Association of SOD2, a mitochondrial antioxidant enzyme, with gray matter volume shrinkage in alcoholics.

Chronic alcoholism leads to gray matter shrinkage and induces the formation of superoxide anions (O(2)(-)) that can cause neuronal cell death. The mitochondrial superoxide dismutase 2 (SOD2) enzyme is critical in the metabolism of superoxide. An Ala16Val polymorphism putatively affects SOD2 enzyme activity in vivo. Brain volumes of 76 treatment-seeking alcohol-dependent individuals were measured with a 1.5T MRI. Intracranial tissue margins were manually outlined on coronal sections. Gray matter, white matter, sulcal, and ventricular CSF volumes were estimated using intensity-based K-means clustering. Ala16Val (rs4880) and a second haplotype tagging SNP, rs10370, were genotyped. The q-value package was used to correct for multiple comparisons. In the alcoholics, cerebrospinal fluid and intra-cranial volumes showed significant differences across the six diplotype categories. The homozygous Ala16-containing diplotype rs10370TT-rs4880GG was associated with lowest gray matter ratio (greater shrinkage; p=0.005). Presence of one or two copies of the low activity Ala16 allele was a risk factor for lower gray matter volume in alcoholics below the median alcohol consumption (p=0.03) but not in alcoholics above this level. White matter ratio was associated with sex (p=0.002) and lifetime total alcohol consumption (p=0.01) but not with diplotypes. In this exploratory analysis, a putative functional missense variant of SOD2 appears to influence gray matter loss in alcoholics. This may be due to impaired clearance of reactive oxygen species formed as a result of alcohol exposure. The risk/protective effect was observed in alcoholics with lower levels of lifetime alcohol consumption. Highest levels of exposure may overwhelm the protective action of the SOD2 enzyme.

BDNF gene polymorphism (Val66Met) predicts amygdala and anterior hippocampus responses to emotional faces in anxious and depressed adolescents.

A polymorphism of the human Brain Derived Neurotrophic Factor (BDNF) gene that produces a valine-to-methionine substitution at codon 66 (Val66Met) is linked to adult anxiety and mood disorders, possibly through effects on brain circuitry function. Associations between BDNF gene variants and brain activity have not been explored in anxious and depressed adolescents. The current study investigated the association between BDNF genotype and amygdala-hippocampal responses to emotional stimuli in adolescents with anxiety disorders and/or major depressive disorder (MDD) and in healthy adolescents. Twenty-seven unmedicated patients with acutely-impairing current anxiety disorders and/or MDD and 31 healthy adolescents, matched on age, gender and IQ, rated their fear of fearful, angry, neutral and happy facial expressions during collection of fMRI data on the amygdala and hippocampus. Left and right amygdala and hippocampal responses were analyzed using repeated-measures analyses of variance models, with diagnosis (patients, healthy) and genotype (Met-carriers, Val/Val homozygotes) as between-group factors and facial expression (fearful, angry, neutral, happy) as a within-subject factor. Significant effects of diagnosis and diagnosis-by-genotype interactions (F's>4, p's<0.05) characterized activations in amygdala and anterior hippocampal regions. Greater activations in patients than healthy adolescents were found. Critically, these hyperactivations were modulated by BDNF genotype: Met-carriers showed greater neural responses of emotional faces than Val/Val homozygotes in patients only. These data are first to demonstrate the contribution of BDNF gene variants to the neural correlates of adolescent anxiety and depression. Early "gene-brain" linkages may lay the foundation for longer-term patterns of neural dysfunction in affective disorders.

Serotonin polymorphisms and posttraumatic stress disorder in a trauma exposed African American population.

BACKGROUND: Genetic polymorphisms that influence serotonin (5-hydroxytryptamine, 5HT) neurotransmission are candidates for contributing to susceptibility to posttraumatic stress disorder (PTSD). The objective of our study was to determine if a variable length polymorphism for the promoter regions of the 5HT transporter (5HTTLPR), and/or a substitution polymorphism in the promoter region for the 5HT2A receptor, would be associated with PTSD in a trauma exposed population of adult African-Americans. METHODS: Using a case control design, 118 participants recruited from the primary care clinics and the campus of a historically black university who met inclusion criteria including trauma exposure provided blood samples for genomic DNA. PTSD criteria were determined by the Clinician Assessment of PTSD Scale (CAPS) and criteria for other psychiatric disorders by the Structured Clinical Interview for DSM-IV (SCID). 5HTTLPR and 5HT2A-1438A/G were genotyped using established methods. Associations of genotypes with lifetime PTSD, and models testing associations of allele "dose", were analyzed. RESULTS: Fifty-five (47%) participants met lifetime criteria for PTSD and 26 (22%) met criteria for (mostly comorbid) major depression. The 5HT2A (lower expressing) G allele was significantly associated with PTSD. We did not find significant associations with 5HTTLPR. CONCLUSIONS: Our findings suggest a relationship between genetic variation in the 5HT2A promoter region and PTSD.

