Abstract
Purpose :
To determine the role of βA3/A1-crystallin in autophagy.
Methods :
We have developed a lens-specific βA3 knockout mouse model (named βA3cKO) (PLoS One. 2023, e0281386). The βA3cKO developed congenital cataract as determined by Micron-IV Slit-lamp microscopy. Lens RNA was isolated from both control (Cre-and flip mice) and βA3cKO mice and was used for transcriptome and Ingenuity pathway analyses. Primary lens epithelial cell (LEC) cultures were generated from βA3cKO- and control mice. LECs from control and βA3cKO were transfected with mRFP-green fluorescent protein (GFP)-LC3 adenoviral vector (Addgene) to determine autophagic flux and examined using confocal microscopy.
Results :
As reported previously in the βA3KO mice (PLoS One, 2016), βA3cKO also exhibited autophagy disruption and developed congenital nuclear cataract. The volcano plot was generated from the RNA-seq data containing 29000 genes. Of these, 297 autophagy-associated genes were either significantly upregulated or downregulated. ATG3, which is involved in the initiation phase of autophagy, was significantly downregulated among the autophagy genes. The ATG3 expression levels were quantified using qRT-PCR analysis and were analyzed in the LECs, which showed that their expression was downregulated compared to the control LECs. We also analyzed autophagy flux using mRFP-green fluorescent protein (GFP)-LC3 in LECs. Autophagosome formation would cause an increase in the number of GFP-/mRFP-positive (yellow) fluorescent puncta. In contrast, fluorescent puncta would become GFP-negative/mRFP-positive (red fluorescence) upon lysosome fusion. Control LECs showed red punctate fluorescence when autophagy was initiated and were lost when cells were treated with bafilomycin (disrupts autophagic flux by inhibiting V-ATPase-dependent acidification). In contrast, βA3cKO LECs showed no red fluorescence when autophagy was initiated on serum withdrawal but showed decreased yellow fluorescence. The results suggest that βA3-crystallin plays a role in the acidification of lysosomes.
Conclusions :
An absence of βA3-crystallin in the lenses of βA3cKO mice leads to the development of congenital nuclear cataract. Results from RNA-seq, IHC, qRT-PCR, and autophagy flux suggest βA3 loss results in an inhibition of autophagy-induction and plays a role in the acidification of the lysosomes
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.