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Decreased mRNA and Protein Expression of BDNF, NGF, and their Receptors in the Hippocampus from Suicide: An Analysis in Human Postmortem Brain

Despite the devastating effect of suicide on numerous lives, there is still a lack of knowledge concerning its neurochemical aspects. There is increasing evidence that brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are involved in the pathophysiology and treatment of depression through binding and activating their cognate receptors TrkB and TrkA respectively. The present study was performed to examine whether the expression profiles of BDNF and/or TrkB as well as NGF and/or TrkA were altered in the hippocampus of postmortem brain of the participants, who had committed suicide and whether these alterations were associated with specific psychopathologic conditions. These studies were performed on the hippocampus of 21 suicide victims and 19 non-psychiatric control individuals. The protein and mRNA levels of BDNF, TrkB, NGF, and TrkA were determined by sandwich enzyme-linked immunosorbent assay, Western blot and reverse transcription-PCR. Given the importance of BDNF and NGF and their cognate receptors in mediating physiological functions, including cell survival and synaptic plasticity, our findings of reduced expression of BDNF, TrkB, NGF, and TrkA on both the protein and mRNA levels of postmortem brains of suicide victims suggest that these molecules may play an important role in the pathophysiological aspects of suicidal behavior.


Introduction
Suicide is a major public health concern; however, its neurobiology is unclear. Postmortem brain tissue obtained from suicide victims irrespective of their status of depression and normal controls offers a useful method for studying the neurobiology of suicide. These studies, though preliminary, have offered important insights into the altered neurochemical milieu of suicide. In this article, we focus on the role of neurotrophins in suicide. Similarly to all other regions worldwide, suicide attempts in India have been increasing progressively. Despite the devastating effect of suicide on numerous lives, there is still a lack of knowledge concerning its underlying causes and pathologic mechanisms. Several clinical and epidemiological studies have identified stress as an important risk factor in suicide. 1,2 The role of neurotrophins in directing brain growth and neuronal functioning is being increasingly recognized. Neurotrophins not only play an important role in cellular proliferation, migration, and phenotypic differentiation and/or maintenance in the developing central nervous system, 2,3 but also their presence is required in the adult central nervous system for maintenance of neuronal functions, structural integrity of neurons, and neurogenesis, 2,3 suggesting that neurotrophins are biologically significant over the entire lifespan. In addition, a number of studies have demonstrated that neurotrophic factors regulate structural and synaptic and morphological plasticity to modulate the strength or number of synaptic connections and neurotransmission. 4,5 Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, plays a key role in the development and survival of neurons in the central nervous system. 6 BDNF specifically binds to the tyrosine kinase receptor tropomyosin-related kinase B receptor (TrkB) and regulates many functions related to neuronal development such as neurite outgrowth, synthesis of differentiating factors and morphological plasticity. 6 In adulthood, BDNF is involved in neural homeostasis and in processes related to neuronal plasticity and connectivity, including learning and memory, 7,8 drug addiction, 9 response to social stress, aggressiveness, and anxiety-related behaviors. 10,11 Alterations in BDNF expression in specific neurons may reduce neural plasticity, therefore impairing the ability to respond to stressors and contributing to various neurodegenerative and neuropsychiatric disorders including depression and bipolar disorders. 12 Nerve growth factor (NGF), the prototypical growth factor, is a protein secreted by a neuron's target cell. NGF is critical for the survival and maintenance of sympathetic and sensory neurons. NGF is released from target cells, binds to and activates its high-affinity receptor TrkA on the neuron, and is internalized into the responsive neuron. It also functions as a signaling molecule. 13,14 The NGF/TrkA complex is subsequently trafficked back to the neuron's cell body. This movement of NGF from axon tip to soma is thought to be involved in the long-distance signaling of neurons. NGF appears to play a role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis-mediated stress response, which exceeds its well-known neurotrophic function within the nervous system.
Results of many preclinical and clinical studies demonstrate that several types of stressors regulate the expression of BDNF and NGF in the brain. [15][16][17][18][19][20][21][22] In addition, preclinical and clinical study results indicate that they both may be involved in depression and in the mechanism of action of antidepressants. 23,28 Given the importance of BDNF and NGF in maintenance of the structural integrity and synaptic plasticity of the brain and their involvement in stress and affective disorders, we investigated the correlation of these neurotrophins with suicidal behavior by examining the expression of BDNF and NGF along with their cognate receptors in the postmortem brain of participants who committed suicide (Suicide participants) and nonpsychiatric healthy control individuals. Since BDNF and NGF mediate biological action through the activation of BDNF/TrkB and NGF/TrkA signaling cascades, [29][30][31] we examined alterations in the expression profiles of BDNF and/or TrkB along with NGF and/or TrkA in all suicide participants, irrespective of psychiatric diagnosis compared to healthy control participants.
In the present study, neurotrophins and their cognate receptors at both the protein and mRNA levels were compared in hippocampus obtained from suicide victims and non-psychiatric healthy control individuals.

