**7. Long-term memory depends on epigenetic alterations**

#### **7.1 Contextual fear memory conditioning causes changes in DNA methylation in brain neurons**

Halder et al. [75], in a mouse study, evaluated differently expressed genes and short differentially methylated regions in neurons of the anterior cingulate cortex, a brain region important for associative memory acquisition and maintenance of long-term memory. In the anterior cingulate cortex at 1 hour after contextual fear conditioning, there were 6250 differentially methylated genes with 46,395 differently methylated short regions (700 base pair regions). (Frequently, multiple short differentially methylated regions occurred in a differentially methylated gene.) At 4 weeks after training 1223 differentially methylated genes and 5018 differently methylated short regions persisted. In addition, at 4 weeks after training they found 1700 differentially expressed genes in the anterior cingulate cortex. These findings suggest that long-term memory (4 weeks) is associated with differential methylation of DNA and altered expression of genes.

Halder et al. [75] also evaluated differentially methylated regions and differently expressed genes in the hippocampal CA1 region, a region that is crucial for shortterm memory formation during contextual fear conditioning. They found that, in contrast to the anterior cingulate cortex, in the hippocampus there were 1619 differentially methylated regions after 1 hour, but these changes did not persist, and almost none were present after 4 weeks.

 Also studying the hippocampus, Duke et al. [76], working with rats, found that at 24 hours after contextual fear conditioning there were more than 5000 differentially methylated regions (500 base pair short regions), but less than 20 differentially methylated regions after context change alone. Hypermethylated differentially methylated regions overlapping differentially expressed genes were associated with decreased gene expression, consistent with the concept that cytosine methylation is often a mechanism for suppressing transcription. Also at 24 hours after contextual fear conditioning, there were more than 2000 differentially methylated regions that were associated with 1048 genes having down-regulated expression and 564 genes having up-regulated expression (usually known to be associated with hypomethylated regions). At 24 hours after training, 9.17% of the genes in the rat genome of hippocampus neurons were differentially methylated. Gene Ontology term analysis was performed, and differentially expressed gene enrichment analysis revealed that many of the genes involved in synaptic functions after fear conditioning were up-regulated.

#### **7.2 The role of base excision repair in memory consolidation**

 In both the studies of Halder et al. [75] and Duke [76], above, there were on the order of a thousand demethylations of cytosines in neuron genomes during memory consolidation in the brain after contextual fear conditioning. The two likely processes of demethylating cytosine each depend on base excision repair, as shown in **Figure 3**. These processes were reviewed by Bayraktar and Kreutz [77]. There is considerable evidence for the left hand process illustrated in **Figure 3**. In this process there are two or more fast oxidations by one of the ten-eleven translocation methylcytosine dioxygenases (TET1, TET2, TET3), first altering 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and then producing 5-formylcytosine (5fC) followed by

#### **Figure 3.**

*Demethylation of 5-methylcytosine (5mC) to cytosine (Cyt) in DNA depends on base excision repair (BER) as the final step. In initial steps, the ten-eleven translocation methylcytosine dioxygenase family of enzymes (TET1, TET2, and TET3) each may catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further steps form 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC and 5caC can be excised from DNA by thymine DNA glycosylase (TDG) to form an apyrimidinic site (AP site). AID/APOBEC is a family of cytidine deaminases that can oxidatively deaminate 5mC to 5-hydroxymethyl uracil (5hmU) or 5mC to thymine (Thy). 5hmU can be excised from DNA by TDG, methyl-CpG-binding domain protein 4 (MBD4), endonuclease VIII-like 1 (NEIL1) or single-strand selective monofunctional uracil DNA glycosylase (SMUG1).* 

5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised by thymine DNA glycosylase (TDG), generating an apyrimidinic (AP) site, which is repaired by base excision repair to place cytosine (cyt) in the DNA opposite guanine. However there is some indication that a cytidine deaminase (AID/APOBEC) enzyme can carry out oxidative deamination of 5mC to 5-hydroxymethyluracil, which is then excised by one of the four enzymes shown, to form an AP site. Alternatively, a cytidine deaminase (AID/APOBEC) may carry out an oxidative deamination of 5mC by to thymine, and the mispair of thymine with guanine is then repaired by base excision repair to generate cytosine paired with guanine in DNA.

 Zhang et al. [78] generated homozygous mutant mice deficient in TET1 catalytic activity. These mice were viable and fertile, with no discernible morphological or growth abnormality. The Tet1 deficient mice would be expected to have reduced ability to convert 5mC to cytosine by the TET/base excision repair-dependent pathway. When examined in neural progenitor cells, 478 genes showed elevated promoter DNA methylation levels compared to the wild-type control, while only 32 genes had lower DNA methylation. There was a link between the altered DNA methylation pattern and transcriptional activity. In the neural progenitor cells of TET1 mutant

*The Role of DNA Repair and the Epigenetic Markers Left after Repair in Neurologic Functions… DOI: http://dx.doi.org/10.5772/intechopen.83477* 

mice 1267 genes were down-regulated with respect to transcription and 498 were up-regulated compared to wild-type. In particular, with TET1 mutant mice, 39 genes were found to be both hyper-methylated and down-regulated in neural progenitor cells isolated from the *dentate gyrus* (part of the brain hippocampus). Four-monthold wild-type and TET1 knockout mice were tested in the Morris water maze. The TET1 deficient mice, with reduced ability to use a pathway dependent on base excision repair, showed impairment in spatial learning and short-term memory.
