SECTION ON MOLECULAR PATHOPHYSIOLOGY? ? Medications development: Target discovery and validation.? ? Our group utilizes rodent models, molecular studies and pharmacological approaches to identifying novel mechanisms and treatment targets, and validating them for human development. ? ? Two categories of models for target discovery? ? Pre-existing genetic susceptibility factors? ? Genetic selection for high alcohol preference has the potential to enrich gene variants (alleles) that contribute to high voluntary alcohol intake and intoxication during the initiation phase of alcohol use. This approach is well established in the alcohol field, and has been successfully applied by several laboratories. Our group has over the years collaborated with two of those, that of Hyytia (Helsinki, Finland: Alko Alcohol preferring, or AA line ) and of Ciccocioppo (Camerino, Italy: marchigian-sardinian preferring, or msP line). Although selected for high alcohol preference, AA and msP lines are phenotypically very different, illustrating the multitude of factors that contribute to high voluntary alcohol intake. Behavioral characterization by our laboratory in collaboration with the Finnish and Italian groups has shown that the AA line has a high degree of behavioral disinhibition (impulsivity), while recent work (see below) has shown msP rats to be anxious / stress-sensitive.? ? Long-term neuroadaptations? ? A major development over the last 5 years has been brought about by the realization of the shift in motivational and neural mechanisms that occurs as dependence evolves, and the development of animal models that allow a study of these neuroadaptive processes. Our group has been at the forefront of this development. In a key paper (Rimondini et al. 2002), we demonstrated that prolonged exposure of the rat brain to repeated cycles of intoxication and mild withdrawal results in a long-term up-regulation of voluntary alcohol intake, encoded by long-term changes in gene expression patterns. ? ? Discovery and validation of novel targets? ? Hypothesis driven studies: focus on stress and negative affect? ? The post-dependent state is also increased sensitivity to stress, a major factor triggering relapse both in human alcohol dependent subjects and in experimental animals. Recent findings from our and other laboratories point to several stress-related neuropeptide systems as a key category of targets, which are selectively sensitive in the post-dependent state. ? ? Corticotropin-Releasing Hormone (CRH) and its CRH1 receptor? ? We have recently found that both the elevated self-administration of alcohol and the increased behavioral sensitivity to stress in the post-dependent state is in large part mediated by an up-regulation of the CRH1 subtype of CRH receptors in the amygdala. This converges with our recent findings of a stress-sensitive / anxious behavioral phenotype in the msP rat, accompanied by an innate up-regulation of CRH1 receptors in the amygdala and several other brain regions in this line. The up-regulated CRH1 gene expression in msP rats is driven by a genetic polymorphism in the promoter region of the receptor gene in this line. Using the selective CRH1 antagonist antalarmin we have shown that this accounts both for the elevated self-administration of alcohol and the anxious phenotype of msP rats. These findings validate the CRH1 receptor as a treatment target. Based on this, we have developed a collaborative research and development agreement (CRADA) with a major pharmaceutical company, and are developing a molecule with suitable pharmaceutical properties for clinical use.? ? Neuropeptide Y (NPY)? ? NPY is a potent endogenous anti-stress compound and counteracts the behavioral stress effects of CRH. Recent studies have shown that potentiation of NPY signaling, by NPY-Y1 receptor agonists, or by blockade of presynaptic Y2 autoreceptors, suppresses post-dependent drinking, while leaving basal intake of alcohol in non-dependent animals unaffected. We are currently evaluating non-peptide compounds targeting the Y2 receptor in order to identify molecules that could be developed for clinical use.? ? Endocannabinoid (EC) system and the CB1 receptor? ? In the post-dependent state, there is an increased sensitivity of alcohol self-administration to CB1 blockade. We recently found that pre-existing genetic factors can emulate this situation. Hansson et al (2006) found a genetically impaired expression and function of the EC metabolizing enzyme, FAAH, in the alcohol preferring AA line. Microinjection experiments with the CB1 antagonist rimonabant showed that the resulting increased EC drive is what underlies the high alcohol self-administration in AA rats, through actions at CB1 receptors in the prefrontal cortex. Pharmacological blockade of FAAH in this region in regular, non-selected Wistar rats produces an increase in self-administration that emulates the AA phenotype. These data combine with a rapidely growing body of evidence that provides a rationale for evaluating the ability of rimonabant in humans. ? ? Unbiased approaches to target discovery ? ? A typical strategy has been to subject the models mentioned above to analysis of differential gene expression in key brain areas that are thought to control ethanol self-administration and negative affect. Brain regions of particular interest are the amygdala complex, cingulate cortex and hippocampus. One strategy has employed Affymetrix DNA microarray analysis for a primary screen, followed by in situ and/or quantitative (TaqMan) PCR confirmation. More recently, high-throughput in situ hybridization techniques for panels of candidate transcripts have been employed. These approaches have established several differentially expressed genes/gene clusters:? ? Glutamatergic genes? ? GLAST, essential for removal of extracellular glutamate, is up-regulated in the post-dependent state. An attempt is right now ongoing to study this mechanism using null-mutants for the GLAST-gene. These might provide a useful model for the neuroadapted dependent state. This model can be used to evaluate whether compounds that reduce glutamatergic drive, such as agonists at presynaptic mGluR2/3 autoreceptors that inhibit release of glutamate, are capable of normalizing a hyperglutamatergic state, and through this suppress alcohol intake.? ? Beta arrestin 2 ? ? A DNA-microarray screen in the preferring AA line has identified an over-expression of beta arrestin 2 (BARR2). We subsequently found that this is due to the presence of a unique genetic variant (haplotype) of this gene in the AA rats. BARR2 is crucial for regulation of signalling through a large number of G-protein coupled receptors, and signalling studies are ongoing to clarify the underlying mechanisms.? ? Glutathion transferase alpha 4? ? Overexpression of this transcript was also identified by an Affymetrix screen in the AA-line. Sequencing of the gene found a unique haplotype variant in AA rats, associated with the high expression level. This locus is most likely not related to the high alcohol preference of AA rats, but rather to their altered responses to oxidative stress.? ? Relevance? ? The arrival over the last decade of naltrexone and acamprosate as modern medications for alcohol dependence has in a fundamental way changed the field of alcohol research, by providing proof-of-concept for pharmacotherapy of this disease. A major quantitative change in clinical outcomes in alcoholism will not be brought about by the arrival of any single ?magic bullet? drug, but rather by the cumulative effect of a growing and mechanistically diverse group of compounds, ultimately leading to personalized treatment. Our program has the potential to contribute in major ways to this development.
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