Leonidas Chouliaras

Epigenetic regulation in the pathophysiology of Alzheimer's disease

With the aging of the population, the growing incidence and prevalence of Alzheimers disease (AD) increases the burden on individuals and society as a whole. To date, the pathophysiology of AD is not yet fully understood. Recent studies have suggested that epigenetic mechanisms may play a pivotal role in its course and development. The most frequently studied epigenetic mechanisms are DNA methylation and histone modifications, and investigations relevant to aging and AD are presented in this review. Various studies on human postmortem brain samples and peripheral leukocytes, as well as transgenic animal models and cell culture studies relevant to AD will be discussed. From those, it is clear that aging and AD are associated with epigenetic dysregulation at various levels. Moreover, data on e.g. twin studies in AD support the notion that epigenetic mechanisms mediate the risk for AD. Conversely, it is still not fully clear whether the observed epigenetic changes actually represent a cause or a consequence of the disease. This is mainly due to the fact that most clinical investigations on epigenetics in AD are conducted in samples of patients already in an advanced stage of the disease. Evidently, more research is needed in order to clarify the exact role of epigenetic regulation in the course and development of AD. Research on earlier stages of the disease could provide more insight into its underlying pathophysiology, possibly contributing to the establishment of early diagnosis and the development of more effective treatment strategies.

Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer's disease patients

Epigenetic dysregulation of gene expression is thought to be critically involved in the pathophysiology of Alzheimers disease (AD). Recent studies indicate that DNA methylation and DNA hydroxymethylation are 2 important epigenetic mechanisms that regulate gene expression in the aging brain. However, very little is known about the levels of markers of DNA methylation and hydroxymethylation in the brains of patients with AD, the cell-type specificity of putative AD-related alterations in these markers, as well as the link between epigenetic alterations and the gross pathology of AD. The present quantitative immunohistochemical study investigated the levels of the 2 most important markers of DNA methylation and hydroxymethylation, that is, 5-methylcytidine (5-mC) and 5-hydroxymethylcytidine (5-hmC), in the hippocampus of AD patients (n = 10) and compared these to non-demented, age-matched controls (n = 10). In addition, the levels of 5-hmC in the hippocampus of a pair of monozygotic twins discordant for AD were assessed. The levels of 5-mC and 5-hmC were furthermore analyzed in a cell-type and hippocampal subregion-specific manner, and were correlated with amyloid plaque load and neurofibrillary tangle load. The results showed robust decreases in the hippocampal levels of 5-mC and 5-hmC in AD patients (19.6% and 20.2%, respectively). Similar results were obtained for the twin with AD when compared to the non-demented co-twin. Moreover, levels of 5-mC as well as the levels of 5-hmC showed a significant negative correlation with amyloid plaque load in the hippocampus (r(p) = -0.539, p = 0.021 for 5-mC and r(p) = -0.558, p = 0.016 for 5-hmC). These human postmortem results thus strengthen the notion that AD is associated with alterations in DNA methylation and hydroxymethylation, and provide a basis for further epigenetic studies identifying the exact genetic loci with aberrant epigenetic signatures.

Cross-region reduction in 5-hydroxymethylcytosine in Alzheimer's disease brain

Epigenetic processes play a key role in the central nervous system and altered levels of 5-methylcytosine have been associated with a number of neurologic phenotypes, including Alzheimers disease (AD). Recently, 3 additional cytosine modifications have been identified (5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine), which are thought to be intermediate steps in the demethylation of 5-methylcytosine to unmodified cytosine. Little is known about the frequency of these modifications in the human brain during health or disease. In this study, we used immunofluorescence to confirm the presence of each modification in human brain and investigate their cross-tissue abundance in AD patients and elderly control samples. We identify a significant AD-associated decrease in global 5-hydroxymethylcytosine in entorhinal cortex and cerebellum, and differences in 5-formylcytosine levels between brain regions. Our study further implicates a role for epigenetic alterations in AD.

Caloric restriction attenuates age-related changes of DNA methyltransferase 3a in mouse hippocampus

