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.

Inherited Retinal Degeneration Caused by Dehydrodolichyl Diphosphate Synthase Mutation-Effect of an ALG6 Modifier Variant.

Modern advances in disease genetics have uncovered numerous modifier genes that play a role in the severity of disease expression. One such class of genetic conditions is known as inherited retinal degenerations (IRDs), a collection of retinal degenerative disorders caused by mutations in over 300 genes. A single missense mutation (K42E) in the gene encoding the enzyme dehydrodolichyl diphosphate synthase (DHDDS), which is required for protein N-glycosylation in all cells and tissues, causes DHDDS-IRD (retinitis pigmentosa type 59 (RP59; OMIM #613861)). Apart from a retinal phenotype, however, DHDDS-IRD is surprisingly non-syndromic (i.e., without any systemic manifestations). To explore disease pathology, we selected five glycosylation-related genes for analysis that are suggested to have disease modifier variants. These genes encode glycosyltransferases (ALG6, ALG8), an ER resident protein (DDOST), a high-mannose oligosaccharyl transferase (MPDU1), and a protein N-glycosylation regulatory protein (TNKS). DNA samples from 11 confirmed DHDDS (K42E)-IRD patients were sequenced at the site of each candidate genetic modifier. Quantitative measures of retinal structure and function were performed across five decades of life by evaluating foveal photoreceptor thickness, visual acuity, foveal sensitivity, macular and extramacular rod sensitivity, and kinetic visual field extent. The ALG6 variant, (F304S), was correlated with greater macular cone disease severity and less peripheral rod disease severity. Thus, modifier gene polymorphisms may account for a significant portion of phenotypic variation observed in human genetic disease. However, the consequences of the polymorphisms may be counterintuitively complex in terms of rod and cone populations affected in different regions of the retina.

A simple, rapid fluorescent reporter-based method for detection of ectopic cre recombinase expression in presumed retinal cell type-targeted mouse lines.

Although cell type-specific Cre recombinase-expressing mouse lines are commonly used to generate conditional knockout of genes of interest, germline recombination and ectopic "leakiness" in Cre recombinase expression in non-specific cell types has been observed in several neuronal and glial-specific Cre lines. This often leads to inadvertent loss of conditional mouse lines, requiring rederivation. It is therefore imperative to be able to monitor and validate cell type-specific Cre recombinase-mediated gene editing. Herein, we describe a simple, inexpensive, rapid ZsGreen fluor-reporter-based strategy for genotype-free identification of ectopic leakiness using a custom-designed, 3-D blue LED light box. We assessed cell type-specific expression in several allegedly specific Cre recombinase mouse lines commonly used in vision research: retinal pigment epithelium (RPE)-specific (VMD2 (Best1) Cre, RPE65 Cre); astrocyte-specific (GFAP Cre); as well as photoreceptor-bipolar progenitor cell-specific (CRX Cre). Our standardized workflow allows facile, rapid identification of ectopic and non-specific Cre recombinase expression in any presume specific Cre mouse line, without the need for genotyping and without causing animal distress.

Cells Special Issue: "The Molecular and Cellular Basis of Retinal Diseases".

The recent success in the treatment of hereditary retinal disease caused by defects in the RPE65 gene and the FDA approval of this treatment has established the importance of the study of animal models and the translational impact of these research findings [...].

A Dhdds K42E knock-in RP59 mouse model shows inner retina pathology and defective synaptic transmission.

Retinitis pigmentosa (RP) defines a group of hereditary progressive rod-cone degenerations that exhibit a common phenotype caused by variants in over 70 genes. While most variants in the dehydrodolichyl diphosphate synthase (DHDDS) gene result in syndromic abnormalities, some variants cause non-syndromic RP (RP59). DHDDS encodes one subunit of the enzyme cis-prenyltransferase (CPT), which is required for the synthesis of dolichol (Dol), that is a necessary protein glycosylation cofactor. We previously reported the creation and initial characterization of a knock-in (KI) mouse model harboring the most prevalent RP59-associated DHDDS variant (K42E) to understand how defects in DHDDS lead to retina-specific pathology. This model exhibited no profound retinal degeneration, nor protein N-glycosylation defects. Here, we report that the Dol isoprenylogue species in retina, liver, and brain of the K42E mouse model are statistically shorter than in the corresponding tissues of age-matched controls, as reported in blood and urine of RP59 patients. Retinal transcriptome analysis demonstrated elevation of many genes encoding proteins involved in synaptogenesis and synaptic function. Quantitative retinal cell layer thickness measurements demonstrated a significant reduction in the inner nuclear layer (INL) and total retinal thickness (TRT) beginning at postnatal (PN) ∼2 months, progressively increasing to PN 18-mo. Histological analysis revealed cell loss in the INL, outer plexiform layer (OPL) disruption, and ectopic localization of outer nuclear layer (ONL) nuclei into the OPL of K42E mutant retinas, relative to controls. Electroretinograms (ERGs) of mutant mice exhibited reduced b-wave amplitudes beginning at PN 1-mo, progressively declining through PN 18-mo, without appreciable a-wave attenuation, relative to controls. Our results suggest that the underlying cause of DHDDS K42E variant driven RP59 retinal pathology is defective synaptic transmission from outer to inner retina.

