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Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer’s Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer’s Disease
Background: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer’s disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. Objective: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-β (Aβ) accumulation, and cognitive/neuromotor performance in mice. Methods: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AβPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aβ was quantified by western blotting (brains) and by confocal microscopy (cells). Results: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AβPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aβ was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aβ without affecting AβPP. Conclusions: Our findings show that Blmh interacts with AβPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aβ accumulation and cognitive/neuromotor deficits.
Homocysteine metabolites inhibit autophagy, elevate amyloid beta, and induce neuropathy by impairing Phf8/H4K20me1-dependent epigenetic regulation of mTOR in cystathionine β-synthase-deficient mice
AbstractThe loss of cystathionine β‐synthase (CBS), an important homocysteine (Hcy)‐metabolizing enzyme or the loss of PHF8, an important histone demethylase participating in epigenetic regulation, causes severe intellectual disability in humans. Similar neuropathies were also observed in Cbs−/− and Phf8−/− mice. How CBS or PHF8 depletion can cause neuropathy was unknown. To answer this question, we examined a possible interaction between PHF8 and CBS using Cbs−/− mouse and neuroblastoma cell models. We quantified gene expression by RT‐qPCR and western blotting, mTOR‐bound H4K20me1 by chromatin immunoprecipitation (CHIP) assay, and amyloid β (Aβ) by confocal fluorescence microscopy using anti‐Aβ antibody. We found significantly reduced expression of Phf8, increased H4K20me1, increased mTOR expression and phosphorylation, and increased App, both on protein and mRNA levels in brains of Cbs−/− mice versus Cbs+/− sibling controls. Autophagy‐related Becn1, Atg5, and Atg7 were downregulated while p62, Nfl, and Gfap were upregulated on protein and mRNA levels, suggesting reduced autophagy and increased neurodegeneration in Cbs−/− brains. In mouse neuroblastoma N2a or N2a‐APPswe cells, treatments with Hcy‐thiolactone, N‐Hcy‐protein or Hcy, or Cbs gene silencing by RNA interference significantly reduced Phf8 expression and increased total H4K20me1 as well as mTOR promoter‐bound H4K20me1. This led to transcriptional mTOR upregulation, autophagy downregulation, and significantly increased APP and Aβ levels. The Phf8 gene silencing increased Aβ, but not APP, levels. Taken together, our findings identify Phf8 as a regulator of Aβ synthesis and suggest that neuropathy of Cbs deficiency is mediated by Hcy metabolites, which transcriptionally dysregulate the Phf8 → H4K20me1 → mTOR → autophagy pathway thereby increasing Aβ accumulation.
Homocysteine metabolites inhibit autophagy by upregulating miR-21-5p, miR-155-5p, miR-216-5p, and miR-320c-3p in human vascular endothelial cells
AbstractNutritional and genetic deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis, which is a major cause of cardiovascular disease (CVD). Impaired autophagy causes the accumulation of damaged proteins and organelles and is associated with CVD. Biochemically, HHcy is characterized by elevated levels of Hcy and its metabolites, Hcy-thiolactone and N-Hcy-protein. However, whether these metabolites can dysregulate mTOR signaling and autophagy in endothelial cells is not known. Here, we examined the influence of Hcy-thiolactone, N-Hcy-protein, and Hcy on autophagy human umbilical vein endothelial cells. We found that treatments with Hcy-thiolactone, N-Hcy-protein, or Hcy significantly downregulated beclin 1 (BECN1), autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and microtubule-associated protein 1 light chain 3 (LC3) mRNA and protein levels. We also found that these changes were mediated by upregulation by Hcy-thiolactone, N-Hcy-protein, and Hcy of autophagy-targeting microRNA (miR): miR-21, miR-155, miR-216, and miR-320c. The effects of these metabolites on levels of miR targeting autophagy as well as on the levels of BECN1, ATG5, ATG7, and LC3 mRNA and protein were abrogated by treatments with inhibitors of miR-21, miR-155, miR-216, and mir320c. Taken together, our findings show that Hcy metabolites can upregulate miR-21, miR-155, miR-216, and mir320c, which then downregulate autophagy in human endothelial cells, important for vascular homeostasis.
