No abstract

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.

Background The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. Methods and Results Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor αIIbβ3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol-catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47phox and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. Conclusions Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.

Background: Coronavirus disease 2019 (COVID-19) is associated with coagulopathy but the optimal prophylactic anticoagulation therapy remains uncertain and may depend on COVID-19 severity. Objective: To compare outcomes in hospitalized adults with severe COVID-19 treated with standard prophylactic versus intermediate dose enoxaparin. Methods: We conducted a multi-center, open-label, randomized controlled trial comparing standard prophylactic dose versus intermediate dose enoxaparin in adults who were hospitalized with COVID-19 and admitted to an intensive care unit (ICU) and/ or had laboratory evidence of coagulopathy. Patients were randomly assigned in a 1:1 ratio to receive standard prophylactic dose enoxaparin or intermediate weightadjusted dose enoxaparin. The primary outcome was all-cause mortality at 30 days.Secondary outcomes included arterial or venous thromboembolism and major bleeding.Results: A total of 176 patients (99 males and 77 females) underwent randomization. In the intention-to-treat population, all-cause mortality at 30 days was 15% for intermediate dose enoxaparin and 21% for standard prophylactic dose enoxaparin (odds ratio, 0.66; 95% confidence interval, 0.30-1.45; P = .31 by Chi-square test). Unadjusted Cox proportional hazards modeling demonstrated no significant difference in mortality between intermediate and standard dose enoxaparin (hazard ratio, 0.67; 95% confidence interval, 0.33-1.37; P = .28). Arterial or venous thrombosis occurred in 13% of patients assigned to intermediate dose enoxaparin and 9% of patients assigned to standard dose enoxaparin. Major bleeding occurred in 2% of patients in each arm. Conclusion:In hospitalized adults with severe COVID-19, standard prophylactic dose and intermediate dose enoxaparin did not differ significantly in preventing death or thrombosis at 30 days.