Amygdala function and 5-HTT gene variants in adolescent anxiety and major depressive disorder.

BACKGROUND: Associations between a functional polymorphism in the serotonin transporter gene and amygdala activation have been found in healthy, depressed, and anxious adults. This study explored these gene-brain associations in adolescents by examining predictive effects of serotonin transporter gene variants (S and L(G) allele carriers vs. L(A) allele homozygotes) and their interaction with diagnosis (healthy vs. patients) on amygdala responses to emotional faces. METHODS: Functional magnetic resonance data were collected from 33 healthy adolescents (mean age: 13.71, 55% female) and 31 medication-free adolescents with current anxiety or depressive disorders (or both; mean age: 13.58, 56% female) while viewing fearful, angry, happy, and neutral facial expressions under varying attention states. RESULTS: A significant three-way genotype-by-diagnosis-by-face-emotion interaction characterized right amygdala activity while subjects monitored internal fear levels. This interaction was decomposed to map differential gene-brain associations in healthy and affected adolescents. First, consistent with healthy adult data, healthy adolescents with at least one copy of the S or L(G) allele showed stronger amygdala responses to fearful faces than healthy adolescents without these alleles. Second, patients with two copies of the L(A) allele exhibited greater amygdala responses to fearful faces relative to patients with S or L(G) alleles. Third, although weaker, genotype differences on amygdala responses in patients extended to happy faces. All effects were restricted to the fear-monitoring attention state. CONCLUSIONS: S/L(G) alleles in healthy adolescents, as in healthy adults, predict enhanced amygdala activation to fearful faces. Contrary findings of increased activation in patients with L(A)L(A) relative to the S or L(G) alleles require further exploration.

Genetic variation in human NPY expression affects stress response and emotion.

Understanding inter-individual differences in stress response requires the explanation of genetic influences at multiple phenotypic levels, including complex behaviours and the metabolic responses of brain regions to emotional stimuli. Neuropeptide Y (NPY) is anxiolytic and its release is induced by stress. NPY is abundantly expressed in regions of the limbic system that are implicated in arousal and in the assignment of emotional valences to stimuli and memories. Here we show that haplotype-driven NPY expression predicts brain responses to emotional and stress challenges and also inversely correlates with trait anxiety. NPY haplotypes predicted levels of NPY messenger RNA in post-mortem brain and lymphoblasts, and levels of plasma NPY. Lower haplotype-driven NPY expression predicted higher emotion-induced activation of the amygdala, as well as diminished resiliency as assessed by pain/stress-induced activations of endogenous opioid neurotransmission in various brain regions. A single nucleotide polymorphism (SNP rs16147) located in the promoter region alters NPY expression in vitro and seems to account for more than half of the variation in expression in vivo. These convergent findings are consistent with the function of NPY as an anxiolytic peptide and help to explain inter-individual variation in resiliency to stress, a risk factor for many diseases.

The impact of tryptophan depletion and 5-HTTLPR genotype on passive avoidance and response reversal instrumental learning tasks.

Transient reductions in serotonin levels during tryptophan depletion (TD) are thought to impair reward processing in healthy volunteers, while another facet of the serotonergic system, the serotonin transporter (5-HTTLPR) short allele polymorphism, is implicated in augmented processing of aversive stimuli. We examined the impact and interactions of TD and the serotonin promoter polymorphism genotype on reward and punishment via two forms of instrumental learning: passive avoidance and response reversal. In this study, healthy volunteers (n=35) underwent rapid TD or control procedures and genotyping (n=26) of the 5-HTTLPR for long and short allele variants. In the passive avoidance task, tryptophan-depleted volunteers failed to respond sufficiently to rewarded stimuli compared to the control group. Additionally, long allele homozygous individuals (n=11) were slower to learn to avoid punished stimuli compared to short allele carriers (n=15). TD alone did not produce measurable deficits in probabilistic response reversal errors. However, a significant drug group by genotype interaction was found indicating that in comparison to short allele carriers, tryptophan-depleted individuals homozygous for the long allele failed to appropriately use punishment information to guide responding. These findings extend prior reports of impaired reward processing in TD to include instrumental learning. Furthermore, they demonstrate behavioral differences in responses to punishing stimuli between long allele homozygotes and short allele carriers when serotonin levels are acutely reduced.

Impaired recognition of fear facial expressions in 5-HTTLPR S-polymorphism carriers following tryptophan depletion.

