Filters
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.
Depletion of Paraoxonase 1 (Pon1) Dysregulates mTOR, Autophagy, and Accelerates Amyloid Beta Accumulation in Mice
Paraoxonase 1 (PON1), a homocysteine (Hcy)-thiolactone detoxifying enzyme, has been associated with Alzheimer’s disease (AD), suggesting that PON1 plays an important protective role in the brain. To study the involvement of PON1 in the development of AD and to elucidate the mechanism involved, we generated a new mouse model of AD, the Pon1−/−xFAD mouse, and examined how Pon1 depletion affects mTOR signaling, autophagy, and amyloid beta (Aβ) accumulation. To elucidate the mechanism involved, we examined these processes in N2a-APPswe cells. We found that Pon1 depletion significantly downregulated Phf8 and upregulated H4K20me1; mTOR, phospho-mTOR, and App were upregulated while autophagy markers Bcln1, Atg5, and Atg7 were downregulated at the protein and mRNA levels in the brains of Pon1─/─5xFAD vs. Pon1+/+5xFAD mice. Pon1 depletion in N2a-APPswe cells by RNA interference led to downregulation of Phf8 and upregulation of mTOR due to increased H4K20me1-mTOR promoter binding. This led to autophagy downregulation and significantly increased APP and Aβ levels. Phf8 depletion by RNA interference or treatments with Hcy-thiolactone or N-Hcy-protein metabolites similarly increased Aβ levels in N2a-APPswe cells. Taken together, our findings define a neuroprotective mechanism by which Pon1 prevents Aβ generation.