Near Infrared Autofluorescence Lifetime Imaging of Human Retinal Pigment Epithelium Using Adaptive Optics Scanning Light Ophthalmoscopy.

Purpose: To demonstrate the first near-infrared adaptive optics fluorescence lifetime imaging ophthalmoscopy (NIR-AOFLIO) measurements in vivo of the human retinal pigment epithelial (RPE) cellular mosaic and to visualize lifetime changes at different retinal eccentricities. Methods: NIR reflectance and autofluorescence were captured using a custom adaptive optics scanning light ophthalmoscope in 10 healthy subjects (23-64 years old) at seven eccentricities and in two eyes with retinal abnormalities. Repeatability was assessed across two visits up to 8 weeks apart. Endogenous retinal fluorophores and hydrophobic whole retinal extracts of Abca4-/- pigmented and albino mice were imaged to probe the fluorescence origin of NIR-AOFLIO. Results: The RPE mosaic was resolved at all locations in five of seven younger subjects (<35 years old). The mean lifetime across near-peripheral regions (8° and 12°) was longer compared to near-foveal regions (0° and 2°). Repeatability across two visits showed moderate to excellent correlation (intraclass correlation: 0.88 [τm], 0.75 [τ1], 0.65 [τ2], 0.98 [a1]). The mean lifetime across drusen-containing eyes was longer than in age-matched healthy eyes. Fluorescence was observed in only the extracts from pigmented Abca4-/- mouse. Conclusions: NIR-AOFLIO was repeatable and allowed visualization of the RPE cellular mosaic. An observed signal in only the pigmented mouse extract infers the fluorescence signal originates predominantly from melanin. Variations observed across the retina with intermediate age-related macular degeneration suggest NIR-AOFLIO may act as a functional measure of a biomarker for in vivo monitoring of early alterations in retinal health.

Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (APOB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic depletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor-associated cholesterol deposits, and photoreceptor cell death, and loss of rod but not cone function. RPE-specific reduction in Mttp had no significant effect on plasma lipids and lipoproteins. While APOB was decreased in the RPE, most ocular retinoids remained unchanged, with the exception of the storage form of retinoid, retinyl ester. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but is not directly involved in plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

Unraveling the mystery of ocular retinoid turnover: Insights from albino mice and the role of STRA6.

A delicate balance between photon absorption for vision and the protection of photoreceptors from light damage is pivotal for ocular health. This equilibrium is governed by the light-absorbing 11-cis-retinylidene chromophore of visual pigments, which, upon bleaching, transforms into all-trans-retinal and undergoes regeneration through an enzymatic pathway, named the visual cycle. Chemical side reactions of retinaldehyde during the recycling process can generate by-products that may result in a depletion of retinoids. In our study, we have clarified the crucial roles played by melanin pigmentation and the retinoid transporter STRA6 in preventing this loss and preserving the integrity of the visual cycle. Our experiments initially confirmed that consecutive green and blue light bleaching of isolated bovine rhodopsin produced 9-cis and 13-cis retinal. The same unusual retinoids were found in the retinas of mice exposed to intense light, with elevated concentrations observed in albino mice. Examining the metabolic fate of these visual cycle byproducts revealed that 9-cis-retinal, but not 13-cis-retinal, was recycled back to all-trans-retinal through an intermediate called isorhodopsin. However, investigations in Stra6 knockout mice unveiled that the generation of these visual cycle byproducts correlated with a light-induced loss of ocular retinoids and visual impairment. Collectively, our findings uncover important novel aspects of visual cycle dynamics, with implications for ocular health and photoreceptor integrity.

Microsomal triglyceride transfer protein is necessary to maintain lipid homeostasis and retinal function.

Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or to age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (apoB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor -associated cholesterol deposits and photoreceptor cell death, and loss of rod but not cone function. RPE-specific ablation of Mttp had no significant effect on plasma lipids and lipoproteins. While, apoB was decreased in the RPE, ocular retinoid concentrations remained unchanged. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but not directly involved in ocular retinoid and plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.

Primary versus Secondary Elevations in Fundus Autofluorescence.

