Neuroscience and Behavioral Physiology, Vol. 44, No. 4, May, 2014

Involvement of Anomalous Apoptosis in Impairments to Synaptic Plasticity in Post-Traumatic Stress Disorder G. M. Mkrtchyan,1 A. S. Boyadzhyan,1 D. G. Avetyan,1 and S. G. Sukiasyan2

Translated from Zhurnal Nevrologii i Psikhiatrii imeni S. S. Korsakova, Vol. 113, No. 1, Iss. I, pp. 26–29, January, 2013. Enzyme-linked immunosorbent assay was used to measure levels of apoptosis and synaptic plasticity marker proteins, i.e., annexin A5 and complexin 2 respectively, as well as the proinflammatory cytokine tumor necrosis factor α (TNF-α ), in serum from patients with post-traumatic stress disorder (PTSD) in comparison with healthy subjects. Correlations between these parameters were studied. The results obtained here showed that annexin A5 and complexin 2 concentrations in PTSD patients were significantly lower than normal, while TNF-α levels were higher. PTSD patients showed a positive correlation between annexin A5 and complexin 2 levels on the one hand, and a negative correlation between annexin A5 and TNF-α levels on the other. These data lead to the conclusion that the pathogenesis of PTSD is characterized by reduced apoptosis associated with defects in synaptic plasticity. It is suggested that anomalous apoptosis may also be among the factors supporting the development of the chronic inflammation typical of the pathogenesis of PTSD. Keywords: apoptosis, annexin A5, complexin 2, tumor necrosis factor α, synaptic plasticity, post-traumatic stress disorder.

Plasticity, i.e., the ability of synapses to undergo functional and morphological rearrangements during activity, is an important and unavoidable property of synaptic contacts in both the central and peripheral nervous system. This property allows synapses to perform many different physiological functions in the body. Transient forms of synaptic plasticity (SP), lasting seconds and minutes, are known, as are long-term forms, lasting hours, months, and even years. The long-term types of plasticity form on the basis of the short-term forms and provide the basis for the cognitive functions of the nervous system – learning, memory, attention, psychomotor coordination, etc. [5, 39, 43]. The mechanisms and regulation of SP are currently subject to intense study by molecular psychiatrists, as injuries cause impairments to both cognitive functions and the

development of mental disorders, including depressive states and post-traumatic stress disorder (PTSD) [15, 25, 34]. The molecular mechanisms underlying impairments to SP in PTSD [29, 31] have received insufficient study. We have suggested that anomalous apoptosis may be one of the factors promoting the development of such disorders; this is known to be a major regulator of SP [1, 21, 36]. This suggestion is based on data [20, 48] showing that the pathogenesis of PTSD includes several sluggish inflammatory processes, which are generally associated with anomalous apoptosis [35, 38]. Inflammatory processes in PTSD are accompanied by hyperproduction of chemokines and proinflammatory cytokines, such as interleukins IL-1β and IL-6 and tumor necrosis factor α (TNF-α) [2, 3, 26]. Cytokines play an important role in controlling and triggering apoptosis signal pathways [10, 24]. Increases in proinflammatory cytokines have been shown to activate apoptotic processes [17, 23, 49]. Thus, binding of TNF-α with type 1 TNF-α receptors (TNF-R1) on cell membranes triggers apoptosis. In addition, cysteine

1 Institute

of Molecular Biology, National Academy of Sciences of the Republic of Armenia, Erevan; e-mail: [email protected]. 2 ArtMed Medical Rehabilitation Center, Erevan, Republic of Armenia.

