International Journal of Neuropsychopharmacology (2007), 10, 621–629. Copyright f 2006 CINP doi:10.1017/S1461145706007231
Cyclic AMP response element-binding protein in post-mortem brain of teenage suicide victims: specific decrease in the prefrontal cortex but not the hippocampus
ARTICLE
CINP
Ghanshyam N. Pandey1, Yogesh Dwivedi1, Xinguo Ren1, Hooriyah S. Rizavi1, Rosalinda C. Roberts2 and Robert R. Conley2 1 2
The Psychiatric Institute, Department of Psychiatry, University of Illinois at Chicago, Chicago, IL, USA Maryland Psychiatric Research Center, Baltimore, MD, USA
Abstract Abnormalities in both adenylyl cyclase (AC) and phosphoinositide (PI) signalling systems have been observed in the post-mortem brain of suicide victims. Cyclic AMP response element-binding protein (CREB) is a transcription factor that is activated by phosphorylating enzymes such as protein kinase A (PKA) and protein kinase C (PKC), which suggests that both AC and PI signalling systems converge at the level of CREB. CREB is involved in the transcription of many neuronally expressed genes that have been implicated in the pathophysiology of depression and suicide. Since we observed abnormalities of both PKA and PKC in the post-mortem brain of teenage suicide victims, we examined if these abnormalities are also associated with abnormalities of CREB, which is activated by these phosphorylating enzymes. We determined CRE–DNA binding using the gel shift assay, as well as protein expression of CREB using the Western blot technique, and the mRNA expression of CREB using a quantitative reverse transcriptase– polymerase chain reaction (RT–PCR) technique in the prefrontal cortex (PFC), and hippocampus obtained from 17 teenage suicide victims and 17 matched normal control subjects. We observed that the CRE–DNA binding and the protein expression of CREB were significantly decreased in the PFC of teenage suicide victims compared with controls. There was also a significant decrease in mRNA expression of CREB in the PFC of teenage suicide victims compared with control subjects. However, there were no significant differences in CRE–DNA binding or the protein and mRNA expression of CREB in the hippocampus of teenage suicide victims compared with control subjects. These results suggest that the abnormalities of PKA, and of PKC, observed in teenage suicide victims are also associated with abnormalities of the transcription factor CREB, and that this may also cause alterations of important neuronally expressed genes, and provide further support of the signal transduction of abnormalities in suicide. Received 27 April 2006 ; Reviewed 8 June 2006 ; Revised 8 August 2006 ; Accepted 10 August 2006; First published online 18 September 2006 Key words : CREB, DNA binding, immunolabelling, mRNA, post-mortem brain, suicide, teenage.
Introduction Teenage suicide is a major public health concern in the USA. It is the third leading cause of death after automobile accidents and homicide (Moscicki et al., 1988). Whereas there is some understanding of the psychosocial factors associated with teenage suicide, the Address for correspondence : G. N. Pandey, Ph.D., The Psychiatric Institute, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA. Tel. : (312) 413-4540 Fax : (312) 413-4547 E-mail :
[email protected]
neurobiology of teenage suicide has been relatively unexplored. Some recent studies suggest that the risk factors and the characteristics of teenage suicide may be similar in some respects to those of adult suicide, but different in others (Apter et al., 1995 ; Brent et al., 1993). Although impulsive-aggressive behaviour is a risk factor for both adult and teenage suicide, adolescent suicide may be more driven, in part, by impulsive-aggressive behaviour, since adolescent suicide completers have more impulsive-aggressive personality disorders and higher aggression ratings than control subjects (Apter et al., 1995; Brent et al., 1993).