The method of quantitative fundus autofluorescence (qAF) can be used to assess the levels of bisretinoids in retinal pigment epithelium (RPE) cells so as to aid the interpretation and management of a variety of retinal conditions. In this review, we focused on seven retinal diseases to highlight the possible pathways to increased fundus autofluorescence. ABCA4- and RDH12-associated diseases benefit from known mechanisms whereby gene malfunctioning leads to elevated bisretinoid levels in RPE cells. On the other hand, peripherin2/RDS-associated disease (PRPH2/RDS), retinitis pigmentosa (RP), central serous chorioretinopathy (CSC), acute zonal occult outer retinopathy (AZOOR), and ceramide kinase like (CERKL)-associated retinal degeneration all express abnormally high fundus autofluorescence levels without a demonstrated pathophysiological pathway for bisretinoid elevation. We suggest that, while a known link from gene mutation to increased production of bisretinoids (as in ABCA4- and RDH12-associated diseases) causes primary elevation in fundus autofluorescence, a secondary autofluorescence elevation also exists, where an impairment and degeneration of photoreceptor cells by various causes leads to an increase in bisretinoid levels in RPE cells.

Spatiotemporal control of genome engineering in cone photoreceptors.

BACKGROUND: Cones are essential for color recognition, high resolution, and central vision; therefore cone death causes blindness. Understanding the pathophysiology of each cell type in the retina is key to developing therapies for retinal diseases. However, studying the biology of cone cells in the rod-dominant mammalian retina is particularly challenging. In this study, we used a bacterial artificial chromosome (BAC) recombineering method to knock in the "CreERT2" sequence into the Gnat2 and Arr3 genes, respectively and generated three novel inducible CreERT2 mice with different cone cell specificities. RESULTS: These models (Gnat2CreERT2, Arr3T2ACreERT2, and Arr3P2ACreERT2) express temporally controllable Cre recombinase that achieves conditional alleles in cone photoreceptors. Cre-LoxP recombination can be induced as early as postnatal day (PD) two upon tamoxifen injection at varying efficiencies, ranging from 10 to 15% in Gnat2CreERT2, 40% in Arr3T2ACreERT2, and 100% in Arr3P2ACreERT2. Notably, knocking in the P2A-CreERT2 cassette does not affect cone cell morphology and functionality. Most cone-phototransduction enzymes, including Opsins, CNGA3, etc. are not altered except for a reduction in the Arr3 transcript. CONCLUSIONS: The Arr3P2ACreERT2 mouse, an inducible cone-specific Cre driver, is a valuable line in studying cone cell biology, function, as well as its relationship with rod and other retinal cells. Moreover, the Cre activity can be induced by delivering tamoxifen intragastrically as early as PD2, which will be useful for studying retinal development or in rapid degenerative mouse models.

CRISPR editing demonstrates rs10490924 raised oxidative stress in iPSC-derived retinal cells from patients with ARMS2/HTRA1-related AMD.

Genome-wide association studies (GWAS) have identified genetic risk loci for age-related macular degeneration (AMD) on the chromosome 10q26 (Chr10) locus and are tightly linked: the A69S (G>T) rs10490924 single-nucleotide variant (SNV) and the AATAA-rich insertion-deletion (indel, del443/ins54), which are found in the age-related maculopathy susceptibility 2 (ARMS2) gene, and the G512A (G>A) rs11200638 SNV, which is found in the high-temperature requirement A serine peptidase 1 (HTRA1) promoter. The fourth variant is Y402H complement factor H (CFH), which directs CFH signaling. CRISPR manipulation of retinal pigment epithelium (RPE) cells may allow one to isolate the effects of the individual SNV and thus identify SNV-specific effects on cell phenotype. Clustered regularly interspaced short palindromic repeats (CRISPR) editing demonstrates that rs10490924 raised oxidative stress in induced pluripotent stem cell (iPSC)-derived retinal cells from patients with AMD. Sodium phenylbutyrate preferentially reverses the cell death caused by ARMS2 rs10490924 but not HTRA1 rs11200638. This study serves as a proof of concept for the use of patient-specific iPSCs for functional annotation of tightly linked GWAS to study the etiology of a late-onset disease phenotype. More importantly, we demonstrate that antioxidant administration may be useful for reducing reactive oxidative stress in AMD, a prevalent late-onset neurodegenerative disorder.

A high fat diet fosters elevated bisretinoids.

