The Lrat−/− Rat: CRISPR/Cas9 Construction and Phenotyping of a New Animal Model for RP

Céline Koster, Koen T. van den Hurk, Colby F. Lewallen, Mays Talib, Jacoline B. ten Brink, Camiel J. F. Boon, Arthur A. Bergen | International Journal of Molecular Sciences | July 5, 2021 | Vol. 22, Issue 13 | 7234 | doi: 10.3390/ijms22137234




Abstract

Purpose: We developed and phenotyped a pigmented knockout rat model for lecithin retinol acyltransferase (LRAT) using CRISPR/Cas9. The introduced mutation (c.12delA) is based on a patient group harboring a homologous homozygous frameshift mutation in the LRAT gene (c.12delC), causing a dysfunctional visual (retinoid) cycle. Methods: The introduced mutation was confirmed by DNA and RNA sequencing. The expression of Lrat was determined on both the RNA and protein level in wildtype and knockout animals using RT-PCR and immunohistochemistry. The retinal structure and function, as well as the visual behavior of the Lrat−/− and control rats, were characterized using scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), electroretinography (ERG) and vision-based behavioral assays.


Results: Wildtype animals had high Lrat mRNA expression in multiple tissues, including the eye and liver. In contrast, hardly any expression was detected in Lrat−/− animals. LRAT protein was abundantly present in wildtype animals and absent in Lrat−/− animals. Lrat−/− animals showed progressively reduced ERG potentials compared to wildtype controls from two weeks of age onwards. Vison-based behavioral assays confirmed reduced vision. Structural abnormalities, such as overall retinal thinning, were observed in Lrat−/− animals. The retinal thickness in knockout rats was decreased to roughly 80% by four months of age. No functional or structural differences were observed between wildtype and heterozygote animals. Conclusions: Our Lrat−/− rat is a new animal model for retinal dystrophy, especially for the LRAT-subtype of early-onset retinal dystrophies. This model has advantages over the existing mouse models and the RCS rat strain and can be used for translational studies of retinal dystrophies.


Introduction

Retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and retinitis punctata albescens (RPA) are severe early-onset retinal dystrophies that cause visual impairment, nystagmus, progressive nyctalopia, and finally, blindness. This heterogeneous retinal dystrophy disease group is characterized by damage to the retinal pigment epithelium (RPE)–photoreceptor (PR) complex. This results usually in progressive dysfunction of the rod photoreceptor cells, often followed by progressive cone degeneration. RP, LCA, and RPA are caused by mutations in virtually all genes encoding proteins acting in the retinoid cycle (1,2,3,4). Indeed, for normal vision, a functionally valid retinoid cycle is essential: In the healthy situation, vitamin A (retinol) is the primary substrate for several functional retinoids’ biosynthesis in the retinoid cycle. Then, the vitamin A-derivatives are shuttled from the RPE to the PRs. There, opsins are light-activated and the visual pigments transform the light energy in a cellular signal, initiating the visual cascade and resulting in a physiological response in the PR cell. After light activation, the cycle regenerates the visual pigments that are used after light activation of rhodopsin (see Figure 1). Upon photoactivation, a configurational change of the visual pigment 11-cis-retinal to all-trans-retinal is induced in the PR cells’ outer segments. Subsequently, all-trans-retinal is reduced to all-trans-retinol and diffuses from the PRs back to to the RPE cells. In the RPE, all-trans-retinol is esterified to all-trans-retinyl-ester by the enzyme lecithin:retinol acetyltransferase (LRAT), after which all-trans-retinyl-ester is subsequently the substrate for the enzyme retinal pigment epithelium-specific protein 65 kDa (RPE65). RPE65 converts all-trans-retinyl-ester to 11-cis-retinol, after which 11-cis-retinol is oxidized by retinol dehydrogenase (RDH) enzymes to 11-cis-retinal. Finally, to complete the cycle, 11-cis-retinal is shuttled back to the PRs, where it can be used for a new round of phototransduction.


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