RATIONALE: Genotype at the 5' promoter region (5-HTTLPR) of the serotonin transporter has been implicated in moderating the effects of acute tryptophan depletion on neurocognitive functioning. Acute tryptophan depletion has been associated with the processing of fear-relevant cues, such as emotional expressions, but the effect of genotype at the 5-HTTLPR has not been assessed. OBJECTIVE: The present study investigated the effects of acute tryptophan depletion on the recognition of standardized facial expressions of emotions in healthy volunteers classified as ll homozygotes or s carriers. MATERIALS AND METHODS: A double-blind between-groups design was used with volunteers randomly selected to ingest capsules containing an amino acid mixture specifically lacking tryptophan, or placebo capsules containing lactose. 5 h after capsule ingestion, subjects were required to identify anger, disgust, fear, happiness, sadness, and surprise expressions that progressed from neutral to each full emotional expression in 5% steps. RESULTS: Tryptophan depletion significantly impaired the recognition of fearful facial expressions in s carriers but not ll homozygotes. This impairment was specific to fear expressions. No significant differences in the recognition of other expressions were found. Free tryptophan levels were correlated with fear recognition in s carriers but not ll homozygotes. CONCLUSIONS: The effects of acute tryptophan depletion on the processing of emotional expressions varies as a function of genotype at the 5-HTTLPR. Depletion impairs the recognition of fear in s carriers but not ll homozygotes. This finding reinforces the importance of considering genotype when assessing the behavioral effects of pharmacologic modulation.

Blockade of nociceptin/orphanin FQ transmission attenuates symptoms and neurodegeneration associated with Parkinson's disease.

The opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are expressed in the substantia nigra (SN), a brain area containing dopamine neurons that degenerate in Parkinson's disease. Endogenous N/OFQ facilitates nigral glutamate release and inhibits nigrostriatal dopamine transmission and motor behavior. Here, we present evidence suggesting that endogenous N/OFQ may contribute to Parkinson's disease. Pharmacological blockade of the SN N/OFQ-NOP receptor system attenuated parkinsonian-like akinesia/hypokinesia in 6-hydroxydopamine hemilesioned or haloperidol-treated rats, whereas deletion of the NOP receptor gene conferred mice partial protection from haloperidol-induced motor depression. The antiparkinsonian action of NOP receptor antagonists was associated with reduction of glutamate release in the SN. In 6-hydroxydopamine hemilesioned rats, enhancement of N/OFQ expression and release was detected in the lesioned compared with the unlesioned SN, indicating that parkinsonism may be associated with overactivation of the N/OFQ-NOP receptor system in the SN. Finally, deletion of the N/OFQ gene conferred mice partial protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced loss of SN dopamine neurons. Based on these data, we propose that NOP receptor antagonists may represent a novel approach for combined (symptomatic and neuroprotective) therapy of Parkinson's disease.

Haplotype-based analysis of alpha 2A, 2B, and 2C adrenergic receptor genes captures information on common functional loci at each gene.

The alpha 2-adrenergic receptors (alpha2-AR) mediate physiological effects of epinephrine and norepinephrine. Three genes encode alpha2-AR subtypes carrying common functional polymorphisms (ADRA2A Asn251Lys, ADRA2B Ins/Del301-303 and ADRA2C Ins/Del322-325). We genotyped these functional markers plus a panel of single nucleotide polymorphisms evenly spaced over the gene regions to identify gene haplotype block structure. A total of 24 markers were genotyped in 96 Caucasians and 96 African Americans. ADRA2A and ADRA2B each had a single haplotype block at least 11 and 16 kb in size, respectively, in both populations. ADRA2C had one haplotype block of 10 kb in Caucasians only. For the three genes, haplotype diversity and the number of common haplotypes were highest in African Americans, but a similar number of markers (3-6) per block was sufficient to capture maximum diversity in either population. For each of the three genes, the haplotype was capable of capturing the information content of the known functional locus even when that locus was not genotyped. The alpha2-AR haplotype maps and marker panels are useful tools for genetic linkage studies to detect effects of known and unknown alpha2-AR functional loci.

Haplotype structure of the beta adrenergic receptor genes in US Caucasians and African Americans.

The beta-adrenergic receptors (beta-AR) are G protein-coupled receptors activated by epinephrine and norepinephrine and are involved in a variety of their physiological functions. Previously, three beta-AR genes (ADRB1, ADRB2 and ADRB3) were resequenced, identifying polymorphisms that were used in genetic association studies of cardiovascular and metabolic disorders. These studies have produced intriguing but inconsistent results, potentially because the known functional variants: ADRB1 Arg389Gly and Gly49Ser, ADRB2 Arg16Gly and Gln27Glu, and ADRB3 Arg64Trp provided an incomplete picture of the total functional diversity at these genes. Therefore, we created marker panels for each beta-AR gene that included the known functional markers and also other markers evenly spaced and with sufficient density to identify haplotype block structure and to maximize haplotype diversity. A total of 27 markers were genotyped in 96 US Caucasians and 96 African Americans. In both populations and for each gene, a single block with little evidence of historical recombination was observed. For each gene, haplotype captured most of the information content of each functional locus, even if that locus was not genotyped, and presumably haplotype would capture the signal from unknown functional loci whose alleles are of moderate abundance. This study demonstrates the utility of using beta-AR gene haplotype maps and marker panels as tools for linkage studies on beta-AR function.

