Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart

Abstract

Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity , the functions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, α and α We observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in α than α mutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.

Transgenic Zebrafish Models Reveal Distinct Molecular Mechanisms for Cataract-linked αA-Crystallin Mutants

“…abnormalities that were readily visible starting at 3dpf ( Figure 1B), without affecting the overall morphology of the embryos ( Figure 1A). The nature of these defects was similar to those previously described for α-crystallin deficient [32,37] or γD-crystallin mutant transgenic lines [33]. Defective lenses exhibited phenotypic features that were either spherical, shiny crystal-like droplets spread sporadically across the lens (minor defects), or frequent droplets covering a large fraction of the lens as well as occasional large irregular protuberances located in the center of the lens (major defects), all of which could lead to opacity and changes in light scattering.…”

“…Embryos were staged according to their ages (dpf; days post-fertilization). The following mutant and transgenic fish lines were used: cryaa vu532 [32], cryaba vu612 ; cryabb vu613 [37], Tg(cryaa:Rno.Cryaa_R49C,myl7:Cerulean) vu615Tg , Tg(cryaa:Rno.Cryaa_R116C,myl7:Cerulean) vu616Tg , Tg(cryaa:Mmu.Cryab_R120G,myl7:Cerulean) vu617Tg (this study), Tg(cryaa:Gal4vp16) mw46Tg ; Tg(UAS:GFP) kca33Tg [42]. All animal procedures were verified and approved by the Vanderbilt University Institutional Animal Care and Use Committee.…”

“…As expected αB-R120G induced lens defects with a moderate penetrance but the severity of lens phenotypes was mostly of the minor class ( Figure 5A). Reducing endogenous αB-crystallin levels by crossing αB-R120G into zebrafish αB-crystallin null mutants (cryaba -/-; cryabb -/-) [37] led to significant enhancement of lens defects in both penetrance and severity ( Figure 5A).…”

“…The structure and function of the zebrafish adult lens parallel those of the mature human lens with respect to symmetry, refraction, transparency and optical function [29,35,36]. Previous work from our laboratory demonstrated that both a-crystallins are required for lens development [32,37]. Furthermore, we established the utility of the zebrafish as a model for lens proteostasis by demonstrating a correlation between the level of gross lens defects and the expression of progressively destabilized mutants of human gD-crystallin [33].…”

Proteasome subunitPSMC3variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress

“…(Cvekl and Zhang, 2017) Pathogenic variations or knockdown of genes implicated in human cataract and/or deafness in zebrafish have been shown to recapitulate similar phenotypes. (Gao et al, 2017;Mishra et al, 2018;Yousaf et al, 2018;Takamiya et al, 2016;Busch-Nentwich, 2004) Using two strategies (morpholino and CRISPR/Cas9) the fish developed a cataract and ear phenotype that could be rescued. We did not observe other obvious anomalies.…”