Alzheimer's disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-␤ peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of B␣-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of ␤-secretase-cleaved APP fragments and amyloid-␤ peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective ␣-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-␤-synthase ϩ/Ϫ mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Abstract-Hyperhomocysteinemia is associated with increased risk for cardiovascular events, but it is not certain whether it is a mediator of vascular dysfunction or a marker for another risk factor. Homocysteine levels are regulated by folate bioavailability and also by the methyl donor S-adenosylmethionine (SAM) and its metabolite S-adenosylhomocysteine (SAH). We tested the hypotheses that endothelial dysfunction occurs in hyperhomocysteinemic mice in the absence of folate deficiency and that levels of SAM and SAH are altered in mice with dysfunction. Heterozygous cystathionine ␤-synthase-deficient (CBS ϩ/-) and wild-type (CBS ϩ/ϩ ) mice were fed a folate-replete, methionine-enriched diet. Plasma levels of total homocysteine were elevated in CBS ϩ/-mice compared with CBS ϩ/ϩ mice after 7 weeks (27.1Ϯ5.2 versus 8.8Ϯ1.1 mol/L; PϽ0.001) and 15 weeks (23.9Ϯ3.0 versus 13.0Ϯ2.3 mol/L; PϽ0.01). After 15 weeks, but not 7 weeks, relaxation of aortic rings to acetylcholine was selectively impaired by 35% (PϽ0.05) and thrombomodulin anticoagulant activity was decreased by 20% (PϽ0.05) in CBS ϩ/-mice. Plasma levels of folate did not differ between groups. Levels of SAH were elevated Ϸ2-fold in liver and brain of CBS ϩ/-mice, and correlations were observed between plasma total homocysteine and SAH in liver (rϭ0.54; PϽ0.001) and brain (rϭ0.67; PϽ0.001). These results indicate that endothelial dysfunction occurs in hyperhomocysteinemic mice even in the absence of folate deficiency. Endothelial dysfunction in CBS ϩ/-mice was associated with increased tissue levels of SAH, which suggests that altered SAM-dependent methylation may contribute to vascular dysfunction in hyperhomocysteinemia.

Altered folate homeostasis is associated with many clinical and pathological manifestations in the CNS. Notably, folate-mediated onecarbon metabolism is essential for methyltransferase-dependent cellular methylation reactions. Biogenesis of protein phosphatase 2A (PP2A) holoenzyme containing the regulatory B␣ subunit, a major brain tau phosphatase, is controlled by methylation. Here, we show that folate deprivation in neuroblastoma cells induces downregulation of PP2A leucine carboxyl methyltransferase-1 (LCMT-1) expression, resulting in progressive accumulation of newly synthesized demethylated PP2A pools, concomitant loss of B␣, and ultimately cell death. These effects are further accentuated by overexpression of PP2A methylesterase (PME-1) but cannot be rescued by PME-1 knockdown. Overexpression of either LCMT-1 or B␣ is sufficient to protect cells against the accumulation of demethylated PP2A, increased tau phosphorylation, and cell death induced by folate starvation. Conversely, knockdown of either protein accelerates folate deficiencyevoked cell toxicity. Significantly, mice maintained for 2 months on low-folate or folate-deficient diets have brain-region-specific alterations in metabolites of the methylation pathway. Those are associated with downregulation of LCMT-1, methylated PP2A, and B␣ expression and enhanced tau phosphorylation in susceptible brain regions. Our studies provide novel mechanistic insights into the regulation of PP2A methylation and tau. They establish LCMT-1-and B␣-containing PP2A holoenzymes as key mediators of the role of folate in the brain. Our results suggest that counteracting the neuronal loss of LCMT-1 and B␣ could be beneficial for all tauopathies and folate-dependent disorders of the CNS.

