Epigenetics and Memory Running head: EPIGENETICS AND MEMORY

Neuroepigenetics of Learning and Memory

A Thesis Submitted to the Faculty of Baylor University In Partial Fulfillment of the Requirements for the Honors Program

By Travis Chapman

Waco, Texas May 2011

Epigenetics and Memory Abstract Long-term memory formation requires changes in gene expression. One mechanism for altering gene expression involves chemical modifications of DNA or its associated histone molecules. These epigenetic tags have long been studied by developmental biologists for their role in cell differentiation, but recent evidence suggests they also coordinate behavior in terminally differentiated neurons. Epigenetic chemical modifications include DNA methylation as well as histone methylation, acetylation, phosphorylation, ubiquitylation, and sumoylation. DNA methylation and histone modificationsin particular acetylation, methylation, and phosphorylationplay a key role in regulating memory-related behavior. Moreover, neuroscientists investigating epigenetics have identified potential targets for therapeutic intervention in diseases like Alzheimers, especially with regard to histone deacetylases (HDACs).

Epigenetics and Memory Table of Contents

Introduction ....4 What is epigenetics? ...........5 Histone Acetylation ..........12 Histone Phosphorylation and Methylation ...26 DNA Methylation .............................................................................................................31 Challenges and Future Research ......................................................................................38 References ........................................................................................................................41

Epigenetics and Memory

Epigenetics is a contentious topic in contemporary biology. On one hand, it is the subject of increasing media attention and excitement among scientists: both PBS and the BBC recently had specials highlighting the revolutionary new field. Many scientists are optimistic that understanding epigenetics will lead to a holistic picture of how the environment and our genes interact. On the other hand, there is no clear consensus among scientists about what epigenetics is, much less about its implications for molecular biology or medicine. Yet the hype surrounding the epigenome continues, even as Wren (2009) shows that about 37% of genes in the human transcriptome have no publications characterizing their functionsa potential problem for understanding epigenetic regulation of these genes. Though further research will tell if the mainstream medias popularization of epigenetics is justified by data, this new approach is already changing how many scientific disciplines look at complex problems. One such discipline, neuroscience, is turning to epigenetics seeking a molecular explanation for memory formation, storage, and retrieval as well as understanding disorders of memory like Alzheimers. As this paper will outline, the epigenetic approach to memory has made significant progress in recent yearseven as a young sub-discipline. The main questions to address in order to understand this progress are as follows: 1. What is epigenetics and how have memory and dementia researchers approached it? 2. How does epigenetics affect memory and dementia? 3. What are some challenges in this research and what are future directions for it?

Epigenetics and Memory CHAPTER ONE What is epigenetics? Definitions for epigenetics are varied and ambiguous. As early as 1957, Conrad Hal Waddington proposed a definition for epigenetics, though he obviously was not as

familiar with its molecular mechanisms as scientists are now. In his book The Strategy of the Genes, he related epigenetics to epigenesis, or how genotypes give rise to phenotypes throughout development. This definition does not incorporate contemporary molecular biology research, so it has become largely irrelevant. Bird (2007) cites Arthur Riggs and colleagues for proposing a more contemporary definition in 1996. Epigenetics, they claim, is the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in the DNA sequence (p. 396). However, this definition is problematic because it leaves mechanisms for these changes open to interpretation and even makes heritability a necessity. Simply put, this is not consistent with contemporary usage of the word epigenetics. Bird (2007) subsequently proposes a very broad definition: the structural adaptation of chromosomal regions so as to register, signal, or perpetuate altered activity states (p. 398). Levenson and Sweatt (2005), on the other hand, argue that epigenetics is the mechanism for the stable maintenance of gene expression that involves physically marking DNA or its associated proteins (p. 109). Broadly speaking, then, epigenetics involves relatively stable chemical modifications to DNA or histones that in turn affect gene expression. Moreover, epigenetics does not involve changes in the DNA sequence itself. It is also important to note that the above definition is somewhat controversial, as many insist epigenetics should include only heritable supra-genetic changes in the cellas opposed to metastable or transient

Epigenetics and Memory chemical markings (Bonasio, Tu, & Reinberg, 2010). This standard would exclude most neuroscience research from epigenetics proper since neurons are terminally differentiated, postmitotic cells. Epigenetic signals can be either cis or trans as well. Cis

signals are inherited by chromosome segregation and physically associated with DNA or chromatin. Trans signals, on the other hand, are maintained in the cytosol by feedback loops independent of the inherited chromatin structure (Bonasio et al., 2010). With this definition of epigenetics as a heuristic, it is now necessary to discuss the main chemical modifications studied by neuroscientists. DNA methylation plays a key role in epigenetic regulation of gene expression; and the relatively well-characterized histone modifications involved in gene regulation are histone methylation, acetylation, and phosphorylation. This discussion of epigenetics will be limited to the aforementioned chemical marks, though various small RNAs (sRNAs) and even transcription factors are sometimes considered epigenetic gene regulators. DNA Methylation DNA methylation occurs when a methyl (--CH3) group attaches to the 5-position of a cytosine residue (Santi, Garrett, & Barr, 1983; Chen et al., 1991). There are four known enzymescalled DNA methyltransferases (Dnmts)that catalyze this reaction in mammals: Dnmt1 (Bestor, Laudano, Mattaliano, & Ingram, 1988), Dnmt2 (Yoder & Bestor, 1998), Dnmt3A, and Dnmt3B (Okano, Xie, & Li, 1998). Interestingly, a recently reported enzyme called Gadd45b has demethylase activity in neurogenesis. Given Gadd45bs close relationship to memory research, it will be discussed extensively later in this review. The roles of these Dnmts are varied, overlapping, and depend on the cellular

Epigenetics and Memory context at any given time. That said, their functions will be discussed later. It is notable, though, that sequence analysis has shown identical cells from the same lineage expressing disparate DNA methylation patterns, suggesting DNA methylation patterns are not replicated as judiciously as the nucleotides themselves (Silva, Ward, & White, 1993). This could give rise to significant variations in phenotype for individual cells or groups of cells. Moreover, DNA methylation generally occurs at cytosine residues followed by guanineknown as CpG dinucleotides. This is distinct from the term for CpG-rich areas called CpG islands. CpG islands are proximal to (or in) promoters for many housekeeping genes like actin (Gardiner-Garden & Frommer, 1987). It logically follows that DNA methylation is associated with transcriptional silencing. For instance, the protein methyl CpG-binding protein 2 (MECP2) binds to methylated DNA

(independent of the underlying DNA sequence) and has been implicated in transcriptional silencing by recruiting histone remodeling proteins (Jones et al., 1998). Some transcription factorserythroblastosis 1 (ETS1), for instancebind to unmethylated DNA and eschew the methylated variety (Maier, Colbert, Fitzsimmons, Clark, & Hagman, 2003). About 70% of CpG dinucleotides are methylated (Cooper & Krawczak, 1989). Finally, methylation may involve either de novo DNA methyltransferases or maintenance DNA methyltransferases. The former initiate the transfer of a methyl group from S-adenosyl methionine to non-methylated cytosine nucleosides while the latter add a methyl group to the complimentary strand of hemi-methylated DNA (i.e. DNA with no methyl group on one strand) (Day & Sweatt, 2010). Histone Modifications

Epigenetics and Memory Before discussing histone modifications, it is important to review the structure of

histone molecules. Core histones are organized in an octamer of four subunits, with each nucleosome containing two copies of every subunit: H2A, H2B, H3, and H4. In addition, each subunit has N-terminus tails that project beyond the rest of the protein. These tails are important in transcriptional regulation and memory, which will be addressed later. The two linker histones, H1 and H5, hold the histone octamer and DNA together as a nucleosome, but these will be discussed less than the others in this essay. The DNA wraps around each octamer with about 150 bp and may be either tightly or loosely wrapped depending on the chemical environment. This packaging results in two different kinds of chromatin: heterochromatin and euchromatin. Constitutive heterochromatin is generally tightly packed, found at centromeres and telomeres, and transcriptionally silent. Euchromatin, on the other hand, is the loosely packed form of chromatin associated with transcriptional activation. Another kind, facultative heterochromatin, silences gene expression in some cell types and activates it in others. Major histone modifications include methylation, acetylation, phosphorylation, sumoylation, and ubiquitylation. SUMO and ubiquitin are small proteins that attach to histone and other molecules as post-translational modifiers. Their roles in memory regulation via histone modification remain mysterious, although there are numerous examples of post-translational changes on memory-related molecules. For example, ubiquitin-mediated proteolysis degrades the regulatory subunit of PKA, causing deficits in long term memory (Chain, Schwartz, & Hegde, 1999). Methylation, acetylation, and phosphorylation involve the addition of a methyl (CH3) group, acetyl (COCH3) group, or phosphate (PO4) group, respectively, to certain amino acids of histone tails. For instance,

Epigenetics and Memory molecules called histone acetyltransferases (HATs) transfer an acetyl group from acetylCoA to lysine residues on histones. Complementarily, histone deacetylases (HDACs) catalyze the removal of acetyl groups on lysine residues. There are also histone methyltransferases (HMTs) and demethylases for transfer of a methyl group to or from histones, respectively. Kinases and phosphatases add and remove phosphate groups, respectively. Drugs called HDAC inhibitors pharmacologically inhibit the activity of HDACswhich has therapeutic potential since increasing histone acetylation (and thus gene expression) may help ameliorate memory loss. Table 1 and Figure 1 summarize known HDAC isoforms, HDAC inhibitors associated with them, and the sites of DNA and histone modifications (image credit: Roth & Sweatt, 2009). These basic molecular changes will be discussed throughout this essay; below is a specific example of epigenetic change in neural development that shows the enormous impact of epigenetics in the nervous system. Table 1.

