Compromised Phagosome Maturation Underlies RPE Pathology in Cell Culture and Whole Animal Models of Smith-Lemli-Opitz Syndrome

Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed. © 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group

Retinal Degeneration Caused by Rod-Specific Dhdds Ablation Occurs without Concomitant Inhibition of ProteinN-Glycosylation

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes the committed step indolichol synthesis. Recessive mutations inDHDDScause retinitis pigmentosa(RP59), resulting in blindness. We hypothesized that rod photoreceptor-specificablation ofDhddswould cause retinal degeneration due to diminished dolichol-dependent proteinN-glycosylation.Dhddsflx/flxmice were crossed with rod-spe-cific Cre recombinase-expressing (Rho-iCre75) mice to generate rod-specificDhddsknockout mice (Dhddsflx/flxiCre+).In vivomorphological and electrophys-iological evaluation ofDhddsflx/flxiCre+retinas revealed mild retinal dysfunctionat postnatal (PN) 4 weeks, compared with age-matched controls; however, rapidphotoreceptor degeneration ensued, resulting in almost complete loss of rodsand cones by PN 6 weeks. Retina dolichol levels were markedly decreased byPN 4 weeks inDhddsflx/flxiCre+mice, relative to controls; despite this,N-glycosyl-ation of retinal proteins, including opsin (the dominant rod-specific glycoprotein),persisted inDhddsflx/flxiCre+mice. These findings challenge the conventionalmechanistic view of RP59 as a congenital disorder of glycosylation

Bottlenecks in the Investigation of Retinal Sterol Homeostasis

Sterol homeostasis in mammalian cells and tissues involves balancing three fundamental processes: de novo sterol biosynthesis; sterol import (e.g., from blood-borne lipoproteins); and sterol export. In complex tissues, composed of multiple different cell types (such as the retina), import and export also may involve intratissue, intercellular sterol exchange. Disruption of any of these processes can result in pathologies that impact the normal structure and function of the retina. Here, we provide a brief overview of what is known currently about sterol homeostasis in the vertebrate retina and offer a proposed path for future experimental work to further our understanding of these processes, with relevance to the development of novel therapeutic interventions for human diseases involving defective sterol homeostasis

Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59

Dehydrodolichyl diphosphate synthase (DHDDS) is required for protein N-glycosylation in eukaryotic cells. A K42E point mutation in the DHDDS gene causes an autosomal recessive form of retinitis pigmentosa (RP59), which has been classified as a congenital disease of glycosylation (CDG). We generated K42E Dhdds knock-in mice as a potential model for RP59. Mice heterozygous for the Dhdds K42E mutation were generated using CRISPR/Cas9 technology and crossed to generate DhddsK42E/K42E homozygous mice. Spectral domain-optical coherence tomography (SD-OCT) was performed to assess retinal structure, relative to age-matched wild type (WT) controls. Immunohistochemistry against glial fibrillary acidic protein (GFAP) and opsin (1D4 epitope) was performed on retinal frozen sections to monitor gliosis and opsin localization, respectively, while lectin cytochemistry, plus and minus PNGase-F treatment, was performed to assess protein glycosylation status. Retinas of DhddsK42E/K42E mice exhibited grossly normal histological organization from 1 to 12 months of age. Anti-GFAP immunoreactivity was markedly increased in DhddsK42E/K42E mice, relative to controls. However, opsin immunolocalization, ConA labeling and PNGase-F sensitivity were comparable in mutant and control retinas. Hence, retinas of DhddsK42E/K42E mice exhibited no overt signs of degeneration, yet were markedly gliotic, but without evidence of compromised protein N-glycosylation. These results challenge the notion of RP59 as a DHDDS loss-of-function CDG and highlight the need to investigate unexplored RP59 disease mechanisms

