Steven Nusinowitz

Retinal degeneration mutants in the mouse.

The Jackson Laboratory, having the worlds largest collection of mouse mutant stocks and genetically diverse inbred strains, is an ideal place to look for genetically determined eye variations and disorders. Through ophthalmoscopy, electroretinography and histology, we have discovered disorders affecting all aspects of the eye including the lid, cornea, iris, lens and retina, resulting in corneal disorders, cataracts, glaucoma and retinal degenerations. Mouse models of retinal degeneration have been investigated for many years in the hope of understanding the causes of photoreceptor cell death. Sixteen naturally occurring mouse mutants that manifest degeneration of photoreceptors in the retina with preservation of all other retinal cell types have been found: retinal degeneration (formerly rd, identical with rodless retina, r, now Pde6b(rd1)); Purkinje cell degeneration (pcd); nervous (nr); retinal degeneration slow (rds, now Prph(Rd2)); retinal degeneration 3 (rd3); motor neuron degeneration (mnd); retinal degeneration 4 (Rd4); retinal degeneration 5 (rd5, now tub); vitiligo (vit, now Mitf(mi-vit)); retinal degeneration 6 (rd6); retinal degeneration 7 (rd7, now Nr2e3(rd7)); neuronal ceroid lipofuscinosis (nclf); retinal degeneration 8 (rd8); retinal degeneration 9 (Rd9); retinal degeneration 10 (rd10, now Pde6b(rd10)); and cone photoreceptor function loss (cpfl1). In this report, we first review the genotypes and phenotypes of these mutants and second, list the mouse strains that carry each mutation. We will also provide detailed information about the cpfl1 mutation. The phenotypic characteristics of cpfl1 mice are similar to those observed in patients with complete achromatopsia (ACHM2, OMIM 216900) and the cpfl1 mutation is the first naturally-arising mutation in mice to cause cone-specific photoreceptor function loss. cpfl1 mice may provide a model for congenital achromatopsia in humans.

Rescue of retinal degeneration by intravitreally injected adult bone marrow–derived lineage-negative hematopoietic stem cells

Inherited retinal degenerations afflict 1 in 3,500 individuals and are a heterogeneous group of diseases that result in profound vision loss, usually the result of retinal neuronal apoptosis. Atrophic changes in the retinal vasculature are also observed in many of these degenerations. While it is thought that this atrophy is secondary to diminished metabolic demand in the face of retinal degeneration, the precise relationship between the retinal neuronal and vascular degeneration is not clear. In this study we demonstrate that whenever a fraction of mouse or human adult bone marrow-derived stem cells (lineage-negative hematopoietic stem cells [Lin- HSCs]) containing endothelial precursors stabilizes and rescues retinal blood vessels that would ordinarily completely degenerate, a dramatic neurotrophic rescue effect is also observed. Retinal nuclear layers are preserved in 2 mouse models of retinal degeneration, rd1 and rd10, and detectable, albeit severely abnormal, electroretinogram recordings are observed in rescued mice at times when they are never observed in control-treated or untreated eyes. The normal mouse retina consists predominantly of rods, but the rescued cells after treatment with Lin- HSCs are nearly all cones. Microarray analysis of rescued retinas demonstrates significant upregulation of many antiapoptotic genes, including small heat shock proteins and transcription factors. These results suggest a new paradigm for thinking about the relationship between vasculature and associated retinal neuronal tissue as well as a potential treatment for delaying the progression of vision loss associated with retinal degeneration regardless of the underlying genetic defect.

Treatment with isotretinoin inhibits lipofuscin accumulation in a mouse model of recessive Stargardt's macular degeneration

Recessive Stargardts macular degeneration is an inherited blinding disease of children caused by mutations in the ABCR gene. The primary pathologic defect in Stargardts disease is accumulation of toxic lipofuscin pigments such as N-retinylidene-N-retinylethanolamine (A2E) in cells of the retinal pigment epithelium. This accumulation appears to be responsible for the photoreceptor death and severe visual loss in Stargardts patients. Here, we tested a therapeutic strategy to inhibit lipofuscin accumulation in a mouse model of recessive Stargardts disease. Isotretinoin (Accutane) has been shown to slow the synthesis of 11-cis-retinaldehyde and regeneration of rhodopsin by inhibiting 11-cis-retinol dehydrogenase in the visual cycle. Light activation of rhodopsin results in its release of all-trans-retinaldehyde, which constitutes the first reactant in A2E biosynthesis. Accordingly, we tested the effects of isotretinoin on lipofuscin accumulation in abcr−/− knockout mice. Isotretinoin blocked the formation of A2E biochemically and the accumulation of lipofuscin pigments by electron microscopy. We observed no significant visual loss in treated abcr−/− mice by electroretinography. Isotretinoin also blocked the slower, age-dependent accumulation of lipofuscin in wild-type mice. These results corroborate the proposed mechanism of A2E biogenesis. Further, they suggest that treatment with isotretinoin may inhibit lipofuscin accumulation and thus delay the onset of visual loss in Stargardts patients. Finally, the results suggest that isotretinoin may be an effective treatment for other forms of retinal or macular degeneration associated with lipofuscin accumulation.