Epigenetics and Memory Figure 1.

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Neural cell differentiation is an example of the potent effects of chromatin remodeling. Clearly neurons have striking differences from other cells in the body. Besides maintaining an ionic gradient conducive to action potential firing, they have a distinctive molecular toolkit involved in excitability and synapse function. So neurons must develop this functional distinctiveness from other cell types. Promoter regions for neuronal genes contain a neuron-restrictive silencer element (NRSE) (Maue, Kraner, Goodman, & Mandel, 1990; Mori, Schoenherr, Vandenbergh, & Anderson, 1992). Other

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cell types ubiquitously express a transcription factor, RE-1-silencing transcription factor (also known as REST or NRSF) that silences expression of neuronal genes (Chong et al., 1995). Transcriptional repression with REST involves changes in chromatin structure. Two proteins, SIN3A and CoREST, bind to REST and affect HDAC1 and HDAC2, respectively (Huang, Myers, & Dingledine, 1999). Although expression of SIN3A mirrors expression levels of REST, CoREST is more restrictedperhaps because SIN3A mediates most NRSE-associated gene silencing whereas CoREST serves a particular role in subtypes of neural cells (Grimes et al., 2000). Both SIN3A and CoREST increase histone deacetylase activity by their interaction with the aforementioned HDACs. The REST/CoREST complex is also associated with increased DNA methylation, and it targets not only genes with the NRSE but also genes proximal to an NRSE (Lunyak et al., 2002). So silencing of neural genes in non-neural cells occurs by the activity of a chromatin remodeling complex that decreases histone acetylation and increases DNA methylation. Epigenetic markings thus have an enormous impact on neural phenotype. The specific neuroepigenetic correlates of learning and memory will be discussed extensively throughout this review. Particular attention will be given to the bettercharacterized correlates of learning and memory, including DNA methylation, histone acetylation, histone phosphorylation, and ubiquitylation.

Epigenetics and Memory CHAPTER TWO How does epigenetics affect memory and dementia? Histone Acetylation

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The best-characterized epigenetic modifications in memory research are histone acetylation, phosphorylation, and methylation as well as DNA methylation. Thus, these will be addressed in detail throughout this review. Two influential studies by Alarcn et al. (2004) and Korzus et al. (2004) addressed the importance of altered histone acetylation patterns in memory formation. Scientists used a model of Rubinstein-Taybi syndrome (RTS) to investigate the epigenetic cause of the severe mental retardation seen in RTS. This model originally piqued the interest of researchers in learning and memory because RTS is caused by a mutation in the CREB binding protein (CBP) gene, and CREB has been reported as a major protein involved in the late phase of long-term potentiation (LTP). First, researchers investigated the phenotype of a heterozygous (cbp+/-) mutant mouse, which shows symptoms similar to RTS in humans. Despite no change in baseline motor activity, anxiety, or short-term memory, the mice showed significant deficits in long-term memory as measured by contextual fear conditioning, cued fear conditioning, and novel object recognition paradigms (Alarcon et al., 2004). However, they showed normal performance in the Morris water maze taskthe authors suggest this is due to practice in this task (in contrast, fear conditioning depended on one noxious event). Moreover, there were electrophysiological signs of late phase LTP deficits in the cbp+/- mice at Schaffer collateral synapses. Mutant mice expressing a constitutively active form of CREB did not show normal memory capacity, suggesting there is another mechanism involved. Alarcn

Epigenetics and Memory and colleagues suggest histone acetylation as a possible mechanism. They tested their hypothesis pharmacologically, giving cbp +/- mice the histone deacetylase (HDAC) inhibitor SAHA. In addition to causing increased H2B acetylation, SAHA restored contextual memory and electrophysiological response to wild-type levels. Korzus et al.

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(2004) investigated this phenomenon further with a different mouse model of RTS. Given that the aforementioned mutant mice are susceptible to developmental abnormalities that could confound results, Korzus and colleagues (2004) developed a mouse with spatial and temporal restriction of CBP deficits. In particular, CA1 and dentate gyrus hippocampal neurons express a form of CBP without histone acetyltransferase (HAT) activity only after treatment with tetracycline. These (CBP{HAT+/-}) mice are substantially impaired in their ability to perform a visual-paired comparison task as well as the Morris water maze task (for spatial memory). However, additional practice restored performance on the Morris task, lending credence to the hypothesis Alarcon et al. (2004) gave about their RTS models Morris task abilities. Termination of the tetracycline diet as well as treatment with the HDAC inhibitor trichostatin A (TSA) ameliorated the memory-related symptoms of the RTS model mice, indicating that the HAT activity of CBP is critical to memory formation (Korzus, Rosenfeld, & Mayford, 2004). These results have been further confirmed and elaborated on. Although TSA was assumed to work by nonspecifically increasing gene expression, it has actually been shown to regulate expression of specific genes in a CREB:CBP-dependent mechanism (Vecsey et al., 2007). Researchers at Baylor College of Medicine validated a critical role for histone acetylation in memory later that year. Levenson et al. (2004) first trained mice in a

Epigenetics and Memory contextual fear conditioning paradigm and collected tissue samples from the hippocampus either 1 hour or 24 hours afterwards. Compared to control subjects, fear-

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conditioned mice showed a two-fold increase in acetylation of histone H3 (Lys-14) after 1 hour. However, the increase was transient; they observed decreased acetylation (normal levels) after 24 hours. Mice unable to form an association between the unconditioned stimulus and the context in which it was received also showed normal acetylation levels (a well-characterized phenomenon called latent inhibition). This indicates that memory formation requires increased acetylation in certain areas of the chromosomesa transformation of heterochromatin. Their findings are also persuasive because they treated other mice with NMDA or MEK antagonists, relating canonical LTP pathways to the putative mechanism of chromatin remodeling. As predicted by other studies, acetylation levels were normal and mice failed to consolidate significant fear conditioning when treated with NMDA or MEK antagonists. In addition, treatment of mice with HDAC inhibitors (TSA or sodium butyrate) enhanced induction of LTP, demonstrated as an increase in freezing in the fear conditioning paradigm. This enhancement of LTP was due, at least in part, to a three to five-fold increase in acetylation of histone H4. They also found electrophysiological hallmarks similar to Alarcn et al. (2004) at Schaffer collateral synapses (Levenson et al., 2004). The dependence of latent inhibition on H4 changes and fear conditioning on H3 changes suggests a histone code for specific types of memories (Levenson & Sweatt, 2005). Understanding HDAC inhibition as an area for therapeutic intervention in learning and memory processes will likely require basic knowledge of the roles of HDACs themselves. As mentioned before, there are 11 known HDAC isoforms, so

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identifying discrete roles for each of them is itself a daunting task. HDAC1 research will be addressed later, as it ties in with a neurodegeneration study by Fischer and colleagues (2007). HDAC2 and HDAC3, however, have been characterized as negative regulators of learning and memory. Guan et al. (2009) implicates HDAC2, and excludes HDAC1, in negative regulation of memory formation and synaptic plasticity. First, they created mutant mouse lines overexpressing HDAC1 and HDAC2 (called HDAC1OE mice and HDAC2OE, respectively) in areas important for memory, such as pyramidal neurons in the hippocampus. The HDAC1 and HDAC2 coding sequences were placed in frame with endogenous Tau, with Tau mutants showing no memory deficits compared to wild type mice. HDAC2OE mice had about 70% less histone 4 lysine 12 (H4K12) acetylation compared to wild type mice as well as significantly less H4K5 acetylation but no change in Ac-H4K14. HDAC1OE mice also had significantly less overall acetylated lysine, but there was no effect on H4K12 and H4K5 as in HDAC2OE mice. HDAC1OE mice had no deficits in associative conditioning (freezing in tone-dependent and contextual fear conditioning). On the other hand, HDAC2OE mice showed ~20% less freezing in tonedependent conditioning as well as ~45% less freezing in contextual fear conditioning experiments. HDAC1OE mice also had similar escape latency and quadrant affiliation as wild type mice in the Morris water maze task. HDAC2OE mice had much higher escape latency than HDAC1OE and wild type mice in addition to spending much less time in the quadrant containing the platform. Correspondingly, HDAC2 knockout mice had more histone acetylation as well as increased freezing behavior. Gross anatomical analysis of HDAC KO and OE mice indicated no developmental abnormalities as well. With regard