Perspectives on Retinal Dolichol Metabolism, and Visual Deficits in Dolichol Metabolism-Associated Inherited Disorders.

De novo synthesis of dolichol (Dol) and dolichyl phosphate (Dol-P) is essential for protein glycosylation. Herein, we provide a brief overview of Dol and Dol-P synthesis and the maintenance of their cellular content. Retinal Dol metabolism and the requirement of Dol-linked oligosaccharide synthesis in the neural retina also are discussed. There are recently discovered and an emerging class of rare congenital disorders that affect Dol metabolism, involving the genes DHDDS, NUS1, SRD5A3, and DOLK. Further understanding of these congenital disorders is evolving, based upon studies utilizing yeast and murine models, as well as clinical reports of these rare disorders. We summarize the known visual deficits associated with Dol metabolism disorders, and identify the need for generation and characterization of suitable animal models of these disorders to elucidate the underlying molecular and cellular mechanisms of the associated retinopathies.

MAP4Ks inhibition promotes retinal neuron regeneration from Müller glia in adult mice.

Mammalian Müller glia (MG) possess limited regenerative capacities. However, the intrinsic capacity of mammalian MG to transdifferentiate to generate mature neurons without transgenic manipulations remains speculative. Here we show that MAP4K4, MAP4K6 and MAP4K7, which are conserved Misshapen subfamily of ste20 kinases homologs, repress YAP activity in mammalian MG and therefore restrict their ability to be reprogrammed. However, by treating with a small molecule inhibitor of MAP4K4/6/7, mouse MG regain their ability to proliferate and enter into a retinal progenitor cell (RPC)-like state after NMDA-induced retinal damage; such plasticity was lost in YAP knockout MG. Moreover, spontaneous trans-differentiation of MG into retinal neurons expressing both amacrine and retinal ganglion cell (RGC) markers occurs after inhibitor withdrawal. Taken together, these findings suggest that MAP4Ks block the reprogramming capacity of MG in a YAP-dependent manner in adult mammals, which provides a novel avenue for the pharmaceutical induction of retinal regeneration in vivo.

Retinal gliosis and phenotypic diversity of intermediate filament induction and remodeling upon acoustic blast overpressure (ABO) exposure to the rat eye.

Traumatic brain injury (TBI) caused by acoustic blast overpressure (ABO) is frequently associated with chronic visual deficits in military personnel and civilians. In this study, we characterized retinal gliotic response in adult male rats following a single ABO exposure directed to one side of the head. Expression of gliosis markers and intermediate filaments was assessed at 48 h and 1 wk post-ABO exposure, in comparison to age-matched non-exposed control retina. In response to a single ABO exposure, type III IF, glial fibrillary acidic protein (GFAP) was variably induced in a subpopulation of retinal Müller glia in ipsilateral eyes. ABO-exposed eyes exhibited radial Müller glial GFAP filament extension through the inner plexiform layer (IPL) and the inner nuclear layer (INL) through the retina in both the nasal quadrant and juxta-optic nerve head (jONH) eye regions at 1 wk post-ABO. We observed an ∼6-fold increase (p ≤ 0.05) in radial glial GFAP immunolabeling in the IPL in both eye regions, in comparison to regionally matched controls. Similarly, GFAP extension through the INL into the outer retina was elevated ∼3-fold, p ≤ 0.05 in the nasal retina, but exhibited wider variability in the jONH retina. In contrast, constitutive type III IF vimentin exhibited greater remodeling in retinal Müller glia through the jONH retina compared to the nasal retina in response to ABO. We observed areas of lateral vimentin remodeling through the Müller glial end-feet, and greater mid-outer retinal radial vimentin IF extension in a subpopulation of glia at 1 wk post-ABO. We also observed a significant increase in total retinal levels of the type III IF desmin in ABO-exposed retina vs. controls (∼1.6-fold, p ≤ 0.01). In addition, ABO-exposure elicited varied glial induction of developmentally regulated type VI family IFs (nestin and synemin) in subpopulations of Müller cells at 48 h and 1 wk post-ABO. We demonstrate that multiple glial phenotypes emerge in the rat retina following a single ABO exposure, rather than a global homogeneous retinal glial response, involving less well characterized IF protein forms which warrant further investigation in the context of ABO-induced retinal gliosis.

