Erland Arning

Protein Phosphatase 2A Methyltransferase Links Homocysteine Metabolism with Tau and Amyloid Precursor Protein Regulation

Alzheimers disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-β 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 Bα-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of β-secretase-cleaved APP fragments and amyloid-β 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 α-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-β-synthase +/− 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.

Cerebral Vascular Dysfunction Mediated by Superoxide in Hyperhomocysteinemic Mice

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 β-synthase gene (Cbs +/−) and their wild type littermates (Cbs +/+) 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 Cbs +/+ (7.9±1.0 versus 5.0±0.5 μmol/L; P <0.05) and Cbs +/− (20.5±3.1 versus 8.2±0.9 μmol/L; P <0.001) mice. Dilatation of cerebral arterioles (≈30 μm baseline diameter) was measured in vivo in response to the endothelium-dependent 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 Cbs +/+ and Cbs +/− mice (P <0.01). Dilatation of arterioles to acetylcholine was restored toward normal by the superoxide scavenger tiron (P <0.05). Vasodilatation to nitroprusside was not influenced by Cbs genotype or diet. Dihydroethidium (DHE) staining for vascular superoxide was elevated in Cbs +/− mice fed the high-methionine diet and was inhibited by apocynin or Nω-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.

Mild Hyperhomocyst(e)inemia: A Possible Risk Factor for Cervical Artery Dissection

Background and Purpose— The pathogenesis of cervical artery dissection (CAD) remains unknown in most cases. Hyperhomocyst(e)inemia [hyperH(e)], an independent risk factor for cerebrovascular disease, induces damage in endothelial cells in animal cell culture. Consecutive patients with CAD and age-matched control subjects have been studied by serum levels of homocyst(e)ine and the genotype of 5,10-methylenetetrahydrofolate reductase (MTHFR). Methods— Twenty-six patients with CAD, admitted to our Stroke Unit (15 men and 11 women; 16 vertebral arteries, 10 internal carotid arteries), were compared with age-matched control subjects. All patients underwent duplex ultrasound, MR angiography, and/or conventional angiography. Results— Mean plasma homocyst(e)ine level was 17.88 &mgr;mol/L (range 5.95 to 40.0 &mgr;mol/L) for patients with CAD and 6.0±0.99 &mgr;mol/L for controls (P <0.001). The genetic analysis for the thermolabile form of MTHFR in CAD patients showed heterozygosity in 54% and homozygosity in 27%; comparable figures for controls were 40% (P =0.4) and 10% (P =0.1), respectively. Conclusions— Mild hyperH(e) might represent a risk factor for cervical artery dissection. The MTHFR mutation is not significantly associated with CAD. An interaction between different genetic and environmental factors probably takes place in the cascade of pathogenetic events leading to arterial wall damage.

Deficiency of Glutathione Peroxidase-1 Sensitizes Hyperhomocysteinemic Mice to Endothelial Dysfunction

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+/+), heterozygous (Gpx1+/−), or homozygous (Gpx1−/−) 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 (23±3 versus 6±0.3 &mgr;mol/L, respectively;P <0.001) 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 (10−5 mol/L) was similar in Gpx1+/+, Gpx1+/−, and Gpx1−/− mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in Gpx1−/− mice (P <0.05 versus Gpx1+/+ mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in Gpx1−/− mice (maximal relaxation 73±6% in Gpx1−/− mice versus 90±2% in Gpx1+/+ mice, P <0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between Gpx1+/+ and Gpx1−/− mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in Gpx1−/− mice fed the high-methionine diet (P <0.05 versus Gpx1+/+ 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.

Overexpression of Dimethylarginine Dimethylaminohydrolase Inhibits Asymmetric Dimethylarginine–Induced Endothelial Dysfunction in the Cerebral Circulation