Epigenetics and Memory to spine density in the CA1 region of the hippocampus, HDAC2OE mice had significantly less than wild type mice while HDAC2 KO mice had much more. An

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immunoreactive synaptophysin assay also showed less synapse formation in HDAC2OE mice and more synapse formation in HDAC2 KO mice. HDAC inhibition (HDACi) with SAHA restored memory functioning in HDAC2OE mice. Finally, HDAC2 KO mice had increased electrophysiological signs of LTP (fEPSPs) over 40 minutes of testing while HDAC2OE mice showed decreased LTP. In addition, a variety a memory-related genes interact with HDAC2 at their promoter regions, and HDAC2 preferentially associates with CoREST. This could pave the way for a potential mechanism by which HDAC2 functions. Given the important role CoREST plays in neuronal gene repression (outlined above), it is plausible that HDAC2 mediates learning and memory by interacting with basic neuronal regulatory mechanisms. Taken together, this indicates a critical role for HDAC2 in memory formation, particularly as a negative regulator of molecular, behavioral, and electrophysiological signs of memory in mice (Guan et al., 2009). HDAC3 performs a similar role as HDAC2, according to McQuown et al. (2011). Researchers probed the role of HDAC3 using a genetic and pharmacological approach. Using a combination of mutant mice and viral infection, they created homozygous deletions of HDAC3 in area CA1 in the dorsal hippocampus. Moreover, they used the selective inhibitor RGFP136 to pharmacologically block normal HDAC3 functioning in the hippocampus. Assayed by immunohistochemistry, both conditions increased histone acetylation in CA1. As expected, genetic and pharmacological HDAC3 manipulation enhanced performance in long-term memory tests. There was also increased hippocampal expression of the memory-related genes nuclear receptor subfamily 4 group A, member 2

Epigenetics and Memory (Nr4a2) and c-fos. However, hippocampus-specific delivery of small interfering RNA (siRNA) targeting Nr4a2 blocked the memory enhancements associated with HDAC3 deletion (but not pharmacological inhibition), suggesting a potential mechanism for HDAC3s negative regulation of long-term memory (McQuown et al., 2011). Besides a role in associative and spatial memory formation, histone acetylation

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changes are also implicated in animal models of Alzheimers and neurodegeneration. It has been previously demonstrated that recovery of learning and memory in mice with significant brain atrophy and loss of memory is associated with environmental enrichment (EE). Findings by Fischer, Sananbenesi, Wang, Dobbin, and Tsai (2007) show that histone proteins are involved in synaptic plasticity and mediate the memoryinfluencing effects of environmental enrichment for mice. Fischer et al. (2007) used CK-p25 Tg micein which a doxycycline diet induces expression of the neurotoxic protein p25to induce symptoms of dementia to compare with a control. Neuronal populations were cut in half in the cingulate cortexassayed by the presence of NeuN, a neuron-specific, nuclear protein. The transgenic mice were split into two groups: those reared with EE and those reared without EE. The EE group recovered long-term memories significantly better than the non-EE group, with about 30% savings for the water maze and fear conditioning taskseven when p25-induced changes in anxiety and locomotor activity were taken into account. In addition, the EE and non-EE transgenic groups showed similar brain atrophy levels, suggesting EE leads to the recovery of long-term memories. Finally, environmental enrichment resulted in significant lysine acetylation increases on histones H3 and H4 in both the cortex and hippocampus. The acetylation effect continued over 2 weeks of measurements after

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behavioral memory tests. Levels of synaptophysin assessed by an immunoreactivity assay in the anterior cingulate cortex indicated significantly more synapses in environmentally enriched animals as well. To investigate whether chromatin remodeling mimics the effects of environmental enrichment, Fischer et al. (2007) used the HDAC inhibitor sodium butyrate (SB). Some Tg mice were given daily injections of SB to induce chromatin remodeling while others were treated with saline solution. Similar to the EE and non-EE groups, the SB group showed about 45% savings for fear conditioning and about 30% savings for the water maze task compared to mice injected with saline solution. Given a period of 4 weeks for consolidation of a fear-conditioning task, the SB-injected mice had similar improvement over mice injected with saline solution. In many tests SB-treated mice performed as well as or nearly as well as wild type mice. Moreover, there were more synapses and upregulated histone acetylation of H3 and H4 lysine residues despite no apparent increase in the number of neurons. This supports the hypothesis that increasing histone-tail acetylation mimics the memory-enhancing effects of environmental enrichment, induces synaptic rewiring, and even leads to the recovery of inaccessible long-term memories in mice (Fischer et al., 2007). Given the importance of characterizing HDAC functioning to understand how HDAC inhibition works, researchers also considered the effects of the CK-p25 Tg mouse model on HDAC1. Cell cycle changes and DNA damage are becoming important areas of interest in neurodegeneration research, and expression of p25 initiates many hallmarks of aberrant cell cycle activity and DNA damage before atrophy occurs. Messenger RNA levels of many cell cycle markers are up-regulated by p25 toxicity in the hippocampus,

Epigenetics and Memory including cyclin B, cyclin E, cdc25a, p21, MCM3, MCM6, and MCM7. In addition,

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protein levels of the DNA damage markers gH2AX and Rad51 were greatly up-regulated in the forebrain after p25 induction. Increased comet tails on a micrograph and increased immunoreactivity of damage markers in fluorescence imaging also indicated DNA damage. Loss of HDAC1 by RNAi or pharmacological inhibition of HDAC1 activity with the class I HDACi MS-275 yielded similar results as p25 toxicity (i.e. DNA damage, aberrant cell cycle activity, and atrophy). More directly linking p25 cell damage and HDAC1 function, overexpression of HDAC1 in p25 cells protected against cell toxicity and DNA damage. This approach to ameliorate p25 damage was effective in cultured neurons as well as in an in vivo ischemia model (Kim et al., 2008). It is therefore clear HDAC1 plays a role in neurodegeneration in the p25 model of cell toxicity, but researchers are also interested in traditional Alzheimers pathology. The traditional molecular hallmarks of AD, amyloid plaques and neurofibrillary tangles, are also important to incorporate in an epigenetic perspective on memory. Although the data is somewhat preliminary given the relative youth of epigenetics as a discipline, there is accumulating evidence that directly implicates epigenetics in canonical Alzheimers pathways. Amyloid- precursor protein (APP) is cleaved in the transmembrane domain by -secretase. This cleavage results in amyloid plaques on the extracellular side as well as a more mysterious domain on the intracellular side. Cao and Sdhof (2001) investigated the APP cytoplasmic (intracellular) domain using a two-hybrid system. The two-hybrid system allows researchers to investigate interactions among proteins by taking advantage of the distinct functional units, or domains, in transcription factors. Transcription factors typically have a DNA binding

Epigenetics and Memory domain (DBD) as well as an activation domain (AD). To accomplish the two-hybrid

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approach, researchers first attach a protein of interest to the DBD of a transcription factor (this fusion protein is called the bait). Second, a protein that presumably interacts with the domain of interest is attached to the AD of a transcription factor (this protein is called the prey). If there is, in fact, an interaction between the bait and prey proteins, transcription will be greatly up-regulated on a reporter gene (usually lacZ). This is because the interacting proteins bring the DBD and AD in such close proximity that they can function similarly to the normal transcription factor and thus initiate transcription. Of course, if there is little or no interaction between two proteins there will be little or no upregulation of transcription of the reporter gene. The most common transcription factors used in the two-hybrid approach are yeast Gal4 and bacterial LexA (Little, 2010). Cao and Sdhof (2001) characterized protein-protein interactions of the APP cytoplasmic domain using cell culture models (PC12, HEK293, COS, and HeLa cells). The DBDs of Gal4 or LexA were fused with full length, intracellular APP695. They also cotransfected the cells with a Gal4/LexA-dependent plasmid containing luciferase (the reporter gene) to analyze transcription changes. There was a small change in transcription levels in the Gal4/LexA transcription factor fusion APP, but >2000-fold transcription increases occurred in cells cotransfected with Fe65. This indicates interaction between exogenous APP and Fe65. Additionally, the increase in transcription did not occur in APP with a point mutation that blocks Fe65 binding; nor was there an increase in transcription when the prey was Mint1/X11 (i.e. the effect is specific to Fe65). Cao and Sdhof (2001) reported similar results in all cell types they tested. Both PTB1 and PTB2 were required domains for Fe65 functioning in their assay. Additionally, the same two-

