Txnip deletions and missense alleles prolong the survival of cones in a retinitis pigmentosa mouse model.

Retinitis pigmentosa (RP) is an inherited retinal disease in which there is a loss of cone-mediated daylight vision. As there are >100 disease genes, our goal is to preserve cone vision in a disease gene-agnostic manner. Previously we showed that overexpressing TXNIP, an α-arrestin protein, prolonged cone vision in RP mouse models, using an AAV to express it only in cones. Here, we expressed different alleles of Txnip in the retinal pigmented epithelium (RPE), a support layer for cones. Our goal was to learn more of TXNIP's structure-function relationships for cone survival, as well as determine the optimal cell type expression pattern for cone survival. The C-terminal half of TXNIP was found to be sufficient to remove GLUT1 from the cell surface, and improved RP cone survival, when expressed in the RPE, but not in cones. Knock-down of HSP90AB1, a TXNIP-interactor which regulates metabolism, improved the survival of cones alone and was additive for cone survival when combined with TXNIP. From these and other results, it is likely that TXNIP interacts with several proteins in the RPE to indirectly support cone survival, with some of these interactions different from those that lead to cone survival when expressed only in cones.

Chemically induced cone degeneration in the 13-lined ground squirrel.

Animal models of retinal degeneration are critical for understanding disease and testing potential therapies. Inducing degeneration commonly involves the administration of chemicals that kill photoreceptors by disrupting metabolic pathways, signaling pathways, or protein synthesis. While chemically induced degeneration has been demonstrated in a variety of animals (mice, rats, rabbits, felines, 13-lined ground squirrels (13-LGS), pigs, chicks), few studies have used noninvasive high-resolution retinal imaging to monitor the in vivo cellular effects. Here, we used longitudinal scanning light ophthalmoscopy (SLO), optical coherence tomography, and adaptive optics SLO imaging in the euthermic, cone-dominant 13-LGS (46 animals, 52 eyes) to examine retinal structure following intravitreal injections of chemicals, which were previously shown to induce photoreceptor degeneration, throughout the active season of 2019 and 2020. We found that iodoacetic acid induced severe pan-retinal damage in all but one eye, which received the lowest concentration. While sodium nitroprusside successfully induced degeneration of the outer retinal layers, the results were variable, and damage was also observed in 50% of contralateral control eyes. Adenosine triphosphate and tunicamycin induced outer retinal specific damage with varying results, while eyes injected with thapsigargin did not show signs of degeneration. Given the variability of damage we observed, follow-up studies examining the possible physiological origins of this variability are critical. These additional studies should further advance the utility of chemically induced photoreceptor degeneration models in the cone-dominant 13-LGS.

Rescue of cone and rod photoreceptor function in a CDHR1-model of age-related retinal degeneration.

Age-related macular degeneration (AMD) is the most common cause of untreatable blindness in the developed world. Recently, CDHR1 has been identified as the cause of a subset of AMD that has the appearance of the "dry" form, or geographic atrophy. Biallelic variants in CDHR1-a specialized protocadherin highly expressed in cone and rod photoreceptors-result in blindness from shortened photoreceptor outer segments and progressive photoreceptor cell death. Here we demonstrate long-term morphological, ultrastructural, functional, and behavioral rescue following CDHR1 gene therapy in a relevant murine model, sustained to 23-months after injection. This represents the first demonstration of rescue of a monogenic cadherinopathy in vivo. Moreover, the durability of CDHR1 gene therapy seems to be near complete-with morphological findings of the rescued retina not obviously different from wildtype throughout the lifespan of the mouse model. A follow-on clinical trial in patients with CDHR1-associated retinal degeneration is warranted. Hypomorphic CDHR1 variants may mimic advanced dry AMD. Accurate clinical classification is now critical, as their pathogenesis and treatment are distinct.

Novel ATF6 homozygous variant in a Chinese patient with achromatopsia.

