Abstract
Purpose :
Autophagy plays a major role in maintaining lens transparency by degrading organelles and misfolded proteins through the lysosome. CRYβA1/A3, a lens structural protein, is also expressed in lysosomes of retinal astrocytes and the pigment epithelium to regulate autophagy. To study the similar role of CRYβA1/A3 in the lens, we employed a mouse model of human congenital nuclear cataract, CRYβA1/A3-Gly91Del (named βA3ΔG91) to examine organelle-degradation in its lenses.
Methods :
The βA3ΔG91 mouse model was generated using CRISPR-Cas9 technology at the UAB Transgenic & Genetically Engineered Models Core facility. Transmission Electron Microscopy (TEM) was performed on one-month-old βA3ΔG91- and WT lenses to visualize autophagic vesicles and the degradation of nuclei, mitochondria, and endoplasmic reticulum (ER) in the lens epithelium, cortex, and nuclear regions. The results were correlated with immunohistochemical (IHC) analyses using DAPI, COX IV (mitochondrial marker), and HSPA5 (ER marker) antibodies.
Results :
TEM analyses revealed several single and double-membrane vesicles scattered throughout βA3ΔG91 lenses relative to the WT lenses. In addition, the lens epithelium displayed healthy nuclei, mitochondria, and ER in the WT and βA3ΔG91 lenses. In the outer cortex, both WT- and βA3ΔG91- lenses began to show signs of mitochondrial degradation, together with few breaks in the nuclear envelope but the ER was still present, although very few. The βA3ΔG91 inner cortex exhibited unhealthy-looking and fragmented nuclei and few mitochondria but no visible ER. In contrast, the WT inner cortex was completely devoid of nuclei and ER but had very few mitochondria with double membrane breaks and disorganized cristae. Additionally, the βA3ΔG91 nuclear region contained remnants of condensed nuclear materials, and many double-membrane vesicles which were completely absent in the WT nuclear region. IHC analyses of βA3ΔG91 lenses showed slightly delayed clearance of mitochondria and retention of nuclei in the lens nucleus but a decrease in ER compared to WT lenses.
Conclusions :
Relative to WT lenses, βA3ΔG91 lenses showed attenuation of nuclei clearance and greater number of double-membrane vesicles representing autophagosomes, which suggests that autophagy may be disrupted in these lenses.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.