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.

The 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 vs. 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. This article is protected by copyright. All rights reserved.