BACKGROUND: ATF6-associated Achromatopsia (ACHM) is a rare autosomal recessive disorder characterized by reduction of visual acuity, photophobia, nystagmus, and poor color vision. METHODS: Detailed ophthalmological examinations were performed in a Chinese patient with ACHM. Whole exome sequencing and Sanger sequencing were performed to detect the disease-causing gene in the patient. RESULTS: A 6-year-old girl presented photophobia, low vision and reduced color discrimination. Small yellow lesion in the macula of both eyes was observed. FAF demonstrated hypofluorescence in the macular fovea. OCT images revealed interruption of ellipsoid and interdigitation zone in the foveal area and a loss of the foveal pit. ERG showed relatively normal rod responses and unrecordable cone responses. Sequencing result identified a novel splicing variant c.354 + 6T>C in the ATF6 gene (NM_007348.4). CONCLUSIONS: We reported detailed clinical features and genetic analysis of a new Chinese ATF6-associated patient with ACHM.

Cone-Driven, Geniculocortical Responses in Canine Models of Outer Retinal Disease.

Purpose: Canine models of inherited retinal degeneration are used for proof of concept of emerging gene and cell-based therapies that aim to produce functional restoration of cone-mediated vision. We examined functional magnetic resonance imaging (MRI) measures of the postretinal response to cone-directed stimulation in wild-type (WT) dogs, and in three different retinal disease models. Methods: Temporal spectral modulation of a uniform field of light around a photopic background was used to target the canine L/M (hereafter "L") and S cones and rods. Stimuli were designed to separately target the postreceptoral luminance (L+S) and chrominance (L-S) pathways, the rods, and all photoreceptors jointly (light flux). These stimuli were presented to WT, and mutant PDE6B-RCD1, RPGR-XLPRA2, and NPHP5-CRD2 dogs during pupillometry and functional MRI (fMRI). Results: Pupil responses in WT dogs to light flux, L+S, and rod-directed stimuli were consistent with responses being driven by cone signals alone. For WT animals, both luminance and chromatic (L-S) stimuli evoked fMRI responses in the lateral geniculate nucleus or visual cortex; RCD1 animals with predominant rod loss had similar responses. Responses to cone-directed stimulation were reduced in XLPRA2 and absent in CRD2. NPHP5 gene augmentation restored the cortical response to luminance stimulation in a CRD2 animal. Conclusions: Cone-directed stimulation during fMRI can be used to measure the integrity of luminance and chrominance responses in the dog visual system. The NPHP5-CRD2 model is appealing for studies of recovered cone function. Translational Relevance: fMRI assessment of cone-driven cortical response provides a tool to translate cell/gene therapies for vision restoration.

Syndromic retinitis pigmentosa caused by biallelic SCAPER frameshift variant.

PURPOSE: Mutations in the SCAPER gene have previously been reported to be a rare cause of syndromic and non-syndromic autosomal recessive retinitis pigmentosa (RP). We report a case of syndromic RP caused by a frameshift heterozygous mutation in SCAPER. Our case has a relatively mild ocular phenotype with the presence of cone involvement noted on full field electroretinogram (ffERG) without impacting central or color vision. MATERIALS AND METHODS: A 17-year-old male presented with progressive nyctalopia in both eyes. He underwent ophthalmic examination and multimodal imaging. A complete retinal degeneration panel consisting of 322 genes was used to screen for molecular causes of retinal dystrophy in this patient along with family segregation analysis. RESULTS: Fundus examination of the proband revealed mild RP phenotype with waxy pallor of optic discs, attenuated retinal arterioles, and single bone spicule like pigmentary change in the mid-periphery bilaterally. Multimodal imaging and ffERG demonstrated a picture of RP with cone dysfunction without impacting central or color vision bilaterally. Examined family members were found to be normal. The proband was found to be heterozygous for two novel frameshift pathogenic variants in SCAPER c.3781del, p. (Val1261Serfs*26), c.868_869del, p. (Glu290Serfs*7) both leading to predicted premature termination. The family members tested were found to be heterozygous for SCAPER c.868_869del, p. (Glu290Serfs*7) pathogenic variant confirming their carrier status. CONCLUSION: We report a case of a syndromic RP of previously unreported ocular phenotype associated with SCAPER pathogenic variant, which will add to the phenotypic spectrum of retinopathy and systemic features associated with pathogenic variants in SCAPER.

Deep phenotyping of PROM1-associated retinal degeneration.

