Abstract
Purpose :
Photoreceptors are vulnerable to dysfunction from metabolic perturbations. This project deciphers the roles of AMP-activated kinase in photoreceptors by elucidating the functions of its catalytic isoforms, Prkaa1 and Prkaa2, which are largely considered redundant or difficult to study due to biased expression in each tissue. However, the retina uniquely expresses both isoforms equivalently, thus establishing a novel premise to study the function of each isoform.
Methods :
Rod photoreceptor specific knockouts of either Prkaa1 or Prkaa2 were generated by crossing mice harboring the rhodopsin-iCre75 transgene with either Prkaa1 or Prkaa2 floxed mice respectively. Experiments to assess functional differences include in vivo electroretinography (ERG), extracellular flux analyses (EFA), metabolomics, bulk RNA sequencing, and phosphoproteomics. Mice were also enucleated and eyes were processed to undergo transmission electron microscopy (TEM), chromogenic in-situ hybridization, or immunofluorescent imaging. Mice were also assessed via ERG and EFA after induction of streptozotocin (STZ) injection diabetes.
Results :
Prkaa2 knockouts demonstrate ERG deficits (n = 8), higher glycolytic capacity on EFA (n = 8), increased levels of ATP and GTP but decreased levels of cGMP by metabolomics (n = 10); however, Prkaa1 knockouts do not recapitulate any of the same changes. TEM shows disorganized outer segments only in Prkaa2 knockout eyes (n = 4). Bulk RNA sequencing of Prkaa1 knockout retinas show decreased gene expression changes related to eye development whereas those of Prkaa2 knockout retinas show decreased changes related to catabolic processes (n = 4). Phosphoproteomics of Prkaa2 rod photoreceptors show notable downregulated species related to phototransduction or metabolic function (n = 4). STZ injected Prkaa1 mice exhibited ERG deficits and increased glycolytic flux from extracted retinas (n = 4), while STZ injected Prkaa2 mice demonstrated no ERG changes and no exacerbated glycolytic flux (n = 4).
Conclusions :
Prkaa1 is dispensable for proper rod photoreceptor function while Prkaa2 is necessary in homeostasis. Prkaa2 deficient rod photoreceptors exhibit a perturbed metabolome and phosphoproteome that is most associated with dysfunction in GTP homeostasis and phototransduction. However, diabetes causes Prkaa1 knockouts to develop dysfunction unseen in Prkaa2 knockouts.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.