Christine A. Curcio - US grants

retina; aging;

A major question is vision research is how human visual performance is limited by the architecture of the neural tissue which subserves it. Our goal is to establish a reliable and internally consistent anatomical database for comparing the anatomy of the human photoreceptor (PR) mosaic to visual function. In this proposal we continue to determine the extent to which age- related declines in visual function can be explained by loss of PR and ganglion cells (GC) in the retina. We use an unstained retinal whole mount and computer-assisted morphometry to characterize the spatial density of cones, rods, GC, and the size spectrum of GC somata in donor eyes over 70 years of age. We will also use these techniques to study a small number of eyes from elderly person with well documented visual function. Data on these cell types can serve as a base line for other studies of age-related maculopathy and glaucoma, major blinding disease of the elderly. We also propose to extend our work on the spatial distribution of cones in young retina to more detailed characterization of morphological substrates underlying sampling, sensitivity, and connectivity properties of the human cone mosaic. We will use Voronoi-based spatial statistics and Fast Fourier methods to analyze cone packing geometry as a function of eccentricity. We will investigate the spatial distribution of blue cones in the human retina using a recently developed antibody specific to the blue cone opsin. The ability of cones to gather light efficiently, is determined partly by inner and outer segment morphology. We plan to quantify this morphology using computer reconstruction and graphic display of optical sections through the long axis of clones. Finally, we will investigate the pattern of cone-cone connectivity in the peripheral retina by injecting a patch of adjacent cones in an in vitro preparation and examining the patterns of connections made by cone telodendria. We will also determine the projection of visual field onto the human GC layer by accounting for the lateral offset of foveal GC. Our previously collected data on the spatial density of GC can then be expressed in terms of cells/deg2 as a function of eccentricity in visual degrees. These data can be compared with the distribution of cones in the same eyes and with published estimates of the human cortical magnification factor.

Improved treatments for age-related maculopathy (ARM), the leading cause of untreatable vision loss among the elderly in the developed world, lie in better understanding of early ARM. On the basis of our preliminary data, we hypothesize that early age-related maculopathy is an ocular form of atherosclerosis. According to this hypothesis, the pathophysiology of ARM involves two mechanisms similar to those that occur during aging and atherosclerosis in the inner wall of the large arteries: According to this hypothesis, the inner wall of the large arteries: an age-related deposition of serum-lipoprotein-derived esterified cholesterol and unesterified cholesterol (EC and UC) in association with extracellular matrix proteins; and a disease-related deposition of a UC-rich material with the participation of local cells. Using a unique resource for human donor eyes and a well-established animal model for experimental atherosclerosis, we propose three aims to test several predictions of this hypothesis. 1) In human eyes, we will determine how the EC and UC content of Bruch's membrane varies with age and retinal location, using filipin fluorescence and digital microscopy. We will identify EC-rich particles in Bruch's membrane by determining how solvents affect filipin fluorescence and ultra-structure. We will characterize the lipid composition of isolated Bruch's membrane using an enzymatic assay and gas-chromatography-mass spectrometry. 2) In human eyes, we will determine the morphology of membranous debris occurs in peripheral retina. We will determine the UC content of membranous debris and photoreceptor outer segments using filipin fluorescence, digital microscopy, and ultrastructural stereology. 3) In rabbit eyes, we will determine if an extended diet of cholesterol supplementation results in deposition of EC in Bruch's membrane EC, using autoradiography to localized systemically injected radiolabeled tyramine-cellubiose-low density lipoprotein in tissue sections. Results from these aims will be valuable in assessing the extent to which ARM and atherosclerosis share pathogenetic mechanisms with regard to the accumulation and source of extracellular cholesterol. This information is required in order to determine if effective treatments for atherosclerosis should be considered for the treatment of early ARM.

Abstract Age-related macular degeneration (AMD) causes vision loss in millions worldwide. Central to AMD initiation and progression is the retinal pigment epithelium (RPE), which is clinically visualized via the massed autofluorescence (AF) of its lipofuscin and melanolipofuscin granules. We hypothesize, based on our imaging and pathology studies, that AMD can be staged and monitored by the expression and distribution of unique RPE fluorophores. We identified and characterized ex vivo spectral signatures of distinct fluorophore families in normal RPE, Bruch's membrane and drusen, AMD's hallmark lesion. However, the molecular sources of these spectra in the macula are now uncertain due to seminal imaging mass spectrometry (IMS) studies showing that a major lipofuscin fluorophore, A2E, is abundant in the periphery. Thus, the long-term goal of this 6-investigator collaboration is to develop AMD diagnostics based on hyperspectral AF for spectral, molecular biopsy of the RPE, linking clinical pathology to underlying molecular composition. Hyperspectral AF imaging, unlike conventional AF imaging, acquires 3-dimensional ?hypercubes? of data (2 spatial coordinates ? x, y - and 1 spectral - wavelength). We explored imaging data with novel tensor-based tools exploiting multiple excitation wavelengths to discover RPE spectral signatures and their spatial distributions. We propose to link fluorophores to granules, RPE cells, tissue, and AMD stages in 3 aims using a common human tissue source. Aim 1 uses hyperspectral AF tissue mapping to understand AMD pathology at the spectral level in eyes with AMD and unaffected control eyes. We will map RPE flat-mounts by hyperspectral AF microscopy linked to a pathology grading system. Spectral AF components will be recovered mathematically and assigned to subcellular and extracellular features. Aim 2 will quantify AMD pathology at the subcellular level by enumerating fluorophore-containing granules using structured illumination microscopy and 3-dimensional electron microscopy. Aim 3 uses hyperspectral fluorophore identification to understand AMD pathology at the molecular level. We will determine candidate molecules for the major spectral components discovered in Aim 1 by hyperspectral thin layer chromatography and will verify their spatial distributions by IMS. Synthetic authentic standards will ensure spectral validation. Biological validation at this level is unprecedented for clinical ophthalmology, yet warranted by the size of the AMD patient population, the enormity of knowledge gap about major RPE fluorophores in human eyes, the availability of donor tissue, and the proven success of validating other imaging technologies. Our results will directly translate to clinical hyperspectral AF imaging for noninvasive, spatially precise early detection and longitudinal AMD follow-up, in vivo target discovery, and immediate extensions beyond AMD. From our discoveries will flow a huge range of experiments in outer retinal cell biology to deepen understanding of retinal degenerations.