Cystathionine β‐synthase gene inactivation dysregulates major urinary protein biogenesis and impairs sexual signaling in mice
Association of Metallic and Nonmetallic Elements with Fibrin Clot Properties and Ischemic Stroke
Objectives—Metallic elements and fibrin clot properties have been linked to stroke. We examined metallic and nonmetallic elements, fibrin clot lysis time (CLT), and maximum absorbance (Absmax) in relation to ischemic stroke. Design—A case–control study of ischemic stroke patients vs. healthy individuals. Subjects and Methods—Plasma and serum were collected from 260 ischemic stroke patients (45.0% women; age, 68 ± 12 years) and 291 healthy controls (59.7% women; age, 50 ± 17 years). Fibrin CLT and Absmax were measured using a validated turbidimetric assay. Serum elements were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and optical emission spectrometry (ICP-OES). Data were analyzed by bivariate correlations and multiple or logistic regression. Results—In female stroke patients, copper, lithium, and aluminum were significantly lower compared with controls; in male stroke patients, potassium was lower, and beryllium was elevated. In female and male stroke patients, iron, zinc, nickel, calcium, magnesium, sodium, and silicon were significantly lower, while strontium was elevated. Positive correlations between fibrin clot properties and metals, observed in healthy controls, were lost in ischemic stroke patients. In multivariate regression analysis, fibrin CLT and/or Absmax was associated with zinc, calcium, potassium, beryllium, and silicon in stroke patients and with sodium, potassium, beryllium, and aluminum in controls. In logistic regression analysis, stroke was independently associated with lithium, nickel, beryllium, strontium, boron, and silicon and with sodium, potassium, calcium, and aluminum but not with fibrin CLT/Absmax. Conclusions—Various elements were associated with fibrin clot properties and the risk of ischemic stroke. Lithium, sodium, calcium, and aluminum abrogated the association of fibrin clot properties with ischemic stroke.
The Molecular Bases of Anti-Oxidative and Anti-Inflammatory Properties of Paraoxonase 1
The anti-oxidative and anti-inflammatory properties of high-density lipoprotein (HDL) are thought to be mediated by paraoxonase 1 (PON1), a calcium-dependent hydrolytic enzyme carried on a subfraction of HDL that also carries other anti-oxidative and anti-inflammatory proteins. In humans and mice, low PON1 activity is associated with elevated oxidized lipids and homocysteine (Hcy)-thiolactone, as well as proteins that are modified by these metabolites, which can cause oxidative stress and inflammation. PON1-dependent metabolic changes can lead to atherothrombotic cardiovascular disease, Alzheimer’s disease, and cancer. The molecular bases underlying these associations are not fully understood. Biochemical, proteomic, and metabolic studies have significantly expanded our understanding of the mechanisms by which low PON1 leads to disease and high PON1 is protective. The studies discussed in this review highlight the changes in gene expression affecting proteostasis as a cause of the pro-oxidative and pro-inflammatory phenotypes associated with attenuated PON1 activity. Accumulating evidence supports the conclusion that PON1 regulates the expression of anti-oxidative and anti-inflammatory proteins, and that the disruption of these processes leads to disease.
Homocysteine Thiolactone Detoxifying Enzymes and Alzheimer’s Disease
Elevated levels of homocysteine (Hcy) and related metabolites are associated with Alzheimer’s disease (AD). Severe hyperhomocysteinemia causes neurological deficits and worsens behavioral and biochemical traits associated with AD. Although Hcy is precluded from entering the Genetic Code by proofreading mechanisms of aminoacyl-tRNA synthetases, and thus is a non-protein amino acid, it can be attached to proteins via an N-homocysteinylation reaction mediated by Hcy-thiolactone. Because N-homocysteinylation is detrimental to a protein’s function and biological integrity, Hcy-thiolactone-detoxifying enzymes—PON1, BLMH, BPHL—have evolved. This narrative review provides an account of the biological function of these enzymes and of the consequences of their impairments, leading to the phenotype characteristic of AD. Overall, accumulating evidence discussed in this review supports a hypothesis that Hcy-thiolactone contributes to neurodegeneration associated with a dysregulated Hcy metabolism.
