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
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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
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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.
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