BACKGROUND/AIMS: The purpose of this study was to investigate retinal structure in detail of subjects with autosomal-dominant (AD) and autosomal-recessive (AR) PROM1-associated retinal degeneration (PROM1-RD), study design: institutional, cross-sectional study. METHODS: Four eyes from four subjects (three with AD and one with AR) PROM1-RD were investigated by ophthalmic examination including best-corrected visual acuity (BCVA) and multimodal retinal imaging: fundus autofluorescence (FAF), spectral-domain optical coherence tomography (SD-OCT) and adaptive optics scanning light ophthalmoscopy. Quantitative assessment of atrophic lesions determined by FAF, thickness of individual retinal layers and cone photoreceptor quantification was performed. RESULTS: BCVA ranged from 20/16 to 20/200. Initial pathological changes included the presence of hyperautofluorescent spots on FAF imaging, while later stages demonstrated discrete areas of atrophy. In all patients, thinning of the outer retinal layers on SD-OCT with varying degrees of atrophy could be detected depending on disease-causing variants and age. Cone density was quantified both in central and/or at different eccentricities from the fovea. Longitudinal assessments were possible in two patients. CONCLUSIONS: PROM1-RD comprises a wide range of clinical phenotypes. Depending on the stage of disease, the cone mosaic in PROM1-RD is relatively preserved and can potentially be targeted by cone-directed interventions.

Reduced cone photoreceptor function and subtle systemic manifestations in two siblings with loss of SCLT1.

BACKGROUND: The sodium channel and clathrin linker 1 gene (SCLT1) has been involved in the pathogenesis of various ciliopathy disorders such as Bardet-Biedl syndrome, orofaciodigital syndrome type IX, and Senior-Løken syndrome. Detailed exams are warranted to outline all clinical features. Here, we present a family with a milder phenotype of SCLT1-related disease. MATERIAL AND METHODS: Comprehensive eye examination including fundus images, OCT, color vision, visual fields and electroretinography were performed. Affected individuals were assessed by a pediatrician and a medical geneticist for systemic features of ciliopathy. Investigations included echocardiography, abdominal ultrasonography, blood work-up for diabetes, liver and kidney function. Genetic testing included NGS retinal dystrophy panel, segregation analysis and transcriptome sequencing. RESULTS: Two male children, age 10 and 8 years, were affected with attention deficit hyperactivity disorder (ADHD), obesity and mild photophobia. The ophthalmic exam revealed reduced best-corrected visual acuity (BCVA), strabismus, hyperopia, astigmatism and moderate red-green defects. Milder changes suggesting photoreceptors disease were found on retinal imaging. Electroretinogram confirmed cone photoreceptors dysfunction. Genetic testing revealed a homozygous likely pathogenic, splice-site variant in SCLT1 gene NM_144643.3: c.1439 + 1del in the proband and in the affected brother. The unaffected parents were heterozygous for the SCLT1 variant. Transcriptome sequencing showed retention of intron 16 in the proband. CONCLUSIONS: In this report, we highlight the importance of further extensive diagnostics in patients with unexplained reduced vision, strabismus, refractive errors and ADHD spectrum disorders. SCLT1-related retinal degeneration is very rare and isolated reduced function of cone photoreceptors has not previously been observed.

Spatially-dependent model for rods and cones in the retina.

We develop a mathematical model for photoreceptors in the retina. We focus on rod and cone outer segment dynamics and interactions with a nutrient source associated with the retinal pigment epithelium cells. Rod and cone densities (number per unit area of retinal surface) are known to have significant spatial dependence in the retina with cones located primarily near the fovea and the rods located primarily away from the fovea. Our model accounts for this spatial dependence of the rod and cone photoreceptor density as well as for the possibility of nutrient diffusion. We present equilibrium and dynamic solutions, discuss their relation to existing models, and estimate model parameters through comparisons with available experimental measurements of both spatial and temporal photoreceptor characteristics. Our model compares well with existing data on spatially-dependent regrowth of photoreceptor outer segments in the macular region of Rhesus Monkeys. Our predictions are also consistent with existing data on the spatial dependence of photoreceptor outer segment length near the fovea in healthy human subjects. We focus primarily on the healthy eye but our model could be the basis for future efforts designed to explore various retinal pathologies, eye-related injuries, and treatments of these conditions.

Histological changes in retinal detachment: A systematic review for the clinician.

