The effects of alcohol use disorders exert a tremendous impact on the central nervous system of adult humans
[1, 2, 17]. Alcohol exposure affects the normal processes of neuronal proliferation, repair, and apoptosis in humans
[10, 18] and in animal models
[19, 20]. The p53-signaling pathway regulates each of these processes
 and has been implicated in ethanol-induced changes in the central nervous system
[15, 22]. The current study focuses on examination of the expression of p53-related genes in human and rat PBLs and identifies sets of ethanol-responsive genes that are correlated with changes in brain structure and function.
One of our major findings is that the expression of the p53 gene and six p53-related genes are decreased in the blood of human subjects with AUD. Furthermore the expression levels of several of these genes (particularly Ercc1, Mcm5, and p53) are positively correlated with the volumes of several cortical and subcortical brain regions, (particularly the left parietal supramarginal gyrus) as well as several measures of significantly affected neuropsychological variables. When combined with our results from the mouse NSCs and rat PBLs, this suggests that alterations in p53 signaling may be occurring within the brain of subjects with AUD. Support for this possibility comes from studies of rat pups exposed to ethanol, which demonstrated reduced p53 expression in the developing cortex
, as well as gene expression studies of postmortem human brain. Specifically, one study of the frontal cortex of adult human alcoholics identified changes in 35 neurogenesis-related transcripts and 11 apoptosis-related transcripts – both of which are highly regulated by p53
Our findings regarding Ercc1 levels are particularly noteworthy for several reasons. The Ercc1 gene-product is involved in nucleotide excision repair (NER) of damaged DNA
. This process is potentially highly relevant to understanding the effects of ethanol on genomic integrity. For example, the process of NER is a critical means for removing harmful DNA adducts that can form after exposure to ethanol or its metabolites. Indeed, in cancer therapy, NER is viewed as the primary mechanism whereby platinum-DNA adducts are removed from the DNA of tumor cells following cisplatin treatment. Accordingly, Ercc1 activity levels have been viewed as a potentially important prognostic biomarker for tumor responsiveness to cisplastin
[24, 25]. In AUD subjects Ercc1 expression was nominally correlated with the age of onset of the alcohol use disorder and robustly and significantly correlated with the volume of the supramarginal gyrus in the left inferior parietal lobe region. Thus, the importance of Ercc1 in repairing ethanol-associated DNA damage may increase over the lifespan - particularly for this brain region, which is now well-established as involved in several aspects of language processing (e.g.,
). Several recent studies lend even stronger support for this notion. For example, the Ercc1 null mouse has been used as a model of accelerated aging (progeria) because it exhibits shunted growth, wasting, ataxia, and premature death by 1-2 months of age
[27, 28]. Mice with reduced expression of Ercc1, however, show a less rapid progeria phenotype, surviving until 4-6 months of age, but exhibit clear signs of metabolic, neurologic, and cognitive decline (including learning and memory deficits) along with neurodegenerative changes (elevated expression of markers that indicate reactive astrocytosis and apoptosis)
[29, 30]. A recent follow up study of these mice by Vegh and colleagues
 revealed that even before these types of changes are seen in the hippocampus, there are significant reductions in the expression levels of numerous proteins involved in synaptic function during the early stages of accelerated aging due to reductions in Ercc1 expression, which they proposed were the cause of the learning and memory deficits in these mice. Thus, DNA damage repair processes may be critical for preventing age-dependent cognitive decline in otherwise normal mice. Given that our AUD subjects showed significantly reduced Ercc1 expression and performance on several standardized measures of verbal function, the present study strongly suggests a role for DNA repair processes and Ercc1 in preventing alcohol-dependent cognitive decline as well. Clearly, the relationship between ethanol consumption, Ercc1 expression, brain volume, neuropsychological function, aging, and cancer risk merits further investigation. In addition, there may be other explanations for the changes in Ercc1 levels that we have not considered.
Our findings regarding Mcm5 are also highly novel and may relate to the Ercc1 findings in terms of the relationship with the volume of the left inferior parietal supramarginal gyrus. The Mcm5 gene product is a chromatin-binding protein that regulates the initiation of the cell cycle at the G0-G1/S transition
 and is negatively regulated by p53. Not only is MCM5 down-regulated in human PBLs, it is also down-regulated in the frontal cortex of human alcoholics
[8, 9] and in mouse neural stem cells exposed to ethanol (11-fold decrease; 15). Such changes could lead to a reduction in cell proliferation. If such effects are also present in the brain of subjects exposed to ethanol during sensitive developmental periods, this could help explain why alcohol-induced decreases in Mcm5 are correlated with reductions in the volumes of several brain regions, such as the left inferior parietal supramarginal gyrus of AUD subjects.
Together, the Tp53, Ercc1, and Mcm5 genes represent intertwined pathways altered by ethanol exposure. Alcohol-induced down-regulation of Mcm5 and other cell cycle-related transcripts (Myc, Cdk4, Pttg1) may lead to checkpoint restriction at both G0 and G2/M. This interruption of proliferation is necessary for activation of DNA-repair mechanisms (i.e. Hus1, Mutyh, Ercc1) secondary to alcohol-induced oxidative stress
. The master regulator of these processes is Tp53, a gene that controls cell fate through the orchestration of repair and proliferation and the initiation of apoptosis
. We can now confirm that the p53-pathway is affected by alcohol in both the CNS and peripheral blood of humans and animal models. Moreover, this study has identified specific players within the p53-pathway that serve as biomarkers for the deleterious effects of alcohol. These genes are not only correlated with changes in brain volume and function, but may also play an integral role in the pathophysiology of alcohol-induced CNS damage.
There are a number of notable limitations to the present study that should be mentioned. First, we did not measure the expression of genes in the brains of AUD subjects since this was not a postmortem study. Nonetheless, our results on p53-related genes are consistent with results from prior postmortem studies. Second, the significant changes seen in the rat and human PBLs appeared to be largely distinct, although both sets of data clearly supported our hypothesis that p53-related genes would be affected, and there was significant association between the rat PBL results and mouse NSC results. Third, we did not assess brain volumes or brain function in the rat drinking model, and thus far we have only established the relationship between changes in p53-related genes in a single drinking paradigm. Finally, it is possible that some of the differences we are ascribing to ethanol that were found between AUD and control subjects may be influenced by the fact that our controls appeared to be very high functioning relative to the general population on several neuropsychological measures. Although this potential bias may be difficult to avoid when selecting non-drinking subjects for inclusion in this type of study, it also argues in favor of additional studies.