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Neurobiological studies suggest abnormalities of the phosphoinositide (PI) and the adenylyl cyclase (AC) signalling systems, as well as several of their components, in the post-mortem brain of suicide victims (Arango et al., 1990 ; Dwivedi et al., 2002a,b, 2004 ; Gross-Isseroff et al., 1998 ; Jope et al., 1996 ; Mann et al., 1986 ; Pachecho et al., 1996 ; Pandey and Dwivedi, 2005 ; Pandey et al., 1997, 1999, 2004). Activation of transcription factors is the final step in the signal transduction pathway, which is mediated by the binding of a cell surface receptor with an agonist. One of the mechanisms by which transcription factors are activated is phosphorylation and dephosphorylation (Nestler and Greengard, 1994). The phosphorylating enzymes protein kinase A (PKA) and protein kinase C (PKC) in the AC and the PI signalling systems, respectively, cause the phosphorylation of several transcription factors, including the AP-1 family of transcription factors (Jun-B, Jun-D) and cyclic AMP (cAMP) response element-binding (CREB) protein (Nichols et al., 1992 ; Riabolow et al., 1988 ; Xie and Rothstein, 1995). The evidence that changes in PKC may result in alterations of CREB activity and phosphorylation was also provided by a recent study of Alkan et al. (2005). They found that inhibition of PKC by safingol in chronic lymphocytic leukaemia (CLL) cells not only decreased the protein and gene expression of PKC isozymes but also decreased CREB phosphorylation and CRE–DNA binding activity. Recently, we have reported that PKA activity, cAMP-binding sites as well as the protein and mRNA expression of PKA catalytic and regulatory subunits are altered in the post-mortem brain of adult (Dwivedi et al., 2002a,b, 2004) as well as teenage suicide victims (Pandey et al., 2005). We also reported that the PKC activity and protein and mRNA expression of some specific isozymes of PKC are decreased in the postmortem brain of teenage suicide victims compared with control subjects (Pandey et al., 2004). CREB is a member of the basic leucine zipper family of transcription factors (Borrelli et al., 1992). Phosphorylation of CREB at serine 133 leads to its dimerization and activation by binding to cAMP response element (CRE) at the consensus motif 5k-TGACGTCA which is found in many neuronally expressed genes (Lee and Masson, 1993 ; Montminy et al., 1990). In its active form, the phosphorylated form, CREB regulates the transcription of many genes that are involved in several aspects of neuronal functioning, including the excitation of neuronal cells (Marshall and Dragunow, 2000 ; Moore et al., 1996), CNS development (Imaki et al., 1994), and long-term
synaptic plasticity (Silva et al., 1998). Since we found that both PKA and PKC are altered in the post-mortem brain of teenage suicide victims, and since these two kinases activate the transcription factor CREB, we hypothesize that the observed PKA and PKC decrease may lead to decreased expression and/or functional characteristics of CREB in the post-mortem brain of teenage suicide victims. In order to test this hypothesis, in the present investigation we determined the protein and mRNA expression of CREB in the prefrontal cortex (PFC) and hippocampus of teenage suicide victims. We also determined the CRE–DNA binding activity in these brain areas of teenage suicide victims and matched control subjects. Materials and methods Acquisition of human post-mortem brain samples Brain tissues were obtained from the Maryland Brain Collection at the Maryland Psychiatric Research Center, Baltimore, MD, in collaboration with the Office of the Chief Medical Examiner of the State of Maryland. Tissue samples were obtained from 17 teenage suicide victims and from 17 teenage control subjects (Table 1). Toxicological data were obtained by analysis of urine and blood samples from these subjects. All procedures were approved by the University of Maryland IRB. All subjects in this study were diagnosed using the Diagnostic Evaluation After Death (DEAD) and the Schedule for Clinical Interviews for DSM-IV (SCID). Family members gave permission for the use of brain tissue for research, clinical records to be obtained from mental health treatment providers when there was a prior history of mental health treatment, or suicide. Two senior psychiatrists provided independent DSM-IV diagnoses. Similarly, normal controls were verified as free from mental illnesses using such consensus diagnostic procedures. Preparation of nuclear fractions The preparation of nuclear fractions followed the protocol from Pierce Biotechnology Inc. (Rockford, IL, USA). Briefly, tissue was homogenized in ice-cold cytoplasmic extraction reagent I (CER I) containing 0.5 mg/ml benzamidine, 2 mg/ml aprotinin, 2 mg/ml leupeptin, and 0.75 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was added to cytoplasmic extraction reagent II (CER II) and then centrifuged at 16 000 g for 15 min. The resulting pellet was suspended in ice-cold nuclear extraction reagent (NER) containing 0.5 mg/ml benazmide, 2 mg/ml aprotinin,
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Table 1. Demographic characteristics of teenage suicide and normal control subjects Group and subject
Age (yr)
Sex
Race
PMI (h)
pH
Diagnosis
Control 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
19 13 16 19 16 18 19 17 16 18 17 13 14 18 16 17 18
Male Male Male Male Male Male Male Male Male Male Male Male Male Male Male Female Male
Black White Black Black Black White Black Black White Black Black Black Black Black White Black White
6 n.a. 6 9 8 8 12 11 10 11 10 22 18 27 21 26 19
6.14 5.19 6.54 6.85 5.64 6.7 5.9 6.52 5.42 6.16 5.9 6.07 5.73 6.09 5.97 6.23 6
Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control Control
Mean
16.71
16 Male, 1 Female
5 White, 12 Black
13.97
6.04
7.1
0.41
S.D.
