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.

Bioenergetic Impairment in Animal and Cellular Models of Alzheimer's Disease: PARP-1 Inhibition Rescues Metabolic Dysfunctions.

Amyloid-beta peptide accumulation in the brain is one of the main hallmarks of Alzheimer's disease. The amyloid aggregation process is associated with the generation of free radical species responsible for mitochondrial impairment and DNA damage that in turn activates poly(ADP-ribose)polymerase 1 (PARP-1). PARP-1 catalyzes the poly(ADP-ribosylation), a post-translational modification of proteins, cleaving the substrate NAD+ and transferring the ADP-ribose moieties to the enzyme itself or to an acceptor protein to form branched polymers of ADP-ribose. In this paper, we demonstrate that a mitochondrial dysfunction occurs in Alzheimer's transgenic mice TgCRND8, in SH-SY5Y treated with amyloid-beta and in 7PA2 cells. Moreover, PARP-1 activation contributes to the functional energetic decline affecting cytochrome oxidase IV protein levels, oxygen consumption rates, and membrane potential, resulting in cellular bioenergetic deficit. We also observed, for the first time, an increase of pyruvate kinase 2 expression, suggesting a modulation of the glycolytic pathway by PARP-1. PARP-1 inhibitors are able to restore both mitochondrial impairment and pyruvate kinase 2 expression. The overall data here presented indicate a pivotal role for this enzyme in the bioenergetic network of neuronal cells and open new perspectives for investigating molecular mechanisms underlying energy charge decline in Alzheimer's disease. In this scenario, PARP-1 inhibitors might represent a novel therapeutic intervention to rescue cellular energetic metabolism.

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.

PARP-1 modulates amyloid beta peptide-induced neuronal damage.

Amyloid beta peptide (Aß) causes neurodegeneration by several mechanisms including oxidative stress, which is known to induce DNA damage with the consequent activation of poly (ADP-ribose) polymerase (PARP-1). To elucidate the role of PARP-1 in the neurodegenerative process, SH-SY5Y neuroblastoma cells were treated with Aß25-35 fragment in the presence or absence of MC2050, a new PARP-1 inhibitor. Aß25-35 induces an enhancement of PARP activity which is prevented by cell pre-treatment with MC2050. These data were confirmed by measuring PARP-1 activity in CHO cells transfected with amylod precursor protein and in vivo in brains specimens of TgCRND8 transgenic mice overproducing the amyloid peptide. Following Aß25-35 exposure a significant increase in intracellular ROS was observed. These data were supported by the finding that Aß25-35 induces DNA damage which in turn activates PARP-1. Challenge with Aß25-35 is also able to activate NF-kB via PARP-1, as demonstrated by NF-kB impairment upon MC2050 treatment. Moreover, Aß25-35 via PARP-1 induces a significant increase in the p53 protein level and a parallel decrease in the anti-apoptotic Bcl-2 protein. These overall data support the hypothesis of PARP-1 involvment in cellular responses induced by Aß and hence a possible rationale for the implication of PARP-1 in neurodegeneration is discussed.

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.

Urokinase expression in course of benign and malignant mammary lesions: comparison between nodular and healthy tissues.

PURPOSE: Increased expression of urokinase (uPA), a member of the serine protease family, is an effector of metastatic cascade and has been reported in various malignancies, including breast cancer. uPA overexpression in cancer tissues was correlated with a more aggressive phenotype and it is considered a strong and independent unfavorable prognostic factor in breast cancer. METHODS: Using real-time PCR assay, we analyzed uPA expression of malignant and benign breast nodular lesions versus healthy tissues (normal breast and lymphocytes). RESULTS: We found that besides breast cancer nodule, normal mammary gland and lymphocytes overexpressed uPA too. Tissues obtained from women with benign lesions expressed homogeneous and lower uPA. CONCLUSIONS: In conclusion, although uPA overexpression is typical of cancer tissues, it could be considered as a feature of the whole organism affected by cancer. On the basis of these first results, uPA could be considered for further studies as a possible useful therapeutic target in breast cancer.

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.

S-adenosylmethionine inhibits ubiquitin-proteasome system in vitro and on rat vascular smooth muscle cells.

S-adenosylmethionine is a metabolite regulating many biological processes; S-adenosylmethionine effect on ubiquitin-proteasome system (UPS) has not been studied yet. We investigated S-adenosylmethionine effects on UPS activity both in vitro, by inhibitor screening assay, and in rat vascular smooth muscle cells, by Western Blot of proteasomal targets. We found that S-adenosylmethionine inhibited UPS activity.

gamma-Secretase is differentially modulated by alterations of homocysteine cycle in neuroblastoma and glioblastoma cells.