Vertebrate Animal Models of RP59: Current Status and Future Prospects

Retinitis pigmentosa-59 (RP59) is a rare, recessive form of RP, caused by mutations in the gene encoding DHDDS (dehydrodolichyl diphosphate synthase). DHDDS forms a heterotetrameric complex with Nogo-B receptor (NgBR; gene NUS1) to form a cis-prenyltransferase (CPT) enzyme complex, which is required for the synthesis of dolichol, which in turn is required for protein N-glycosylation as well as other glycosylation reactions in eukaryotic cells. Herein, we review the published phenotypic characteristics of RP59 models extant, with an emphasis on their ocular phenotypes, based primarily upon knock-in of known RP59-associated DHDDS mutations as well as cell type- and tissue-specific knockout of DHDDS alleles in mice. We also briefly review findings in RP59 patients with retinal disease and other patients with DHDDS mutations causing epilepsy and other neurologic disease. We discuss these findings in the context of addressing “knowledge gaps” in our current understanding of the underlying pathobiology mechanism of RP59, as well as their potential utility for developing therapeutic interventions to block the onset or to dampen the severity or progression of RP59

Prevention of Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome

Abstract Smith-Lemli-Opitz Syndrome (SLOS) is a recessive human disease caused by defective cholesterol (CHOL) synthesis at the level of DHCR7 (7-dehydrocholesterol reductase), which normally catalyzes the conversion of 7-dehydrocholesterol (7DHC) to CHOL. Formation and abnormal accumulation of 7DHC and 7DHC-derived oxysterols occur in SLOS patients and in rats treated with the DHCR7 inhibitor AY9944. The rat SLOS model exhibits progressive and irreversible retinal dysfunction and degeneration, which is only partially ameliorated by dietary CHOL supplementation. We hypothesized that 7DHC-derived oxysterols are causally involved in this retinal degeneration, and that blocking or reducing their formation should minimize the phenotype. Here, using the SLOS rat model, we demonstrate that combined dietary supplementation with CHOL plus antioxidants (vitamins E and C, plus sodium selenite) provides better outcomes than dietary CHOL supplementation alone with regard to preservation of retinal structure and function and lowering 7DHC-derived oxysterol formation. These proof-of-principle findings provide a translational, pre-clinical framework for designing clinical trials using CHOL-antioxidant combination therapy as an improved therapeutic intervention over the current standard of care for the treatment of SLOS

Selective Ablation of Dehydrodolichyl Diphosphate Synthase in Murine Retinal Pigment Epithelium (RPE) Causes RPE Atrophy and Retinal Degeneration

Patients with certain defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene (RP59; OMIM #613861) exhibit classic symptoms of retinitis pigmentosa, as well as macular changes, suggestive of retinal pigment epithelium (RPE) involvement. The DHDDS enzyme is ubiquitously required for several pathways of protein glycosylation. We wish to understand the basis for selective ocular pathology associated with certain DHDDS mutations and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. To circumvent embryonic lethality associated with Dhdds knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). We used targeted Cre expression to study the importance of the enzyme in the RPE. Structural alterations of the RPE and retina including reduction in outer retinal thickness, cell layer disruption, and increased RPE hyper-reflectivity were apparent at one postnatal month. At three months, RPE and photoreceptor disruption was observed non-uniformly across the retina as well as RPE transmigration into the photoreceptor layer, external limiting membrane descent towards the RPE, and patchy loss of photoreceptors. Functional loss measured by electroretinography was consistent with structural loss showing scotopic a- and b-wave reductions of 83% and 77%, respectively, at three months. These results indicate that RPE dysfunction contributes to DHDDS mutation-mediated pathology and suggests a more complicated disease mechanism than simply disruption of glycosylation

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/– mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/– mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/– mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knockin mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for cotranslational expression of CreERT2. Cre+/– mice were able to recombine a stringent Cre reporter allele with more than 99% efficiency and absolute RPE specificity upon tamoxifen induction at both postnatal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/– mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knockin allele, visual cycle function was normal in Cre+/– mice. These data indicate that Rpe65CreERT2 mice are well suited for studies of gene function and pathophysiology in the RPE