High dietary fat intake is associated with metabolic dysregulation, but little is known regarding the effects of a high fat diet (HFD) on photoreceptor cell functioning. We explored the intersection of an HFD and the visual cycle adducts that form in photoreceptor cells by nonenzymatic reactions. In black C57BL/6J mice and albino C57BL/6Jc2j mice raised on an HFD until age 3, 6, or 12 months, chromatographically quantified bisretinoids were increased relative to mice on a standard diet. In vivo measurement of fundus autofluorescence, the source of which is bisretinoid, also revealed a significant increase in the HFD mice. Additionally, mice provided with a diet high in fat presented with elevated retinol-binding protein 4, the protein responsible for transporting retinol in plasma. Vitamin A was elevated in plasma although not in ocular tissue. Bisretinoids form in photoreceptor cell outer segments by random reactions of retinaldehyde with phosphatidylethanolamine. We found that the latter phospholipid was significantly increased in mice fed an HFD versus mice on a control diet. In leptin-deficient ob/ob mice, a genetic model of obesity, plasma levels of retinol-binding protein 4 were higher but bisretinoids in retina were not elevated. Photoreceptor cell viability measured as outer nuclear layer thickness was reduced in the ob/ob mice relative to WT. The accelerated formation of bisretinoid we observed in diet-induced obese mice is related to the high fat intake and to increased delivery of vitamin A to the visual cycle.

Monitoring Lesion Area Progression in Stargardt Disease: A Comparison of En Face Optical Coherence Tomography and Fundus Autofluorescence.

Purpose: To compare longitudinal changes in en face spectral domain-optical coherence tomography (SD-OCT) measurements of ellipsoid zone (EZ) and retinal pigment epithelium (RPE) loss to changes in the hypoautofluorescent and hyperautofluorescent (AF) areas detected with short-wavelength (SW)-AF in ABCA4-associated retinopathy. Methods: SD-OCT volume scans were obtained from 20 patients (20 eyes) over 2.6 ± 1.2 years (range 1-5 years). The EZ, and RPE/Bruch's membrane boundaries were segmented, and en face slab images generated. SubRPE and EZ slab images were used to measure areas of atrophic RPE and EZ loss. These were compared to longitudinal measurements of the hypo- and abnormal AF (hypoAF and surrounding hyperAF) areas. Results: At baseline, the en face area of EZ loss was significantly larger than the subRPE atrophic area, and the abnormal AF area was significantly larger than the hypoAF area. The median rate of EZ loss was significantly greater than the rate of increase in the subRPE atrophic area (1.2 mm2/yr compared to 0.5 mm2/yr). The median rate of increase in the abnormal AF area was significantly greater than the increase in the hypoAF area (1.6 mm2/yr compared to 0.6 mm2/yr). Conclusions: En face SD-OCT can be used to quantify changes in RPE atrophy and photoreceptor integrity. It can be a complementary or alternative technique to SW-AF with the advantage of monitoring EZ loss. The SW-AF results emphasize the importance of measuring changes in the hypo- and abnormal AF areas. Translational Relevance: The findings are relevant to the selection of outcome measures for monitoring ABCA4-associated retinopathy.

Insights Into PROM1-Macular Disease Using Multimodal Imaging.

Purpose: To describe the features of genetically confirmed PROM1-macular dystrophy in multimodal images. Methods: Thirty-six (36) eyes of 18 patients (5-66 years; mean age, 42.4 years) were prospectively studied by clinical examination and multimodal imaging. Short-wavelength autofluorescence (SW-AF) and quantitative fundus autofluorescence (qAF) images were acquired with a scanning laser ophthalmoscope (HRA+OCT, Heidelberg Engineering) modified by insertion of an internal autofluorescent reference. Further clinical testing included near-infrared autofluorescence (NIR-AF; HRA2, Heidelberg Engineering) with semiquantitative analysis, spectral domain-optical coherence tomography (HRA+OCT) and full-field electroretinography. All patients were genetically confirmed by exome sequencing. Results: All 18 patients presented with varying degrees of maculopathy. One family with individuals affected across two generations exhibited granular fleck-like deposits across the posterior pole. Areas of granular deposition in SW-AF and NIR-AF corresponded to intermittent loss of the ellipsoid zone, whereas discrete regions of hypoautofluorescence corresponded with a loss of outer retinal layers in spectral-domain optical coherence tomography scans. For 18 of the 20 eyes, qAF levels within the macula were within the 95% confidence intervals of healthy age-matched individuals; nor was the mean NIR-AF signal increased relative to healthy eyes. Conclusions: Although PROM1-macular dystrophy (Stargardt disease 4) can exhibit phenotypic overlap with recessive Stargardt disease, significantly increased SW-AF levels were not detected. As such, elevated bisretinoid lipofuscin may not be a feature of the pathophysiology of PROM1 disease. The qAF approach could serve as a method of early differential diagnosis and may help to identify appropriate disease targets as therapeutics become available to treat inherited retinal disease.

