al., 1995. The RNA component of human telomerase. Science 269 (5228), 1236–1241 (New York, N.Y.). Hartmann, N., et al., 2010. Telomere length of patients with major depression is shortened but independent from therapy and severity of the disease. Depress. Anxiety 27 (12), 1111–1116. Heidinger, B.J., et al., 2012. Telomere length in early life predicts lifespan. Proc.Natl. Acad. Sci. USA 109 (5), 1743–1748.
5 Hoen, P.W., et al., 2011. Depression and leukocyte telomere length in patients with coronary heart disease: data from the heart and soul study. Psychosom. Med. 73 (7), 541–547. Ilmonen, P., Kotrschal, A., Penn, D.J., 2008. Telomere attrition due to infection. PLoS One 3 (5), e2143. Kinser, P.A., Lyon, D.E., 2013. Major depressive disorder and measures of cellular aging: an integrative review. Nurs. Res. Pract. 2013, 469070. Kotrschal, A., Ilmonen, P., Penn, D.J., 2007. Stress impacts telomere dynamics. Biol. Lett. 3 (2), 128–130. Machado-Vieira, R., et al., 2009. The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. Bipolar Disord. 11 (Suppl 2), 92–109. Martinsson, L., et al., 2013. Long-term lithium treatment in bipolar disorder is associated with longer leukocyte telomeres. Transl. Psychiatry 3, e261. Penninx, B.W., et al., 1998. Chronically depressed mood and cancer risk in older persons. J. Natl. Cancer Inst. 90 (24), 1888–1893. Price, L.H., et al., 2013. Telomeres and early-life stress: an overview. Biol. Psychiatry 73 (1), 15–23. Rizzo, L.B., et al., 2013. Immunosenescence is associated with human cytomegalovirus and shortened telomeres in type I bipolar disorder. Bipolar Disord. 15 (8), 832–838. Rubio, M.A., Davalos, A.R., Campisi, J., 2004. Telomere length mediates the effects of telomerase on the cellular response to genotoxic stress. Exp. Cell Res. 298 (1), 17–27. Shlush, L.I., et al., 2011. Telomere elongation followed by telomere length reduction, in leukocytes from divers exposed to intense oxidative stress–implications for tissue and organismal aging. Mech. Ageing Dev. 132 (3), 123–130. Simon, N.M., et al., 2006. Telomere shortening and mood disorders: preliminary support for a chronic stress model of accelerated aging. Biol. Psychiatry 60 (5), 432–435. Sodhi, S.K., et al., 2012. Evidence for accelerated vascular aging in bipolar disorder. J. Psychosom. Res. 73 (3), 175–179. Sung, Y.H., et al., 2005. The pleiotropy of telomerase against cell death. Mol. Cells 19 (3), 303–309. Wassertheil-Smoller, S., et al., 2004. Depression and cardiovascular sequelae in postmenopausal women. The Women's Health Initiative (WHI). Arch. Intern. Med. 164 (3), 289–298. Wikgren, M., et al., 2012. Short telomeres in depression and the general population are associated with a hypocortisolemic state. Biol. Psychiatry 71 (4), 294–300. Wolkowitz, O.M., et al., 2010. Depression gets old fast: do stress and depression accelerate cell aging? Depress. Anxiety 27 (4), 327–338. Wolkowitz, O.M., et al., 2012. Resting leukocyte telomerase activity is elevated in major depression and predicts treatment response. Mol. Psychiatry 17 (2), 164–172. Wolkowitz, O.M., Mellon, S.H., et al., 2011a. Leukocyte telomere length in major depression: correlations with chronicity, inflammation and oxidative stress – preliminary findings. PLoS One 6 (3), e17837. Wolkowitz, O.M., Reus, V.I., Mellon, S.H., 2011b. Of sound mind and body: depression, disease, and accelerated aging. Dialogues Clin. Neurosci. 13 (1), 25–39. Wong, J.M.Y., Collins, K., 2003. Telomere maintenance and disease. Lancet 362 (9388), 983–988. Yatham, L.N., et al., 2013. Canadian Network for Mood and Anxiety Treatments (CANMAT) and International Society for Bipolar Disorders (ISBD) collaborative update of CANMAT guidelines for the management of patients with bipolar disorder: update 2013. Bipolar Disord. 15 (1), 1–44. Zglinicki, von, T., 2002. Oxidative stress shortens telomeres. Trends Biochem. Sci. 27 (7), 339–344. Zhou, Q.-G., et al., 2011. Hippocampal telomerase is involved in the modulation of depressive behaviors. J. Neurosci. 31 (34), 12258–12269.
Please cite this article as: Soeiro-de-Souza, M.G., et al., Leukocyte telomerase activity and antidepressant efficacy in bipolar disorder. European Neuropsychopharmacology (2014), http://dx.doi.org/10.1016/j.euroneuro.2014.03.005