Recent studies have suggested that DNA methylation is implicated in age-related changes in gene expression as well as in cognition. DNA methyltransferase 3a (Dnmt3a), which catalyzes DNA methylation, is essential for memory formation and underlying changes in neuronal and synaptic plasticity. Because caloric restriction (CR) and upregulation of antioxidants have been suggested as strategies to attenuate age-related alterations in the brain, we hypothesized that both a diet restricted in calories and transgenic overexpression of normal human Cu/Zn superoxide dismutase 1 (SOD) attenuate age-related changes in Dnmt3a in the aging mouse hippocampus. For this purpose, we performed qualitative and quantitative analyses of Dnmt3a-immunoreactivity (IR) for the hippocampal dentate gyrus (DG), CA3 and CA1-2 regions in 12- and 24-month-old mice from 4 groups, i.e. (1) wild-type (WT) mice on a control diet (WT-CD), (2) SOD-CD mice, (3) WT mice on CR (WT-CR), and (4) SOD-CR. Qualitative analyses revealed two types of Dnmt3a immunoreactive cells: type I cells--present throughout all hippocampal cell layers showing moderate levels of nuclear Dnmt3a-IR, and type II cells--a subpopulation of hippocampal cells showing very intense nuclear Dnmt3a-IR, and colocalization with Bromodeoxyuridine. Quantitative analyses indicated that the age-related increase in Dnmt3a-IR within the CA3 and CA1-2 in type I cells was attenuated by CR, but not by SOD overexpression. In contrast, the density of type II Dnmt3a immunoreactive cells showed an age-related reduction, without significant effects of both CR and SOD. These changes in Dnmt3a levels in the mouse hippocampus may have a significant impact on gene expression and associated cognitive functioning.

Prevention of age-related changes in hippocampal levels of 5-methylcytidine by caloric restriction

Aberrant DNA methylation patterns have been linked to molecular and cellular alterations in the aging brain. Caloric restriction (CR) and upregulation of antioxidants have been proposed as interventions to prevent or delay age-related brain pathology. Previously, we have shown in large cohorts of aging mice, that age-related increases in DNA methyltransferase 3a (Dnmt3a) immunoreactivity in the mouse hippocampus were attenuated by CR, but not by overexpression of superoxide dismutase 1 (SOD1). Here, we investigated age-related alterations of 5-methylcytidine (5-mC), a marker of DNA methylation levels, in a hippocampal subregion-specific manner. Examination of 5-mC immunoreactivity in 12- and 24-month-old wild type (WT) mice on control diet, mice overexpressing SOD1 on control diet, wild type mice on CR, and SOD1 mice on CR, indicated an age-related increase in 5-mC immunoreactivity in the hippocampal dentate gyrus, CA3, and CA1-2 regions, which was prevented by CR but not by SOD1 overexpression. Moreover, positive correlations between 5-mC and Dnmt3a immunoreactivity were observed in the CA3 and CA1-2. These findings suggest a crucial role for DNA methylation in hippocampal aging and in the mediation of the beneficial effects of CR on aging.

Behavioral and neurobiological effects of prenatal stress exposure in male and female APPswe/PS1dE9 mice

Epidemiological evidence implies a role for chronic stress and stress-related disorders in the etiopathogenesis of sporadic Alzheimers disease (AD). Although chronic stress exposure during various stages of life has been shown to exacerbate AD-related cognitive deficits and neuropathology in AD mouse models, the role of stress exposure during the prenatal period on AD development and progression remained to be investigated. The present study therefore explored the effects of prenatal maternal stress (PMS) in both male and female APPswe/PS1dE9 mouse offspring in terms of cognition, affect, and AD-related neuropathology. As prenatal perturbations are likely to mediate their effects via alterations in epigenetic regulation, changes in hippocampal DNA methyltransferase 3a, 5-methylcytosine and 5-hydroxymethylcytosine levels were assessed as underlying mechanisms. Repetitive restraint stress during the first week of gestation exerted a sex-dependent effect, with male PMS mice showing spatial memory deficits and a blunted hypothalamus-pituitary-adrenal axis response, while female PMS mice showed improved spatial memory performance, increased depressive-like behavior, as well as a decrease in hippocampal plaque load. In addition, sex differences were observed among APPswe/PS1dE9 mice, independent of PMS (i.e., female mice showed impaired spatial memory performance, higher hippocampal plaque load, altered amyloid precursor protein processing in the CA3 and lower DNA methyltransferase 3a immunoreactivity in the dentate gyrus when compared with male mice of the same age). In conclusion, PMS exposure impacts on the behavioral phenotype and neuropathology of APPswe/PS1dE9 mice. Moreover, given the remarkable sex differences observed, one should not overlook the impact of sex-specific responses to environmental exposures when investigating gene-environment interactions in AD.

