John P. Spence

α-Synuclein maps to a quantitative trait locus for alcohol preference and is differentially expressed in alcohol-preferring and -nonpreferring rats

Total gene expression analysis (TOGA) was used to identify genes that are differentially expressed in brain regions between the alcohol-naïve, inbred alcohol-preferring (iP), and -nonpreferring (iNP) rats. α-Synuclein, expressed at >2-fold higher levels in the hippocampus of the iP than the iNP rat, was prioritized for further study. In situ hybridization was used to determine specific brain regions and cells expressing α-synuclein in the iP and iNP rats. Similar to α-synuclein mRNA levels, protein levels in the hippocampus were higher in iP rats than iNP rats. Higher protein levels were also observed in the caudate putamen of iP rats compared with iNP rats. Sequence analysis identified two single nucleotide polymorphisms in the 3′ UTR of the cDNA. The polymorphism was used to map the gene, by using recombination-based methods, to chromosome 4, within a quantitative trait locus for alcohol consumption that was identified in the iP and iNP rats. A nucleotide exchange in the iNP 3′ UTR reduced expression of the luciferase reporter gene in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the α-synuclein gene may contribute to alcohol preference in the iP rats.

Association of ALDH1 promoter polymorphisms with alcohol-related phenotypes in southwest California Indians.

BACKGROUND Cytosolic aldehyde dehydrogenase (ALDH1A1) is an important enzyme in the metabolism of acetaldehyde and the synthesis of retinoic acid. Two polymorphisms in the promoter region of ALDH1A1-ALDH1A1*2 and ALDH1A1*3-have recently been identified and described in small samples of Asian, Caucasian, and African individuals. The aim of this study was to determine the prevalence of these polymorphisms in a sample of Southwest California Indians and to test for associations with alcohol dependence and other substance-related behaviors. METHODS The participants in this study were 463 adult men and women recruited from 8 contiguous Indian reservations. A structured interview was used to gather information on demographics, psychiatric diagnoses, and personal drinking and drug use history. A blood sample was obtained from each participant, and leukocyte DNA was extracted and used to genotype for the presence of the ALDH1A1 promoter polymorphisms. RESULTS Twenty-seven participants (6%) possessed ALDH1A1*2 (frequency, 0.03), two participants possessed ALDH1A1*3, and one participant displayed both of these alleles. Individuals with an ALDH1A1*2 allele had lower rates of alcohol dependence and regular tobacco use than those without this allele. Individuals with ALDH1A1*2 also reported a significantly lower maximum number of drinks ever consumed in a 24-hr period, reported drinking fewer drinks per occasion when they first started drinking regularly, and reported lower expectations of alcohols effects compared with individuals without this allele. CONCLUSIONS Results from this study suggest that ALDH1A1*2 may be associated with protection from the development of alcohol and other substance use disorders.

Evaluation of Aldehyde Dehydrogenase 1 Promoter Polymorphisms Identified in Human Populations

BACKGROUND Cytosolic aldehyde dehydrogenase, or ALDH1A1, functions in ethanol detoxification, metabolism of neurotransmitters, and synthesis of retinoic acid. Because the promoter region of a gene can influence gene expression, the ALDH1A1 promoter regions were studied to identify polymorphism, to assess their functional significance, and to determine whether they were associated with a risk for developing alcoholism. METHODS Sequence analysis was performed in the promoter region by using Asian, Caucasian, and African American subjects. The resulting polymorphisms were assessed for frequency in Asian, Caucasian, Jewish, and African American populations and tested for associations with alcohol dependence in Asian and African American populations of alcoholics and controls. The functional significance of each polymorphism was determined through in vitro expression analysis by using HeLa and HepG2 cells. RESULTS Two polymorphisms, a 17 base pair (bp) deletion (-416/-432) and a 3 bp insertion (-524), were discovered in the ALDH1A1 promoter region: ALDH1A1*2 and ALDH1A1*3, respectively. ALDH1A1*2 was observed at frequencies of 0.035, 0.023, 0.023, and 0.012 in the Asian, Caucasian, Jewish, and African American populations, respectively. ALDH1A1*3 was observed only in the African American population, at a frequency of 0.029. By using HeLa and HepG2 cells for in vitro expression, the activity of the luciferase reporter gene was significantly decreased after transient transfection of ALDH1A1*3-luciferase compared with the wild-type construct ALDH1A1*1-luciferase. In an African American population, a trend for higher frequencies of the ALDH1A1*2 and ALDH1A1*3 alleles was observed in a population of alcoholics (p = 0.03 and f = 0.12, respectively) compared with the control population. CONCLUSIONS ALDH1A1*2 and ALDH1A1*3 may influence ALDH1A1 gene expression. Both ALDH1A1*2 and ALDH1A1*3 produce a trend in an African American population that may be indicative of an association with alcoholism; however, more samples are required to validate this observation. The underlying mechanisms contributing to these trends are still unknown.

Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase.

Children with neurofibromatosis type 1 (NF1) are increasingly recognized as having a high prevalence of social difficulties and autism spectrum disorders (ASDs). We demonstrated a selective social learning deficit in mice with deletion of a single Nf1 allele (Nf1+/−), along with greater activation of the mitogen-activated protein kinase pathway in neurons from the amygdala and frontal cortex, structures that are relevant to social behaviors. The Nf1+/− mice showed aberrant amygdala glutamate and GABA neurotransmission, deficits in long-term potentiation and specific disruptions in the expression of two proteins that are associated with glutamate and GABA neurotransmission: a disintegrin and metalloprotease domain 22 (Adam22) and heat shock protein 70 (Hsp70), respectively. All of these amygdala disruptions were normalized by the additional deletion of the p21 protein-activated kinase (Pak1) gene. We also rescued the social behavior deficits in Nf1+/− mice with pharmacological blockade of Pak1 directly in the amygdala. These findings provide insights and therapeutic targets for patients with NF1 and ASDs.

From QTL to Candidate Gene: A Genetic Approach to Alcoholism Research

A major focus of research in alcohol-related disorders is to identify the genes and pathways that modulate alcohol-seeking behavior. In light of this, animal models have been established to study various aspects of alcohol dependence. The selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines were developed from Wistar rats to model high and low voluntary alcohol consumption, respectively. Using inbred P and NP strains, a strong QTL (LOD-9.2) for alcohol consumption was identified on rat chromosome 4. To search for candidate genes that underlie this chromosomal region, complementary molecular-based strategies were implemented to identify genetic targets that likely contribute to the linkage signal. In an attempt to validate these genetic targets, corroborative studies have been utilized including pharmacological studies, knock-out/transgenic models as well as human association studies. Thus far, three candidate genes, neuropeptide Y (Npy), alpha-synuclein (Snca), and corticotrophin-releasing factor receptor 2 (Crhr2), have been identified that may account for the linkage signal. With the recent advancements in bioinformatics and molecular biology, QTL analysis combined with molecular-based strategies provides a systematic approach to identify candidate genes that contribute to various aspects of addictive behavior.

Effect of polymorphism on expression of the neuropeptide Y gene in inbred alcohol-preferring and -nonpreferring rats.

Using animal models of alcoholism, previous studies suggest that neuropeptide Y (NPY) may be implicated in alcohol preference and consumption due to its role in the modulation of feeding and anxiety. Quantitative trait loci (QTL) analysis previously identified an interval on rat chromosome 4 that is highly associated with alcohol preference and consumption using an F2 population derived from inbred alcohol-preferring (iP) and -nonpreferring (iNP) rats. NPY mapped to the peak of this QTL region and was prioritized as a candidate gene for alcohol-seeking behavior in the iP and iNP rats. In order to identify a potential mechanism for reduced NPY protein levels documented in the iP rat, genetic and molecular components that influence NPY expression were analyzed between iP and iNP rats. Comparing the iP rat to the iNP rat, quantitative real-time polymerase chain reaction detected significantly decreased levels of NPY mRNA expression in the iP rat in the six brain regions tested: nucleus accumbens, frontal cortex, amygdala, hippocampus, caudate-putamen, and hypothalamus. In addition, the functional significance of three previously identified polymorphisms was assessed using in vitro expression analysis. The polymorphism defined by microsatellite marker D4Mit7 in iP rats reduced luciferase reporter gene expression in SK-N-SH neuroblastoma cells. These results suggest that differential expression of the NPY gene resulting from the D4mit7 marker polymorphism may contribute to reduced levels of NPY in discrete brain regions in the iP rats.

Restraint stress and repeated corticotrophin-releasing factor receptor activation in the amygdala both increase amyloid-β precursor protein and amyloid-β peptide but have divergent effects on brain-derived neurotrophic factor and pre-synaptic proteins in the prefrontal cortex of rats.

