GSK3β 5’-flanking DNA methylation and expression in Alzheimer’s disease patients

Background: The GSK3β has been associated to pathological functions in neurodegenerative diseases. This kinase is involved in hyperphosphorylation of microtubule-associated tau protein, leading to aggregation andformation of NFTs. It has clearly been shown that GSK3β is regulated at posttranslational level: phosphorylation at Tyr 216 activates kinase, while phosphorylation at Ser 9 is essential to inhibit its activity. Objectives: At present, there are contradictory findings about the possibility that GSK3β may be regulated at gene level. Previous data showed overexpression of GSK3β mRNA in hypomethylating conditions, pointing out to the existence of epigenetic mechanisms responsible for GSK3β gene regulation. Analysis of human GSK3β promoter through bisulphite modification, both in neuroblastoma cells and in postmortem frontal cortex from AD patients (AD patients both at Braak stages I-II and at stages V-VI) , allowed us to characterize the methylation pattern of a putative CpG islands in human GSK3β 5’- flanking region. Results: The analysis evidenced overall hypomethylation of CpG and non-CpG cytosine residues both in cells and in human brain (AD patients and control subjects). We found that GSK3β mRNA was overexpressed only in patients with initial AD, with no effect on the levels of the protein. On the other hand, we unexpectedly observed the decrease of the inactive GSK3β in cortex from AD patients at Braak stages I-II, whereas considerable increase was observed in AD patients at stages V-VI compared to the control subjects. Conclusions: These results point out that GSK3β hyperactivity, and then NFTs formation, could come into function at an early stage of the disease and then turn off at the last stages

IS RAGE THE SO FAR UNIDENTIFIED TRAIT D'UNION BETWEEN VASCULAR RISK FACTORS AND ALZHEIMER'S DISEASE?

889 IS RAGE THE SO FAR UNIDENTIFIED TRAIT D'UNION BETWEEN VASCULAR RISK FACTORS AND ALZHEIMER'S DISEASE? Rita Businaro1, L. Capriotti1, M. Corsi1, M. Leopizzi1, V. Nicolia2, A. Fuso2 1Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, 2Surgery 'P. Valdoni', Sapienza University of Rome, Rome, Italy Aim: The receptor for advanced glycation end products (RAGE), is considered to be a key mediator of atherogenesis. Recent findings indicate the important role played by RAGE in the progression of Alzheimer's disease (AD). It has been shown that RAGE signaling contributes to the production of proinflammatory cytokines, leading to the impairment of neuronal functions and to the amyloid accumulation. Moreover, atherosclerosis, stroke and cardiac disease in the aging individual, could result in cerebrovascular dysfunction and trigger AD pathology. It is known that diet depleted in folate and vitamins B6 and B12 promotes an enhanced expression of RAGE and leads to hyperhomocysteinemia, a well-known risk factor for the development of cardiovascular disease as well as for late onset AD. This work aims at evaluating the effects of hyperhomocysteinemia, induced by B vitamin deficiency, on RAGE expression. Methods: TgCNRD8 mice carrying a Indiana/Swedish mutated APP transgene, an animal model of AD, were grown either with control or B vitamin deficient diet. We measured RAGE by immunohistochemistry, western blots, real-time PCR. Results: B vitamin deficiency enhances RAGE expression in particular at the level of frontal and parietal microvasculature. Both neurons and endothelium in the hippocampus were stained, showing an increase in the number of positive cells as well as in the amount of reaction, compared to the animals fed with a standard diet. Conclusions: B vitamin deficiency and hyperomocysteinemia are able to modulate RAGE expression, promoting in this way atherosclerosis progression and the transport of beta Amyloid across the BBB

One-Carbon Metabolism Alteration Affects Brain Proteome Profile in a Mouse Model of Alzheimer's Disease

Late Onset Alzheimer's Disease (LOAD) can be associated to high homocysteine level and alteration of one-carbon metabolism. We previously demonstrated in the TgCRND8 mice strain, over-expressing human amyloid-β protein precursor, that B vitamin deficiency causes alteration of one-carbon metabolism, together with unbalance of S-adenosylmethionine/S-adenosylhomocysteine levels, and is associated with AD like hallmarks as increased amyloid-β plaque deposition, hyperhomocysteinemia, and oxidative stress. The same model of nutritional deficit was used here to study the variation of the brain protein expression profile associated to B vitamin deficiency. A group of proteins mainly involved in neuronal plasticity and mitochondrial functions was identified as modulated by one-carbon metabolism. These findings are consistent with increasing data about the pivotal role of mitochondrial abnormalities in AD patho-physiology. The identified proteins might represent new potential biomarkers of LOAD to be further investigated

Genotype, age and gender interaction in regulating BDNF expression in TgCRND8 Alzheimer's disease mice

