Lens-specific βA3/A1-conditional knockout mice: Phenotypic characteristics and calpain activation causing protein degradation and insolubilization

**Background** βA3/A1-crystallin is a structural lens protein essential for maintaining transparency through interactions with other crystallins. While its functions in the retina are well studied, its roles in the lens beyond structural support remain unclear. Mutations in CRYBA1 are associated with congenital cataracts, but the molecular mechanisms are not fully understood. Previous work with total βA3/A1 knockout mice showed nuclear cataracts, defective autophagy, and increased calpain-3 activity. To further investigate lens-specific functions, the authors generated a lens-specific βA3/A1 conditional knockout (βA3cKO) mouse model.

**Methods** βA3cKO mice were generated by crossing homozygous floxed Cryba1 mice (exon 4 flanked by loxP sites) with MLR10 Cre transgenic mice, which express Cre recombinase specifically in the developing lens from approximately E10.5. This deleted exon 4, which encodes amino acids critical for the Greek key motif 2. Phenotypic characterization included slit-lamp microscopy, H&E staining, immunohistochemistry, and qRT-PCR. Lens proteins from 1-month-old wild-type (WT), heterozygous (HET), and βA3cKO mice were fractionated by sequential centrifugation at 800 rpm (70 × g) and 5000 rpm (2744 × g). Protein fragments <20 kDa in the insoluble pellets were identified by mass spectrometry. Western blotting assessed levels of β-tubulin, phakinin, αA-crystallin, and calpain-3. Calpain activity was measured using the substrate N-Succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin, with and without 3 mM calcium. In vitro calpain-3 activation was tested by incubating WT lens homogenates with 100 μM Ca²⁺ for 2–3 hours at 37°C, followed by analysis of α-spectrin degradation and protein insolubilization.

**Key Results** βA3cKO mice developed congenital nuclear cataracts at birth, confirmed by slit-lamp imaging and H&E staining showing anomalous nuclear structure. qRT-PCR confirmed no βA3-crystallin mRNA expression in lenses, and immunohistochemistry showed loss of βA3 protein in the lens but preserved expression in retinal ganglion cells and RPE. Mass spectrometry of insoluble pellets after 800 rpm centrifugation identified 176 protein fragments in βA3cKO lenses (vs. 149 in WT and 151 in HET). After 5000 rpm, 176 fragments were found in βA3cKO (vs. 200 in WT and 165 in HET). Unique fragments in βA3cKO lenses included multiple β-tubulin chains (e.g., Tubb1, Tubb2a, Tubb3, Tubb5) and calpain-3. Western blotting showed a 40% reduction in β-tubulin levels in βA3cKO lenses compared to WT (GAPDH loading control). Increased fragmentation of phakinin, αA-crystallin, and calpain-3 was observed in βA3cKO lenses. Calpain activity assays showed a 10% increase in basal activity in βA3cKO homogenates vs. WT; with 3 mM Ca²⁺, activity was 10% lower in βA3cKO, suggesting prior activation. The ratio of total calpain-3 to full-length calpain-3 was 2.4 in WT and 1.7 in βA3cKO, indicating a 35% increase in autolyzed fragments. Immunostaining showed calpain-3 localized to the outer cortex in WT but associated with nuclei in βA3cKO lenses. In vitro, 100 μM Ca²⁺ activated calpain-3 in WT homogenates, causing α-spectrin degradation (250 kDa band lost without EGTA) and increased protein insolubilization, but did not directly proteolyze β-tubulin or αA-crystallin.

**Clinical Implications** This study demonstrates that lens-specific loss of βA3/A1-crystallin leads to calpain-3 activation, which degrades and insolubilizes key cytoskeletal and structural proteins (β-tubulin, phakinin, αA-crystallin), disrupting microtubules and contributing to congenital nuclear cataract formation. The findings suggest that βA3/A1-crystallin normally suppresses calpain activity, possibly via calcium binding or lysosomal function. Targeting calpain-3 or restoring βA3/A1 function could represent therapeutic strategies for congenital cataracts associated with CRYBA1 mutations. The results also highlight parallels with age-related diseases where protein aggregation and calpain dysregulation occur, such as macular degeneration and Alzheimer's disease.