Although there have been numerous innovations in the management of retinal detachment (RD) over the past decades, there is still limited understanding of the pathophysiological processes that take place before and after repair. Summarizing key concepts using animal studies may allow for a better assessment of common pre- and postoperative microstructural abnormalities in RD. We performed a systematic literature review on Ovid MEDLINE, EMBASE, and Cochrane Controlled Register of Trials from January 1968 to January 2022, searching animal or human studies reporting retinal histologic changes following primary or induced RD. Thirty-two studies were included. Main cellular events were summarized: photoceptor apoptosis occurs as early as 12 hours after RD and, although most cells survive, there is extensive remodeling. Outer segments progressively degenerate, while inner segments are reorganized. Rod and cone opsins are redistributed, and rod axons retract while cones undergo changes in shape. Second- and third-order neurons rearrange their dendritic processes, and Müller cells become hypertrophic, growing into the subretinal space. Finally, retinal pigment epithelium cells undergo a change in their morphology. Acknowledging critical morphologic changes following RD is crucial in understanding why anatomical and functional outcomes can vary. Insights from histological studies, together with high-resolution imaging, may be key in identifying novel biomarkers in RD.

Deep Density Estimation for Cone Counting and Diagnosis of Genetic Eye Diseases From Adaptive Optics Scanning Light Ophthalmoscope Images.

Purpose: Adaptive optics scanning light ophthalmoscope (AOSLO) imaging offers a microscopic view of the living retina, holding promise for diagnosing and researching eye diseases like retinitis pigmentosa and Stargardt's disease. The technology's clinical impact of AOSLO hinges on early detection through automated analysis tools. Methods: We introduce Cone Density Estimation (CoDE) and CoDE for Diagnosis (CoDED). CoDE is a deep density estimation model for cone counting that estimates a density function whose integral is equal to the number of cones. CoDED is an integration of CoDE with deep image classifiers for diagnosis. We use two AOSLO image datasets to train and evaluate the performance of cone density estimation and classification models for retinitis pigmentosa and Stargardt's disease. Results: Bland-Altman plots show that CoDE outperforms state-of-the-art models for cone density estimation. CoDED reported an F1 score of 0.770 ± 0.04 for disease classification, outperforming traditional convolutional networks. Conclusions: CoDE shows promise in classifying the retinitis pigmentosa and Stargardt's disease cases from a single AOSLO image. Our preliminary results suggest the potential role of analyzing patterns in the retinal cellular mosaic to aid in the diagnosis of genetic eye diseases. Translational Relevance: Our study explores the potential of deep density estimation models to aid in the analysis of AOSLO images. Although the initial results are encouraging, more research is needed to fully realize the potential of such methods in the treatment and study of genetic retinal pathologies.

Achromatopsia-Visual Cortex Stability and Plasticity in the Absence of Functional Cones.

Purpose: Achromatopsia is a rare inherited disorder rendering retinal cone photoreceptors nonfunctional. As a consequence, the sizable foveal representation in the visual cortex is congenitally deprived of visual input, which prompts a fundamental question: is the cortical representation of the central visual field in patients with achromatopsia remapped to take up processing of paracentral inputs? Such remapping might interfere with gene therapeutic treatments aimed at restoring cone function. Methods: We conducted a multicenter study to explore the nature and plasticity of vision in the absence of functional cones in a cohort of 17 individuals affected by autosomal recessive achromatopsia and confirmed biallelic disease-causing CNGA3 or CNGB3 mutations. Specifically, we tested the hypothesis of foveal remapping in human achromatopsia. For this purpose, we applied two independent functional magnetic resonance imaging (fMRI)-based mapping approaches, i.e. conventional phase-encoded eccentricity and population receptive field mapping, to separate data sets. Results: Both fMRI approaches produced the same result in the group comparison of achromatopsia versus healthy controls: sizable remapping of the representation of the central visual field in the primary visual cortex was not apparent. Conclusions: Remapping of the cortical representation of the central visual field is not a general feature in achromatopsia. It is concluded that plasticity of the human primary visual cortex is less pronounced than previously assumed. A pretherapeutic imaging workup is proposed to optimize interventions.

Achromatopsia Showing Compound Heterozygous Mutations in ATF6 by Whole Exome Sequencing: A Rare Case Report.