HIF2α activation and mitochondrial deficit due to iron chelation cause retinal atrophy.

Iron accumulation causes cell death and disrupts tissue functions, which necessitates chelation therapy to reduce iron overload. However, clinical utilization of deferoxamine (DFO), an iron chelator, has been documented to give rise to systemic adverse effects, including ocular toxicity. This study provided the pathogenic and molecular basis for DFO-related retinopathy and identified retinal pigment epithelium (RPE) as the target tissue in DFO-related retinopathy. Our modeling demonstrated the susceptibility of RPE to DFO compared with the neuroretina. Intriguingly, we established upregulation of hypoxia inducible factor (HIF) 2α and mitochondrial deficit as the most prominent pathogenesis underlying the RPE atrophy. Moreover, suppressing hyperactivity of HIF2α and preserving mitochondrial dysfunction by α-ketoglutarate (AKG) protects the RPE against lesions both in vitro and in vivo. This supported our observation that AKG supplementation alleviates visual impairment in a patient undergoing DFO-chelation therapy. Overall, our study established a significant role of iron deficiency in initiating DFO-related RPE atrophy. Inhibiting HIF2α and rescuing mitochondrial function by AKG protect RPE cells and can potentially ameliorate patients' visual function.

Vitamin A deficiency and the retinal "double carrot" sign with optical coherence tomography.

BACKGROUND: Spectral-domain optical coherence tomography (SD-OCT) and full-field electroretinography (ERG) allow retinal assessment with vitamin A deficiency (VAD). Using SD-OCT, this study aimed to characterize and follow a novel retinal abnormality in patients with VAD and intramuscular supplementation. METHODS: Patients with VAD were retrospectively reviewed, including SD-OCT and electroretinography. RESULTS: Three patients had VAD following bariatric or colon surgery and varying supplementation. All had nyctalopia, extinguished scotopic rod-specific function with ERG, and decreased serum vitamin A. None demonstrated surface abnormalities. All received intramuscular vitamin A with subjective resolution of symptoms. On SD-OCT, four of six eyes exhibited homogenous foveal hyperreflectivity anterior to retinal pigment epithelium-Bruch complex, reminiscent of a "double carrot", which improved following supplementation. ERG findings demonstrated improved scotopic rod-specific function in all cases; however, photopic function remained diminished in two cases. CONCLUSIONS: Structural improvement of the proposed "double carrot" sign occurs soon after vitamin A supplementation. While scotopic function improves rapidly following supplementation, cone function recovers more slowly. Therefore, foveal changes such as the "double carrot" sign suggest that structural recovery of cones precedes functional recovery.

Products of the visual cycle are detected in mice lacking retinol binding protein 4, the only known vitamin A carrier in plasma.

Efficient delivery of vitamin A to the retinal pigment epithelium is vital to the production of the light-sensitive visual chromophore 11-cis-retinal. Nevertheless, retinol binding protein 4 (RBP4) is the only known carrier of vitamin A in plasma. Here, we present new findings that further characterize the visual cycle in the presence of Rbp4 deficiency. In the face of impaired delivery of retinol in Rbp4-/- mice, we determined that 11-cis-retinaldehyde reached levels that were ∼60% of WT at 4 months of age and all-trans-retinyl ester was 18% of normal yet photoreceptor cell loss was apparent by 8 months of age. The lack of Rbp4 appeared to have a greater impact on scotopic rod-mediated responses than on cone function at early ages. Also, despite severely impaired delivery of retinol, bisretinoid lipofuscin that forms as a byproduct of the visual cycle was measurable by HPLC and by quantitative fundus autofluorescence. In mice carrying an Rpe65 amino acid variant that slows visual cycle kinetics, Rbp4 deficiency had a less pronounced effect on 11-cis-retinal levels. Finally, we found that ocular retinoids were not altered in mice expressing elevated adipose-derived total Rbp4 protein (hRBP4+/+AdiCre+/-). In conclusion, our findings are consistent with a model in which vitamin A can be delivered to the retina by Rbp4-independent pathways.