442 0097-0549/14/4404-0442 ©2014 Springer Science+Business Media New York

Involvement of Anomalous Apoptosis in Impairments to Synaptic Plasticity

Fig. 1. Serum annexin A5 levels in PTSD patients and healthy subjects. The ordinate shows concentrations, ng/ml. Here and in Fig. 2 – boxwhiskers show interquartile distances (range from 25th to 75th percentiles); vertical bars outside boxes show ranges from the 10th to the 90th percentiles. The horizontal line shows the median.

proteases, which are related to interleukin-1β-convertase, have a central position in controlling apoptosis. It has been suggested that binding of TNF-α with TNF-R1 leads to activation of these proteases via a multistep process of protein interactions which ultimately triggers apoptosis [33, 41, 44]. The aim of the present work was to seek experimental support for this suggestion by determining the blood levels of apoptosis and SP marker proteins, i.e., annexin A5 [8] and complexin 2 [28, 51] respectively, and the proinflammatory cytokine tumor necrosis factor α in patients with PTSD as compared with healthy subjects, as well as to test correlations between these values. MATERIALS AND METHODS A total of 37 war veterans with PTSD (DSM-IV-TR 309.81) [13], mean age (M ± δ) 47 ± 8 years, mean duration of illness 17 years, were studied. The control group consisted of somatically and mentally healthy subjects without inherited burden of mental illness; these were 35 academic staff (mean age 46 ± 3 years). PTSD patients and controls were male. Diagnoses of patients’ status were made and the severity and frequency of clinical psychopathological signs (symptoms) of PTSD were analyzed at the Armenian “Stress” medical psychiatric center in terms of DSM-IV-TR criteria [13], structured clinical interview for DSM-IV criteria (SCDI-I) [16], and the PTSD clinical diagnosis scale CAPS [7]. Doctors explained the study to all subjects and all gave informed consent for blood sampling. The investigation was

443

approved by the Ethics Committee of the Institute of Molecular Biology, National Academy of Sciences of the Republic of Armenia. Blood was collected from the ulnar vein at 09:00–10:00 on an empty stomach. Blood samples were immediately placed on ice and then centrifuged at 3000g for 10 min; serum was harvested and used in subsequent experiments. Serum samples were stored at –30°C. All samples were analyzed in two parallel repeats. Serum annexin A5 and complexin 2 levels were measured by enzyme-linked immunosorbent assay (ELISA) using commercial reagent kits (USCN Life Sciences, Inc.) following the manufacturer’s instructions, with the results being expressed in ng/ml and pg/ml of serum, respectively. Serum TNF-α concentrations were measured by ELISA using commercial reagent kits (Gen-Probe, France) following the manufacturer’s instructions, with results expressed in pg/ml of serum. Data were analyzed statistically in GraphPad Prism 3.03 (GraphPad Software Inc., USA) running the Mann– Whitney U test and correlation analysis, including calculation of the Spearman rank correlation coefficient (Rs). Values of p < 0.05 were taken as identifying statistical significance. RESULTS AND DISCUSSION Serum annexin A5 and complexin 2 levels in PTSD patients were, on average, 2.3 and 1.2 times (p < 0.0001 and p < 0.05, respectively) times lower than in normal subjects. The results are presented in Figs. 1 and 2. TNF-α levels were a mean of 1.3 times greater in PTSD patients than normal subjects, the difference being statistically significant (p < 0.03), confirming the results obtained from studies published previously both by ourselves [2, 3] and other groups [26]. Correlation analysis identified a statistically significant positive correlation between annexin A5 and complexin 2 levels in PTSD patients (Rs = 0.38, p < 0.045). No statistically significant correlation was seen between these parameters in healthy subjects (Rs = –0.14, p > 0.425). In addition, PTSD patients showed a statistically significant negative correlation between annexin A5 and TNF-α levels (Rs = –0.35, p < 0.047). Much attention in recent years has been paid to the biological activity of proteins of the annexins family. Annexin A5, like others, is not extracted from normal cells; the source of extracellular (soluble) annexin A5 is apoptotic and ruptured cells [37]. The ability of annexins to bind with negatively charged phospholipids, including phosphatidylserine, plays an important role in the mechanism of action of annexin A5 – exposure of phosphatidylserine on cell membranes is one of the early signs of apoptosis [42, 46]. In addition, studies in recent years have shown that annexin A5 has anticoagulant and anti-inflammatory properties: it binds to phosphatidylserine molecules exposed on the surfaces of

