S-Adenosylmethionine and Superoxide Dismutase 1 Synergistically Counteract Alzheimer's Disease Features Progression in TgCRND8 Mice.

Recent evidence emphasizes the role of dysregulated one-carbon metabolism in Alzheimer's Disease (AD). Exploiting a nutritional B-vitamin deficiency paradigm, we have previously shown that PSEN1 and BACE1 activity is modulated by one-carbon metabolism, leading to increased amyloid production. We have also demonstrated that S-adenosylmethionine (SAM) supplementation contrasted the AD-like features, induced by B-vitamin deficiency. In the present study, we expanded these observations by investigating the effects of SAM and SOD (Superoxide dismutase) association. TgCRND8 AD mice were fed either with a control or B-vitamin deficient diet, with or without oral supplementation of SAM + SOD. We measured oxidative stress by lipid peroxidation assay, PSEN1 and BACE1 expression by Real-Time Polymerase Chain Reaction (PCR), amyloid deposition by ELISA assays and immunohistochemistry. We found that SAM + SOD supplementation prevents the exacerbation of AD-like features induced by B vitamin deficiency, showing synergistic effects compared to either SAM or SOD alone. SAM + SOD supplementation also contrasts the amyloid deposition typically observed in TgCRND8 mice. Although the mechanisms underlying the beneficial effect of exogenous SOD remain to be elucidated, our findings identify that the combination of SAM + SOD could be carefully considered as co-adjuvant of current AD therapies.

GSK3ß 5'-flanking DNA Methylation and Expression in Alzheimer's Disease Patients.

BACKGROUND: The GSK3ß 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 GSK3ß is regulated at posttranslational level: phosphorylation at Tyr216 activates kinase, while phosphorylation at Ser9 is essential to inhibit its activity. OBJECTIVES: At present, there are contradictory findings about the possibility that GSK3ß may be regulated at gene level. Previous data showed overexpression of GSK3ß mRNA in hypomethylating conditions, pointing out to the existence of epigenetic mechanisms responsible for GSK3ß gene regulation. Analysis of human GSK3ß 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 GSK3ß 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 GSK3ß 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 GSK3ß 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 GSK3ß hyperactivity, and then NFTs formation, could come into function at an early stage of the disease and then turn off at the last stages.

Sexually Dimorphic Expression of Reelin in the Brain of a Mouse Model of Alzheimer Disease.

Recent evidence highlights the protective role of reelin against amyloid ß (Aß)-induced synaptic dysfunction and cognitive impairment in Alzheimer disease (AD). In this study, exploiting TgCRND8 mice that overexpress a mutant form of amyloid ß precursor protein (AßPP) and display an early onset of AD neuropathological signs, we addressed the question whether changes of reelin expression eventually precede the appearance of Aß-plaques in a sex-dependent manner. We show that sex-associated and brain region-specific differences in reelin expression appear long before Aß-plaque formation. However, in spite of a downregulation of reelin expression compared to males, TgCRND8 females display fewer Aß-plaques, suggesting that additional factors, other than sex and reelin level, influence amyloidosis in this mouse model.

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-ß 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.

S-adenosylmethionine reduces the progress of the Alzheimer-like features induced by B-vitamin deficiency in mice.

Methylation reactions linked to homocysteine in the one-carbon metabolism are increasingly elicited in Alzheimer's disease, although the association of hyperhomocysteinemia and of low B vitamin levels with the disease is still debated. We previously demonstrated that hyperhomocysteinemia and DNA hypomethylation induced by B vitamin deficiency are associated with PSEN1 and BACE1 overexpression and amyloid production. The present study is aimed at assessing S-adenosylmethionine effects in mice kept under a condition of B vitamin deficiency. To this end, TgCRND8 mice and wild-type littermates were assigned to control or B vitamin deficient diet, with or without S-adenosylmethionine supplementation. We found that S-adenosylmethionine reduced amyloid production, increased spatial memory in TgCRND8 mice and inhibited the upregulation of B vitamin deficiency-induced PSEN1 and BACE1 expression and Tau phosphorylation in TgCRND8 and wild-type mice. Furthermore, S-adenosylmethionine treatment reduced plaque spreading independently on B vitamin deficiency. These results strengthen our previous observations on the possible role of one-carbon metabolism in Alzheimer's disease, highlighting hyperhomocysteinemia-related mechanisms in dementia onset/progression and encourage further studies aimed at evaluating the use of S-adenosylmethionine as a potential candidate drug for the treatment of the disease.

DNA methylase and demethylase activities are modulated by one-carbon metabolism in Alzheimer's disease models.

Late-onset Alzheimer's disease seems to be a multi-factorial disease with both genetic and non-genetic, environmental, possible causes. Recently, epigenomics is achieving a major role in Alzheimer's research due to its involvement in different molecular pathways leading to neurodegeneration. Among the different epigenetic modifications, DNA methylation is one of the most relevant to the disease. We previously demonstrated that presenilin1 (PSEN1), a gene involved in amyloidogenesis, is modulated by DNA methylation in neuroblastoma cells and Alzheimer's mice in an experimental model of nutritionally altered one-carbon metabolism. This alteration, obtained by nutritional deficiency of B vitamins (folate, B12 and B6) hampered S-adenosylmethionine (SAM)-dependent methylation reactions. The aim of the present paper was to investigate the regulation of DNA methylation machinery in response to hypomethylating (B vitamin deficiency) and hypermethylating (SAM supplementation) alterations of the one-carbon metabolism. We found that DNA methylases (DNMT1, 3a and 3b) and a putative demethylase (MBD2) were differently modulated, in line with the previously observed changes of PSEN1 methylation pattern in the same experimental conditions.