Alzheimer'd Disease (AD) and other aging-associated neurodegenerative disorders may be induced by dysregulation of neurotrophins. Among these, Brain Derived Neurotrophic Factor (BDNF) is responsible for maturation and function of neurons in both peripheral and central nervous systems in the developing brain and has been shown to play a critical role in neuronal survival and in functional and structural synaptic plasticity. A direct role for BDNF/TrkB signalling in Aβ production, tau hyperphosphorylation and cognition decline in AD has been postulated. Aim of this work was to compare BDNF expression profiles in neurodevelopment and aging in an AD model. To this end, TgCRND8 mice (carrying the Indiana/Swedish double-mutated human APP transgene) and 129sv WT littermates were sacrificed at E14.5 and at PN30 and PN90 (i.e. before and after amyloid plaques deposition). BDNF expression in several brain areas was assessed by Real-Time PCR and by immunohistochemistry with particular attention to regional and sub-regional differences. BDNF promoter methylation was assessed by bisulfite modification of genomic DN

DNA methylation profiles of selected pro-inflammatory cytokines in Alzheimer disease

By means of functional genomics analysis, we recently described the mRNA expression profiles of various genes involved in the neuroinflammatory response in the brains of subjects with late-onset Alzheimer Disease (LOAD). Some of these genes, namely interleukin (IL)-1b and IL-6, showed distinct expression profiles with peak expression during the first stages of the disease and control-like levels at later stages. IL-1b and IL-6 genes are modulated by DNA methylation in different chronic and degenerative diseases; it is also well known that LOAD may have an epigenetic basis. Indeed, we and others have previously reported gene-specific DNA methylation alterations in LOAD and in related animal models. Based on these data, we studied the DNA methylation profiles, at single cytosine resolution, of IL-1b and IL-6 5'-flanking region by bisulphite modification in the cortex of healthy controls and LOAD patients at 2 different disease stages: Braak I-II/A and Braak V-VI/C. Our analysis provides evidence that neuroinflammation in LOAD is associated with (and possibly mediated by) epigenetic modifications

Plasma thiols levels in Alzheimer's disease mice under diet-induced hyperhomocysteinemia: effect of S-adenosylmethionine and superoxide-dismutase supplementation.

Widely confirmed reports were published on association between hyperhomocysteinemia, B vitamin deficiency, oxidative stress, and amyloid-β in Alzheimer's disease (AD). Homocysteine, cysteine, cysteinylglycine and glutathione are metabolically interrelated thiols that may be potential indicators of health status and disease risk; they all participate in the metabolic pathway of homocysteine. Previous data obtained in one of our laboratories showed that B vitamin deficiency induced exacerbation of AD-like features in TgCRND8 AD mice; these effects were counteracted by S-adenosylmethionine (SAM) supplementation, through the modulation of DNA methylation and antioxidant pathways. Since the cellular response to oxidative stress typically involves alteration in thiols content, a rapid and sensitive HPLC method with fluorescence detection was here used to evaluate the effect of SAM and superoxide-dismutase (SOD) supplementation on thiols level in plasma, in TgCRND8 mice. The quantitative data obtained from HPLC analysis of mice plasma samples showed significant decrease of thiols level when the B vitamin deficient diet was supplemented with SAM + SOD and SOD alone, the latter showing the greatest effect. All these considerations point out the measurement of plasma thiols concentration as a powerful tool of relevance for all clinical purposes involving the evaluation of oxidative stress. The coupling of HPLC with fluorimetric detection, here used, provided a strong method sensitivity allowing thiols determination at very low levels

CpG and non-CpG Presenilin1 methylation pattern in course of neurodevelopment and neurodegeneration is associated with gene expression in human and murine brain

The Presenilin1 (PSEN1) gene encodes the catalytic peptide of the γ-secretase complex, a key enzyme that cleaves the amyloid-β protein precursor (AβPP), to generate the amyloid-β (Aβ) peptides, involved in Alzheimer’s Disease (AD). Other substrates of the γ-secretase, such as E-cadherin and Notch1, are involved in neurodevelopment and haematopoiesis. Gene-specific DNA methylation influences PSEN1 expression in AD animal models. Here we evaluated canonical and non-canonical cytosine methylation patterns of the PSEN1 5ʹ-flanking during brain development and AD progression, in DNA extracted from the frontal cortex of AD transgenic mice (TgCRND8) and post-mortem human brain. Mapping CpG and non-CpG methylation revealed different methylation profiles in mice and humans. PSEN1 expression only correlated with DNA methylation in adult female mice. However, in post-mortem human brain, lower methylation, both at CpG and non-CpG sites, correlated closely with higher PSEN1 expression during brain development and in disease progression. PSEN1 methylation in blood DNA was significantly lower in AD patients than in controls. The present study is the first to demonstrate a temporal correlation between dynamic changes in PSEN1 CpG and non-CpG methylation patterns and mRNA expression during neurodevelopment and AD neurodegeneration. These observations were made possible by the use of an improved bisulphite methylation assay employing primers that are not biased towards non-CpG methylation. Our findings deepen the understanding of γ-secretase regulation and support the hypothesis that epigenetic changes can promote the pathophysiology of AD. Moreover, they suggest that PSEN1 DNA methylation in peripheral blood may provide a biomarker for AD