Achromatopsia, inherited in an autosomal recessive manner, is a rare condition featured by dysfunction of cone photoreceptors responsible for high-acuity vision in daylight. To date, its pathogenesis and genetic mechanism are still not well defined due to the rarity of cases. In this study, the authors describe a patient with achromatopsia who was diagnosed based on the combination of whole exome sequencing, ocular examination, fundus photography, and fundus fluorescein angiography. A 1-year-old girl presented due to absence of the foveal reflex, severe photophobia, and pigment mottling. Fundus photography and fundus fluorescein angiography were performed on admission. Blood samples were extracted from the proband and her parents. Whole exome sequencing detected two ATF6 variants (c.533C>A and c.82+1G>T), which were confirmed through Sanger sequencing. According to the American College of Medical Genetics guidelines, both c.533C>A and c.82+1G>T variants in ATF6 were predicted as pathogenic mutations (PVS1, PM2, PM3). The patient was diagnosed as having achromatopsia with pathogenicity of ATF6 variants (c.533C>A and c.82+1G>T). [J Pediatr Ophthalmol Strabismus. 2023;60(5):e65-e69.].

Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia.

PURPOSE: CNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential. METHODS: We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification. RESULTS: We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%. CONCLUSION: Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.

Enhanced S-cone Syndrome, a Mini-review.

Enhanced S-cone Syndrome (ESCS) is an autosomal recessive inherited retinal disease mostly associated with disease-causing variants in the NR2E3 gene. During retinal development in ESCS, rod photoreceptor precursors are misdirected to form photoreceptors similar to short-wavelength cones, or S-cones. Compared to a normal human retina, patients with ESCS have no rods and significantly increased numbers of S-cones. Night blindness is the main visual symptom, and visual acuity and color vision can be normal at early disease stages. Histology of donor eyes and adaptive optics imaging revealed increased S-cone density outside of the fovea compared to normal. Visual function testing reveals absent rod function and abnormally enhanced sensitivity to short-wavelength light. Unlike most retinal degenerative diseases, ESCS results in a gain in S-cone photoreceptor function. Research involving ESCS could improve understanding of this rare retinal condition and also shed light on the role of NR2E3 expression in photoreceptor survival.

Single-Cell Transcriptomic Profiling of Müller Glia in the rd10 Retina.

Müller glia are the principal macroglia of the retina and support retinal neurons both in health and disease. In retinitis pigmentosa (RP), a highly heterogeneous inherited retinal disorder, the most common form of pathology involves primary rod degeneration, followed by secondary cone death. To investigate Müller glia responses to rod degeneration, we performed droplet-based single-cell RNA sequencing in the rd10 mouse model of RP during primary rod degeneration. We confirmed known MG behavior on gliosis, metabolic, and immune functions. Pde6brd10 Müller glia also exhibited an increased expression of histocompatibility complex members, which might arise from a novel immune function of Müller glia in RP. We also describe a possible decrease in glial lipid biogenesis, which might affect degenerating photoreceptors.

Comparative study of PRPH2 D2 loop mutants reveals divergent disease mechanism in rods and cones.

Mutations in the photoreceptor-specific tetraspanin gene peripherin-2 (PRPH2) lead to widely varying forms of retinal degeneration ranging from retinitis pigmentosa to macular dystrophy. Both inter- and intra-familial phenotypic heterogeneity has led to much interest in uncovering the complex pathogenic mechanisms of PRPH2-associated disease. Majority of disease-causing mutations in PRPH2 reside in the second intradiscal loop, wherein seven cysteines control protein folding and oligomerization. Here, we utilize knockin models to evaluate the role of three D2 loop cysteine mutants (Y141C, C213Y and C150S), alone or in combination. We elucidated how these mutations affect PRPH2 properties, including oligomerization and subcellular localization, and contribute to disease processes. Results from our structural, functional and molecular studies revealed that, in contrast to our understanding from prior investigations, rods are highly affected by PRPH2 mutations interfering with oligomerization and not merely by the haploinsufficiency associated with these mutations. On the other hand, cones are less affected by the toxicity of the mutant protein and significantly reduced protein levels, suggesting that knockdown therapeutic strategies may sustain cone functionality for a longer period. This observation provides useful data to guide and simplify the current development of effective therapeutic approaches for PRPH2-associated diseases that combine knockdown with high levels of gene supplementation needed to generate prolonged rod improvement.

Modeling rod and cone photoreceptor cell survival in vivo using optical coherence tomography.

