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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 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.
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.
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.
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.