Hepatic and renal AAV9-mediated expression of codon-optimized rice OsDJ-1C in aged mice installs a GSH-independent ...

IF a single intravenous dose of AAV9 encoding a human-codon-optimized OsDJ-1C transgene under a constitutive CAG promoter (1×10¹¹ vg/mouse) is administered to 18-month-old male and female C57BL/6J mice carrying established dicarbonyl damage (elevated tissue MG-H1, CEdG DNA adducts, and a depleted GSH/GSSG ratio consistent with aged tissue),

THEN quantitative LC-MS/MS measurement at 6 months post-injection will reveal ≥40% reduction in hepatic and renal tissue-free MGO concentrations, ≥30% reduction in MG-H1 adduct density on short-to-medium turnover proteins (assessed by targeted proteomics), a measurable decrease in CEdG DNA adduct burden in liver (assessed by immunoslot-blot and LC-MS), and partial restoration of endogenous PARK7/DJ-1 deglycase activity on long-lived glycated protein substrates, compared with age-matched AAV9-GFP controls,

BECAUSE the following causal chain operates in aged mammalian tissue:

1. Aged tissue suffers a "double-hit" collapse of the canonical glyoxalase system: GLO1 activity declines transcriptionally in liver, kidney, and brain with advancing age, and simultaneously the cellular GSH pool is depleted and the GSH/GSSG ratio falls, creating a kinetic bottleneck wherein even residual GLO1 protein cannot convert MGO to S-D-lactoylglutathione at physiological rates. Free reactive MGO therefore accumulates above the nanomolar steady-state, driving accelerating AGE formation and direct DNA mutagenesis (as reviewed in the Evidence Set: "the 'double hit' of reduced enzyme availability and insufficient cofactor supply leads to a supraphysiological accumulation of MGO, driving the formation of DNA crosslinks and protein adducts associated with cellular senescence").

2. OsDJ-1C provides a biochemically orthogonal, GSH-independent detoxification pathway: Rice OsDJ-1C, a member of the DJ-1/PfpI superfamily, catalyzes the direct, single-step isomerization of free methylglyoxal to D-lactate using a conserved catalytic cysteine nucleophile (Cys111), without any requirement for glutathione as cofactor or co-substrate. This pathway is therefore fully active under the GSH-depleted, oxidized-redox conditions of aged tissue where GLO1 is non-functional. Codon-optimized expression in mammalian hepatocytes via AAV9 (which exhibits strong hepatotropism) reconstitutes this enzymatic activity in the cytosol (as detailed in the Evidence Set enzymatic characterization of OsDJ-1C and the feasibility analysis of heterologous expression).

3. Reduction of free cytosolic MGO concentration relieves the substrate burden on endogenous PARK7/DJ-1 deglycase activity [SPECULATIVE but mechanistically grounded]: Human PARK7/DJ-1 has evolutionarily diverged from its plant counterpart to function primarily as a deglycase — repairing already-formed MGO-glycation adducts on proteins and nucleic acids — rather than scavenging free MGO. In aged tissue, the chronic supraphysiological MGO load overwhelms DJ-1's deglycase capacity, resulting in ...

SENS category: GlycoSENS

Key references: • doi.org/10.1016/j.cmet.2017.07.010]. • doi.org/10.1101/2024.06.18.599500]. • doi.org/10.1016/j.cmet.2017.07.010] • doi.org/10.1101/2024.06.18.599500]