Epigenetics and Memory hybrid assay was used with a different reporter in COS cells to examine interactions

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between Fe65 and a HAT reported in many cancer studies, Tip60. There was a dramatic transcriptional increase when Gal4-Tip60 was co-expressed with Fe65 and APP, but either no increase or very little increase with mutant APP, Fe65 alone, or APP alone. This implies that Tip60 histone acetyltransferase forms a complex with both Fe65 and the cytoplasmic domain of cleaved APP to remodel chromatin and increase gene expressiondirectly linking epigenetics and canonical Alzheimers pathways (Cao & Sdhof, 2001). Since epigenetic modifications happen in the nucleus (where chromosomes are localized), Cao and Sdhof (2001) determined by fluorescence microscopy that Tip60 is in the nucleus under every condition tested while Fe65 is nuclear only in the absence of APP (or in the presence of mutant APP). APP was localized in the cytoplasm, but because of assay limitations this did not preclude the possibility that some (<5%) APP might be in the nucleus. There is additional evidence suggesting the APP intracellular domain (AICD) functions in a similar manner to the Notch intracellular domain (NICD) (Sastre et al., 2001; Kimberly, Zheng, Guenette, & Selkoe, 2001). However, an important study notes that the AICD also has a pathway of its own and regulates its own precursors transcription via the multiprotein complex described by Cao and Sdhof (2001) (von Rotz et al., 2004). Using a combination of confocal microscopy with inducible fluorescence tagging and co-immunoprecipitation, von Rotz and colleagues (2004) determined that Tip60 localized in the nucleus. However, fluorescent AICD localized in the nucleus with Tip60 only in the presence of Fe65 in HEK293 cells. The AICD-Fe65Tip60 complex (called AFT) had structural damage in a mutant of Tip60, abolishing the

Epigenetics and Memory spherical nuclear spots that indicated nuclear localization of AFT. Gamma-secretase

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inhibitors also blocked formation of the AFT complex. Besides the AFT complex, Tip60 and AICD also form an association with the APP adaptor protein Jip1b. The AICD-Jip1bTip60 (AJT) complex serves a similar function as AFT, with Jip1b aiding in transport of AICD to the nucleus and docking it to Tip60. Consistent with Cao and Sdhof (2001), Mint1/X11 trapped AICD in the cytosol. Finally, there is an apparent positive feedback mechanism involved in AICD signaling. Induced expression of AICD resulted in increased expression of APP and Tip60, but not the Notch-effector gene Hes1 (von Rotz et al., 2004). Although present studies of APP intracellular signaling are far from providing a comprehensive view of how epigenetics and amyloid- are connected, there is already evidence that epigenetic intervention in APP signaling could lead to better treatments of Alzheimers. Additional evidence for epigenetic involvement in APP signaling comes from a mutant mouse model, APP/PS1dE9. APPswe/PS1dE9 mice have contextual memory deficits after 6 months of life due to overexpression of presenilin-1 (PS1) and the presence of a Swedish mutation, which is linked to early-onset AD. Kilgore et al. (2010) demonstrated the beneficial effects of HDACi on class I HDACs in the aforementioned AD model mouse. Chronic (2-3 week), intraperitoneal injection (IP) of sodium valproate, sodium butyrate, or vorinostat (SAHA) into the mutant mouse enhanced histone acetylation and contextual memory task performance to wild type levels. Amelioration of memory function in the mutant mice happened without affecting baseline exploratory behavior or immediate freezing. Thus, state-dependent effects were not an issue and HDACi of class I HDACs is a potential therapeutic pathway for Alzheimers researchers.

Epigenetics and Memory Most strikingly, SAHA affected each class of HDAC to achieve its therapeutic effect. Given that vorinostat is already a T-cell lymphoma drug marketed as Zolinza, clinical trials in AD would be much easier to pursue. As expected, memory improvement correlated with histone acetylation increases (Kilgore et al., 2010). Besides its relationship with APP biology, Alzheimers is also an age-related

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disease diagnosed most frequently in people over 65 years of age. Given the likelihood of memory-related problems occurring later in life, Peleg et al. (2010) designed an experiment to relate hippocampal histone acetylation changes to aging in mice. Levels of histone tail acetylation in three-month-old mice were compared to sixteen-month-old mice in a contextual fear conditioning task. Given that mice live 26-28 months on average, 16-month-old mice could be considered elderly. Behaviorally, 16-month-old mice performed significantly less freezing and had increased platform-locating latency in the Morris task (besides spending significantly less time in the target quadrant). The lysine acetylation assay was performed in control mice as well as mice 10 min., 30 min., 60 min., and 24 hours after fear conditioning training. Residues analyzed for acetylation changes include H3K9, H3K14, H4K5, H4K8, H4K12, and H4K16. The histone acetylation profile of every group was similar with two notable exceptions. H4K5 showed increased acetylation at 30 min. after fear conditioning in 16-month-old mice while there was no such increase in 3-month-olds. Since other time points showed a similar acetylation profile in young and aged mice at H4K5, this result is much less eyecatching than the following one. H4K12 acetylation did not increase in 16-month-old mice as a response to fear conditioning at any time point, whereas in 3-month-old mice every fear-conditioned group had increased acetylation. This happened without any

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change in baseline HAT/HDAC or H4 levels in the cell. This led researchers to conclude that deregulation of H4K12 in aged rats after fear conditioning leads to their impaired memory performance. Furthermore, microarray analysis of hippocampal gene expression 1 hour after fear conditioning showed 2229 gene transcript changes (increase or decrease) in 3-month-old mice, with 1539 of those genes previously linked to associative learning. On the other hand, 16-month-old mice had only 6 gene expression changes after fear conditioning. Some gene expression profiles deciphered by data mining were also verified with real-time PCR. Baseline gene expression was virtually identical in 3-month vs. 16-month control groups. Taken together, this means deregulation of H4K12 acetylation following fear conditioning in aged mice results in drastically different gene expression profiles than young mice. Similar to other studies, SAHA treatment of 16month-old mice increased freezing behavior in an associative learning task. Interestingly, H4K12 acetylation increased significantly in response to SAHA treatment whereas no other lysine residues had a statistically significant increase, including H4K5 (which had increased acetylation at the 30 min. time point in the old group). Finally, H4K12 acetylation increased at the promoter regions of the formin 2 and Prkca genes, and a variety of other genes had increased H4K12 acetylation at coding regions. Increased H4K12 acetylation at coding regions correlated with increased gene expression as well. There was no change in the 16-month-old groups ability to find a visible platform, exploratory behavior, or response to foot shock; various markers of hippocampal plasticity and integrity were similar between young and old mice. Thus, agings negative effect on memory capacity in mice is related to H4K12 acetylation changes rather than loss of structural integrity, exploratory behavior, or response to foot shock (Peleg et al.,

Epigenetics and Memory 2010). Epigenetics could, therefore, offer insight into the cognitive decline seen with natural aging.

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Epigenetics and Memory CHAPTER THREE How does epigenetics affect memory and dementia? Histone Phosphorylation and Methylation There has recently been an explosion of knowledge gathered about the relationship between histone acetylation, DNA methylation, and memory, with

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comparatively little work done on histone phosphorylation and methylation. At the very least, however, the latter two chemical alterations appear important in early hippocampal memory consolidation. Notably, there is currently no work published on prefrontal cortex changes with respect to histone phosphorylation and methylation, which puts this research significantly behind other epigenetic approaches. That is not to say work is not currently being done on this. The Lubin lab at the University of Alabama has already given oral presentations of forthcoming work relating histone methylation to prefrontal cortex memory consolidation. It has been previously reported that cellular stress and activation of immediate early genes correlates with increases in histone phosphorylation (Thomson, Clayton, & Mahadevan, 2001). Two early studies have also indicated the importance of histone phosphorylation in early memory consolidation in the hippocampus. Crosio, Heitz, Allis, Borrelli, and Corsi (2003) demonstrated that multiple signaling pathways can trigger histone phosphorylation in the hippocampus. Researchers used a dopaminergic receptor agonist (SKF82958), mACh receptor agonist (pilocarpine), and a kainate glutamate receptor agonist (kainic acid), all of which induced chromatin remodeling in hippocampal neurons. Marks of chromatin remodeling included H3 serine 10 phosphorylation and lysine 14 acetylation. Validating previous results, activation of these signaling pathways

Epigenetics and Memory also increased c-fos transcription (Crosio et al., 2003). However, these results do not necessarily indicate that histone phosphorylation is important in learning and memory. They only show that hippocampal neurons use it in response to stimulation.

