Kristine Wiren - US grants

The proposed project takes a novel approach to identifying the genes of importance in modulating the severity of alcohol withdrawal in mice by identifying mRNAs expressed and regulated during chronic ethanol exposure. The underlying hypothesis is that changes in gene expression are likely to occur in the brain after chronic exposure to ethanol, and that any gene whose expression is affected by chronic ethanol is a good candidate for having a functionally important role in ethanol neuroadaptation. Genes regulated by chronic ethanol exposure might promote withdrawal risk, or expert protection against withdrawal. We propose to identify novel candidate genes of importance, without bias about potential roles, using the technique of differential display. Inbred WSP-2 mice will be made physically dependent by inhalation, and mRNA from the brains of exposed and control mice will be analyzed by differential display. Dependent and control RNAs will be hybridized to modified oligo-dT primers and reverse transcribed. The reverse transcribed cDNA sequences will be amplified by PCR with subsets of random primers, and the products labeled and separated on a DNA sequencing gel. By comparing the banding patterns for the different groups, bands with similar intensities in both the control and treated samples will be ignored, leaving a number of gene products differentially displayed (i.e., either up- or down-regulated) presumably as a result of an effect of ethanol on gene expression.

Chronic ethanol exposure results in the development of physical dependence. After ethanol removal, a withdrawal syndrome is exhibited that can be life threatening. Aspects of the withdrawal syndrome are also influenced by gender. Using microarray analysis, we propose to identify specific genes of importance in modulating the severity of the withdrawal syndrome in both genders by identifying mRNAs regulated by chronic alcohol exposure and withdrawal over an extended time course. We will employ unique models of withdrawal severity, the Withdrawal Seizure- Prone and -Resistant lines (WSP and WSR), that represent alleles from an eight-way cross of inbred mouse lines selectively bred over generations for high or low withdrawal severity. The hypothesis to be tested is that altered brain gene expression that results from withdrawal is a critical factor mediating neuroadaptation and physical dependence. Genes regulated following chronic ethanol exposure might promote withdrawal risk, or exert protection against withdrawal. We also hypothesize that gene profiles will show sex-specific regulation. Due to the inverse genetic relationship between voluntary ethanol drinking and withdrawal severity, comparison between control WSP and WSR animals may identify genes modulating alcohol consumption. Male and female WSP or WSR mice will be exposed to intoxicating levels of ethanol and withdrawn. Prefrontal cortex will be harvested from mice in the presence or absence of severe withdrawal; temporal patterns in gene expression will be identified over an extended time course using microarray analysis. Specific Aim 1 will identify mRNA transcripts that may mediate neuroadaptative processes that underlie risk for the development of physical dependence and aspects of the withdrawal syndrome in both males and females by characterizing differences in mRNA expression in WSP mice. Specific Aim 2 will identify mRNA transcripts that may mediate neuroadaptative processes that protect against the development of physical dependence and aspects of the withdrawal syndrome in both sexes by characterizing differences in mRNA expression in WSR mice. Specific Aim 3 will identify mRNA transcripts that are putative candidates for mediating voluntary alcohol consumption by characterizing differences in mRNA expression between control male and female WSP and WSR. The long-term goal of this research is to understand mechanisms underlying how deleterious responses to chronic ethanol exposure (i.e. physical dependence leading to withdrawal seizures) are regulated at the genetic level. This knowledge may help in the development of new strategies for the treatment of alcohol dependence.

