Homocysteine Metabolites Inhibit Autophagy and Elevate Amyloid Beta by Impairing Phf8/H4K20me1-dependent Epigenetic Regulation of mTOR in Cystathionine Beta-Synthase-Deficient Mice

Abstract

The loss of cystathionine {beta}-synthase (CBS), an important homocysteine (Hcy)-metabolizing enzyme or the loss of PHF8, an important histone demethylase participating in epigenetic regulation, causes severe mental retardation 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 {beta} (A{beta}) by confocal fluorescence microscopy using anti-A{beta} 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 vs. Cbs+/- sibling controls. Autophagy-related proteins Becn1, Atg5, and Atg7 were downregulated while p62 was upregulated on protein and mRNA levels, suggesting impaired autophagy 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 A{beta} levels. The Phf8 gene silencing increased A{beta}, but not App, levels. Taken together, our findings identify Phf8 as a regulator of A{beta} synthesis and suggest that neuropathy of Cbs deficiency is mediated by Hcy metabolites, which transcriptionally dysregulate the Phf8->H4K20me1->mTOR->autophagy pathway thereby increasing A{beta} accumulation.