Haploinsufficient Bmp4 Ocular Phenotypes Include Anterior Segment Dysgenesis with Elevated Intraocular Pressure

BackgroundGlaucoma is a blinding disease usually associated with high intraocular pressure (IOP). In some families, abnormal anterior segment development contributes to glaucoma. The genes causing anterior segment dysgenesis and glaucoma in most of these families are not identified and the affected developmental processes are poorly understood. Bone morphogenetic proteins (BMPs) participate in various developmental processes. We tested the importance of Bmp4 gene dosage for ocular development and developmental glaucoma.ResultsBmp4+/- mice have anterior segment abnormalities including malformed, absent or blocked trabecular meshwork and Schlemms canal drainage structures. Mice with severe drainage structure abnormalities, over 80% or more of their angles extent, have elevated IOP. The penetrance and severity of abnormalities is strongly influenced by genetic background, being most severe on the C57BL/6J background and absent on some other backgrounds. On the C57BL/6J background there is also persistence of the hyaloid vasculature, diminished numbers of inner retinal cells, and absence of the optic nerve.ConclusionsWe demonstrate that heterozygous deficiency of BMP4 results in anterior segment dysgenesis and elevated IOP. The abnormalities are similar to those in human patients with developmental glaucoma. Thus, BMP4 is a strong candidate to contribute to Axenfeld-Rieger anomaly and other developmental conditions associated with human glaucoma. BMP4 also participates in posterior segment development and wild-type levels are usually critical for optic nerve development on the C57BL/6J background. Bmp4+/- mice are useful for studying various components of ocular development, and may allow identification of strain specific modifiers affecting a variety of ocular phenotypes.

Accelerated Accumulation of Lipofuscin Pigments in the RPE of a Mouse Model for ABCA4-Mediated Retinal Dystrophies following Vitamin A Supplementation

PURPOSE Dietary supplementation with vitamin A is sometimes prescribed as a treatment for retinitis pigmentosa, a group of inherited retinal degenerations that cause progressive blindness. Loss-of-function mutations in the ABCA4 gene are responsible for a subset of recessive retinitis pigmentosa. Other mutant alleles of ABCA4 cause the related diseases, recessive cone-rod dystrophy, and recessive Stargardt macular degeneration. Mice with a knockout mutation in the abca4 gene massively accumulate toxic lipofuscin pigments in the retinal pigment epithelium. Treatment of these mice with fenretinide, an inhibitor of vitamin A delivery to the eye, blocks formation of these toxic pigments. Here the authors tested the hypothesis that dietary supplementation with vitamin A may accelerate lipofuscin pigment formation in abca4(-/-) mice. METHODS Wild-type and abca4(-/-) mice were fed normal or vitamin A-supplemented diets. Tissues from these mice were analyzed biochemically for retinoids and lipofuscin pigments. Eyes from these mice were analyzed morphologically for lipofuscin in the retinal pigment epithelium and for degeneration of photoreceptors. Visual function in these mice was analyzed by electroretinography. RESULTS Mice that received vitamin A supplementation had dramatically higher levels of retinyl esters in the liver and retinal pigment epithelium. Lipofuscin pigments were significantly increased by biochemical and morphologic analysis in wild-type and abca4(-/-) mice fed the vitamin A-supplemented diet. Photoreceptor degeneration was observed in 11-month-old albino, but not pigmented, abca4(-/-) mice on both diets. CONCLUSIONS Vitamin A supplementation should be avoided in patients with ABCA4 mutations or other retinal or macular dystrophies associated with lipofuscin accumulation in the retinal pigment epithelium.

Blindness and auditory impairment caused by loss of the sodium bicarbonate cotransporter NBC3

Normal sensory transduction requires the efficient disposal of acid (H+) generated by neuronal and sensory receptor activity. Multiple highly sensitive transport mechanisms have evolved in prokaryotic and eukaryotic organisms to maintain acidity within strict limits. It is currently assumed that the multiplicity of these processes provides a biological robustness. Here we report that the visual and auditory systems have a specific requirement for H+ disposal mediated by the sodium bicarbonate cotransporter NBC3 (refs. 7,8). Mice lacking NBC3 develop blindness and auditory impairment because of degeneration of sensory receptors in the eye and inner ear as in Usher syndrome. Our results indicate that in certain sensory organs, in which the requirement to transduce specific environmental signals with speed, sensitivity and reliability is paramount, the choice of the H+ disposal mechanism used is limited.