444

Fig. 2. Serum complexin 2 contents in PTSD patients and healthy subjects. The ordinate shows concentrations, pg/ml.

apoptotic cells and inhibits the procoagulant and proinflammatory activity of dying cells [4, 37]. Thus, the decreased serum levels of soluble annexin A5 in PTSD patients as compared with normal subjects provide evidence of the reduced apoptotic function characteristic of these patients, which may among the causes of the development of the sluggish systemic inflammation in this disease. In this regard, it is interesting to note that, as demonstrated by results from recent experiments, prolonged use of neuroleptics leads to increases in annexin A5 levels, correlating with decreases in TNF-α levels in patients with chronic schizophrenia [18]. Complexin 2 is a presynaptic protein expressed mainly by excitatory neurons. Decreased levels of expression of this protein, leading to a functional deficit of synaptic transmission, make a significant contribution to the etiology, pathogenesis, and progression of schizophrenia, Huntington’s disease, Parkinson’s disease, Alzheimer’s disease, depression, bipolar disorder, craniocerebral trauma, Wernicke’s encephalopathy, and fetal alcohol syndrome [9, 14, 19, 22, 45]. Thus, decreases in the serum level of this protein in PTSD patients provide evidence of SP deficit in this pathology, as previously noted by other authors [29, 31]. In addition, a number of polymorphic complexin 2 genes, which can be detected in terms of a reduction in the level of this protein in peripheral blood, have been shown to be associated with impairments to cognitive functions [6]. These data lead to the suggestion that that low complexin 2 levels in the blood of PTSD patients are inherited and reflect the cognitive dysfunction characteristic of this pathology [25, 34]. Further studies of the association between functional polymorphism in the complexin 2 gene with PTSD will clarify this issue.

Mkrtchyan, Boyadzhyan, Avetyan, and Sukiasyan Both hypo- and hyperfunction of apoptosis lead to disturbance to homeostasis and are involved in the development of many diseases in humans [27, 40], including mental disorders [18, 47, 50]. Blood annexin A5 concentrations in patients with schizophrenia and Alzheimer’s disease are significantly increased compared with normal subjects [18, 50]. Data have also been obtained showing low levels of this protein in the cerebrospinal fluid of patients with Parkinsonism, as compared with normal [47]. Studies in recent years have shown that impairments at the level of apoptosis make a significant contribution to synaptic dysfunction and lead to changes in the structuralfunctional integrity of neural networks. This process is regarded as a major regulator of SP [11, 21, 30, 32]. In PTSD, the correlation we found between the low level of the apoptosis marker annexin A5 and decreased levels of the SP indicator complexin 2 provides evidence of the involvement of anomalous apoptosis in the SP impairments characteristic of PTSD. Clarification of the molecular and cellular bases for the interaction between impairments at the level of apoptosis and SP in PTSD requires further study. In addition, as noted above, PTSD patients have a sluggish inflammatory process [20, 48]. The mechanisms involved in the development of this process in PTSD are not clear. The results of the present studies demonstrated a negative correlation between blood levels of the proinflammatory cytokine TNF-α and the apoptosis marker annexin A5 protein in PTSD patients. On the basis of these data, we suggest that the low annexin A5 level in PTSD patients may be responsible for the reduced level of apoptosis of immunocompetent cells, as seen in autoinflammatory diseases [48] and, thus, makes a significant contribution to the development of PTSD-associated chronic inflammation. This suggestion is consistent with published data [12, 20, 48] on chronic increases in blood leukocyte levels in PTSD patients. Thus, the results obtained here lead to the view that the pathogenesis of PTSD is characterized by hypofunction of apoptosis correlating with defects to SP and TNF-α levels. Anomalous apoptosis in PTSD may be a factor promoting the development of the chronic inflammation associated with this pathology. This study was supported by the State Science Committee of the Republic of Armenia. REFERENCES 1.