Background and Purpose-Hyperhomocysteinemia is an emerging risk factor for stroke, but little is known about effects of hyperhomocysteinemia on cerebral vascular function. We tested the hypothesis that chronic hyperhomocysteinemia in mice causes endothelial dysfunction in cerebral arterioles through a mechanism that involves superoxide. Methods-Mice heterozygous for a targeted disruption of the cystathionine ␤-synthase gene (Cbsϩ/Ϫ) and their wild type littermates (Cbsϩ/ϩ) were fed either a control diet or a high-methionine diet for 10 to 12 months. Results-Plasma total homocysteine was elevated with the high-methionine diet compared with the control diet for both Cbsϩ/ϩ (7.9Ϯ1.0 versus 5.0Ϯ0.5 mol/L; PϽ0.05) and Cbsϩ/Ϫ (20.5Ϯ3.1 versus 8.2Ϯ0.9 mol/L; PϽ0.001) mice. Dilatation of cerebral arterioles (Ϸ30 m baseline diameter) was measured in vivo in response to the endotheliumdependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Vasodilatation to acetylcholine was impaired with the high-methionine diet compared with the control diet for both Cbsϩ/ϩ and Cbsϩ/Ϫ mice (PϽ0.01). Dilatation of arterioles to acetylcholine was restored toward normal by the superoxide scavenger tiron (PϽ0.05). Vasodilatation to nitroprusside was not influenced by Cbs genotype or diet. Dihydroethidium (DHE) staining for vascular superoxide was elevated in Cbsϩ/Ϫ mice fed the high-methionine diet and was inhibited by apocynin or N-nitro-L-arginine methyl ester, implicating NAD(P)H oxidase and nitric oxide synthase as potential sources of superoxide. Conclusions-Superoxide is a key mediator of endothelial dysfunction in the cerebral circulation during diet-induced hyperhomocysteinemia.

Deficiency of methylenetetrahydrofolate reductase (MTHFR) predisposes to hyperhomocysteinemia and vascular disease. We tested the hypothesis that heterozygous disruption of the Mthfr gene sensitizes mice to diet-induced hyperhomocysteinemia and endothelial dysfunction. Mthfr ؉/؊ and Mthfr ؉/؉ mice were fed 1 of 4 diets: control, high methionine (HM), low folate (LF), or high methionine/low folate (HM/LF). Plasma total homocysteine (tHcy) was higher with the LF and HM/LF diets than the control (P < .01) or HM (P < .05) diets, and Mthfr ؉/؊ mice had higher tHcy than Mthfr ؉/؉ mice (P < .05). With the control diet, the S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio was lower in the liver and brain of Mthfr ؉/؊ mice than Mthfr ؉/؉ mice (P < .05). SAM/SAH ratios decreased further in Mthfr ؉/؉ or Mthfr ؉/؊ mice fed LF or LF/HM diets (P < .05). In cerebral arterioles, endothelium-dependent dilation to 1 or 10 M acetylcholine was markedly and selectively impaired with the HM/LF diet compared with the control diet for both Mthfr ؉/؉ (maximum dilation 5% ؎ 2% versus 21% ؎ 4%; P < .01) and Mthfr ؉/؊ (6% ؎ 2% versus 21% ؎ 3%; P < .01) mice. These findings demonstrate that the Mthfr ؉/؊ genotype sensitizes mice to diet-induced hyperhomocysteinemia and that hyperhomocysteinemia alters tissue methylation capacity and impairs endothelial function in cerebral microvessels. IntroductionElevation of plasma total homocysteine (tHcy) is associated with increased risk of stroke, myocardial infarction, and venous thrombosis. 1,2 In a recent meta-analysis, a 25% elevation in plasma tHcy was associated with a 10% higher risk of cardiovascular disease and a 20% higher risk of stroke after adjustment for other known risk factors. 3 The vascular pathogenic effects of homocysteine have not been fully characterized but are thought to involve oxidative inactivation of endothelium-derived nitric oxide and vascular inflammation. [4][5][6] Impaired endothelium-dependent vasodilation has been observed in human subjects with acute hyperhomocysteinemia induced by oral methionine loading [7][8][9][10][11] and animal models of diet-induced chronic hyperhomocysteinemia. 12,13 Mice with hyperhomocysteinemia produced by heterozygosity for a targeted disruption of the cystathionine ␤-synthase (Cbs) gene have enhanced sensitivity to endothelial dysfunction in both the aorta 14-16 and mesenteric arterioles. 14,17 Some pathogenic effects of hyperhomocysteinemia may be related to the metabolic link between homocysteine and methionine. Within the methionine cycle, methionine is converted to S-adenosylmethionine (SAM), which serves as a methyl donor for numerous methyl acceptors, including DNA, RNA, protein, histones, neurotransmitters, and phospholipids. 18 S-adenosylhomocysteine (SAH) is produced as a by-product of methyl donation, and homocysteine is formed through the (reversible) liberation of adenosine from SAH. During hyperhomocysteinemia, intracellular concentrations of SAH may increase, resulting in a lower SAM/ SAH ratio and dimini...