Background and Purpose— Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase (NOS). An elevation of plasma ADMA levels is associated with cardiovascular disease. ADMA is hydrolyzed by dimethylarginine dimethylaminohydrolases (DDAHs). The goal of this study was to determine whether overexpression of human DDAH-1 in transgenic (DDAH-1–Tg) mice inhibits the vascular effects of ADMA. Methods— Using nontransgenic (non-Tg) and DDAH-1–Tg mice, we compared responses of the carotid artery and aorta (in vitro) and of the cerebral arterioles (in vivo) in the absence or presence of ADMA. DDAH-1 expression and plasma levels of ADMA were also measured. Results— Western blotting indicated that vascular expression of DDAH-1 was increased markedly in DDAH-1–Tg mice. Plasma levels of ADMA were reduced by ≈50% in DDAH-1–Tg mice compared with non-Tg mice (0.19±0.02 vs 0.37±0.04 &mgr;mol/L, P<0.05). Contraction of the aorta to nitro-l-arginine methyl ester (an inhibitor of NOS), an index of basal production of NO, was increased in DDAH-1–Tg mice compared with controls (50±4% vs 34±4%, P<0.05). Relaxation of the carotid artery to acetylcholine (an endothelium-dependent agonist) was enhanced in DDAH-1–Tg animals compared with control mice (relaxation of 74±6% vs 59±5%, respectively, in response to 10 &mgr;mol/L acetylcholine, P<0.05). ADMA (100 &mgr;mol/L) impaired the vascular response to acetylcholine in both non-Tg and DDAH-1–Tg mice, but the relative difference between the 2 strains remained. Responses to the endothelium-independent NO donor nitroprusside were similar in all groups. In vivo, ADMA (10 &mgr;mol/L) reduced responses of the cerebral arterioles to acetylcholine by ≈70% in non-Tg mice (P<0.05), and this inhibitory effect was largely absent in DDAH-1–Tg mice. Conclusions— These findings provide the first evidence that overexpression of DDAH-1 increases basal levels of vascular NO and protects against ADMA-induced endothelial dysfunction in the cerebral circulation.

Folate Deficiency Induces In Vitro and Mouse Brain Region-Specific Downregulation of Leucine Carboxyl Methyltransferase-1 and Protein Phosphatase 2A Bα Subunit Expression That Correlate with Enhanced Tau Phosphorylation

Altered folate homeostasis is associated with many clinical and pathological manifestations in the CNS. Notably, folate-mediated one-carbon metabolism is essential for methyltransferase-dependent cellular methylation reactions. Biogenesis of protein phosphatase 2A (PP2A) holoenzyme containing the regulatory Bα 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 Bα, 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 Bα 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 deficiency-evoked 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 Bα 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 Bα-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 Bα could be beneficial for all tauopathies and folate-dependent disorders of the CNS.

Cerebral Vascular Dysfunction in Methionine Synthase–Deficient Mice

Background—Methionine synthase (MS) catalyzes the folate-dependent remethylation of homocysteine to methionine. We tested the hypothesis that deficiency of MS impairs endothelial function in mice heterozygous for disruption of the Mtr gene, which encodes MS. Methods and Results—Plasma total homocysteine was similar in wild-type (Mtr+/+) and heterozygous (Mtr+/−) mice fed a control diet (4.5±0.3 and 5.3±0.4 &mgr;mol/L, respectively) and mildly elevated in Mtr+/+ and Mtr+/− mice fed a low-folate (LF) diet (7.5±0.7 and 9.6±1.2 &mgr;mol/L, respectively; P<0.001 versus control diet). Dilatation of cerebral arterioles to the endothelium-dependent dilator, acetylcholine (10 &mgr;mol/L) was blunted in Mtr+/− mice compared with Mtr+/+ mice fed the control diet (21±4 versus 32±4%; P<0.05). Both Mtr+/+ and Mtr+/− mice exhibited impaired dilatation of cerebral arterioles to acetylcholine when they were fed the LF diet (12±2 and 14±2%, respectively; P<0.01 versus Mtr+/+ mice fed the control diet). Elevated levels of superoxide and hydrogen peroxide were detected by confocal microscopy in cerebral arterioles of Mtr+/− mice fed the control diet and in both Mtr+/+ and Mtr+/− mice fed the LF diet. Conclusions—These findings demonstrate that defective homocysteine remethylation caused by deficiency of either MS or folate produces oxidative stress and endothelial dysfunction in the cerebral microcirculation of mice.

