Mechanistically Distinct Mouse Models for <i>CRX</i>-Associated Retinopathy

<div><p>Cone-rod homeobox (CRX) protein is a “paired-like” homeodomain transcription factor that is essential for regulating rod and cone photoreceptor transcription. Mutations in human <i>CRX</i> are associated with the dominant retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congenital Amaurosis (LCA), with variable severity. Heterozygous <i>Crx Knock</i>-<i>Out</i> (<i>KO</i>) mice (“<i>+/−</i>”) have normal vision as adults and fail to model the dominant human disease. To investigate how different mutant CRX proteins produce distinct disease pathologies, we generated two <i>Crx Knock</i>-<i>IN (K-IN)</i> mouse models: <i>Crx^E168d2</i> (<i>“E168d2”</i>) and <i>Crx^R90W</i> (<i>“R90W”</i>). <i>E168d2</i> mice carry a frameshift mutation in the CRX activation domain, <i>Glu168del2</i>, which is associated with severe dominant CoRD or LCA in humans. <i>R90W</i> mice carry a substitution mutation in the CRX homeodomain, <i>Arg90Trp</i>, which is associated with dominant mild late-onset CoRD and recessive LCA. As seen in human patients, heterozygous <i>E168d2</i> (“<i>E168d2/+</i>”) but not <i>R90W</i> (<i>“R90W/+”</i>) mice show severely impaired retinal function, while mice homozygous for either mutation are blind and undergo rapid photoreceptor degeneration. <i>E168d2/+</i> mice also display abnormal rod/cone morphology, greater impairment of CRX target gene expression than <i>R90W/+</i> or <i>+/−</i> mice, and undergo progressive photoreceptor degeneration. Surprisingly, <i>E168d2/+</i> mice express more mutant CRX protein than wild-type CRX. <i>E168d2neo/+</i>, a subline of <i>E168d2</i> with reduced mutant allele expression, displays a much milder retinal phenotype, demonstrating the impact of <i>Crx</i> expression level on disease severity. Both CRX^[E168d2] and CRX^[R90W] proteins fail to activate transcription <i>in vitro</i>, but CRX^[E168d2] interferes more strongly with the function of wild type (WT) CRX, supporting an antimorphic mechanism. <i>E168d2</i> and <i>R90W</i> are mechanistically distinct mouse models for <i>CRX</i>-associated disease that will allow the elucidation of molecular mechanisms and testing of novel therapeutic approaches for different forms of <i>CRX-</i>associated disease.</p>