Osteoporosis is a metabolic bone disease with low bone mass and compromised skeletal microarchitecture that increases bone fragility and, consequently, fracture risk. Total bone mass acquired during the active growth phases early in life is an important determinant of the risk of disease development, with bone quality being another important determinant. Osteoporosis is often coupled with a hypogonadal state in both men and women but the influence of androgen and estrogen on the skeleton remains poorly characterized. Estrogens are thought to act through an inhibition of bone resorption by the osteoclast, i.e. as anti-resorptive agents, which protect the skeleton from further loss of bone. Non-aromatizable androgens such as Salpha- dihydrotestosterohe (DHT), are anabolic agents that increase bone mass by stimulation of bone formation, and thus represent an important therapeutic class. One target of androgen action is the periosteal compartment, with activation leading to an increase in bone size believed to underlie differences in skeletal size observed between males and females, but the mechanisms remain controversial. We hypothesize that androgens influence bone formation and bone size through actions mediated by the androgen receptor (AR) in the osteoblastic lineage. We have developed an AR-transgenic animal model as a tool to better identify the important biological consequences of androgen action. AR-transgenic lines exhibit overexpressionof the AR targeted to distinct osteoblastic populations through the use of two different promoters;co!3.6 AR- transgenic mice with AR overexpression in the periosteum and throughout the osteoblast lineage vs. co!2.3 AR-transgenic mice with overexpression restricted to mineralizing mature osteoblasts. We proposethat differences between controls and selectively targeted AR-transgenic lines will provide a novel model to characterize androgen responsiveness in bone without systemic administration of hormone. In Specific Aim 1, we will define the contribution of AR signaling to the developing skeleton in mice with enhanced sensitivity to androgen in distinct osteoblast populations. In Specific Aim 2, we will characterize molecular and cellular events influenced by androgen and estrogen in osteoblast models, including periosteal and endosteal cells. These studies will identify specific molecular events/pathways influenced by androgen treatment in bone, and should lead to an improved understanding of mechanisms that influence adult bone size and quality.

DESCRIPTION (provided by applicant): This proposal explores consequences of chronic alcohol (ethanol;EtOH) exposure and withdrawal on sex- specific astrocyte activation, expression and function. Chronic EtOH abuse is associated with neurotoxicity and reactive astrogliosis, but the mechanisms and cell types involved remain poorly characterized. Furthermore, brain damage associated with alcoholism is sexually dimorphic, yet the sex-specific effects of chronic EtOH exposure have not been fully explored. This proposal extends our previous analysis that compared males vs. females using gene expression transcriptional profiling, to characterize neuroadaptive changes that are associated with withdrawal from chronic EtOH exposure. Notably, these studies demonstrated that sex is a more powerful determinant of neuroadaptation and the transcriptional response after chronic exposure than genotype and/or withdrawal severity phenotype. Biological confirmation of gene expression differences revealed that females were more vulnerable to the resulting alcohol-induced brain damage, consistent with some (but not all) clinical studies. Among the genes significantly regulated by chronic EtOH in the prefrontal cortex are some that are predominantly or exclusively expressed by astrocytes. Furthermore, EtOH-induced brain damage is associated with excitotoxicity resulting from increased glutamate release, likely from astrocyte sources. GABA receptors are also a target of neuroadaptation to chronic EtOH abuse, and have been implicated in astrocyte function. Because astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse, investigations into the effects of chronic EtOH on astrocyte function are timely and important. Given the evidence that EtOH withdrawal differentially damages female vs. male brain, the present application examines the hypothesis that chronic alcohol exposure and withdrawal targets astrocyte function in a sex- specific fashion, resulting in changes in gene expression and function, resulting in elevated excitotoxic signaling. Aim 1 characterizes the cellular effects of chronic ethanol ex vivo on cortical astrocyte cultures from males vs. females. This aim will develop primary single sex cortical astrocyte culture methodologies to directly test the response to chronic EtOH exposure and withdrawal in male vs. female astrocyte populations in a controlled environment. This aim also characterizes astrocyte proliferation, apoptosis, glutamate uptake and release, GABAergic signaling and the contribution of calcium stores after chronic ethanol exposure and withdrawal. Aim 2 will define sex-specific gene expression differences in astrocytes after chronic EtOH insult. Methods include qRT-PCR analysis of transcripts important in glutamatergic and GABAergic signaling with Western or immunocytochemical analysis for confirmation of expression differences. Utilizing the primary cortical astrocyte male vs. female ex vivo culture methodologies and chronic EtOH treatment paradigms developed in this developmental R21 application, these studies will lead to a better understanding of the specific effects of alcohol on astrocyte function to potentially identify therapeutic targets for the sex-specific treatment of brain damage associated with chronic EtOH abuse in both males and females. PUBLIC HEALTH RELEVANCE: Chronic alcohol abuse is a major public health problem. Although many abused drugs, including alcohol, cause neurotoxicity and brain damage, the mechanisms and the contribution of specific cell types involved are poorly characterized. In addition, there are many differences between males and females with regard to EtOH behaviors and sex-specific brain damage has been reported, but there is little analysis of the pathways involved. Astrocytes are able to regulate neuronal activity and synaptic neurotransmission (both excitatory and inhibitory signals) to influence neurotoxicity, and astrocyte activation is observed with alcohol abuse. However, the influence of chronic alcohol exposure and withdrawal on astrocyte expression and function has not been determined. The present application utilizes sex-specific primary cortical astrocyte cell culture as an ex vivo model to elucidate how alcohol influences astrocyte viability, gene expression and function in males vs. females, with a focus on glutamatergic and GABAergic signaling. These studies will provide for a better understanding of the consequences of chronic alcohol exposure and withdrawal on astrocyte function in both males and females, and may lead to novel targets for the sex-specific treatment or amelioration of brain damage associated with chronic ethanol abuse.

