POST MORTEM STUDIES ON THE HUMAN ALCOHOLIC BRAIN: DNA METHYLATION AND MOLECULAR RESPONSES

Chronic alcoholism is a multi-factorial psychiatric disease manifested by re-occurring periods of relapse with frequent symptoms of cognitive impairments. There is no explanation to the extreme vulnerability to relapse, since this often occurs without evident molecular or biochemical changes remaining at the time of relapse. Epigenetic marks, such as DNA methylation, may represent a memory of molecular manifestations beyond evident genetic or biochemical signaling, in addition to its role as a gene activity regulator. The alcoholic genome could potentially possess an altered DNA methylation pattern by 1) DNA-methyltransferase blockage by acetaldehyde or 2) folate deficiency and aberrant methyl-donation by S-adenosyl-methionine (SAM). This thesis describes molecular and DNA methylation alterations in multiple alcoholic brain regions, with focus on a brain region important for reward related behavior and cognition – the prefrontal cortex (PFC). First, an optimal normalization for quantitative expression analysis was established by analysis of a panel of endogenous control genes. This study demonstrated the importance of brain region specific normalization genes due to differences in expression stability between brain regions. Moreover, it reveals expression differences in a number of commonly used normalizing genes between controls and alcoholics. Furthermore, components involved in programmed cell death/cell survival were analyzed to examine a possible involvement in alcoholism. Several key proand antiapoptotic genes and proteins were found changed in the alcoholic PFC in a direction that supports a neuronal protective adaptation rather than an active cell death. This thesis further shows how the genome of alcoholics is deprived of DNA methylation. This is demonstrated to occur globally both in humans and in a rat model of alcohol dependence, by a technique developed during the thesis work, LUminometric Methylation Assay (LUMA), but it also occurs with gene specificity as shown in humans using a microarray based methylation technology. The results further indicate a specificity to brain regions involved in reward and cognition – PFC and the striatum. DNA hypomethylation was also shown to be functionally important to investigate since a correlation (p = 0.00001) between DNA hypomethylation and gene expression was found in 14 genes analyzed. Moreover, genes of expected functional groups, including alcohol metabolizing enzymes and genes regulating methylation processes were found differentially methylated, as well as the previously reported insulin signaling pathway and acetylcholine receptors. Finally, a neuronal atrophy pathway was associated to alcoholism by comparison analysis between DNA methylation microarray and previously reported gene expression microarrays. This pathway, Dentatorubral-pallidoluysian atrophy (DRPLA), is responsible for an autosomal neuronal atrophy disorder with clinical symptoms of epilepsy, cerebellar ataxia, dementia and involuntary reflexes and movements symptoms also frequently found in alcoholics. LIST OF PUBLICATIONS I. Johansson S *, Fuchs A, Okvist A, Karimi M, Harper C, Garrik T, Sheedy D, Hurd Y, Bakalkin G, Ekstrom T.J. Validataion of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics. Brain Research, 2007, 1132(1):20-28. II. Johansson S, Ekstrom T.J, Marinova Z, Okvist A, Sheedy D, Garrick T, Harper C, Kuzmin A, Yakovleva T and Bakalkin G *. Dysregulation of cell death machinery in the prefrontal cortex in human chronic alcoholics. International Journal of Neuropsychopharmacology, under revision. III. Karimi M, Johansson S, Stach D, Corcoran M, Grander D, Shalling M, Bakalkin G, Lyko F, Larsson C, Ekstrom T.J *. LUMA (LUminometric Methylation Assay)--a high throughput method to the analysis of genomic DNA methylation. Experimental Cell Research, 2006, 312(11):1989-95. IV. Johansson S *, Merino Martinez R, Thompson R.F, Rimondini R, Sheedy D, Garrick T, Harper C, Bakalkin G, Hurd Y.L, Greally J.M, Hatchwell E, Flores A, Dunn J.J, Ekstrom T.J. Global and gene specific DNA hypomethylation in the alcoholic brain with implication for biological function. Submitted for publication. * Corresponding author RELATED PUBLICATIONS I. Okvist A, Johansson S, Kuzmin A, Bazov I, Merino-Martinez R, Ponomarev I, Mayfield R.D, Harris A.R, Sheedy D, Garrick T, Harper C, Hurd Y.L, Terenius L, Ekstrom T.J, Bakalkin G and Yakovleva T. Neuroadaptations in Human Chronic Alcoholics: Dysregulation of the NFB System. PLoS ONE. 2007 Sep 26; 2(9):e930. II. Stenvinkel P, Karimi M, Johansson S, Axelsson J, Suliman M, Lindholm B, Heimburger O, Barany P, Alvestrand A, Nordfors L, Qureshi A.R, Ekstrom T.J, Schalling M. Impact of inflammation on epigenetic DNA methylation a novel risk factor for cardiovascular disease? J Intern Med. 2007 May;261(5):488-99.