Abstract
Purpose :
The mGluR6-TRPM1 signalplex produces light-evoked post-synaptic ON-bipolar cell (ON-BC) depolarization. Despite its importance to vision, virtually nothing is known about how this signalplex is regulated. Mechanistic target of rapamycin (mTOR) is a ubiquitous signaling hub kinase that controls the balance between many metabolic and anabolic processes. mTOR's role in vision is not known. Protein kinase C alpha (PKCα) is highly expressed in rod ON-BC and was suggested to be necessary for light-evoked rod ON-BC depolarization; however, Pkca knockout mice exhibited normal electroretinogram (ERG) b-wave amplitudes. Classical PKC (cPKC) isoforms are stabilized by mTOR complex 2 (mTORC2)-mediated phosphorylation.
Methods :
Conditional knockout (cKO) of the Rictor gene (encoding an essential component mTORC2) in the entire neural retina was accomplished by crossing Rictor-floxed mice with the Six3Cre driver mouse. mGluR6Cre mice were used for Rictor cKO specifically in rod ON-BC. Western blotting of whole retina lysates and immunoflourescence (IF) analysis of frozen retina sections were used to determine cPKC isoform protein expression. Flash ERG a-wave and b-wave responses were measured in dark-adapted retinas.
Results :
mTORC2-deficient retinas exhibited normal morphology and rod ON-BC number. In contrast, scotopic ERG (0.001-32 cd.s/m2) b-wave amplitudes were reduced by 84-50% (p<0.0001) in Six3Cre/Rictorfl/fl mice, and by 53-36% (p<0.0001) in mGluR6Cre/Rictorfl/fl mice, compared to their respective controls. Six3Cre-mediated Rictor cKO reduced photopic (10-100 cd.s/m2) b-wave amplitudes by 71-70% (p<0.0001), while mGluR6Cre-mediated cKO reduced photopic b-wave amplitudes by approximately 30% (p<0.05). In contrast, a-wave amplitudes were not significantly affected by mTORC2 deficiency. Both Six3Cre/Rictorfl/fl and mGluR6Cre/Rictorfl/fl mice exhibited greatly reduced retinal PKCα protein expression, which was nearly undetectable in rod ON-BC by IF. Other classical PCK isoforms, PKCβII and PKCγ, were similarly reduced.
Conclusions :
The results suggest that mTORC2 function is necessary for normal rod ON-BC depolarization in response to light, perhaps by promoting classical PKC protein stability. Effects on photopic b-waves and quantitatively different effects of Rictor cKO in the entire neural retina and in rod ON-BC suggest that mTORC2 is necessary in other ON-BC as well.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.