Chorioretinal atrophy following voretigene neparvovec despite the presence of fundus autofluorescence.

INTRODUCTION: Leber congenital amaurosis (LCA) type 2, due to disease-causing variants in RPE65, is characterized by severe visual loss in early infancy. Current treatments include voretigene neparvovec-rzyl (VN) for RPE65-associated LCA. Herein, we present the long-term follow-up of a patient treated with VN using quantitative autofluorescence (488 nm excitation). CASE REPORT: A 9-year-old girl with a diagnosis of LCA with biallelic variants in RPE65 presented for evaluation. The patient underwent VN treatment at the age of 11. The patient returned to clinic at age of 19 at which time imaging revealed evidence of chorioretinal atrophy. Quantitative autofluorescence performed prior to gene therapy and at 6- and 8-year follow-up revealed a central area of fundus autofluorescence. DISCUSSION: This case report demonstrates acquisition of fundus autofluorescence at 6- and 8-year follow-up despite the development of chorioretinal atrophy.

Choroideremia Carriers: Dark-Adapted Perimetry and Retinal Structures.

Purpose: In choroideremia (CHM) carriers, scotopic sensitivity was assessed by dark adapted chromatic perimetry (DACP) and outer retinal structure was evaluated by multimodal imaging. Methods: Nine carriers (18 eyes) and 13 healthy controls (13 eyes) underwent DACP testing with cyan and red stimuli. Analysis addressed peripapillary (4 test locations closest to the optic disc), macular (52 locations), and peripheral (60 locations outside the macula) regions. Responses were considered to be rod-mediated when cyan relative to red sensitivity was >5 dB. Fundus imaging included spectral domain optical coherence tomography (SD-OCT), short-wavelength (SW-AF), near-infrared (NIR-AF), ultrawide-field (200 degrees) pseudocolor fundus imaging, and quantitative (qAF) fundus autofluorescence. Results: Detection of the cyan stimulus was rod mediated in essentially all test locations (99.7%). In the macular and peripheral areas, DACP sensitivity values were not significantly different from healthy eyes. In the peripapillary area, sensitivities were significantly decreased (P < 0.05). SD-OCT imaging ranged from hyper-reflective lesions and discontinuities of the outer retinal bands to hypertransmission of signal. SW-AF and NIR-AF images presented with peripapillary atrophy in seven patients (14 eyes). Mosaicism was detectable in SW-AF images in seven patients and in NIR-AF images in five patients. Frank hypo-autofluorescence was visible in eight patients with distinct chorioretinopathy in seven patients. The qAF values were below the 95% confidence interval (CI) of healthy age-matched individuals in 12 eyes. Conclusions: Rod mediated scotopic sensitivity was comparable to that in control eyes in macular and peripheral areas but was decreased in the peripapillary area where changes in retinal structure were also most severe.

Vitamin A aldehyde-taurine adducts function in photoreceptor cells.