Traumatic brain injury induces nociceptin/orphanin FQ expression in neurons of the rat cerebral cortex.

Nociceptin/orphanin FQ (N/OFQ) is a recently identified opioid-related neuropeptide. Earlier in vitro studies revealed regulation of N/OFQ expression by injury-induced factors, such as ciliary neurotrophic factor, inflammatory cytokines, and reactive oxygen species. We have extended our studies to in vivo experiments investigating the effect of traumatic brain injury on N/OFQ gene expression and peptide levels in the rat brain. Stab wound injury to the rat cerebral cortex led to a significant increase in N/OFQ mRNA levels in the vicinity of the injury, with the largest induction being seen at 24 h post-injury. Quantitative in situ hybridization revealed an almost twofold increase in the number of cells expressing N/OFQ, and the signal intensities within cells were also elevated. Stab wound injury leads to proliferation and hypertrophy of astrocytes, which respond to injury-related factors in vitro by up-regulating N/OFQ expression. However, in vivo N/OFQ co-localized exclusively with the neuronal marker, NeuN, following injury. N/OFQ expression was not detected in caspase-3-positive neurons undergoing apoptosis following injury, and increased N/OFQ expression was spatially more extended than the secondary injury-induced responses, such as astrogliosis and neuronal degeneration. Elevation of N/OFQ immunoreactivity closely followed the increase in N/OFQ gene expression as determined by immunohistochemistry. N/OFQ selectively activates the NOP receptor (ORL-1), but we did not detect parallel changes in levels of NOP receptor mRNA following injury, indicating regulation of the nociceptin system at the peptide and not the receptor level. In summary, a profound and prolonged up-regulation of N/OFQ expression in neurons surrounding a stab wound lesion to cerebral cortex was detected. The function of N/OFQ up-regulation in injury-induced responses in the brain is currently under investigation.

Regulation of nociceptin/orphanin FQ gene expression in astrocytes by ceramide.

Ceramide is generated in response to inflammatory mediators and oxidative stress. Since these stimuli dramatically increase nociceptin/orphanin FQ (N/OFQ) gene expression in astrocytes we evaluated the regulation of N/OFQ by ceramide and the signaling mechanisms involved. We found that ceramide induced N/OFQ mRNA levels 22-fold after 24 h. In astrocytes ceramide stimulated the JNK, p38 and ERK MAP kinase pathways and also led to the activation of the transcription factors CREB and NFkappaB. Using specific inhibitors of signaling pathways we determined that N/OFQ gene induction is mediated by ERK and p38 MAP kinases. ERK activation may be induced by protein kinase C and it leads to CREB phosphorylation. The NFkappaB pathway also appears to be crucial for ceramide-induced N/OFQ gene expression.

Inflammatory mediators increase the expression of nociceptin/orphanin FQ in rat astrocytes in culture.

In the central nervous system, glial cells play an important role in inflammatory and immune responses, and opioid peptides have been identified as essential mediators between the nervous and the immune systems. We report the profound upregulation of the opioid-related nociceptin/orphanin FQ (N/OFQ) by inflammatory mediators in astrocytes. The bacterial endotoxin, lipopolysaccharide (LPS), and the proinflammatory cytokines, interleukin-beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), induced levels of N/OFQ mRNA and immunoreactivity. HPLC analysis of the immunoreactivity in astrocyte extracts revealed that a large molecular weight precursor for N/OFQ is being synthesized and released in response to LPS and astrocytes appear to lack the enzymes required to process the precursor protein. Western blot analysis showed that LPS treatment elicited the activation of ERK 1/2 and p38 MAP kinases. Blockade of the p38 or the ERK MAP kinase pathways prevented the LPS-induced increase in N/OFQ mRNA levels indicating a role for these cascades in the regulation of N/OFQ genes in response to LPS. Regulation of N/OFQ gene expression by ERK and p38 activation may be mediated through the transcription factor CREB. We observed CREB phosphorylation in response to LPS, which was also prevented by SB202190 and PD98059. The NFkappaB pathway also appears to be involved in the induction of N/OFQ transcription by LPS, since NFkappaB inhibitors antagonized the effect of LPS on N/OFQ expression. Regulation of N/OFQ by inflammatory mediators in astrocytes may suggest a role for N/OFQ in neural-glial communication and in inflammatory responses in certain neuropathophysiological conditions.