1.93
Suicide 18 19 20 21 22
20 19 15 12 15
Female Male Female Male Female
White White White Black White
11 12 7 10 11
6.5 5.9 5.48 5.91 6.44
Schizophrenia Drug abuse Alcohol abuse Major depression No mental disorder
23 24 25
17 16 13
Male Male Male
White Black White
11 12 18
5.9 5.4 6
26 27 28
13 15 15
Male Male Female
Black White White
11 11 17
5.37 5.33 5.58
29 30 31
16 17 15
Male Female Male
Hispanic White White
20 25 27
6.17 5.55 6.08
32 33 34
16 16 16
Female Female Male
White White White
18 33 24
6.31 6.61 6.81
Adjustment disorder No mental disorder Hyperactive and attention deficit Undetermined Major depression Major depression and hyperactive and attention deficit No mental disorder Adjustment disorder Adjustment disorder and major depression No mental disorder Adjustment disorder Adjustment and conduct disorder
15.65
10 Male, 7 Female
13 White, 3 Black, 1 Hispanic
16.12
5.96
7.54
0.47
Mean
S.D.
1.99
Drug toxicity
– – – – – – – – – – – – – – – – –
Cause of death
GSW Accident GSW GSW GSW Stabbing GSW GSW Stabbing GSW GSW GSW GSW Drowning Accidental hanging Multiple injuries Drowning
None None Ethanol None Phenylpropanolamine, chlorophenylamine, codeine, salicylate, acetaminophene None None Ritalin
Jump from height Hanging GSW Hanging Drug overdose
None None Imipramine, desipramine
GSW Asphyxia Drug overdose
None Verapamil Pseudoephedrine, phenylpropanol amine Amitriptyline None None
Hanging Drug overdose GSW
PMI, Post-mortem interval ; GSW, gunshot wound ; n.a., not available ; S.D., standard deviation.
Electrocution GSW Hanging
GSW GSW Hanging
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2 mg/ml leupeptin, and 2 mM PMSF and incubated for 40 min on ice with frequent agitation. The nuclear extracts were separated by centrifugation at 16 000 g for 10 min. The protein content of the nuclear fraction was determined by the method of Lowry et al. (1951). This nuclear fraction was used to determine the protein expression of CREB and CRE–DNA binding activity. Quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) analysis of CREB mRNA levels Total RNA was isolated by means of caesium chloride ultracentrifugation. The yield of total RNA was determined by measuring the absorbency of an aliquot of the precipitated stock at a wavelength of 260/280 nm. We assessed the degradation of mRNA using denaturing agarose gel electrophoresis and evaluating the sharpness of 28S and 18S rRNA bands. Internal standards were designed to introduce a BglII restriction site midway between the amplification primers so that the digestion of the amplicon would generate two fragments of approximately equal molecular size. Each of the internal standards was synthesized in two PCR steps, starting with a complementary DNA (cDNA) template reverse transcribed from the human brain RNA. In-vitro cRNA synthesis was accomplished by linearizing the templates with SspI, which cuts 601 base pairs (bp) downstream of the EcoRI 3k cloning site. The cRNA corresponding to the sense strand was synthesized using 4–8 mg of the linearized template with SP6 RNA polymerase. CREB mRNA was analysed by adding decreasing concentrations of internal standard cRNA to 1 mg total RNA. The RNA/cRNA mixtures were denatured at 80 xC for 6 min and then reverse transcribed with cloned M-MLV reverse transcriptase in RT buffer containing 50 mM Tris–HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 mM dNTPs using random hexamers (2.5 mM) and ribonuclease inhibitor (28 U) in a 20 ml volume. The RT mixture was incubated at 37 xC for 60 min to promote cDNA synthesis. The reaction was terminated by heating the samples at 98 xC for 5 min. Competitive PCR amplification of cDNA aliquots containing reverse transcribed material was performed with Hot Tub DNA polymerase (Amersham, Arlington Heights, IL, USA). The amplification mixture contained cDNA, 0.5 mM specific primer pairs, 200 mM dNTPs, 1.5 mM MgCl2, 50 mM Tris–HCl (pH 9.0), 20 mM ammonium sulphate, 15 mM KCl, and 1.5 U of Hot Tub DNA polymerase in a 50 ml volume. Trace amounts of [32P]dCTP (0.5 mCi) were included during the PCR step for subsequent quantification.