To facilitate the movement of retinoids through the visual cycle and to limit nonspecific chemical reaction, multiple mechanisms are utilized to handle these molecules when not contained within the binding pocket of opsin. Vitamin A aldehyde is sequestered by reversible Schiff base formation with phosphatidylethanolamine (PE) and subsequently undergoes NADPH-dependent reduction. Otherwise inefficient handling of retinaldehyde can lead to the formation of fluorescent di-retinal compounds within the outer segments of photoreceptor cells. These bisretinoid fluorophores initiate photooxidative processes having adverse consequences for retina. Various carrier proteins confer water solubility and maintain the 11-cis-retinoid configuration. Mechanisms for sequestration of retinoid include the formation of a reversible Schiff base between retinaldehyde and taurine (A1-taurine, A1T), the most abundant amino acid in photoreceptor cells. Here we have undertaken to examine the effects of taurine depletion using the transport inhibitors guanidinoethyl sulfonate (GES) and ß-alanine. Oral treatment of BALB/cJ mice with ß-alanine reduced ocular A1T and the mice exhibited significantly lower scotopic and photopic a-wave amplitudes. As a secondary effect of retinal degeneration, A1T was not detected and taurine was significantly reduced in mice carrying a P23H opsin mutation. The thinning of ONL that is indicative of reduced photoreceptor cell viability in albino Abca4-/- mice was more pronounced in ß-alanine treated mice. Treatment of agouti and albino Abca4-/- mice with ß-alanine and GES was associated with reduced bisretinoid measured chromatographically. Consistent with a reduction in carbonyl scavenging activity by taurine, methylglyoxal-adducts were also increased in the presence of ß-alanine. Taken together these findings support the postulate that A1T serves as a reservoir of vitamin A aldehyde, with diminished A1T explaining reduced photoreceptor light-sensitivity, accentuated ONL thinning in Abca4-/- mice and attenuated bisretinoid formation.

Intrinsic differences in rod and cone membrane composition: implications for cone degeneration.

PURPOSE: In many retinal pathological conditions, rod and cone degeneration differs. For example, the early-onset maculopathy Stargardts disease type 1 (STGD1) is typified by loss of cones while rods are often less affected. We wanted to examine whether there exist intrinsic membrane differences between rods and cones that might explain such features. METHODS: Abca4 mRNA and protein levels were quantified in rod- and cone-enriched samples from wild-type and Nrl-/- mice retinas; rod- and cone-enriched outer segments (ROS and COS respectively) were prepared from pig retinas, and total lipids were analyzed by flame ionization, chromatography, and tandem mass spectrometry. Immunohistochemical staining of cone-rich rodent Arvicanthis ansorgei retinas was conducted, and ultra-high performance liquid chromatography of lipid species in porcine ROS and COS was performed. RESULTS: Abca4 mRNA and Abca4 protein content was significantly higher (50-300%) in cone compared to rod-enriched samples. ROS and COS displayed dramatic differences in several lipids, including very long chain poly-unsaturated fatty acids (VLC-PUFAs), especially docosahexaenoic acid (DHA, 22:6n-3): ROS 20.6% DHA, COS 3.3% (p < 0.001). VLC-PUFAs (> 50 total carbons) were virtually absent from COS. COS were impoverished (> 6× less) in phosphatidylethanolamine compared to ROS. ELOVL4 ("ELOngation of Very Long chain fatty acids 4") antibody labelled Arvicanthis cones only very weakly compared to rods. Finally, there were large amounts (905 a.u.) of the bisretinoid A2PE in ROS, whereas it was much lower (121 a.u., ~ 7.5-fold less) in COS fractions. In contrast, COS contained fivefold higher amounts of all-trans-retinal dimer (115 a.u. compared to 22 a.u. in rods). CONCLUSIONS: Compared to rods, cones expressed higher levels of Abca4 mRNA and Abca4 protein, were highly impoverished in PUFA (especially DHA) and phosphatidylethanolamine, and contained significant amounts of all-trans-retinal dimer. Based on these and other data, we propose that in contrast to rods, cones are preferentially vulnerable to stress and may die through direct cellular toxicity in pathologies such as STGD1.

Expanding the phenotype of TTLL5-associated retinal dystrophy: a case series.

BACKGROUND: Inherited retinal dystrophies describe a heterogeneous group of retinal diseases that lead to the irreversible degeneration of rod and cone photoreceptors and eventual blindness. Recessive loss-of-function mutations in Tubulin Tyrosine Ligase Like 5 (TTLL5) represent a recently described cause of inherited cone-rod and cone dystrophy. This study describes the unusual phenotypes of three patients with autosomal recessive mutations in TTLL5. Examination of these patients included funduscopic evaluation, spectral-domain optical coherence tomography, short-wavelength autofluorescence, and full-field electroretinography (ffERG). Genetic diagnoses were confirmed using whole exome capture. Protein modeling of the identified variants was performed to explore potential genotype-phenotype correlations. RESULTS: Genetic testing revealed five novel variants in TTLL5 in three unrelated patients with retinal dystrophy. Clinical imaging demonstrated features of sectoral cone-rod dystrophy and cone dystrophy, with phenotypic variability seen across all three patients. One patient also developed high-frequency hearing loss during a similar time period as the onset of retinal disease, potentially suggestive of a syndromic disorder. Retinal structure findings were corroborated with functional measures including ffERG findings that supported these diagnoses. Modeling of the five variants suggest that they cause different effects on protein function, providing a potential reason for genotype-phenotype correlation in these patients. CONCLUSIONS: The authors report retinal phenotypic findings in three unrelated patients with novel mutations causing autosomal recessive TTLL5-mediated retinal dystrophy. These findings broaden the understanding of the phenotypes associated with TTLL5-mediated retinal disease and suggest that mutations in TTLL5 should be considered as a potential cause of sectoral retinal dystrophy in addition to cone-rod and cone dystrophies.

