Abstract
Purpose :
Reactive dicarbonyls, glyoxal (GO) and methylglyoxal (MGO), react with proteins and nucleotides, eventually leading to the formation of advanced glycation end products (AGEs). AGEs are associated with aging-related diseases, including age-related macular degeneration and cataracts. We recently identified GATD3A as a novel mitochondrial specific deglycase, with similar activity to the Parkinsonism associated deglycase DJ-1/Park7. Notably, loss of DJ-1 results in retinal associated phenotypes. This project aimed to validate GATD3A’s biochemical activity and elucidate its necessity for retinal function.
Methods :
Subcellular fractionation was utilized to determine organelle compartmentalization. Enzyme activity assays using recombinant GATD3A were used to confirm deglycation of amino species. A novel CRISPR-mediated Gat3da knockout mouse was generated, and complete loss of mature protein was confirmed via retinal and heart immunoblot. Retinal function was assessed via scotopic and photopic ERG. Histological and electron microscopy imaging were used to examine both retinal and heart structure. Using mouse embryonic fibroblasts (MEFs) from the Gatd3a knockout mice, mitochondrial biogenesis was quantified with qPCR and mitochondrial morphology was examined using confocal image-based morphometric analysis.
Results :
GATD3A has glyoxalase activity primarily against GO and localized to the mitochondrial matrix. Gatd3a knockout resulted in disrupted mitochondrial dynamics in the heart and MEFs, with mitochondria displaying more rounded morphology and electron lucent cristae. Gatd3a knockout mice displayed a 3-4-fold increase in dicarbonyl and AGE immunoreactivity in the heart. Knockout mice exhibited left ventricular heart fibrosis. Surprisingly, 4-month-old and 18-month-old retinas did not display any reduced functionality in either scotopic or photopic ERG. The retina did not display changes in morphology or levels of fibrosis.
Conclusions :
GATD3A exhibits glyoxalase activity which functions in the prevention of AGE accumulation and fibrosis in the heart. However, Gatd3a knockout displayed no detectable changes in the retina under normal physiological conditions. Future directions include stress induction via provision of a high-glycemic diet in order to increase the prevalence of AGEs and exacerbate previously identified phenotypes in Gatd3a knockout mice. We will further quantify AGE species using LC-MS/MS based methods.
This is a 2021 ARVO Annual Meeting abstract.