Both environmental stress and anxiety may represent important risk factors for Alzheimers disease (AD) pathogenesis. Previous studies demonstrate that restraint stress is associated with increased amyloid beta (Aβ) and decreased brain-derived neurotrophic factor (BDNF) levels in the brain. Aβ deposition, synaptic loss, and neurodegeneration define major hallmarks of AD, and BDNF is responsible for the maintenance of neurons. In contrast to restraint stress, repeated injections of sub-anxiogenic doses of the corticotrophin releasing factor receptor agonist urocortin1 (Ucn1) administered in the basolateral amygdala (BLA) of rats elicits persistent anxiety-like responses. We hypothesized that both restraint stress and Ucn1-induced anxiety would contribute to a neurobiological abnormality that would change the levels of Aβ precursor protein (APP) and Aβ as well as BDNF and pre-synaptic markers. In the first experiment, adult male Wister rats (n=5) were subjected to 3-h restraint, as compared to unstressed controls. In the second experiment, adult male Wistar rats (n=6) were subjected to sub-anxiogenic doses of Ucn1 (6 fmol/100 nl) administered in the BLA for 5 consecutive days, as compared to controls. Following each respective treatment, the social interaction (SI) test was performed to measure anxiety-like behavior. Protein studies were then conducted to quantify levels of APP, Aβ, BDNF and presynaptic proteins in the prefrontal cortex (PFC). In both experiments, we detected differences in either corticosterone levels or the SI test associated with a stress response. Furthermore, our findings indicate that both restraint stress and Ucn1 administration in the BLA lead to increased APP and Aβ deposition. However, restraint-induced stress leads to reductions in the levels of BDNF and presynaptic markers, while Ucn1-induced anxiety is associated with increases in the levels of each respective protein. This demonstrates a convergent role for stress response and Ucn1-induced anxiety in the regulation of APP and Aβ, but opposing roles for each respective treatment in the regulation of BDNF and presynaptic markers.

Analyses of Quantitative Trait Loci Contributing to Alcohol Preference in HAD1/LAD1 and HAD2/LAD2 Rats

BACKGROUND The high-alcohol-drinking (HAD1/HAD2) and low-alcohol-drinking (LAD1/LAD2) rat lines, derived from the N/NIH rat, were developed by using a within-family selection and rotational breeding design for alcohol preference and alcohol consumption. Previously, a 20-cM genome screen identified quantitative trait loci (QTLs) on chromosomes 5, 10, 12, and 16 by using F2 progeny from HAD1 and LAD1 animals. METHODS A total of 459 F2 HAD1 x LAD1 animals had been previously genotyped, and 428 HAD2 x LAD2 F2 animals were genotyped for microsatellite markers within the identified QTL regions. Linkage analyses were performed with the program QTL Express, a recently developed Web-based interface that implements a least-squares method. RESULTS The linkage peaks previously identified in the HAD1 x LAD1 genome scan relied on one or two markers. Placement of additional markers in and around the QTL regions provided further support for each of the QTLs. Two of the QTLs on chromosomes 10 and 16 were confirmed in the replicate line; these QTLs exhibited linkage in both the HAD1/LAD1 and HAD2/LAD2 studies. CONCLUSIONS This study demonstrated the importance of confirmation of QTLs in a replicate line, as well as the complexity of the genetic contribution to alcohol preference. Assessing these QTL regions in the inbred HAD/LAD animals will further facilitate characterization of these regions.

Confirmation of alcohol preference quantitative trait loci in the replicate high alcohol drinking and low alcohol drinking rat lines.

Objective Selective breeding has been employed to develop replicate high-alcohol-drinking (HAD1 and HAD2) and low-alcohol-drinking (LAD1 and LAD2) rat lines from the heterogeneous N/Nih rat. Within-family selection and a rotational breeding design were used to discourage inbreeding (Li et al., 1993). A genome screen was previously performed using 459 HAD1×LAD1 F2 progeny to identify quantitative trait loci (QTLs) on rat chromosomes 5, 10, 12 and 16 that contribute to alcohol preference and consumption in these non-inbred rat models of alcoholism. Methods To confirm these QTLs in the replicate lines, 16 HAD2 and 16 LAD2 rats were genotyped for microsatellite markers within each of these QTL intervals. Results Review of the genotypic data support confirmation of the QTLs on chromosomes 5 and 10; several markers in the QTL region display different alleles in the HAD2 and LAD2 rats, suggesting linkage disequilibrium between the microsatellite markers and the QTL. Although the QTL on chromosome 12 had the highest LOD score in the HAD1 and LAD1 studies, little evidence supported confirmation of this QTL based on the genotyped markers. Conclusions Further evaluation of each of these QTL regions is ongoing in a sample of HAD2×LAD2 F2 progeny currently being generated that will be used to assess the evidence of linkage in each of these QTL regions.

Expression profiling and QTL analysis: a powerful complementary strategy in drug abuse research.

Alcoholism is a complex disease exhibiting a multifactorial mode of transmission. To simplify the genetic and phenotypic complexity of the alcoholic phenotype, alcohol‐preferring (P) and ‐non‐preferring (NP) rats were developed on the basis of alcohol preference and consumption as an animal model of alcoholism. Total gene expression analysis (TOGA) and quantitative trait loci (QTL) analysis were applied to selectively bred, inbred P and NP rats as complementary studies to identify genetic factors that contribute to alcohol preference and consumption. TOGA analysis was utilized to screen for differential expression in several brain regions involved in the mesocorticolimbic dopamine (DA) system. Genes exhibiting differences in expression were then screened for an association to the alcohol preference phenotype, the quantitative trait of a previously identified QTL. By evaluating differences in gene expression for linkage to a quantitative trait, this combined approach was implemented to identify α‐synuclein, a candidate gene for alcohol preference.