Mechanisms of blindness: animal models provide insight into distinct CRX-associated retinopathies

Abstract

Background: The homeodomain transcription factor CRX is a crucial regulator of mammalian photoreceptor gene expression. Mutations in the human CRX gene are associated with dominant inherited retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD), and Leber Congenital Amaurosis (LCA), of varying severity. In vitro and in vivo assessment of mutant CRX proteins have revealed pathogenic mechanisms for several mutations, but no comprehensive mutation-disease correlation has yet been reported.

Results: Here we describe four different classes of disease-causing CRX mutations, characterized by mutation type, pathogenetic mechanism, and the molecular activity of the mutant protein: (1) hypomorphic missense mutations with reduced DNA binding, (2) antimorphic missense mutations with variable DNA binding, (3) antimorphic frameshift/nonsense mutations with intact DNA binding, and (4) antimorphic frameshift mutations with reduced DNA binding. Mammalian models representing three of these classes have been characterized.

Conclusions: Models carrying Class I mutations display a mild dominant retinal phenotype and recessive LCA, while models carrying Class III and IV mutations display characteristically distinct dominant LCA phenotypes. These animal models also reveal unexpected pathogenic mechanisms underlying CRX-associated retinopathies. The complexity of genotype-phenotype correlation for CRX-associated diseases highlights the value of developing comprehensive "true-to-disease" animal models for understanding pathologic mechanisms and testing novel therapeutic approaches.

Keywords: antimorph; disease models; dominant-negative; gene expression; human genetics; hypomorph; neural development; neuronal degeneration; photoreceptors; retina; transcription factors.

Figure 1. Schematic of CRX protein showing locations of co-segregating human mutations and mutations in associated animal models

The CRX protein is represented by a rectangle, conserved domains are shaded. The locations of human mutations that co-segregate with disease are shown above the schematic. Based on mutation type and molecular function, mutations fall into four classes: I) hypomorphic missense mutations with reduced DNA binding (gold text), II) antimorphic missense mutations with variable DNA binding (purple text), III) antimorphic frameshift/nonsense mutations with intact DNA binding (red text) and IV) antimorphic frameshift mutations with reduced DNA binding (blue text). While both classes of frameshift mutations are predicted to have antimorphic activity, they act through different mechanisms. Currently, there are five mouse models and one cat model carrying mutations in Crx (bottom): 1) the Crx KO mouse (grey text) does not express CRX protein; 2) the R90W K-IN mouse (gold text) carries a Class I mutation; 3) the E168d2 K-IN mouse and Rdy cat (red text) both carry Class III mutations; 4) the Rip mouse (blue text) carries a Class IV mutation; 5) the tvrm65 mouse (grey text) carries a nonsense mutation that does not match any reported disease-causing human mutation classes.

Figure 2. Schematic showing the position of out-of-frame stop codons in human Crx mRNA

Human CRX coding sequence (hCRX CDS) and messenger RNA (hCRX mRNA) are shown. Scale below indicates the base pair position in hCRX CDS beginning at the start codon. All out-of-frame stop codons in hCRX mRNA coding region and its 3’UTR up to position 1109 are shown above the schematic. Nucleotide position and stop codon frame are shown in colored text (matched to Table 2) for all predicted termination sites for reported human mutations, non-utilized out-of-frame stop codons are shown in grey text. The positions of all out-of-frame stop codons are indicated in brackets. [-1] and [-2] indicate whether the stop codon is located 1 or 2 bases 5’ of the normal reading frame, respectively.

Figure 3. Expression of CRX protein in mutant mouse retinas

A) Immunoblot analyses showing the expression of CRX protein in P10 E168d2, E168d2neo, R90W and R90Wneo mouse retinas using the rabbit polyclonal CRX 119b-1 antibody and mouse monoclonal anti-β-ACTIN antibody (α-BACT, Sigma-Aldrich) (Tran et al. 2014). Lanes are numbered below for reference. R90W mice express a normal length ~37kDA mutant CRX protein (lanes 6 & 8), E168d2 mice overexpress a truncated ~27kDA mutant CRX protein (lanes 2 & 4) and the intronic neo cassette results in reduced expression of both the R90W (lanes 7 & 9) and E168d2 (lanes 3 &5) proteins. B) Immunoblot analyses showing the expression of CRX protein in 1mo Rip mouse retinas using rabbit polyclonal CRX H-120 antibody (Santa Cruz) and mouse monoclonal anti-β-ACTIN antibody (Actin, Millipore) (Roger et al. 2014). Rip mice express an elongated ~44kDA mutant CRX protein.

Figure 4. Proposed pathogenic mechanisms of mutant CRX proteins in mammalian models

(Adapted from: Tran et al. 2014) A) In WT mice, CRX coordinately regulates transcription by interacting with target DNA and co-factors including the rod-specific transcription factor NRL. B) In E168d2/+ mice and possibly Rdy/+ cats, a Class III mutant CRX is overexpressed and directly competes with WT CRX, producing an LCA phenotype. C) In R90W/+ mice, a Class I mutant CRX is expressed and largely does not interfere with WT CRX producing only a mild late-onset CoRD phenotype. D) In Rip/+ mice, a Class IV mutant CRX is expressed and disrupts the photoreceptor transcription factor network including the loss of NRL expression, producing an LCA phenotype with distinct pathology from E168d2/+ and Rdy/+. E-H) Homozygous Crx KO, E168d2, R90W and Rip mice all produce a LCA phenotype and are completely blind from birth. CRX target gene expression is severely impaired in all homozygous mice with E168d2/d2 and Rip/Rip mice having the strongest changes.