Changes in Presenilin 1 gene methylation pattern in diet-induced B vitamin deficiency.

We have previously shown that a nutritional model of B vitamin deficiency and homocysteine cycle alteration could lead to increased amyloid ß deposition, due to PSEN1 and BACE over-expression and consequent increase in secretase activity. We hypothesize that nutritional factors causing homocysteine cycle alterations (i.e. hyperhomocysteinemia) could induce sequence-specific DNA hypomethylation and "aberrant" gene activation. Aim of present study was to analyze the methylation pattern of PSEN1 promoter in SK-N-BE neuroblastoma cells and TgCRND8 mice, in a B vitamin (folate, B12 and B6) deficiency paradigm. PSEN1 methylation status has been evaluated through bisulphite modification and genomic sequencing. We demonstrate that B vitamin deficiency induces hypomethylation of specific CpG moieties in the 5'-flanking region; S-adenosylmethionine has been supplemented as methyl donor to reverse this effect. PSEN1 promoter methylation status is correlated with gene expression. These findings pinpoint a direct relationship between B vitamin-dependent alteration of homocysteine cycle and DNA methylation and also indicate that PSEN1 promoter is regulated by methylation of specific CpG moieties.

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-ß 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-ß 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.

S-adenosylmethionine prevents oxidative stress and modulates glutathione metabolism in TgCRND8 mice fed a B-vitamin deficient diet.

Oxidative stress, altered glutathione levels, and hyperhomocysteinemia play critical roles in Alzheimer's disease. We studied the relationships between hyperhomocysteinemia, glutathione, and oxidative stress in TgCRND8 mice maintained in conditions of folate, B12, and B6 deficiency and the effect of S-adenosylmethionine supplementation. We found that hyperhomocysteinemia was correlated with increased reduced/oxidized brain glutathione ratio, with decreased glutathione S-transferase activity and increased lipid peroxidation. S-adenosylmethionine potentiated superoxide dismutase and glutathione S-transferase activity and restored altered brain glutathione and erythrocytes lipid peroxidation. These results underline the importance of S-adenosylmethionine as neuroprotective compound, acting both on methylation and oxidation metabolism.

B vitamin deficiency promotes tau phosphorylation through regulation of GSK3beta and PP2A.

Neurofibrillary tangles (NFTs), composed of intracellular filamentous aggregates of hyperphosphorylated protein tau, are one of the pathological hallmarks of Alzheimer's disease (AD). Tau phosphorylation is regulated by the equilibrium between activities of its protein kinases and phosphatases; unbalance of these activities is proposed to be a reasonable causative factor to the disease process. Glycogen synthase kinase 3beta (GSK3beta) is one of the most important protein kinase in regulating tau phosphorylation; overexpression of active GSK3beta causes ADlike hyperphosphorylation of tau. Protein phosphatase 2A (PP2A) is the major phosphatase that dephosphorylates tau; it was demonstrated that highly conserved carboxyl-terminal sequence of PP2A C-subunit is a focal point for phosphatase regulation. This is the site of a reversible methyl esterification reaction that controls AB_{alpha}C heterotrimers formation. Here we demonstrate that GSK3beta and PP2A genes were upregulated by inhibiting methylation reactions through B vitamin deficiency. In this condition, methylated catalytic subunit PP2Ac was decreased, leading to reduced PP2A activity. By contrast, we observed GSK3beta protein increase and a modulation in phosphorylation sites that regulate GSK3beta activity. Therefore, one-carbon metabolism alteration seems to be a cause of deregulation of the equilibrium between GSK3beta and PP2A, leading to abnormal hyperphosphorylated tau.

B-vitamin deprivation induces hyperhomocysteinemia and brain S-adenosylhomocysteine, depletes brain S-adenosylmethionine, and enhances PS1 and BACE expression and amyloid-beta deposition in mice.

Etiological and molecular studies on the sporadic form of Alzheimer's disease have yet to determine the underlying mechanisms of neurodegeneration. Hyperhomocysteinemia is associated with Alzheimer's disease, and has been hypothesized to promote neurodegeneration, by inhibiting brain methylation activity. The aim of this work was to determine whether a combined folate, B12 and B6 dietary deficiency, would induce amyloid-beta overproduction, and to study the mechanisms linking vitamin deficiency, hyperhomocysteinemia and amyloidogenesis in TgCRND8 and 129Sv mice. We confirmed that B-vitamin deprivation induces hyperhomocysteinemia and imbalance of S-adenosylmethionine and S-adenosylhomocysteine. This effect was associated with PS1 and BACE up-regulation and amyloid-beta deposition. Finally, we detected intraneuronal amyloid-beta and a slight cognitive impairment in a water maze task at a pre-plaque age, supporting the hypothesis of early pathological function of intracellular amyloid. Collectively, these findings are consistent with the hypothesis that abnormal methylation in association with hyperhomocysteinemia may contribute to Alzheimer's disease.