2.

O. A. Gomazkov, “Apoptosis of neuronal structures and the role of neurotrophic growth factors. Biochemical mechanisms of the efficacy of brain-derived peptide agents,” Zh. Nevrol. Psikhiat., Suppl. Stroke, No. 7, 17–22 (2002). L. P. Oganesyan, G. M. Mkrtchyan, S. G. Sukiasyan, and A. S. Boyadzhyan, “Classical and alternative complement cascades in posttraumatic stress disorder,” Byull. Eksperim. Biol. Med., 147, No. 12, 618–621 (2009).

Involvement of Anomalous Apoptosis in Impairments to Synaptic Plasticity 3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15. 16.

17.

18.

19.

20.

21.

22.

L. P. Oganesyan, G. M. Mkrtchyan, A. S. Boyadzhyan, et al., “Inflammation markers in post-traumatic stress disorder,” Tsitokin. Vospal., No. 11, 1 (2012). N. N. Petrishchev, L. V. Vasina, and A. V. Lugovaya, “Blood levels of soluble apoptosis markers and circulating annexin V-binding apoptotic cells in patients with acute coronary syndrome,” Vestn. St. Peterb. Univers., 11, No. 1, 14–23 (2008). V. V. Semchenko, S. S. Stepanov, and N. N. Bogolepov, Synaptic Plasticity of the Brain (basic and applied aspects), Omsk Regional Typography, Omsk (2008). M. Begemann, S. Grube, S. Papiol, et al., “Modification of cognitive performance in schizophrenia by complexin 2 gene polymorphisms,” Arch. Gen. Psychiatry, 67, No. 9, 879–888 (2010). D. D. Blake, F. W. Weathers, L. M. Nagy, et al., “The development of a clinician administered PTSD scale,” J. Trauma Stress, 8, No. 1, 75–90 (1995). H. H. Boersma, B. L. Kitselaer, L. M. Stolk, et al., “Past, present and future of Annexin A5: from protein discovery to clinical applications,” J. Nucl. Med., 46, No. 12, 2035–2050 (2005). N. Brose, “Altered complexin expression in psychiatric and neurological disorders: cause or consequence?” Mol. Cells., 25, No. 1, 7–19 (2008). J. J. Chae, H. D. Komarow, J. Cheng, et al., “Targeted disruption of pyrin, the FMF protein, causes heightened sensitivity to endotoxin and a defect in macrophage apoptosis,” Mol. Cell., 11, No. 3, 591–604 (2003). S. L. Chan and M. P. Mattson, “Caspase and calpain substrates: roles in synaptic plasticity and cell death,” J. Neurosci. Res., 58, 167–190 (1999). C. S. De Kloet, E. Vermetten, A. Bikker, et al., “Leukocyte glucocorticoid receptor expression and immunoregulation with and without post-traumatic stress disorder,” Mol. Psychiatry, 12, No. 5, 443–453 (2007). Diagnostic and Statistical Manual of Mental Disorders, 4th edition, revised text, American Psychiatric Association, Washington DC (2000). N. A. DiProspero, E. Y. Chen, V. Charles, et al., “Early changes in Huntington’s disease patient brains involve alterations in cytoskeletal and synaptic elements,” J. Neurocytol., 33, No. 5, 517–533 (2004). R. S. Duman, “Pathophysiology of depression: the concept of synaptic plasticity,” Eur. Psychiatry, 17, No. 3, 306–310 (2001). M. B. First, R. L. Spitzer, M. Gibbon, and J. B. Williams, Structured Clinical Interview for the DSM-IV Axis I Disorders, American Psychiatric Press Inc., Washington DC (1996). H. D. Flad, E. Grage-Griebenow, F. Peterson, et al., “The role of cytokines in monocyte apoptosis,” Pathobiology, 67, No. 5–6, 291–293 (1999). L. P. Francesconi, K. M. Ceresér, R. Mascarenhas, et al., “Increased annexin-V and decreased TNF-α serum levels in chronic-medicated patients with schizophrenia,” Neurosci. Lett., 502, No. 3, 143–146 (2011). W. Freeman and A. J. Morton, “Regional and progressive changes in brain expression of complexin II in a mouse transgenic for the Huntington’s disease mutation,” Brain Res. Bull., 63, No. 1, 45–55 (2004). J. M. Gill, L. Saligan, S. Woods, and G. Page, “PTSD is associated with an excess of inflammatory immune activities,” Perspect. Psychiatr. Care, 45, No. 4, 262–277 (2009). C. P. Gilman and M. P. Mattson, “Do apoptotic mechanisms regulate synaptic plasticity and growth-cone motility?” Neuromolec. Med., 2, No. 2, 197–214 (2002). D. Glynn, K. Reim, N. Brose, and A. J. Morton, “Depletion of complexin II does not affect disease progression in a mouse model of Huntington’s disease (HAD); support for role for complexin II in behavioural pathology in a mouse model of HAD,” Brain Res. Bull., 72, No. 2–3, 108–120 (2007).