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More directly implicating histone phosphorylation in learning processes, Chwang, ORiordan, Levenson, and Sweatt (2006) found phosphorylation and acetylation increases (on H3S10 and H3K14) as a result of ERK/MAPK activation in vitro. The process was NMDA receptor-dependent since the NDMA-R anatagonist MK801 blocked the effect. More importantly, though, contextual fear conditioning increased area CA1 histone phosphorylation transiently (1-2 hours after training). MEK works just upstream of ERK in the ERK signaling cascade, and administration of the MEK inhibitor SL327 blocked H3S10 phosphorylation changes after fear conditioning. This result was verified with a phospho-ERK assay, which made sure phosphorylation (activation) of ERK was, in fact, inhibited by SL327 (Chwang et al., 2006). Although phenomenological results are important, it is clinically beneficial to identify target kinases and phosphatases involved in epigenetic modification for therapeutic intervention. ERK/MEK signaling has long been considered an important pathway in modulation of gene expression, but an important study by Lubin and Sweatt (2007) elucidates the role of a well-studied immune response kinase in memory reconsolidation. Memory maintenance requires reconsolidation after recall. In rats, contextual fear conditioning normally involves shocking the animal in a novel context (the conditioning chamber) and testing freezing behavior after the initial training. Reestablishment of contextual conditioned fear (CCF) memory, accomplished by re-exposing the animal to the novel context, can be blocked with inhibition of protein synthesisERK/MAPK

Epigenetics and Memory inhibition, for instance (Lubin & Sweatt, 2007). Lubin and Sweatt (2007) specifically looked at signaling with the transcription factor nuclear factor kappa B (NF-B) to see whether there was an effect on histone phosphorylation during reconsolidation. It is first necessary to review information on NF-B signaling, which has been

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primarily implicated in response to inflammation. NF-B is inhibited by inhibitor kappa B (IB); thus modification of IB is required in order to activate NF-B. IB proteins are marked for degradation when they are phosphorylated by the IB kinase (IKK) complex. The IKK complex has , , and regulatory subunits. Once IKK frees IB from NF-B, NF-B translocates to the nucleus and binds genes with the B consensus sequence in their promoter. However, components of the NF-B signaling pathway have been shown to add phosphate and acyl groups to histones in nonneuronal cells, so the NF-B DNA binding complex is likely not the whole story; this was the impetus for investigating chromatin remodeling by NF-B in hippocampus cells. First, the NF-B inhibitor diethyldithiocarbamate (DDTC) was administered (via IP injection) to a group of rats after a memory test. Although vehicle and DDTC rats exhibited similar freezing behavior during the first memory test, freezing decreased in DDTC-treated rats during tests 2 days and 7 days after training, suggesting NF-B inhibition interferes with CCF memory. Phosphorylated IKK serine 180and not IKK serine 181levels also increased in area CA1 1 hour after training. DDTC treatment blocked the increase. Increased IKK phosphorylation only occurred in rats re-exposed to the context after initial context + shock training, as there was no such increase in animals only exposed to the context (over 1 or 2 days) or only given contextual fear conditioning. DNA binding activity of NF-B assessed by an electrophoretic mobility

Epigenetics and Memory shift assay (EMSA) also rose following conditioning and attenuated in DDTC groups.

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DDCT injection ultimately resulted in decreased H3 phosphorylation after CCF training. Surprisingly, pharmacological inhibition of IKK with sulfasalazine (SSZ) reversed the behavioral and molecular effects of context re-exposure. Another pharmacological tool, SN50, blocks NF-Bs normal interaction with DNA binding sites. SN50 administration decreased freezing behavior in the CCF paradigm, but there was no effect on histone phosphorylation. On the other hand, IKK inhibition decreased phosphorylation at the promoters of the immediate early gene Zif268 as well as IB. Taken together, the results impart a critical role for IB kinase (IKK) in regulation of chromatin structure after CCF memory training in area CA1 of the hippocampus. Regulation of chromatin structure by IKK happens upstream of regulation by NF-Bs transcription complex to affect learning and memory (Lubin & Sweatt, 2007). The kinase activity of IKK thus represents a promising target for therapeutic intervention in diseases of memory. Histone methylations involvement in memory-related processes remains more mysterious than other epigenetic factors. Gupta et al. (2010) was the first to investigate hippocampus-specific histone methylation patterns after fear conditioning. Unlike previous epigenetic marks discussed in this review, researchers demonstrated transcriptional activation and silencing with histone methylation. This is due to the wellcharacterized capacity of lysine residues to be monomethylated, dimethylated, or trimethylated. Each of these result in a different transcriptional hallmark depending on the lysine residue of interest. Tissue collection 1 hour after fear conditioning from area CA1 of the hippocampus showed an increase in both H3 trimethylation at lysine 4 (H3K4me3) as well as H3K9me2. H3K9 dimethylation, however, decreased below

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control levels 24 hours after fear conditioning while H3K4 trimethylation returned to the baseline. Latent inhibition (i.e. exposure to the context before context+shock pairing) decreased freezing behavior and blocked the transient increase in H3K4me3, so proper timing is crucial for transient trimethylation. Deficits in a methyltransferase, Mll, with specific activity on H3K4 resulted in decreased freezing behavior. Finally, promoters regions for Zif268 and BDNF also had an increase in H3K4me3 after fear conditioning. HDAC inhibition with sodium butyrate also helped increase trimethylation and decrease dimethylation, confirming pervious studies. The trimethylation increase correlated with altered DNA methylation at Zif268 promoters, increased mRNA levels, and altered MeCP2 binding (Gupta et al., 2010). Histone methylation therefore functions to alter memory consolidation in area CA1 of the hippocampus in concert with histone acetylation and phosphorylation. However, there is currently no evidence of histone methylation as a mark of memory maintenance. This concludes the discussion of histone modifications in learning and memory, but DNA methylation also impacts learningrelated behavior.

Epigenetics and Memory CHAPTER FOUR How does epigenetics affect memory and dementia? DNA Methylation

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As outlined previously, DNA methylation involves the enzyme-catalyzed addition of a methyl (--CH3) group at the 5 position of cytosine pyrimidine rings. There is increasing evidence that transcriptional regulation by DNA methylation aids in both memory formation in the hippocampus as well as memory maintenance in the cortex. Miller and Sweatt (2007) reported an important role for DNA methylation by DNMTs in contextual fear conditioning experiments. Context + shock animals showed dramatically up-regulated mRNA levels of DNMT3A, DNMT3B, and c-fos 30 minutes following fear conditioning (compared to context only animals) in area CA1 of the hippocampus. Infusion of global DNMT inhibitors 5-azadeoxycytidine (5-aza) or zebularine (zeb) into area CA1 of the hippocampus immediately after training in a contextual fear conditioning task blocked freezing behavior. This effect was not seen in animals injected with a DNMT inhibitor 6 hours after training. Notably, the decrease in freezing was transientDNMT inhibited animals in later tests had similar freezing times as control animals in the test 1 day after training. A CpG island analyzed in the memory suppressor gene protein phosphatase 1 (PP1) was dramatically more methylated than context-only controls 1 hour after learning, and DNMT inhibition occluded this effect while increasing mRNA of PP1 in area CA1. Reelin (Reln) methylation also decreases after fear conditioning; the effect is larger with DNMT inhibition. These results suggest a critical role for covalent modification of DNA in memory formation in CA1 of the hippocampus (Miller & Sweatt, 2007).

