The effect of chronic alcoholism on epigenetic patterns regulating gene expression and neurodegeneration in the human brain

Abstract

Chronic alcoholism causes about 3.2% of all deaths worldwide. It has an association to about 60 different diseases and injuries with enormous social and economical impact. Despite these facts, very few pharmacological treatments exist and we can still not explain the mechanisms behind addiction, craving and cognitive impairments seen in the abuser. Alcohol targets most areas of the brain and produces an overall decline in many basic functions. Cognitive functions, such as memory, decision making and problem solving is often impaired after years of heavy drinking. The frontal lobe region is strongly connected to cognitive functions. It regulates the magnitude of response to an environmental stimulus and determines that based on former experiences and balancing positive and negative consequences in uncertain outcomes. This higher-order function Is what separates humans from primates.

Alcohol abuse can harm the brain in three ways: 1) Acetaldehyde, metabolite of ethanol, is directly toxic to neurons; 2) alcohol challenges glutamate receptors by blocking the binding of the excitatory neurotransmitter glutamate, therefore increasing the numbers of receptors which causes indirect neurotoxicity during alcohol withdrawal; and 3) dietary deficiencies due to lower food intake and deteriorated intestine resorption directly causes altered nucleic acid synthesis (DNA) and methyl group substrates to DNA and proteins.

A decrease of methyl group donation to DNA and proteins will affect the epigenetics within the genome. Methyl-groups attached to the DNA and the histone proteins regulate gene activity. Dynamic regulation of the epigenetic marking is the way the environment can impact on the activity of our genes and consequently the proteins and all functions in the organism.

The aims of this thesis were to evaluate DNA methylation and gene activity changes in the brain of chronic alcoholics. This resulted in optimization of a method to measure global DNA methylation, and establishing the activity of common housekeeping genes as well as selected genes regulating neuronal cell viability, using real-time PCR. We developed the LUMA (LUminometric Methylation Assay), based on restriction enzyme cleavage with HpaII and MspI, cleaving DNA in CpGs, dependently and independently of cytosine methylation, respectively. Detection of nucleotide incorporation and hence level of methylation was performed using Pyrosequencing platform. By using a high-throughput real-time PCR, Low Density Arrays (LDA), we evaluated 16 endogenous control genes and found brain region specific genes for optimal normalization in mRNA quantification assays. The brain regions analyzed, prefrontal and motor cortices had different gene expression stability patterns. We further identified three reference genes which were significantly downregulated in motor cortex of alcoholics compared to controls, and one gene upregulated in the prefrontal cortex. This shows the importance of careful endogenous control validation prior to quantification experiments.

Finally, we show preliminary data of downregulation of genes which activity is related to cell survival, myelination, cell cycle, DNA repair and signaling in the prefrontal cortex, while the motor cortex remained less affected in alcoholics. This suggests a higher vulnerability of alcohol abuse to the frontal cortex region, mainly in the myelin genes that were mostly downregulated. The results from the thesis serve as a good platform to further study gene specific epigenetic changes in chronic alcoholics.