The PCR mixture was amplified for 32 cycles with denaturation (94 xC, 15 s), annealing (60 xC, 30 s), and elongation (72 xC, 30 s) amplification steps. The reaction was terminated with a 5 min final elongation step (72 xC, 5 min). Following amplification, aliquots were digested with BglII and run on 1.5 % agarose gel electrophoresis. External primer sequences, forward : 5k-GTTCAGTCTTCCTGTAAGGAC ; reverse : 3k-GTT-AGCCAGCTGTATTGCTCC. Internal primer sequences, forward : 5k-GAATGACAGATCTTCTGATGCACC ; reverse : 3k-GGTGCATCAGAAGATCTGTCATTC (bold italicized letters indicate the mutated bases). Immunolabelling of CREB The procedure for Western blotting has been described in detail (Dwivedi et al., 2003). Protein samples (30 mg protein) were loaded onto 10 % (w/v) sodium dodecyl sulphate (SDS)–polyacrylamide gel. The gels were run and transferred electrophoretically to an enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham). The membranes were washed with TBST buffer [10 mM Tris-base, 0.15 M NaCl, and 0.05 % (v/v) Tween 20] for 10 min. The blots were blocked by incubating with 5 % (w/v) powdered nonfat milk in TBST, 0.02 % nonidet P-40, and 0.02 % (w/v) SDS (pH 8.0). Then the blots were incubated overnight at 4 xC with primary polyclonal anti-CREB antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) with a dilution of 1 : 3000. The membranes were washed with TBST and incubated with horseradishperoxidase-linked secondary antibody [anti-rabbit immunoglobulin G (IgG) ; 1 : 2000] for 5 h at room temperature. The membranes were extensively washed with TBST and exposed to ECL autoradiography film. The same nitrocellulose membrane was stripped and re-probed with b-actin antibody (Sigma Chemical Co., St. Louis, MO, USA). The bands on the autoradiogram were quantified using the Loats Image Analysis system (Westminster, MD, USA), and the optical density of each sample was corrected by the optical density of the corresponding b-actin band. The values are represented as a percent of control. Determination of CREB–DNA binding activity by gel-mobility shift assay Preparation of DNA probe : commercially available (Stratagene ; La Jolla, CA, USA) oligonucleotides incorporating regulatory elements of the CREB sequence (5k-GATTGGCTGAC-GTCA GAGAGCT) are used. The probes are end-labelled with [c-32P]ATP using T4
CREB in teenage suicide
Statistical analysis Statistical differences in age, post-mortem interval (PMI), and gender, CRE–DNA binding activity, and protein and mRNA expression of CREB between normal controls and suicide victims were evaluated by Student’s t test. The relationships between PMI, age, gender, CRE–DNA binding activity, and protein and mRNA expression of CREB were determined by Pearson product-moment correlation analysis. Differences between suicide victims with and without mental disorders were evaluated by one-way ANOVA. Results CRE–DNA binding activity, and protein and mRNA expression of CREB were determined in the PFC (Brodmann’s area 9) obtained from 17 teenage suicide victims, as well as 17 teenage normal control subjects. The demographic characteristics, as well as the PMI and toxicology and diagnosis of the subjects, are shown in Table 1. CRE–DNA binding activity in the PFC and hippocampus of suicide victims and control subjects We determined the functional status of CREB by determining the CRE–DNA binding activity using a gel mobility shift assay in the PFC and hippocampus of teenage suicide victims. A representative autoradiogram showing CRE–DNA binding activity in the PFC of five suicide victims and five control subjects is shown in Figure 1a. The mean CRE–DNA binding activity in suicide victims was significantly decreased (p