Methods Subjects
Postmortem brain samples from suicide participants who died by suicide with and without major depression and non-psychiatric healthy control participants were obtained from the Calcutta Medical College Hospital under the supervision of the Institutional Ethical Committee. The hippocampal region of the brains were dissected and stored at −80 °C. We determined the selected neurotrophins and their respective receptors mRNA and protein expression in the hippocampal areas obtained from 19 nonpsychiatric healthy control individuals and 21 suicide victims. Detailed demographic characteristics of control and suicide participants are shown in Table 1

Diagnostic methodology
Psychiatric diagnoses of subjects were made using the psychological autopsy method. This technique has been validated for Axis I and II diagnoses. 32,33 Informants included primarily family member who were most familiar with the deceased such as the mother, father, siblings, friends, or relatives to serve as an informer and undergo the interview process. After giving written informed consent, at least one family member was interviewed using the Diagnostic Evaluation After Death (DEAD) 34,35 and the Schedule for Clinical Interviews for the DSM-IV (SCID) 36 by a psychiatrist. Information collected through SCID I and II interviews and from the coroner's notes and medical records were used by interviewers to write a case history for each subject. These case histories were then reviewed by a clinical panel in order to reach a consensus regarding the DSM-IV diagnosis for each subject. Similarly, controls were verified as free from mental illness using similar consensus diagnostic procedures.
The standard deviations (SD) of the mean age of non-psychiatric healthy control and suicide victims were 15.93 and 14.94. The age range was 15 to 80 years for normal participants and 15 to 75 years for suicide victims.
The study was performed on postmortem samples of brain tissue extracted from the hippocampal area obtained from 21 participants who died by suicide and 19 non-psychiatric healthy control participants.
Autoptic samples were collected within 12 to 26 h of the subject's death (postmortem interval information is reported in the Table 1), in the course of autopsy at the Calcutta Medical College Hospital Morgue, and stored at −80 °C. Data on subject's age range, drug history, and cause of death were gathered from the medical records of suicide victims. A detailed description of the subjects analyzed in this study is reported in the Table 1.

extraction of hippocampus from suicide victims and nonpsychiatric healthy controls
The brains of suicide participants and control individuals were removed for isolation of the hippocampal tissues. The hippocampus was cut into 0.5-cm coronal slices of the middle of the hippocampus, which included the dentate gyrus and areas CA1-4.
Quantitative estimation of BDNF and NgF levels in the hippocampus of suicide victims and non-psychiatric healthy controls by sandwich eLISA Endogenous BDNF and NGF levels were measured in hippocampus samples using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Chemicon, Temecula, CA, USA). Hippocampi were immediately extracted the method described above. Rabbit polyclonal antibodies generated against human BDNF and NGF were coated onto two separate microplates, which were used to capture BDNF and NGF, respectively, from the samples. BDNF-specific biotin-conjugated, mouse monoclonal antibodies were used to detect the captured BDNF and NGF separately. After addition of streptavidin-enzyme substrate and stop solution, the amount of BDNF and NGF were determined. The standard curve showed a direct relationship between optical density (OD) and BDNF and NGF concentrations: i.e., a higher OD indicated higher the BDNF and NGF concentrations in the samples. The amount of BDNF and NGF were determined by measuring the absorbance at 450 nm (Tecan Infinite M200, Mäennedorf, Switzerland). A standard curve was plotted, which ranged from 7.8-500 pg/mL of BDNF and NGF. These curves were obtained based on the direct relationship between optical density (OD) and neurotrophin concentrations. Total protein concentration was measured by Bradford method using bovine serum albumin (BSA) as a standard.