Epigenetics and Memory Similar to studies on the native function of HDACs in the chapter on histone

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acetylation, some DNMTs have been analyzed for specific functional properties. Feng et al. (2010) implicates Dnmt1 and Dnmt3a in synaptic function, learning, and memory. Although mice with mutations in Dnmt1, Dnmt3a, or Dnmt3b are not viable, Feng et al. (2010) developed a conditional knockout model for adult forebrain neurons that eschews viability issuesmore specifically they used a CaMKIIa-Cre93 transgene to induce deletion exclusively in postmitotic neurons postnatally. Researchers knocked out Dnmt1, Dnmt3a, or both; no significant gene knockout occurred by postnatal day 3 (P3), but by P14 and into 4 months of life knockout was accomplished. Stereological analysis of double knockout (DKO) mice revealed slightly decreased hippocampus and dentate gyrus volume while single knockout mice had no obvious anatomical differences from control mice. Regardless, optical fractionator analysis indicated that the decrease in total volume was not a result of atrophy but rather a decrease in volume of individual neurons. First, DKO mice had disrupted LTP at Schaffer collateral-CA1 synapses and enhanced LTD in a stimulation protocol that normally fails to induce LTD. Behaviorally, DKO mice took more time to find the platform in the Morris water maze and spent less time in the target quadrant than control mice with no change in baseline swimming speeds. This indicates deficits in spatial learning and memory in DKO mice. Knockout animals also exhibited less freezing behavior 24 hours after contextual fear conditioning, another indicator of learning and memory deficiency. Microarray and real time PCR analysis revealed upregulated immune genes important in learning and memory, including MHC-I and Stat1. Interestingly, Reln and PP1 levels did not change in DKO mice. These genes will be discussed in more detail later. Bisulfite sequencing also showed significantly less

Epigenetics and Memory DNA methylation at the Stat1 promoter from -895 to -1,010 bp in DKO mice. This did not occur in single knockout mice, suggesting Dnmt1 and Dnmt3a compensate for one

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another. In addition, demethylation occurred only in cells that tested positive for NeuN in fluorescence-activated cell sorting (FACS)an indicator of neuronal populations. Finally, demethylation was verified with a variant of mass spectrometry, and bisulfite sequencing found demethylation on the promoters for Dhh, Kcne1, and two regions of Pten. Overall, these results strongly suggest Dnmt1 and Dnmt3a work together to maintain methylation patterns in adult forebrain neurons. Their regulation of DNA methylation ultimately perpetuates proper synaptic function for learning and memory (Feng et al., 2010). One of the most important results with regard to DNA methylation comes from Miller et al (2010). While previous studies investigated transient epigenetic marks after learning in animals, Miller et al. (2010) found long-lasting DNA methylation patterns maintaining memory. Since behavioral memories persist multiple molecular lifetimes, maintaining these memories would likely involve such a self-perpetuating mechanism. Consequently, DNA methylation is a good candidate for maintenance of transcriptional repression. However, one of the most compelling aspects of Miller et al. (2010) is its description of DNA methylation as a potential mechanism for remote memory maintenance in a prefrontal cortical area called the anterior cingulate cortex (ACC). It has been demonstrated previously that the ACC maintains remote contextual fear memory. In particular, activity-dependent expression of c-fos and Zif268 (genes implicated in memory) increases during remote memory maintenance in the ACC. Moreover, a null CaMKII mutation and pharmacological inactivation of the ACC with lidocaine block

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remote memory (Frankland, Bontempi, Talton, Kaczmarek, & Silva, 2004). However, a mechanism for long-term memory storage remains elusive. The prefrontal cortex is a popular candidate for involvement in epigenetic memory maintenancehippocampal epigenetic marks return to baseline levels after a maximum of about one day after learning. There are now results implicating DNA methylation in the consolidation process. Miller et al. (2010) first used immunoprecipitation to examine dorsomedial prefrontal cortex (dmPFC) DNA methylation levels on learning-related genes that have large GC-rich CpG islands in rodents. The immediate early gene Erg1 was demethylated at all time points. On the other hand, reelin (Reln) was hypermethylated 1 hour after training, with levels of hypermethylation decreasing over time. This result is somewhat surprising since reelin is considered a positive regulator of memory and hypermethylation is associated with transcriptional silencing. Finally, methylation of the phosphatase and memory suppressor gene calcineurin (CaN, Ppp3ca) did not change shortly after training. Rather, hypermethylation measured by bisulfite sequencing occurred within 1 day of training and persisted in tests both 7 days and 30 days after training. This is more intuitive than results for Reln since CaN is a memory suppressor gene. Thus, CaN was used as a proxy for global dmPFC DNA methylation changes occurring after contextual fear conditioning. To further confirm that the observed DNA methylation changes represent associative learning persisting in the cortex, a group of rats tested 7 days posttraining were injected with the NMDA antagonist MK-801. Besides blocking acquisition of the fear memory, MK-801 administration interfered with dmPFC hypermethylation of CaN and Reln (with no effect on Erg1), suggesting that specific environmental signals induce hypermethylation of CaN and Reln. Notably, hypermethylation of CaN was

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accompanied by considerable decreases in CaN mRNA and protein levels 30 days after training. But none of this determines whether DNA methylation is a necessary factor in maintaining remote memory. Pharmacological inhibitors of DNMTs (5azadeoxycytidine, zebularine, RG108) administered in the ACC (a subregion of the dmPFC) 30 days after training decreased freezing behavior by 45-60% in the memory test, in addition to reducing CaN DNA methylation levels and increasing CaN mRNA levels. Intra-ACC injections of DNMT inhibitors had no effect on fear memory 2 days after training; nor was there an effect in these rats 30 days after training, so administration of DNMT inhibitors to disrupt remote memory in the ACC must occur after necessary cortical DNA methylation events. Moreover, there was no indication of major damage to the ACC itself (Miller et al., 2010). DNA methylation also appears to be crucial in BDNFs ability to spur dendritic growth and positively regulate learning and memory. Lubin, Roth, and Sweatt (2008) first found a large increase in BDNF transcription in area CA1 of the hippocampus 2 hours after fear conditioning. The increase was specific to exon IV of BDNF, but others increased slightly in context-only controls. Fear conditioning was accompanied by methylation decreases at exons I and IV, and methylation increases at exon VI of BDNF. The appropriate increase or decrease in BDNF exon mRNA was confirmed with real time PCR. Similar to previous studies, DNMT inhibition (with zeb and RG108) reversed these behavioral and molecular hallmarks and changed BDNF expression. Researchers also found alterations in chromatin remodeling as measured by histone acetylation as a result of contextual fear conditioning and DNMT inhibition; and the process was NMDA receptor-dependent (Lubin et al., 2008). Similar results were reported by Martinowich et

Epigenetics and Memory al. (2003) for cultured neurons after depolarization. CpG methylation of the exon IV

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promoter decreased after depolarization while BDNF synthesis increased. This increase in transcription involved changes in a chromatin remodeling complex. The MeCP2HDAC1-mSin3A repression complex dissociated from the BDNF promoter, allowing the increase in transcription (Martinowich et al., 2003). Taken together, Martinowich et al. (2003) and Lubin, Roth, and Sweatt (2008) indicate that BDNFs important role in dendrite survival and learning depends on DNA methylation. As mentioned in the introduction, molecules with DNA demethylase activity have been elusive in mammals. Even the existence of demethylases was the subject of controversy. After all, a large amount of energy is required to break the covalent carbon bonds of methylated DNA, and this could be prohibitive on a realistic biological timescale. There have previously been reports of such proteins in C. elegans, but Ma et al. (2009) was the first to characterize a mammalian protein with specific demethylase activity and a role in adult neurogenesis, Gadd45b. Electroconvulsive treatment (ECT) of adult mice causes upregulation of hippocampal neurogenesis, and Ma et al. (2009) found a large induction of Gadd45b mRNA (growth arrest and DNA-damage-inducible protein 45 beta) 1-3 hours after ECT. Whats more, in situ hybridization also confirmed the increase in Gadd45b after ECT. Even exploration of a novel environment significantly increased Gadd45b expression in the dentate gyrus (measured 1 hour after exploration by immunostaining). Gadd45a had already been demonstrated as a demethylase in human cultured cells, so Gadd45b was a good candidate to investigate for demethylase activity controlling neurogenesis induction. Intriguingly, the dramatic mRNA increase was not seen for Gadd45a and Gadd45g in the hippocampus, implicating Gadd45b specifically in

Epigenetics and Memory the ECT/exploratory response process. The Gadd45b induction process depends on the NMDA receptor, as a NMDAR antagonist (CPP) administered in vivo before ECT

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specifically blocked Gadd45b up-regulation. A cell proliferation assay with BrdU in the dentate gyrus also showed that shRNA-induced knockdown of Gadd45b abolished normal ECT-dependent proliferation. Finally, the frequency of methylation at individual CpG sites of BDNF IX and FGF-1B increased to control levels in Gadd45b KO mice after ECT. The methylation increase was accompanies by decreased dendritic length of samples in the dentate gyrus (Ma et al., 2009). Since Gadd45b modulates neurogenesis and learning-related gene expression with demethylase activity, it will be the subject of more extensive research in the coming years. It is an exciting result because of its potential to give neural DNA methylation research another avenue for therapeutic intervention (similar to HDAC inhibition). This could be an important advance in treatment of Alzheimers disease, as age-specific DNA methylation pattern alterations have already been reported in late-onset AD patients (Wang, Oelze, & Schumacher, 2008). Normal aging even correlates with loss of genomic 5-methyldeoxycytidine (Wilson, Smith, Ma, & Cutler, 1987).