23.

24. 25.

26.

27. 28.

29.

30. 31.

32.

33.

34. 35.

36.

37.

38. 39.

40.

41.

42.

43. 44.

45.

445

L. G. Grunnet, R. Aikin, M. F. Tonnesen, et al., “Proinflammatory cytokines activate the intrinsic apoptotic pathway in beta-cells,” Diabetes, 58, No. 8, 1807–1815 (2009). C. Haanen and I. Vermes, “Apoptosis and inflammation,” Mediators Inflamm., 4, No. 1, 5–15 (1995). J. Hart, T. Kimbrell, P. Fauver, et al., “Cognitive dysfunctions associated with PTSD: evidence from World War II prisoners of war,” J. Neuropsych. Clin. Neurosci., 20, 309–316 (2008). E. A. Hoge, K. Brandstetter, S. Moshier, et al., “Broad spectrum of cytokine abnormalities in panic disorder and post-traumatic stress disorder,” Depress. Anxiety, 26, No. 5, 447–455 (2009). M. Holcik, Apoptosis in Health and Disease: Clinical and Therapeutic Aspects, Cambridge University Press, UK (2005). G. Z. Huang, H. Ujihara, S. Takahashi, et al., “Involvement of complexin II in synaptic plasticity in the CA1 region of the hippocampus: the use of complexin II-lacking mice,” J. Pharmacol. (Jpn), 84, No. 2, 179–187 (2000). G. B. Kaplan, J. J. Vasterling, and P. C. Vedak, “Brain-derived neurotrophic factor in traumatic brain injury, post-traumatic stress disorder, and their comorbid conditions: role in pathogenesis and treatment,” Behav. Pharmacol., 21, No. 5–6, 427–437 (2010). Z. Li and M. Sheng, “Caspases in synaptic plasticity,” Mol. Brain, 14, No. 5, 15 (2012). A. L. Mahan and K. J. Ressler, “Fear conditioning, synaptic plasticity and the amygdala: implications for post-traumatic stress disorder,” Trends Neurosci., 35, No. 1, 24–36 (2012). M. P. Mattson and W. Duan, “‘Apoptotic’ biochemical cascades in synaptic compartments: Roles in adaptive plasticity and neurodegenerative disorders,” J. Neurosci. Res., 58, 152–166 (1999). M. Miura, H. Zhu, R. Rotello, et al., “Induction of apoptosis in fibroblasts by IL-1-converting enzyme, mammalian homolog of the C. elegans cell death gene ced-3,” Cell, 75, 653–660 (1993). S. A. Moore, “Cognitive abnormalities in post-traumatic stress disorder,” Curr. Opin. Psychiatry, 22, No. 1, 19–24 (2009). L. E. Munoz, B. Frey, F. Pausch, et al., “Role of annexin A5 in modulation of the immune response against dying and dead cells,” Curr. Med. Chem., 14, No. 3, 271–277 (2007). C. Nucci, S. Piccirilli, R. Nisticò, et al., “Apoptosis in the mechanisms of neuronal plasticity in the developing visual system,” Neuromolecular Med., 2, No. 2, 197–214 (2002). C. P. M. Reutelingsperger and W. I. Van Heerde, “Annexin V. The regulator of phosphatidylserine-catalyzed inflammation and coagulation during apoptosis,” Cell Mol. Life Sci., 53, 527–532 (1997). J. Savill, “Apoptosis in resolution of inflammation,” J. Leukocyte Biol., 61, 375–380 (1997). A. J. Silva, “Molecular and cellular cognitive studies of the role of synaptic plasticity in memory,” J. Neurobiol., 54, No. 1, 224–237 (2003). N. Sing, “Apoptosis in health and disease and modulation of apoptosis for therapy: an overview,” Indian J. Clin. Biochem., 22, No. 2, 6–16 (2007). M. Sugano, K. Tsuchida, and N. Makino, “Effects of soluble TNFalpha receptor 1 on apoptosis induced by oxidized LDL in endothelial cells,” Mol. Cell. Biochem., 258, 57–63 (2004). M. A. Swairjo, N. O. Concha, M. A. Kaetzel, et al., “Ca(2+)-bridging mechanism and phospholipid group recognition in the membranebinding protein annexin V,” Nat. Struct. Biol., No. 2,968–974 (1995). K. Takayima, “Molecular mechanisms of synaptic plasticity underlying learning and memory,” Seikagaku, 83, No. 11, 1016–1026 (2011). Y. Takeda, H. Watanabe, S. Yonehara, et al., “Rapid acceleration of neutrophil apoptosis by tumor necrosis factor-alpha,” Int. Immunol., 5, 691–694 (1993). R. K. Tannenberg, H. L. Scott, A. E. Tannenberg, and P. R. Dodd, “Selective loss of synaptic proteins in Alzheimer’s disease: evidence for an increased severity with APOE varepsilon4,” Neurochem. Int., 49, No. 7, 631–639 (2006).