Hyperhomocysteinemia is common in Alzheimer's disease and is negatively correlated with cognitive function. Hyperhomocysteinemia can increase S-adenosylhomocysteine (SAH), a potent methyltransferase inhibitor. This study investigates the role of brain SAH in the cognitive and neurological disruption in Alzheimer's disease. SAH was significantly (26%) higher in prefrontal cortex of Alzheimer patients than normals. Brain homogenates from Alzheimer patients inhibited an exogenous methyltransferase 15% more than normal homogenates (P <.001). Brain SAH levels correlated (r=.508) with methyltransferase inhibition by brain homogenates. Methyltransferase inhibition by Alzheimer brain homogenates correlated inversely with cognitive function as determined by MMSE (r=-0.36). Phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) activities were more than 30% lower (P<0.001) in Alzheimer than normal brains. Brain PNMT activity correlated significantly with cognitive function (r=0.243), age of Alzheimer's onset (r=0.272), and choline acetyltransferase activity (r=0.333), but negatively with neurofibrillary tangles (r=-0.332). COMT activity also correlated significantly with cognitive function (r=0.324), age of disease onset (r=0.209), choline acetyltransferase activity (r=0.326), levels of synaptophysin (r=0.506), and negatively with tangles (r=-0.216 P=0.039). Elevated SAH in Alzheimer brain inhibits methyltransferases and is related to markers of disease progression and cognitive impairment.

These findings could reflect several possible mechanisms, including low dietary intake of folate, low GCPII activity, cigarette smoking, and the involvement of folate in the synthesis of neurotransmitters. Additional studies are needed to clarify these findings.

Objective-We tested the hypothesis that deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia. Methods and Results-Mice that were wild type (Gpx1 ϩ/ϩ ), heterozygous (Gpx1 ϩ/Ϫ ), or homozygous (Gpx1 Ϫ/Ϫ ) for the mutated Gpx1 allele were fed a control diet or a high-methionine diet for 17 weeks. Plasma total homocysteine was elevated in mice on the high-methionine diet compared with mice on the control diet (23Ϯ3 versus 6Ϯ0.3 mol/L, respectively; PϽ0.001) and was not influenced by Gpx1 genotype. In mice fed the control diet, maximal relaxation of the aorta in response to the endothelium-dependent dilator acetylcholine (10 Ϫ5 mol/L) was similar in Gpx1 ϩ/ϩ , Gpx1 ϩ/Ϫ , and Gpx1 Ϫ/Ϫ mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in Gpx1 Ϫ/Ϫ mice (PϽ0.05 versus Gpx1 ϩ/ϩ mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in Gpx1 PϽ0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between Gpx1 ϩ/ϩ and Gpx1 Ϫ/Ϫ mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in Gpx1 Ϫ/Ϫ mice fed the high-methionine diet (PϽ0.05 versus Gpx1 ϩ/ϩ mice fed the control diet). Conclusions-These findings demonstrate that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice and provide support for the hypothesis that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism. Key Words: endothelium Ⅲ homocysteine Ⅲ nitric oxide Ⅲ peroxide H yperhomocysteinemia is an emerging risk factor for cardiovascular events and venous thrombosis, 1,2 but the mechanisms responsible for the vascular pathology of hyperhomocysteinemia are still incompletely understood. Like many other cardiovascular risk factors, hyperhomocysteinemia produces endothelial dysfunction, which is possibly due to oxidative inactivation of endothelium-derived NO. 3,4 The oxidative stress hypothesis 4 is supported by the observation that auto-oxidation of homocysteine in vitro generates reactive oxygen species (ROS), including hydrogen peroxide and superoxide, and promotes oxidation of LDL. 5-7 Treatment of cultured endothelial cells with homocysteine decreases bioavailable NO 8,9 and produces cytotoxicity mediated by hydrogen peroxide. 10 Homocysteine also indirectly contributes to oxidative stress by inhibiting the expression of antioxidant enzymes, such as cellular glutathione peroxidase (GPx-1), an effect that may sensitize one to the toxic effects of homocysteine-derived hydrogen peroxides and lipid peroxides. 8,11 However, the role of oxidative stress in the vascular dysfunction of hyperhomocysteinemia in vivo is less clear, and its clinical importance has been questioned. 12 Attempts to demonstrate elevated levels of oxidation products in humans with hyperhomocysteinemia have produced conflicting results. [13][14][15][16] R...