Epigenetics and Memory CHAPTER FIVE Challenges and Future Directions Given that epigenetics is a relatively new discipline, the literature is currently

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somewhat vague. For instance, most epigenetics of memory studies incorporate HDACs like sodium butyrate, which modulates expression of a large number of genes in a shotgun attempt to increase those that actually help memory. However, scientists do not fully understand what all of these genes door in other words why increased expression of a set of genes results in enhanced performance for mice in a fear conditioning task. And getting to that point will take time. It will require advanced knowledge of very specific HDACs, HDAC inhibitors, HATs, DNMTs, etc.knowledge that could require decades of research and substantial funding. Targeting specific histone residues to improve memory is a daunting task in itself, not to mention figuring out which ones improve memory without deleterious side-effects. That is not to say it is a far-fetched idea, though. EnVivo Pharmaceuticals recently announced that a drug similar in structure to SAHA reached phase 2 clinical trials for treatment of Alzheimers, according to private correspondence with one of their consultants, David Sweatt. Given the current intrigue with results like Fischer et al. (2007), in which the memory-related symptoms of neurodegeneration were greatly decreased despite the atrophy, funding may soon be easier to acquire for this research. After all, clinical implications this compelling have certainly received attention from governments seeking decreased medical care costs for the elderly and start-up companies looking for a panacea. While some clinical trials with HDACs have already failed in phase 3, it may only be a matter of time before the right molecules are tested if current mouse models are truly revelatory; the appeal of HDACs

Epigenetics and Memory or HATs for the treatment of diseases may not just be hype. Sananbenesi and Fischer

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(2009) argue that many inherited and sporadic epigenetic deregulations in brain diseases seem causally involved in disease progression unlike any other potential treatment options. This could be why HDAC inhibitors have both neuroprotective and neurodegenerative potentialepigenetics might be the key bottleneck to understanding these diseases. Though the histone code is not nearly as well understood as DNAs traditional code, deciphering it may be challenging and exciting for neuroscience. Indeed, there is even debate about whether the epigenetic marks outlined above integrate to form a histone code at all. Beyond this problem, there are few studies indicating that lifelong memories could be stored by such a histone code. The first studies suggesting this possibility relate long term epigenetic changes to early life stress and nurturing (Roth, Lubin, Funk, & Sweatt, 2009; Weaver et al., 2004). Still, it will take more research to fully answer the question of how lifelong memories are stored. Epigenetic states will even differ between brain regions to integrate behavior, and understanding this could take years of research as well (Day & Sweatt, 2011). Epigenetic modifications could very well act independently to affect gene expression. Another interesting implication of neuroepigenetics is its challenge to physiological psychologys doctrine of Hebbian learningcells that fire together wire together. While it may be true that interactions at thousands of synapses for each cell create neural circuits, epigenetics would require some rethinking of the initial hypothesis. Chemical changes in the nucleus of one cell could completely silence genes involved in plasticity, and thus shut down the neural circuit. So there could be at least two

Epigenetics and Memory mechanisms at play in the process of behavioral plasticityepigenetic and Hebbian (Roth & Sweatt, 2009).

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Regardless of these challenges, one thing remains clear: epigenetic manipulation is one of the most promising avenues for therapeutic intervention in Alzheimers right now. An epigenetic perspective could bridge the gap between known genetic predispositions for Alzheimers and the necessary cellular events that trigger it over time.

Epigenetics and Memory References Alarcon, J. M., Malleret, G., Touzani, K., Vronskaya, S., Ishii, S., Kandel, E. R., et al. (2004). Chromatin acetylation, memory, and LTP are impaired in CBP+/- mice: A model for the cognitive deficit in rubinstein-taybi syndrome and its amelioration. Neuron, 42(6), 947-959. doi:10.1016/j.neuron.2004.05.021

41

Bestor, T., Laudano, A., Mattaliano, R., & Ingram, V. (1988). Cloning and sequencing of a cDNA encoding DNA methyltransferase of mouse cells. the carboxyl-terminal domain of the mammalian enzymes is related to bacterial restriction methyltransferases. Journal of Molecular Biology, 203(4), 971-983. Bird, A. (2007). Perceptions of epigenetics. Nature, 447(7143), 396-398. doi:10.1038/nature05913 Bonasio, R., Tu, S., & Reinberg, D. (2010). Molecular signals of epigenetic states. Science (New York, N.Y.), 330(6004), 612-616. doi:10.1126/science.1191078 Cao, X., & Sudhof, T. C. (2001). A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science (New York, N.Y.), 293(5527), 115-120. doi:10.1126/science.1058783 Chain, D. G., Schwartz, J. H., & Hegde, A. N. (1999). Ubiquitin-mediated proteolysis in learning and memory. Molecular Neurobiology, 20(2-3), 125-142. doi:10.1007/BF02742438

Epigenetics and Memory

42

Chen, L., MacMillan, A. M., Chang, W., Ezaz-Nikpay, K., Lane, W. S., & Verdine, G. L. (1991). Direct identification of the active-site nucleophile in a DNA (cytosine-5)methyltransferase. Biochemistry, 30(46), 11018-11025. Chong, J. A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J. J., Zheng, Y., Boutros, M. C., et al. (1995). REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell, 80(6), 949-957. Chwang, W. B., O'Riordan, K. J., Levenson, J. M., & Sweatt, J. D. (2006). ERK/MAPK regulates hippocampal histone phosphorylation following contextual fear conditioning. Learning & Memory (Cold Spring Harbor, N.Y.), 13(3), 322-328. doi:10.1101/lm.152906 Cooper, D. N., & Krawczak, M. (1989). Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes. Human Genetics, 83(2), 181-188. Crosio, C., Heitz, E., Allis, C. D., Borrelli, E., & Sassone-Corsi, P. (2003). Chromatin remodeling and neuronal response: Multiple signaling pathways induce specific histone H3 modifications and early gene expression in hippocampal neurons. Journal of Cell Science, 116(Pt 24), 4905-4914. doi:10.1242/jcs.00804 Day, J. J., & Sweatt, J. D. (2010). DNA methylation and memory formation. Nature Neuroscience, 13(11), 1319-1323. doi:10.1038/nn.2666 Day, J. J., & Sweatt, J. D. (2011). Epigenetic modifications in neurons are essential for formation and storage of behavioral memory. Neuropsychopharmacology : Official

Epigenetics and Memory

43

Publication of the American College of Neuropsychopharmacology, 36(1), 357-358. doi:10.1038/npp.2010.125 Feng, J., Zhou, Y., Campbell, S. L., Le, T., Li, E., Sweatt, J. D., et al. (2010). Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons. Nature Neuroscience, 13(4), 423-430. doi:10.1038/nn.2514 Fischer, A., Sananbenesi, F., Wang, X., Dobbin, M., & Tsai, L. H. (2007). Recovery of learning and memory is associated with chromatin remodelling. Nature, 447(7141), 178-182. doi:10.1038/nature05772 Frankland, P. W., Bontempi, B., Talton, L. E., Kaczmarek, L., & Silva, A. J. (2004). The involvement of the anterior cingulate cortex in remote contextual fear memory. Science (New York, N.Y.), 304(5672), 881-883. doi:10.1126/science.1094804 Gardiner-Garden, M., & Frommer, M. (1987). CpG islands in vertebrate genomes. Journal of Molecular Biology, 196(2), 261-282. Grimes, J. A., Nielsen, S. J., Battaglioli, E., Miska, E. A., Speh, J. C., Berry, D. L., et al. (2000). The co-repressor mSin3A is a functional component of the REST-CoREST repressor complex. The Journal of Biological Chemistry, 275(13), 9461-9467. Guan, J. S., Haggarty, S. J., Giacometti, E., Dannenberg, J. H., Joseph, N., Gao, J., et al. (2009). HDAC2 negatively regulates memory formation and synaptic plasticity. Nature, 459(7243), 55-60. doi:10.1038/nature07925

Epigenetics and Memory Gupta, S., Kim, S. Y., Artis, S., Molfese, D. L., Schumacher, A., Sweatt, J. D., et al. (2010). Histone methylation regulates memory formation. The Journal of