Age-Related Increase in Levels of 5-Hydroxymethylcytosine in Mouse Hippocampus is Prevented by Caloric Restriction

Aberrations in epigenetic marks have been associated with aging of the brain while caloric restriction (CR) and upregulation of endogenous antioxidants have been suggested as tools to attenuate the aging process. We have recently observed age-related increases in levels of 5-methylcytidine (5-mC) and DNA methyltransferase 3a (Dnmt3a) in the mouse hippocampus. Most of those age-related changes in these epigenetically relevant markers were prevented by CR but not by transgenic overexpression of the endogenous antioxidant superoxide dismutase 1 (SOD1). As recent work has suggested a distinct role for hydroxymethylation in epigenetic regulation of gene expression in the brain, the current study investigated age-related changes of 5-hydroxymethylcytosine (5-hmC) in the mouse hippocampus, and furthermore tested whether CR and transgenic upregulation of SOD1 affected any age-related changes in 5-hmC. Immunohistochemical analyses of 5-hmC in 12- and 24-month-old wild-type and transgenic mice overexpressing SOD1, which were kept under either a control or a calorie restricted diet, revealed an increase of 5-hmC immunoreactivity occurring with aging in the hippocampal dentate gyrus, CA3 and CA1-2 regions. Moreover, CR, but not overexpression of SOD1, prevented the age-related increase in the CA3 region. These findings indicate that the aging process in mice is connected with changes in epigenetic machinery in the hippocampus and suggest that CR acts by influencing epigenetic regulation.

The role of 5-hydroxymethylcytosine in aging and Alzheimer's disease: current status and prospects for future studies.

Epigenetic modifications have been proposed to underlie age-related dysfunction and associated disorders. 5- hydroxymethylcytosine (5-hmC) is a newly described epigenetic modification. It is generated by the oxidation of 5- methylcytosine (5-mC) by the ten-eleven translocation (TET) family of enzymes. Various studies have shown that 5-hmC is present in high levels in the brain. Its lower affinity to methyl-binding proteins as compared to 5-mC suggests that it might have a different role in the regulation of gene expression, while it is also implicated in the DNA demethylation process. Interestingly, various widely used methods for DNA methylation detection fail to discriminate between 5-hmC and 5-mC, while numerous specific techniques are currently being developed. Recent studies have indicated an increase of 5-hmC with age in the mouse brain as well as an age- and gene-expression-level-related enrichment of 5-hmC in genes implicated in neurodegeneration. These findings suggest that 5-hmC may play an important role in the etiology and course of age-related neurodegenerative disorders. The present perspective summarizes the current knowledge on 5-hmC, discusses methodological challenges related to its detection, and suggests future strategies for examining the functional role of this epigenetic modification and its possible implication in aging and Alzheimers disease.

Gene-Environment Interaction Research and Transgenic Mouse Models of Alzheimer's Disease

The etiology of the sporadic form of Alzheimers disease (AD) remains largely unknown. Recent evidence has suggested that gene-environment interactions (GxE) may play a crucial role in its development and progression. Whereas various susceptibility loci have been identified, like the apolipoprotein E4 allele, these cannot fully explain the increasing prevalence of AD observed with aging. In addition to such genetic risk factors, various environmental factors have been proposed to alter the risk of developing AD as well as to affect the rate of cognitive decline in AD patients. Nevertheless, aside from the independent effects of genetic and environmental risk factors, their synergistic participation in increasing the risk of developing AD has been sparsely investigated, even though evidence points towards such a direction. Advances in the genetic manipulation of mice, modeling various aspects of the AD pathology, have provided an excellent tool to dissect the effects of genes, environment, and their interactions. In this paper we present several environmental factors implicated in the etiology of AD that have been tested in transgenic animal models of the disease. The focus lies on the concept of GxE and its importance in a multifactorial disease like AD. Additionally, possible mediating mechanisms and future challenges are discussed.

Considering the senses in the diagnosis and management of dementia

Associations between dementia and impairments in hearing, vision, olfaction and (to a lesser degree) taste have been identified. Hearing impairment has been shown to precede cognitive decline, but it is not clear if the hearing loss is an early marker of dementia or a modifiable risk factor. Olfactory impairment is seen in many neurodegenerative conditions, but it has been shown that those with dementia have particular difficulties with the recognition and identification of odours rather than the detection, suggesting a link to impairment of higher cognitive function. Olfactory impairment has been shown to be predictive of conversion from mild cognitive impairment to Alzheimers disease with 85.2% sensitivity. As cognitive function deteriorates, the world is experienced at a sensory level, with reduced ability to integrate the sensory experiences to understand the context. Thus, people with dementia are very sensitive to sensory experiences and their environment needs to be managed carefully to make it understandable, comfortable, and (if possible) therapeutic. Light can be used to stabilise the circadian rhythm, which may be disturbed in dementia. Music therapy, aromatherapy, massage and multisensory stimulation are recommended by NICE for the management of behavioural and psychological symptoms of dementia (BPSD), although the mechanisms behind such interventions are poorly understood and evidence is limited. Sensory considerations are likely to play a greater role in dementia care in the future, with the development of purpose-built dementia care facilities and the focus on non-pharmacological management strategies for BPSD.