DESCRIPTION (provided by applicant): Both genetic and environmental contributions have crucial roles in the development of a complex disease such as alcoholism. Unfortunately, little progress has been made in identifying the underlying molecular mechanisms altered during abstinence to aid development of novel therapeutics for the maintenance of sobriety. We propose a combined genetic, molecular, pharmacological and behavioral strategy to identify pathways that are altered after a period of abstinence. Neuroadaptations in brain structure, plasticity and gene expression occur with chronic alcohol abuse, but the stability of these expression differences in the abstinent alcoholic is controversial. We have previously reported identification of pathways altered in prefrontal cortex (PFC), a brain region associated with cognitive dysfunction and damage in alcoholics, during a defined period of abstinence. To characterize genetic contributions, both sexes of an animal model with widely divergent responses to alcohol derived by selective breeding, the Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) lines, were analyzed. During a sustained period of abstinence, the transcriptional response correlated with withdrawal phenotype rather than sex. Bioinformatic analysis showed that among the major pathways altered that were the most dimorphic between WSR and WSP mice were 'acetylation' and 'histone deacetylase complex'. Data shows a complex phenotype-specific regulation during abstinence indicating widespread epigenetic reprogramming in the low response WSR but not the high response WSP mice exposed to the same ethanol concentrations. We will identify phenotype-specific regulatory mechanisms in the low response animal model in three specific aims by integrating data from high-throughput targeting technologies including expression profiling, DNaseI-seq and ChIP-seq, with confirmation of involvement of pathways to modulate relapse using pharmacological intervention in our established dependence-induced relapse drinking model. We hypothesize that targetable epigenetic mechanisms maintain expression differences during abstinence and that these differences increase the risk of relapse in the low response to alcohol endophenotype. These studies have high impact because of the morbidity/mortality associated with alcohol abuse, the high incidence of alcohol use disorders in the general population, and the tremendous impact these maladies have on human health. In addition, neuroadaptive changes and altered expression patterns may also play a role in persistent neurotoxicity and brain damage during abstinence with detrimental consequences for learning and memory functions, to play a role in the down-ward cycle of addiction and the self-sustaining nature of alcoholism. Thus, successful completion of these aims will aid in our understanding of the mechanism(s) underlying the risk for relapse and advance our ability to provide therapy for alcohol abuse targeted to the low response endophenotype, through identification of novel pharmacotherapies or to enhance translational applications for currently available therapeutics with previously unrecognized utility.