Many retinal diseases involve the loss of light-sensing photoreceptor cells (rods and cones) over time. The severity and distribution of photoreceptor loss varies widely across diseases and affected individuals, so characterizing the degree and pattern of photoreceptor loss can clarify pathophysiology and prognosis. Currently, in vivo visualization of individual photoreceptors requires technology such as adaptive optics, which has numerous limitations and is not widely used. By contrast, optical coherence tomography (OCT) is nearly ubiquitous in daily clinical practice given its ease of image acquisition and detailed visualization of retinal structure. However, OCT cannot resolve individual photoreceptors, and no OCT-based method exists to distinguish between the loss of rods versus cones. Here, we present a computational model that quantitatively estimates rod versus cone photoreceptor loss from OCT. Using histologic data of human photoreceptor topography, we constructed an OCT-based reference model to simulate outer nuclear layer thinning caused by differential loss of rods and cones. The model was able to estimate rod and cone loss using in vivo OCT data from patients with Stargardt disease and healthy controls. Our model provides a powerful new tool to quantify photoreceptor loss using OCT data alone, with potentially broad applications for research and clinical care.

CDHR1-Related Cone-Rod Dystrophy: Clinical Characteristics, Imaging Findings, and Genetic Test Results-A Case Report.

Background: Cone-rod dystrophies (CRDs) are a heterogeneous group of inherited retinal diseases (IRDs) characterized by cone photoreceptor loss, that is followed by subsequent rod photoreceptor impairment. Case presentation: A 49-year-old man complaining of diminution of vision in both eyes (OU) was referred to our outpatient clinic. He reported visual loss for 5 years, but it was most progressive during the last few months. The best-corrected visual acuity (BCVA) at presentation was 0.4 in the right eye (RE) and 1.0 in the left eye (LE). Fundus fluorescein angiography (FFA) revealed granular hyperfluorescence in the macula and concomitant areas of capillary atrophy. Flash full-field electroretinography (ffERG) showed lowering of a and b waves as well as prolonged peak time in light-adapted conditions. However, outcomes of dark-adapted ERGs were within normal limits. Based on the constellation of clinical, angiographic, and electrophysiological tests findings, a diagnosis of IRD was suspected. Genetic testing showed a homozygous, pathogenic c.783G>A mutation in the cadherin-related family member 1 (CDHR1) gene, which confirmed CRD type 15 (CRD15). Conclusions: We demonstrate the clinical characteristics, retinal imaging outcomes, and genetic test results of a patient with CRD15. Our case contributes to expanding our knowledge of the clinical involvement of the pathogenic mutation c.783G>A in CDHR1 variants.

In Vivo Longitudinal Measurement of Cone Photoreceptor Density in Intermediate Age-Related Macular Degeneration.

PURPOSE: To evaluate cone photoreceptor density in clinically unremarkable retinal regions in patients with age-related macular degeneration (AMD) using adaptive optics scanning laser ophthalmoscopy (AOSLO). DESIGN: Prospective case series with normal comparison group. METHODS: Ten eyes of 7 patients with intermediate AMD were studied, including 4 with predominantly subretinal drusenoid deposits (SDD) and 3 without SDD. Macular regions with a clinical absence of AMD-associated lesions were identified by cone packing structure on AOSLO and optical coherence tomography. Cone density was measured in 1174 clinically unremarkable regions within the central subfield (CSF), the inner (IR), and outer rings (OR) of the Early Treatment Diabetic Retinopathy Study grid over 39.6 ± 3.3 months and compared with age-matched normal values obtained in 17 participants. RESULTS: Cone density decreased at 98.3% of the examined locations over time in the eyes with AMD. In the CSF, IR, and OR, cones declined by -255 ± 135, -133 ± 45, and -59 ± 24 cones/degree2/year, respectively, in eyes with SDD, and by -212 ± 89, -83 ± 37, and -27 ± 18 cones/degree2/year, respectively, in eyes without SDD. The percentage of retinal loci with cone density lower than normal (Z score < -2) increased over the follow-up: from 42% at the baseline to 80% at the last visit in eyes with SDD and from 31% to 70% in eyes without SDD. CONCLUSIONS: AOSLO revealed cone photoreceptor loss in regions that appear otherwise unremarkable clinically. These findings may help explain the loss of mesopic sensitivity reported in these areas in eyes with intermediate AMD.