Multiple aspects of homocysteine metabolism were studied to understand the mechanism responsible for hyperhomocysteinemia toxicity in Alzheimer disease. Besides oxidative stress and vascular damage, homocysteine has also a great importance in regulating DNA methylation through S-adenosylmethionine, the main methyl donor in eukaryotes. Alterations of S-adenosylmethionine and methylation were evidenced in Alzheimer disease and in elderly. In order to clarify whether DNA methylation can provide the basis for amyloid-beta overproduction, we used human SK-N-BE neuroblastoma and A172 glioblastoma cell lines. We tested the effects of folate, B12 and B6 deprivation and S-adenosylmethionine addition on methylation metabolism. Our results indicate that homocysteine accumulation induced through vitamin B deprivation could impair the "Methylation Potential" with consequent presenilin 1, BACE and amyloid-beta upregulation. Moreover, we found that homocysteine alterations had an effect on neuroblastoma but not on glioblastoma cells; this suggests a possible differential role of the two cell types in Alzheimer disease.

Gene silencing through methylation: an epigenetic intervention on Alzheimer disease.

Alzheimer disease (AD) is among the few diseases that may display high homocysteine (HCY) and low B12 and folate in blood. This observation has raised the suspect that amyloid-beta overproduction and accumulation, which may be the cause of the disease, could be due to the loss of epigenetic control in the expression of the genes involved in AbetaPP (amyloid-beta protein precursor) processing. We have shown, in cell culture, that two of the genes responsible for amyloid-beta production are controlled by the methylation of their promoters. The process is strictly related to S-adenosylmethionine (SAM) metabolism. SAM is a natural compound, mainly produced by the liver, which has been found at very low concentrations in AD brains. A further support to this thesis came from the observation that in elderly DNA methylations are consistently lower than in young and mid aged people. We are actually experimenting in transgenic mice the possibility to prevent or to arrest amyloid-beta accumulation, through SAM administration, and therefore its significance and the use of this drug for the treatment of the disease.

The effect of S-adenosylmethionine on CNS gene expression studied by cDNA microarray analysis.

High homocysteine (Hcy) together with low S-adenosylmethionine (SAM) levels are often observed in Alzheimer disease (AD), and this could be a sign of alteration of SAM/Hcy metabolism. It has already been shown that DNA methylation is involved in amyloid-beta-protein precursor (AbetaPP) processing and amyloid-beta(Abeta) production through the regulation of Presenilin 1 (PS1) expression and that exogenous SAM can silence the gene reducing Abeta. To investigate whether SAM administration globally influenced gene expression in the brain, we analysed 588 genes of the central nervous system in SK-N-BE neuroblastoma cells, with cDNA probes derived from untreated (DM; Differentiation Medium) or SAM treated (DM+SAM) cultures. In these conditions only seven genes were modulated by SAM treatment (and therefore by DNA methylation); three were up-regulated and four down-regulated, showing low levels of modulation.

Evaluation of chemical and diastereoisomeric stability of S-adenosylmethionine in aqueous solution by capillary electrophoresis.

Capillary electrophoresis was used for monitoring the stability of S-adenosylmethionine in aqueous solution under different conditions of storage and incubation used for "in vitro" and "in vivo" experiments, by evaluating both the entity of degradation and the possibility of epimerization at the sulfonium group. The determination of S,S-S-adenosylmethionine in presence of its R,S-epimer and degradation products was performed in uncoated capillary of 50 microm ID using 150 mM sodium phosphate buffer at pH 2.5. The analyses were performed in short or long-end injection modes depending if a fast monitoring of the degradation products or the evaluation of the diastereoisomeric ratio were carried out, respectively. In the long-end injection mode the baseline separation of S-adenosylmethionine diastereoisomeric forms and degradation products was obtained in less than 10 min with efficiency values in the range of 172,520-311,439 number of theoretical plates per meter. The results showed that freezing was the optimum storage mode for S-adenosylmethionine aqueous solutions preserving from degradation and diastereoisomeric ratio alterations. Under incubation conditions at 38 degrees C during 14 days period S-adenosylmethionine showed a strong degradation and the formation of three main increasing degradation products. After 7 and 14 days only the 52% and 32% of the initial drug concentration were available and the active S,S-S-adenosylmethionine form was the most affected.

S-adenosylmethionine/homocysteine cycle alterations modify DNA methylation status with consequent deregulation of PS1 and BACE and beta-amyloid production.

Few diseases are characterized by high homocysteine (HCY) and low folate and vitamin B12 blood levels. Alzheimer disease (AD) is among these. It has already been shown that DNA methylation is involved in amyloid precursor protein (APP) processing and beta-amyloid (A beta) production through the regulation of Presenilin1 (PS1) expression and that exogenous S-adenosylmethionine (SAM) can silence the gene reducing A beta production. Here we demonstrate that BACE (beta-secretase), as well as PS1, is regulated by methylation and that the reduction of folate and vitamin B12 in culture medium can cause a reduction of SAM levels with consequent increase in presenilin1 and BACE levels and with increase in A beta production. The simultaneous administration of SAM to the deficient medium can restore the normal gene expression, thus reducing the A beta levels. The use of deprived medium was intended to mimic a mild nutritional deficit involved in the onset of AD.