Homocysteine metabolites impair the PHF8/H4K20me1/mTOR/autophagy pathway by upregulating the expression of histone demethylase PHF8‐targeting microRNAs in human vascular endothelial cells and mice
AbstractThe inability to efficiently metabolize homocysteine (Hcy) due to nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) and endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). PHF8 is a histone demethylase that demethylates H4K20me1, which affects the mammalian target of rapamycin (mTOR) signaling and autophagy, processes that play important roles in CVD. PHF8 is regulated by microRNA (miR) such as miR‐22‐3p and miR‐1229‐3p. Biochemically, HHcy is characterized by elevated levels of Hcy, Hcy‐thiolactone and N‐Hcy‐protein. Here, we examined the effects of these metabolites on miR‐22‐3p, miR‐1229‐3p, and their target PHF8, as well as on the downstream consequences of these effects on H4K20me1, mTOR‐, and autophagy‐related proteins and mRNAs expression in human umbilical vein endothelial cells (HUVEC). We found that treatments with N‐Hcy‐protein, Hcy‐thiolactone, or Hcy upregulated miR‐22‐3p and miR‐1229‐3p, attenuated PHF8 expression, upregulated H4K20me1, mTOR, and phospho‐mTOR. Autophagy‐related proteins (BECN1, ATG5, ATG7, lipidated LC3‐II, and LC3‐II/LC3‐I ratio) were significantly downregulated by at least one of these metabolites. We also found similar changes in the expression of miR‐22‐3p, Phf8, mTOR‐ and autophagy‐related proteins/mRNAs in vivo in hearts of Cbs−/− mice, which show severe HHcy and endothelial dysfunction. Treatments with inhibitors of miR‐22‐3p or miR‐1229‐3p abrogated the effects of Hcy‐thiolactone, N‐Hcy‐protein, and Hcy on miR expression and on PHF8, H4K20me1, mTOR‐, and autophagy‐related proteins/mRNAs in HUVEC. Taken together, these findings show that Hcy metabolites upregulate miR‐22‐3p and miR‐1229‐3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis.
Hydrolaza bleomycyny, metabolizm homocysteiny, epigenetyczna regulacja mTOR, autofagia i choroba Alzheimera
Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke
AbstractHomocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.
Proteomic Exploration of Paraoxonase 1 Function in Health and Disease
High-density lipoprotein (HDL) exhibits cardio- and neuro-protective properties, which are thought to be promoted by paraoxonase 1 (PON1), a hydrolytic enzyme associated with an HDL subfraction also enriched with an anticoagulant protein (PROS1) and amyloid beta-transport protein clusterin (CLU, APOJ). Reduced levels of PON1 activity, characterized biochemically by elevated levels of homocysteine (Hcy)-thiolactone, oxidized lipids, and proteins modified by these metabolites in humans and mice, are associated with pathological abnormalities affecting the cardiovascular system (atherothrombosis) and the central nervous system (cognitive impairment, Alzheimer’s disease). The molecular bases of these abnormalities have been largely unknown. Proteomic and metabolic studies over the past decade have significantly contributed to our understanding of PON1 function and the mechanisms by which PON1 deficiency can lead to disease. Recent studies discussed in this review highlight the involvement of dysregulated proteostasis in the pro-oxidative, pro-atherothrombotic, and pro-amyloidogenic phenotypes associated with low PON1 activity.
Diet-induced hyperhomocysteinemia causes sex-dependent deficiencies in offspring musculature and brain function
Hyperhomocysteinemia (HHcy), characterized by elevated homocysteine (Hcy) levels, is a known risk factor for cardiovascular, renal, and neurological diseases, as well as pregnancy complications. Our study aimed to investigate whether HHcy induced by a high-methionine (high-Met) diet exacerbates cognitive and behavioral deficits in offspring and leads to other breeding problems. Dietary HHcy was induced four weeks before mating and continued throughout gestation and post-delivery. A battery of behavioral tests was conducted on offspring between postnatal days (PNDs) 5 and 30 to assess motor function/activity and cognition. The results were correlated with brain morphometric measurements and quantitative analysis of mammalian target of rapamycin (mTOR)/autophagy markers. The high-Met diet significantly increased parental and offspring urinary tHcy levels and influenced offspring behavior in a sex-dependent manner. Female offspring exhibited impaired cognition, potentially related to morphometric changes observed exclusively in HHcy females. Male HHcy pups demonstrated muscle weakness, evidenced by slower surface righting, reduced hind limb suspension (HLS) hanging time, weaker grip strength, and decreased activity in the beaker test. Western blot analyses indicated the downregulation of autophagy and the upregulation of mTOR activity in HHcy cortexes. HHcy also led to breeding impairments, including reduced breeding rate, in-utero fetal death, lower pups’ body weight, and increased mortality, likely attributed to placental dysfunction associated with HHcy. In conclusion, a high-Met diet impairs memory and cognition in female juveniles and weakens muscle strength in male pups. These effects may stem from abnormal placental function affecting early neurogenesis, the dysregulation of autophagy-related pathways in the cortex, or epigenetic mechanisms of gene regulation triggered by HHcy during embryonic development.