44

Neuroscience : The Official Journal of the Society for Neuroscience, 30(10), 35893599. doi:10.1523/JNEUROSCI.3732-09.2010 Huang, Y., Myers, S. J., & Dingledine, R. (1999). Transcriptional repression by REST: Recruitment of Sin3A and histone deacetylase to neuronal genes. Nature Neuroscience, 2(10), 867-872. doi:10.1038/13165 Jones, P. L., Veenstra, G. J., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., et al. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nature Genetics, 19(2), 187-191. doi:10.1038/561 Kilgore, M., Miller, C. A., Fass, D. M., Hennig, K. M., Haggarty, S. J., Sweatt, J. D., et al. (2010). Inhibitors of class 1 histone deacetylases reverse contextual memory deficits in a mouse model of alzheimer's disease. Neuropsychopharmacology : Official Publication of the American College of Neuropsychopharmacology, 35(4), 870-880. doi:10.1038/npp.2009.197 Kim, D., Frank, C. L., Dobbin, M. M., Tsunemoto, R. K., Tu, W., Peng, P. L., et al. (2008). Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity. Neuron, 60(5), 803817. doi:10.1016/j.neuron.2008.10.015 Kimberly, W. T., Zheng, J. B., Guenette, S. Y., & Selkoe, D. J. (2001). The intracellular domain of the beta-amyloid precursor protein is stabilized by Fe65 and translocates

Epigenetics and Memory

45

to the nucleus in a notch-like manner. The Journal of Biological Chemistry, 276(43), 40288-40292. doi:10.1074/jbc.C100447200 Korzus, E., Rosenfeld, M. G., & Mayford, M. (2004). CBP histone acetyltransferase activity is a critical component of memory consolidation. Neuron, 42(6), 961-972. doi:10.1016/j.neuron.2004.06.002 Levenson, J. M., O'Riordan, K. J., Brown, K. D., Trinh, M. A., Molfese, D. L., & Sweatt, J. D. (2004). Regulation of histone acetylation during memory formation in the hippocampus. The Journal of Biological Chemistry, 279(39), 40545-40559. doi:10.1074/jbc.M402229200 Levenson, J. M., & Sweatt, J. D. (2005). Epigenetic mechanisms in memory formation. Nature Reviews.Neuroscience, 6(2), 108-118. doi:10.1038/nrn1604 Little, J. W. (2010). Two-hybrid system for detecting protein-protein interactions. University of Arizona. Retrieved January 6, 2011 from http://www.biochem.arizona.edu/classes/bioc568/two-hybrid_system.htm Lubin, F. D., Roth, T. L., & Sweatt, J. D. (2008). Epigenetic regulation of BDNF gene transcription in the consolidation of fear memory. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 28(42), 10576-10586. doi:10.1523/JNEUROSCI.1786-08.2008

Epigenetics and Memory Lubin, F. D., & Sweatt, J. D. (2007). The IkappaB kinase regulates chromatin structure during reconsolidation of conditioned fear memories. Neuron, 55(6), 942-957. doi:10.1016/j.neuron.2007.07.039

46

Lunyak, V. V., Burgess, R., Prefontaine, G. G., Nelson, C., Sze, S. H., Chenoweth, J., et al. (2002). Corepressor-dependent silencing of chromosomal regions encoding neuronal genes. Science (New York, N.Y.), 298(5599), 1747-1752. doi:10.1126/science.1076469 Ma, D. K., Jang, M. H., Guo, J. U., Kitabatake, Y., Chang, M. L., Pow-Anpongkul, N., et al. (2009). Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science (New York, N.Y.), 323(5917), 10741077. doi:10.1126/science.1166859 Maier, H., Colbert, J., Fitzsimmons, D., Clark, D. R., & Hagman, J. (2003). Activation of the early B-cell-specific mb-1 (ig-alpha) gene by pax-5 is dependent on an unmethylated ets binding site. Molecular and Cellular Biology, 23(6), 1946-1960. Martinowich, K., Hattori, D., Wu, H., Fouse, S., He, F., Hu, Y., et al. (2003). DNA methylation-related chromatin remodeling in activity-dependent BDNF gene regulation. Science (New York, N.Y.), 302(5646), 890-893. doi:10.1126/science.1090842 Maue, R. A., Kraner, S. D., Goodman, R. H., & Mandel, G. (1990). Neuron-specific expression of the rat brain type II sodium channel gene is directed by upstream regulatory elements. Neuron, 4(2), 223-231.

Epigenetics and Memory

47

McQuown, S. C., Barrett, R. M., Matheos, D. P., Post, R. J., Rogge, G. A., Alenghat, T., et al. (2011). HDAC3 is a critical negative regulator of long-term memory formation. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 31(2), 764-774. doi:10.1523/JNEUROSCI.5052-10.2011 Miller, C. A., Gavin, C. F., White, J. A., Parrish, R. R., Honasoge, A., Yancey, C. R., et al. (2010). Cortical DNA methylation maintains remote memory. Nature Neuroscience, 13(6), 664-666. doi:10.1038/nn.2560 Miller, C. A., & Sweatt, J. D. (2007). Covalent modification of DNA regulates memory formation. Neuron, 53(6), 857-869. doi:10.1016/j.neuron.2007.02.022 Mori, N., Schoenherr, C., Vandenbergh, D. J., & Anderson, D. J. (1992). A common silencer element in the SCG10 and type II na+ channel genes binds a factor present in nonneuronal cells but not in neuronal cells. Neuron, 9(1), 45-54. Okano, M., Xie, S., & Li, E. (1998). Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nature Genetics, 19(3), 219-220. doi:10.1038/890 Peleg, S., Sananbenesi, F., Zovoilis, A., Burkhardt, S., Bahari-Javan, S., Agis-Balboa, R. C., et al. (2010). Altered histone acetylation is associated with age-dependent memory impairment in mice. Science (New York, N.Y.), 328(5979), 753-756. doi:10.1126/science.1186088

Epigenetics and Memory Roth, T. L., Lubin, F. D., Funk, A. J., & Sweatt, J. D. (2009). Lasting epigenetic influence of early-life adversity on the BDNF gene. Biological Psychiatry, 65(9), 760-769. doi:10.1016/j.biopsych.2008.11.028 Roth, T. L., & Sweatt, J. D. (2009). Regulation of chromatin structure in memory formation. Current Opinion in Neurobiology, 19(3), 336-342. doi:10.1016/j.conb.2009.05.011 Sananbenesi, F., & Fischer, A. (2009). The epigenetic bottleneck of neurodegenerative and psychiatric diseases. Biological Chemistry, 390(11), 1145-1153. doi:10.1515/BC.2009.131 Santi, D. V., Garrett, C. E., & Barr, P. J. (1983). On the mechanism of inhibition of DNA-cytosine methyltransferases by cytosine analogs. Cell, 33(1), 9-10. Sastre, M., Steiner, H., Fuchs, K., Capell, A., Multhaup, G., Condron, M. M., et al.

48

(2001). Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of notch. EMBO Reports, 2(9), 835-841. doi:10.1093/embo-reports/kve180 Silva, A. J., Ward, K., & White, R. (1993). Mosaic methylation in clonal tissue. Developmental Biology, 156(2), 391-398. doi:10.1006/dbio.1993.1086 Thomson, S., Clayton, A. L., & Mahadevan, L. C. (2001). Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction. Molecular Cell, 8(6), 1231-1241.

Epigenetics and Memory

49

Vecsey, C. G., Hawk, J. D., Lattal, K. M., Stein, J. M., Fabian, S. A., Attner, M. A., et al. (2007). Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB:CBP-dependent transcriptional activation. The Journal of Neuroscience : The Official Journal of the Society for Neuroscience, 27(23), 6128-6140. doi:10.1523/JNEUROSCI.0296-07.2007 von Rotz, R. C., Kohli, B. M., Bosset, J., Meier, M., Suzuki, T., Nitsch, R. M., et al. (2004). The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor. Journal of Cell Science, 117(Pt 19), 4435-4448. doi:10.1242/jcs.01323 Wang, S. C., Oelze, B., & Schumacher, A. (2008). Age-specific epigenetic drift in lateonset alzheimer's disease. PloS One, 3(7), e2698. doi:10.1371/journal.pone.0002698 Weaver, I. C., Cervoni, N., Champagne, F. A., D'Alessio, A. C., Sharma, S., Seckl, J. R., et al. (2004). Epigenetic programming by maternal behavior. Nature Neuroscience, 7(8), 847-854. doi:10.1038/nn1276 Wilson, V. L., Smith, R. A., Ma, S., & Cutler, R. G. (1987). Genomic 5methyldeoxycytidine decreases with age. The Journal of Biological Chemistry, 262(21), 9948-9951. Wren, J. D. (2009). A global meta-analysis of microarray expression data to predict unknown gene functions and estimate the literature-data divide. Bioinformatics (Oxford, England), 25(13), 1694-1701. doi:10.1093/bioinformatics/btp290

Epigenetics and Memory Yoder, J. A., & Bestor, T. H. (1998). A candidate mammalian DNA methyltransferase related to pmt1p of fission yeast. Human Molecular Genetics, 7(2), 279-284.

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