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.

A Dpagt1 Missense Variant Causes Degenerative Retinopathy without Myasthenic Syndrome in Mice.

Congenital disorders of glycosylation (CDG) are a heterogenous group of primarily autosomal recessive mendelian diseases caused by disruptions in the synthesis of lipid-linked oligosaccharides and their transfer to proteins. CDGs usually affect multiple organ systems and vary in presentation, even within families. There is currently no cure, and treatment is aimed at ameliorating symptoms and improving quality of life. Here, we describe a chemically induced mouse mutant, tvrm76, with early-onset photoreceptor degeneration. The recessive mutation was mapped to Chromosome 9 and associated with a missense mutation in the Dpagt1 gene encoding UDP-N-acetyl-D-glucosamine:dolichyl-phosphate N-acetyl-D-glucosaminephosphotransferase (EC 2.7.8.15). The mutation is predicted to cause a substitution of aspartic acid with glycine at residue 166 of DPAGT1. This represents the first viable animal model of a Dpagt1 mutation and a novel phenotype for a CDG. The increased expression of Ddit3, and elevated levels of HSPA5 (BiP) suggest the presence of early-onset endoplasmic reticulum (ER) stress. These changes were associated with the induction of photoreceptor apoptosis in tvrm76 retinas. Mutations in human DPAGT1 cause myasthenic syndrome-13 and severe forms of a congenital disorder of glycosylation Type Ij. In contrast, Dpagt1tvrm76 homozygous mice present with congenital photoreceptor degeneration without overt muscle or muscular junction involvement. Our results suggest the possibility of DPAGT1 mutations in human patients that present primarily with retinitis pigmentosa, with little or no muscle disease. Variants in DPAGT1 should be considered when evaluating cases of non-syndromic retinal degeneration.

Monitoring basal autophagy in the retina utilizing CAG-mRFP-EGFP-MAP1LC3B reporter mouse: technical and biological considerations.

We describe the utility of a tandem-tagged autophagy reporter mouse model (CAG-RFP-EGFP-MAP1LC3B) in investigating basal macroautophagic/autophagic flux in the neural retina. Western blot, in situ hybridization, immunohistochemistry, and confocal microscopy showed that CAG promoter-driven expression of RFP-EGFP-MAP1LC3B increased "cytosolic" RFP-EGFP-LC3B-I levels, whereas RFP-EGFP-LC3B-II decorates true phagosomes. We verified that the electroretinographic (ERG) responses of tandem-tagged LC3B mice were comparable to those of age-matched controls. Optimized microscope settings detected lipofuscin autofluorescence in retinas of abca4-/- mice. The majority of retinal phagosomes in the reporter mice exhibited only RFP (not EGFP) fluorescence, suggesting rapid maturation of phagosomes. Only ~1.5% of the total phagosome population was EGFP-labeled; RFP-labeled (mature) phagosomes colocalized with lysosomal markers LAMP2 and CTSD. In the outer retina, phagosome sizes were as follows (in µm2, ave ± SEM): RPE, 0.309 ± 0.015; photoreceptor inner segment-myoid, 0.544 ± 0.031; and outer nuclear layer, 0.429 ± 0.011. Detection of RPE phagosomes by fluorescence microscopy is challenging, due to the presence of melanin. Increased lipofuscin autofluorescence, such as observed in the abca4-/- mouse model of Stargardt disease, is a strong confounding factor when attempting to study autophagy in the RPE. In addition to RPE and photoreceptor cells, phagosomes were detected in inner retinal cell types, microglia, astrocytes, and endothelial cells. We conclude that the tandem-tagged LC3B mouse model serves as a useful system for studying autophagy in the retina. This utility, however, is dependent upon several technical and biological factors, including microscope settings, transgene expression, choice of fluorophores, and lipofuscin autofluorescence.Abbreviations: ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATG: autophagy related; CTSD: cathepsin D; DAPI: (4',6-diamido-2-phenylindole); DIC: differential interference contrast; EGFP: enhanced green fluorescent protein; ELM: external limiting membrane; ERG: electroretinography; GCL: ganglion cell layer; GLUL: glutamine-ammonia ligase (glutamine synthetase); INL: inner nuclear layer; IS-E/M: inner segment - ellipsoid/myoid; ISH: in situ hybridization; LAMP2: lysosomal-associated membrane protein 2; L.I.: laser Intensity; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; O.C.T.: optimal cutting temperature; OS: outer segment; ONL: outer nuclear layer; PE: phosphatidylethanolamine; RFP: red fluorescent protein; R.O.I.: region of interest; RPE: retinal pigment epithelium.