Homocysteine thiolactone contributes to the prognostic value of fibrin clot structure/function in coronary artery disease
Fibrin clot structure/function contributes to cardiovascular disease. We examined sulfur-containing metabolites as determinants of fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to outcomes in coronary artery disease (CAD) patients. Effects of B-vitamin/folate therapy on CLT and Absmax were studied. Plasma samples were collected from 1,952 CAD patients randomized in a 2 x 2 factorial design to (i) folic acid, vitamins B12, B6; (ii) folic acid, vitamin B12; (iii) vitamin B6; (iv) placebo for 3.8 years in the Western Norway B-Vitamin Intervention Trial. Clot lysis time (CLT) and maximum absorbance (Absmax) were determined using a validated turbidimetric assay. Acute myocardial infarction (AMI) and mortality were assessed during a 7-year follow-up. Data were analyzed using bivariate and multiple regression. Survival free of events was studied using Kaplan Mayer plots. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazards models. Baseline urinary homocysteine (uHcy)-thiolactone and plasma cysteine (Cys) were significantly associated with CLT while plasma total Hcy was significantly associated with Absmax, independently of fibrinogen, triglycerides, vitamin E, glomerular filtration rate, body mass index, age, sex plasma creatinine, CRP, HDL-C, ApoA1, and previous diseases. B-vitamins/folate did not affect CLT and Absmax. Kaplan-Meier analysis showed associations of increased baseline CLT and Absmax with worse outcomes. In Cox regression analysis, baseline CLT and Absmax (>cutoff) predicted AMI (CLT: HR 1.58, 95% CI 1.10–2.28; P = 0.013. Absmax: HR 3.22, CI 1.19–8.69; P = 0.021) and mortality (CLT: HR 2.54, 95% CI 1.40–4.63; P = 0.002. Absmax: 2.39, 95% CI 1.17–4.92; P = 0.017). After adjustments for other prognostic biomarkers these associations remained significant. Cys and uHcy-thiolactone, but not tHcy, were significant predictors of AMI in Cox regression models that included CLT. Conclusions uHcy-thiolactone and plasma Cys are novel determinants of CLT, an important predictor of adverse CAD outcomes. CLT and Absmax were not affected by B-vitamin/folate therapy, which could account for the lack of efficacy of such therapy in CAD. Trial registration: URL: http://clinicaltrials.gov. Identifier: NCT00354081.
Association of GLOD4 with Alzheimer’s Disease in Humans and Mice
Background: Glyoxalase domain containing protein 4 (GLOD4), a protein of an unknown function, is associated with Alzheimer’s disease (AD). Three GLOD4 isoforms are known. The mechanism underlying GLOD4’s association with AD was unknown. Objective: To assess GLOD4’s role in the central nervous system by studying GLOD4 isoforms expression in human frontal cerebral cortical tissues from AD patients and in brains of Blmh–/–5xFAD mouse AD model of AD. Methods: GLOD4 protein and mRNA were quantified in human and mouse brains by western blotting and RT-qPCR, respectively. Mouse brain amyloid-β (Aβ) was quantified by western blotting. Behavioral assessments of mice were performed by cognitive/neuromotor testing. Glod4 gene in mouse neuroblastoma N2a-APPswe cells was silenced by RNA interference and Glod4, Aβ precursor protein (Aβpp), Atg5, p62, and Lc3 proteins and mRNAs were quantified. Results: GLOD4 mRNA and protein isoforms were downregulated in cortical tissues from AD patients compared to non-AD controls. Glod4 mRNA was downregulated in brains of Blmh–/–5xFAD mice compared to Blmh+/+5xFAD sibling controls, but not in Blmh–/– mice without the 5xFAD transgene compared to Blmh+/+ sibling controls. The 5xFAD transgene downregulated Glod4 mRNA in Blmh–/– mice of both sexes and in Blmh+/+ males but not females. Attenuated Glod4 was associated with elevated Aβ and worsened memory/sensorimotor performance in Blmh–/–5xFAD mice. Glod4 depletion in N2a-APPswe cells upregulated AβPP, and downregulated autophagy-related Atg5, p62, and Lc3 genes. Conclusions: These findings suggest that GLOD4 interacts with AβPP and the autophagy pathway, and that disruption of these interactions leads to Aβ accumulation and cognitive/neurosensory deficits.