446 46.

47.

48.

Mkrtchyan, Boyadzhyan, Avetyan, and Sukiasyan D. M. Vanags, S. Coppola, and D. H. Burgess, “Protease involvement in fodrin cleavage and phosphatidylserine exposure in apoptosis,” Biol. Chem., 271, 31075–31031 (1996). I. Vermes, E. N. Steur, C. Ratelingsperger, and C. Haanen, “Decreased concentration of annexin V in parkinsonian cerebrospinal fluid: speculation on the underlying cause,” Mov. Disord., 14, No. 6, 1008–1010 (1999). R. von Känel, B. Kraemer, R. Traber, et al., “Evidence for low-grade systemic proinflammatory activity in patients with post-traumatic stress disorder,” J. Psychiatr. Res., 41, No. 9, 744–752 (2007).

49.

50.

51.

S. R. Wiley, K. Schooley, P. J. Smolak, et al., “Identification and characterization of a new member of the TNF family that induces apoptosis,” Immunity, 3, No. 6, 673–682 (1995). M. Yamaguchi, Y. Kokai, S. Imai, et al., “Investigation of annexin A5 as a biomarker for Alzheimer’s disease using neuronal cell culture and mouse model,” J. Neurosci. Res., 88, No. 12, 2682–2692 (2010). Y. Yamauchi, L. H. Qin, M. Nishihara, et al., “Vulnerability of synaptic plasticity in the complexin II knockout mouse to maternal deprivation stress,” Brain Res., 1056, No. 1, 59–67 (2005).