Project Summary Age-related macular degeneration (AMD), the leading cause of irreversible vision impairment in the US and third globally, is a disease of the photoreceptor support system involving the retinal pigment epithelium (RPE), Bruch's membrane, and the choriocapillaris, ultimately leading to photoreceptor demise and eventual vision loss. Our research with that of others has clearly documented the selective vulnerability of rod photoreceptors and rod-mediated (scotopic) vision, including delayed rod-mediated dark adaptation (RMDA) and impaired rod-mediated light sensitivity, in aging and early AMD. RMDA is not only more likely to be slower in eyes with early AMD compared to eyes in normal macular health, but also delayed RMDA is a functional biomarker (i.e., risk factor) for incident early AMD. The next frontier is to establish the structural basis of rod-mediated dysfunction in older adults at-risk for AMD and those already converted to early AMD. Our unifying hypothesis across all aims is: Early AMD is a disease of micronutrient deficiency and vascular insufficiency, due to detectable structural changes in the retinoid re-supply route from the choriocapillaris to the photoreceptors, manifest functionally as delayed rod- mediated dark adaptation. These structural disturbances will occur in specific chorioretinal layers and regions reflecting the spatial distribution of disease in the photoreceptor support system. Our multidisciplinary team has expertise in visual psychophysics, epidemiology, histopathology, digital image analysis and interpretation for retinal disease, study design, and biostatistics. Toward our goals, we will execute the 3 specific aims in an exceptionally well phenotyped cohort at aging-early AMD transition, staged by the AREDS 9-step scale, with 3 years of longitudinal follow-up: (1) To examine the abundance and extent of AMD's pathognomonic deposits (drusen and newly recognized subretinal drusenoid deposits) in relationship to scotopic dysfunction via optical coherence tomography (OCT); (2) To examine RPE cell bodies as structural correlates of scotopic dysfunction via quantitative fundus autofluorescence and layer thicknesses via OCT; (3) To measure vascular density (coverage of macular Bruch's membrane by choriocapillaris), a measure of exchange capacity for outer retinal cells, using OCT angiography. An accurate map and timeline of structure-function relationships in aging and early AMD gained from our research, especially the critical transition from aging to disease, will help define major effects that can be developed into future treatments and preventative measures. Our data will help define new endpoints for clinical trials for drugs to treat early AMD, the absence of which has impeded translational research on this prevalent cause of legal blindness. Endpoints are needed more than ever, because causal treatments targeting lipids in drusen and BrM can be pressed forward, thanks to clinical and pre-clinical proof- of-concept studies.

Vision requires an orchestrated coordination between all parts of the eye. Of all the parts, the retina is the most vital for normal perception of an image. It is a precisely layered structure lining the surface of the back of the eye, comprising many millions of cells packed together in a tightly knit network. The optic nerve connects the retina with the brain. The retina not only receives light, but also processes it, and transmits downstream signals to the midbrain and the thalamus. When the retina becomes diseased as in age-related macular degeneration (AMD), the unfortunate result can be blindness which is the most feared disability. Progress in the genetics of AMD has been substantial, yet the translation of these results has been slow to reach the clinic. Reasons for this delay include lack of suitable animal models to perform functional genetics because of anatomical differences with humans, insufficient understanding about the specific cell types involved in the initiation of AMD and an incomplete understanding of human retinal biology. It is challenging to assess if the early pathology in AMD affects diverse cell populations versus highly specific cell types. Recent technologic breakthroughs in single-cell RNA-seq (scRNA-seq) have made it possible to measure gene expression in single cells, paving the way for exploring cellular heterogeneity. Collaborating with the Alabama Eye Bank, we will deeply sample human retinal cells and RPE/choroid, fully characterize cell diversity, and elucidate the functional roles of findings from genome- wide association studies for AMD. We propose the following aims. Aim 1 will generate single and bulk RNA-seq data from eyes of 20 healthy adults, 24 early/intermediate AMD and 6 GA donors. Aim 2 will characterize cell diversity and cell gene expression in normal human retina and RPE/choroid, and compare these results to AMD eyes. Aim 3 will infer cell-type specific eQTLs and integrate these results with AMD GWAS to identify target genes. These pioneering studies leverage novel methods and interdisciplinary expertise to characterize cell type-specific gene expression in human retina and supporting tissues. By detailed characterization of the cell atlases in four geographical areas in human eye, our study will provide novel insights into cell- type specific functions that can power precision therapeutic targeting of AMD.