Long-term vitamin A supplementation in a preclinical mouse model for RhoD190N-associated retinitis pigmentosa.

Retinitis pigmentosa (RP) is caused by one of many possible gene mutations. The National Institutes of Health recommends high daily doses of vitamin A palmitate for RP patients. There is a critical knowledge gap surrounding the therapeutic applicability of vitamin A to patients with the different subtypes of the disease. Here, we present a case report of a patient with RP caused by a p.D190N mutation in Rhodopsin (RHO) associated with abnormally high quantitative autofluorescence values after long-term vitamin A supplementation. We investigated the effects of vitamin A treatment strategy on RP caused by the p.D190N mutation in RHO by exposing Rhodopsin p.D190N (RhoD190N/+) and wild-type (WT) mice to experimental vitamin A-supplemented and standard control diets. The patient's case suggests that the vitamin A treatment strategy should be further studied to determine its effect on RP caused by p.D190N mutation in RHO and other mutations. Our mouse experiments revealed that RhoD190N/+ mice on the vitamin A diet exhibited higher levels of autofluorescence and lipofuscin metabolites compared to WT mice on the same diet and isogenic controls on the standard control diet. Vitamin A supplementation diminished photoreceptor function in RhoD190N/+ mice while preserving cone response in WT mice. Our findings highlight the importance of more investigations into the efficacy of clinical treatments like vitamin A for patients with certain genetic subtypes of disease and of genotyping in the precision care of inherited retinal degenerations.

Comparisons Among Optical Coherence Tomography and Fundus Autofluorescence Modalities as Measurements of Atrophy in ABCA4-Associated Disease.

PURPOSE: In ABCA4-associated retinopathy, central atrophy was assessed by spectral domain optical coherence tomography (SD-OCT) and by short-wavelength (SW-AF) and near-infrared (NIR-AF) autofluorescence. METHODS: Patients exhibited a central atrophic lesion characterized by hypoautofluorescence (hypoAF) surrounded either by hyperautofluorescent (hyperAF) rings in both AF images (group 1, 4 patients); or a hyperAF ring in SW-AF but not in NIR-AF images (group 2, 11 patients); or hyperAF rings in neither AF images (group 3, 11 patients). Choroidal hypertransmission and widths of ellipsoid zone (EZ) loss were measured in foveal SD-OCT scans, and in AF images hypoAF and total hypo+hyperAF widths were measured along the same axis. Bland-Altman and repeated measures analysis of variance with Tukey post hoc were applied. RESULTS: For all groups, hypertransmission widths were significantly smaller than EZ loss widths. In Groups 1 and 2, hypertransmission width was not significantly different than SW-hypoAF width, but hypertransmission was narrower than the width of SW-hypo+hyperAF (groups 1, 2) and NIR-hypo+hyperAF (group 1). In group 3, the hypertransmission width was also significantly less than the width of SW-hypoAF and NIR-hypoAF. The EZ loss widths were not significantly different than measurements of total lesion size, the latter being the widths of SW-hypo+hyperAF and NIR-hypo+hyperAF (group 1); widths of NIR-hypoAF and SW-hypo+hyperAF (group 2); and widths of NIR-hypoAF and SW-hypoAF (group 3). CONCLUSIONS: Hypertransmission and SW-hypoAF (except when reflecting total lesion width) underestimate lesion size detected by EZ loss, SW-hypoAF+hyperAF, and NIR-hypo+hyperAF. TRANSLATIONAL RELEVANCE: The findings are significant to the selection of outcome measures in clinical studies.