Dependence of visual and cognitive outcomes on animal holder configuration in a rodent model of blast overpressure exposure.

Blast-induced traumatic brain injury is the signature injury of modern military conflicts. To more fully understand the effects of blast exposure, we placed rats in different holder configurations, exposed them to blast overpressure, and assessed the degree of eye and brain injury. Anesthetized Long-Evans rats received blast exposures directed at the head (63 kPa, 195 dB-SPL) in either an "open holder" (head and neck exposed; n = 7), or an "enclosed holder" (window for blast exposure to eye; n = 15) and were compared to non-blast exposed (control) rats (n = 22). Outcomes included optomotor response (OMR), electroretinography (ERG), and spectral domain optical coherence tomography (SD-OCT) at 2, 4, and 6 months post-blast, and cognitive function (Y-maze) at 3 months. Spatial frequency and contrast sensitivity were reduced in ipsilateral blast-exposed eyes in both holders (p < 0.01), while contralateral eyes showed greater deficits with the enclosed holder (p < 0.05). Thinner retinas (p < 0.001) and reduced ERG a- and b- wave amplitudes (p < 0.05) were observed for both ipsilateral and contralateral eyes with the enclosed, but not the open, holder. Rats in the open holder showed cognitive deficits compared to rats in the enclosed holder (p < 0.05). Overall, the animal holder configuration used in blast exposure studies can significantly affect outcomes. Enclosed holders may cause secondary damage to the contralateral eye by concussive injury or blast wave reflection off the holder wall. Open holders may damage the brain via rapid head movement (contrecoup injury). These results highlight additional factors to be considered when evaluating patients with blast exposure or developing models of blast injury.

Reducing acetylated tau is neuroprotective in brain injury.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.

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.

Cholesterol homeostasis in the vertebrate retina: biology and pathobiology.

Cholesterol is a quantitatively and biologically significant constituent of all mammalian cell membrane, including those that comprise the retina. Retinal cholesterol homeostasis entails the interplay between de novo synthesis, uptake, intraretinal sterol transport, metabolism, and efflux. Defects in these complex processes are associated with several congenital and age-related disorders of the visual system. Herein, we provide an overview of the following topics: (a) cholesterol synthesis in the neural retina; (b) lipoprotein uptake and intraretinal sterol transport in the neural retina and the retinal pigment epithelium (RPE); (c) cholesterol efflux from the neural retina and the RPE; and (d) biology and pathobiology of defects in sterol synthesis and sterol oxidation in the neural retina and the RPE. We focus, in particular, on studies involving animal models of monogenic disorders pertinent to the above topics, as well as in vitro models using biochemical, metabolic, and omic approaches. We also identify current knowledge gaps and opportunities in the field that beg further research in this topic area.

Transcriptomic Changes Associated with Loss of Cell Viability Induced by Oxysterol Treatment of a Retinal Photoreceptor-Derived Cell Line: An In Vitro Model of Smith-Lemli-Opitz Syndrome.

Smith-Lemli-Opitz Syndrome (SLOS) results from mutations in the gene encoding the enzyme DHCR7, which catalyzes conversion of 7-dehydrocholesterol (7DHC) to cholesterol (CHOL). Rats treated with a DHCR7 inhibitor serve as a SLOS animal model, and exhibit progressive photoreceptor-specific cell death, with accumulation of 7DHC and oxidized sterols. To understand the basis of this cell type specificity, we performed transcriptomic analyses on a photoreceptor-derived cell line (661W), treating cells with two 7DHC-derived oxysterols, which accumulate in tissues and bodily fluids of SLOS patients and in the rat SLOS model, as well as with CHOL (negative control), and evaluated differentially expressed genes (DEGs) for each treatment. Gene enrichment analysis and compilation of DEG sets indicated that endoplasmic reticulum stress, oxidative stress, DNA damage and repair, and autophagy were all highly up-regulated pathways in oxysterol-treated cells. Detailed analysis indicated that the two oxysterols exert their effects via different molecular mechanisms. Changes in expression of key genes in highlighted pathways (Hmox1, Ddit3, Trib3, and Herpud1) were validated by immunofluorescence confocal microscopy. The results extend our understanding of the pathobiology of retinal degeneration and SLOS, identifying potential new druggable targets for therapeutic intervention into these and other related orphan diseases.