Immunoprecipitation of BDNF and NgF
Supernatant containing 100 µg of protein was incubated with antibodies against BDNF and NGF (100:1 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at 4 °C. A suspension of protein-A sepharose beads (Amersham, NJ, USA) in Trisbuffered saline (TBS) were added and incubated at 4 °C for 1 h. The pellet was collected by centrifugation at 2,500 rpm for 30 s at 4 °C and washed four times with TBS containing 0.5 mM Na 3 VO 4 and 0.01 mM phenylmethylsulfonyl fluoride. The pellet was resuspended in 2× sample buffer, boiled for 5 min, and subjected to Western blot analysis.
The OD value of each band was analyzed with the electrophoresis image analysis system (SmartView Pro Imager System, USA). The OD of each protein was determined by using the OD of the corresponding β-actin band. The values are presented as a percent of the control.
The PCR mixture was amplified for 32 cycles with denaturation (94 °C, 15 s), annealing (60 °C, 30 s), and elongation (72 °C, 30 s) amplification steps. The reaction was terminated with a 5-min final elongation step (72 °C, 5 min). The PCR products were observed by electrophoresis on 1.5% agarose gel and the density of each band was analyzed on the gel image analysis system (Smartview 2001, S/N: SV-0002202). The level of the mRNA was determined by calculating the density ratio of each band of BDNF, TrkB, NGF and TrkA mRNA to GAPDH mRNA.

Statistical evaluation
The Statistical Package for the Social Science (SPSS) 15.0 was utilized for statistical analyses. All data are expressed as mean ± standard deviation (SD) of n subjects, and have been statistically analyzed with the Student's t-test, determined correlation-coefficient (r), and regression. P values less than 0.001 and 0.05 were considered statistically significant. Statistical evaluation through analysis of variance was also used to determine the demographic characteristics of suicide victims (n = 21) and non-psychiatric healthy control individuals (n = 19).

Demographic characteristic of postmortem brain of suicide victims and non-psychiatric healthy control individuals
The demographic characteristics of suicide victims (n = 21) and non-psychiatric healthy control individuals (n = 19) was described earlier. The mean postmortem interval was 20.17 ± 5.4 h for control subjects and 21.45 ± 4.46 h for suicide victims. The brain pH for control participants was 6.19 ± 0.53 and for suicide victims was 6.54 ± 0.35. There were no signifi- Quantitative analysis of BDNF and NgF in the hippocampus of suicide victims and non-psychiatric healthy control individuals We quantitatively measured BDNF and NGF concentrations by sandwich ELISA in the hippocampus of suicidal victims and non-psychiatric healthy controls. Suicide victims showed significantly reduced BDNF concentration (t = 20.14; df = 19; P , 0.001; Fig. 1) compared to non-psychiatric healthy control individuals. Similarly, hippocampal NGF concentration was also significantly reduced (t = 13.39; df = 19; P , 0.001; Fig. 1) in suicide victims compared to non-psychiatric healthy control persons. expression of BDNF, NgF, TrkB, and TrkA protein in the hippocampus of suicide victims and non-psychiatric healthy control individuals We analyzed expression profiles of both neurotrophins BDNF and NGF in the hippocampus by Western blotting and observed significantly reduced levels (t BDNF = 15.36; df = 19; P , 0.001 and t NGF = 20.80; df = 19; P , 0.001 respectively; Fig. 2) among suicide victims compared to non-psychiatric healthy controls. Similarly, reduced expression was also observed during the analysis of their receptor, that is t TrkB = 21.99; df = 19; P , 0.001 and t TrkA = 22.23; df = 19; P , 0.001 in Figure 2. The molecular weight of BDNF, NGF, TrkB, TrkA, and β-actin were 14, 13.5, 145, 140, and 42 kDa, respectively. Expression of BDNF, NGF, TrkB, and TrkA proteins were normalized against the internal control β-actin to determine their expression. mrNA expression of BDNF, NgF, TrkB, and TrkA in the hippocampus of suicide victims and non-psychiatric healthy control individuals We also measured mRNA expression of BDNF, NGF, TrkB, and TrkA that were significantly reduced (t BDNF = 9.31; df = 19; P , 0.001; t TrkB = 28.25; df = 19; P , 0.001; t NGF = 19.74; df = 19; P , 0.001 and t TrkA = 26.23; df = 19; P , 0.001; Fig. 3) in the hippocampus of participants who died by suicide and the differences were statistically significant. The lengths of BDNF, NGF, TrkB, TrkA, and GAPDH amplified fragments were 178, 199, 79, 96, and 173 base pairs, respectively. The levels of BDNF, NGF, TrkB, and TrkA mRNA were normalized against GAPDH mRNA levels as an internal control. GAPDH mRNA expression was not altered among the participants of both control and suicide