Tissue-specific downregulation of dimethylarginine dimethylaminohydrolase in hyperhomocysteinemia

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, has been proposed to be a mediator of vascular dysfunction during hyperhomocysteinemia. Levels of ADMA are regulated by dimethylarginine dimethylaminohydrolase (DDAH). Using both in vitro and in vivo approaches, we tested the hypothesis that hyperhomocysteinemia causes downregulation of the two genes encoding DDAH (Ddah1 and Ddah2). In the MS-1 murine endothelial cell line, the addition of homocysteine decreased NO production but did not elevate ADMA or alter levels of Ddah1 or Ddah2 mRNA. Mice heterozygous for cystathionine beta-synthase (Cbs) and their wild-type littermates were fed either a control diet or a high-methionine/low-folate (HM/LF) diet to produce varying degrees of hyperhomocysteinemia. Maximal relaxation of the carotid artery to the endothelium-dependent dilator acetylcholine was decreased by approximately 50% in Cbs(+/-) mice fed the HM/LF diet compared with Cbs(+/+) mice fed the control diet (P < 0.001). Compared with control mice, hyperhomocysteinemic mice had lower levels of Ddah1 mRNA in the liver (P < 0.001) and lower levels of Ddah2 mRNA in the liver, lung, and kidney (P < 0.05). Downregulation of DDAH expression in hyperhomocysteinemic mice did not result in an increase in plasma ADMA, possibly due to a large decrease in hepatic methylation capacity (S-adenosylmethionine-to-S-adenosylhomocysteine ratio). Our findings demonstrate that hyperhomocysteinemia causes tissue-specific decreases in DDAH expression without altering plasma ADMA levels in mice with endothelial dysfunction.

Prothrombotic Effects of Hyperhomocysteinemia and Hypercholesterolemia in ApoE-Deficient Mice

Objective—We tested the hypothesis that hyperhomocysteinemia and hypercholesterolemia promote arterial thrombosis in mice. Methods and Results—Male apolipoprotein E (Apoe)-deficient mice were fed one of four diets: control, hyperhomocysteinemic (HH), high fat (HF), or high fat/hyperhomocysteinemic (HF/HH). Total cholesterol was elevated 2-fold with the HF or HF/HH diets compared with the control or HH diets (P<0.001). Plasma total homocysteine (tHcy) was elevated (12 to 15 &mgr;mol/L) with the HH or HF/HH diets compared with the control or HF diets (4 to 6 &mgr;mol/L; P<0.001). Aortic sinus lesion area correlated strongly with total cholesterol (P<0.001) but was independent of tHcy. At 12 weeks of age, the time to thrombotic occlusion of the carotid artery after photochemical injury was >50% shorter in mice fed the HF diets, with or without hyperhomocysteinemia, compared with the control diet (P<0.05). At 24 weeks of age, carotid artery thrombosis was also accelerated in mice fed the HH diet (P<0.05). Endothelium-dependent nitric oxide–mediated relaxation of carotid artery rings was impaired in mice fed the HF, HH, or HF/HH diets compared with the control diet (P<0.05). Conclusions—Hyperhomocysteinemia and hypercholesterolemia, alone or in combination, produce endothelial dysfunction and increased susceptibility to thrombosis in Apoe-deficient mice.

Plasma total homocysteine levels and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene : a study in an Italian population with dementia

Hyperhomocysteinemia is a known risk factor for vascular disease and commonly occurs in the elderly. Several studies have shown an association between elevated plasma homocysteine levels and cognitive impairment, indicating that it may play a role in the pathophysiology of dementia. We studied plasma homocysteine, folate, vitamin B12 levels and the MTHFR C677T genotype in an Italian population of patients with dementia. We confirmed that elevated plasma tHcy (>14 micromol/l) is common in elderly subjects with dementia. Although we found a high prevalence of the MTHFR TT genotype (21.2%) the allele frequency is not over-represented relative to the control population. We also observed a high incidence of folate deficiency (38%) in subjects with dementia. Elevated homocysteine was associated with low plasma folate (<5.7 nmol/l) and the MTHFR TT genotype. Moderate to severe hyperhomocysteinemia (>26.1 nmol/l) was associated with a significantly lower MMSE score. Hyperhomocysteinemia may be neurotoxic by several different mechanisms affecting cognitive function. Further studies are needed to fully explore the potential of B vitamin supplementation to lower plasma homocysteine and improve cognitive function.