Mouse model of subretinal neovascularization with choroidal anastomosis.

Purpose To characterize the phenotype and report a reliable genetic model of retinal angiogenesis and subretinal neovascularization in the mouse. Methods The mouse phenotype was characterized using ophthalmoscopy, fundus photography, fluorescein angiography, electroretinography, histology, gene sequencing, and linkage analysis. Results Scattered pink-gray retinal lesions were found on ophthalmoscopy and were confirmed to be subretinal neovascularization on fluorescein angiography. On histologic examination, outer plexiform retinal neovascularization with growth into the subretinal space was found as early as postnatal Day 15. On genetic analysis, homozygosity of the Vldlr mutation always segregated with the retinal angiogenesis, whereas normal and heterozygous mice had no neovascularization. The histologic studies 15 to 18 days consistently showed new outer plexiform neovascular vessels drawn to the subretinal space by 20 days, and by 30 to 50 days, subretinal hemorrhages and choroidal anastomoses were common. Mice by 8 months had increased vascularity of the iris and ciliary body. Conclusions The Vldlr mutation in the mouse provides a good model for retinal angiogenesis and subretinal neovascularization. Finding a strong association between retinal angiogenesis and a very low density lipid receptor mutation is new, and study of lipid receptor physiology may broaden the understanding of retinal angiogenesis.

Progressive photoreceptor degeneration, outer segment dysplasia, and rhodopsin mislocalization in mice with targeted disruption of the retinitis pigmentosa-1 (Rp1) gene

Retinitis pigmentosa (RP), a common group of human retinopathic diseases, is characterized by late-onset night blindness, loss of peripheral vision, and diminished or absent electroretinogram (ERG) responses. Mutations in the photoreceptor-specific gene RP1 account for 5–10% of cases of autosomal dominant RP. We generated a mouse model of the RP1 form of RP by targeted disruption of the mouse ortholog (Rp1) of human RP1. In Rp1−/− mice, the number of rod photoreceptors decreased progressively over a period of 1 year, whereas that of cone photoreceptors did not change for at least 10 months. Light and electron microscopic analysis revealed that outer segments of Rp1−/− rods and cones were morphologically abnormal and became progressively shorter in length. Before photoreceptor cell death, rhodopsin was mislocalized in inner segments and cell bodies of Rp1−/− rods. Rod ERG amplitudes of Rp1−/− mice were significantly smaller than those of Rp1+/+ mice over a period of 12 months, whereas those of Rp1+/− mice were intermediate. The decreases in cone ERG amplitudes were slower and less severe than those in rods. These findings demonstrate that Rp1 is required for normal morphogenesis of photoreceptor outer segments and also may play a role in rhodopsin transport to the outer segments. The phenotype of Rp1 mutant mice resembles the human RP1 disease. Thus, these mice provide a useful model for studies of RP1 function, disease pathology, and therapeutic interventions.

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternate visual cycle for regenerating opsins in daylight. Here, we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternate pathway. DES1 is expressed in retinal Müller cells where it co-immunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in rpe65 −/− mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA-interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 possessed very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity.

The Role of Interphotoreceptor Retinoid-Binding Protein on the Translocation of Visual Retinoids and Function of Cone Photoreceptors

The first event in light perception is absorption of a photon by the retinaldehyde chromophore of an opsin pigment in a rod or cone photoreceptor cell. This induces isomerization of the chromophore, rendering the bleached pigment insensitive to light. Restoration of light sensitivity requires chemical reisomerization of retinaldehyde via a multistep enzyme pathway, called the visual cycle, in cells of the retinal pigment epithelium (RPE). Interphotoreceptor retinoid-binding protein (IRBP) is present in the extracellular space between photoreceptors and the RPE. IRBP is known to bind visual retinoids. Previous studies on irbp −/− mice suggested that IRBP plays an insignificant role in opsin-pigment regeneration. However, the mice in these studies were uncontrolled for a severe mutation in the rpe65 gene. Rpe65 catalyzes the rate-limiting step in the visual cycle. Here, we examined the phenotype in irbp −/− mice homozygous for the wild-type (Leu450) rpe65 gene. We show that lack of IRBP causes delayed transfer of newly synthesized chromophore from RPE to photoreceptors. Removal of bleached chromophore from photoreceptors is also delayed in irbp −/− retinas after light exposure. It was previously shown that rods degenerate in irbp −/− mice. Here, we show that cones and rods degenerate at similar rates. However, cones are more affected functionally and show greater reductions in outer segment length than rods in irbp −/− mice. The disproportionate reductions in cone function and outer-segment length appear to result from mistrafficking of cone opsins due to impaired delivery of retinaldehyde chromophore, which functions as a chaperone for cone opsins but not rhodopsin.