Discussion
Depression is a complex disorder that involves multiple neuronal substrates and brain regions. Among several alterations, neurotrophins regulation, particularly low levels of BDNF and NGF, have been suggested to lead to a depression-like state.
In the present study, quantitative evaluations of BDNF, NGF, and their cognate receptors TrkB and TrkA at both the protein and mRNA levels in the hippocampus of 21 participants who died by suicide and 19 non-psychiatric healthy controls were determined by sandwich ELISA, Western blot, and real-time PCR. The main conclusions of our study are: (1) suicide subjects showed a statistically significant reduction of protein levels of BDNF, NGF, and their cognate receptors as investigated by ELISA and Western blot, respectively and (2) we observed a statistically significant decrease in translation of BDNF along with its receptor TrkB as well as NGF along with its receptor TrkA at the mRNA level.
Multiple lines of evidence indicate that BDNF is present at low levels in the blood cells and plasma of depressed patients. Postmortem suicide victims with or without depression and genetic association studies linking BDNF to suicide suggest that suicidal behavior may be associated with a decrease in BDNF function. 37,38 BDNF alone or in combination with other neurotransmitters is known to regulate synaptic plasticity, neurogenesis, and neuronal survival through its specific signaling pathways. 39,40 Additionally, stressful events such as a suicide attempt may alter the responsiveness of the HPA system and stress-induced elevation of glucocorticoids to reduce expression of BDNF in the blood and brain. 41 In the present study, hippocampal BDNF and TrkB contents were significantly reduced among individuals who died by suicide compared to non-psychiatric healthy individuals which agrees with the results of Dwivedi et al. 18 Similarly, when mRNA expression level was determined among these individuals, the mRNA levels of BDNF and TrkB were significantly reduced in the hippocampus of suicide victims compared to in non-psychiatric healthy control individuals. Such brain-derived neurotrophic factors exert their physiological effects by binding with TrkB receptors, which exist in truncated and full-length forms, both of which are important in mediating the functions of BDNF. 42,44 NGF content may be reduced by changes in neuronal activity during stress. In support of this hypothesis, it has been demonstrated that expression of hippocampal NGF is dependent on neuronal activation and release of cholinergic neurotransmitters. 45,46 NGF may be important for counteracting the neurotoxic effects of glucocorticoids, which are elevated during stress. 47 The observed reduction in NGF after a stressful experience may be of pathophysiological relevance. NGF serves as a survival protein for cholinergic neurons; it can reverse cellular damage and reduce vulnerability to toxic influences. 48 Thus, a reduction in NGF content due to stress may have detrimental consequences for the survival of cholinergic neurons. Our study showing a significant reduction in hippocampal NGF and TrkA mRNA expression among the individuals who died by suicide compared to normal controls agree with the observations of Dwivedi et al. 18 The results of the present study indicate significant defective brain neurotrophin milieus in suicide victims, strengthening the role of neurotrophins in the pathophysiology of suicide.
Though the present findings are of particular relevance for understanding the neurochemical basis of suicide, it was beyond the scope of the present investigation conduct an in-depth investigation of the molecular mechanisms of these neurochemical alterations associated with stress. Future studies are needed to not only understand the molecular basis of the disease but also to design future therapeutic modalities targeting those molecular pathways.

Disclosures and ethics
As a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with ICMJE authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. This article was subject to blind, independent, expert peer review. The reviewers reported no competing interests.