Although imaging of the living retina with adaptive optics scanning light ophthalmoscopy (AOSLO) provides microscopic access to individual cells, such as photoreceptors, retinal pigment epithelial cells, and blood cells in the retinal vasculature, other important cell classes, such as retinal ganglion cells, have proven much more challenging to image. The near transparency of inner retinal cells is advantageous for vision, as light must pass through them to reach the photoreceptors, but it has prevented them from being directly imaged in vivo. Here we show that the individual somas of neurons within the retinal ganglion cell (RGC) layer can be imaged with a modification of confocal AOSLO, in both monkeys and humans. Human images of RGC layer neurons did not match the quality of monkey images for several reasons, including safety concerns that limited the light levels permissible for human imaging. We also show that the same technique applied to the photoreceptor layer can resolve ambiguity about cone survival in age-related macular degeneration. The capability to noninvasively image RGC layer neurons in the living eye may one day allow for a better understanding of diseases, such as glaucoma, and accelerate the development of therapeutic strategies that aim to protect these cells. This method may also prove useful for imaging other structures, such as neurons in the brain.

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby's fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid-and protein-rich "drusen-like" composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE-ECM interface.human induced pluripotent stem cells | retinal pigment epithelium | macular dystrophies | drusen | sub-RPE deposits M aculopathies are a major cause of blindness, with agerelated macular degeneration (AMD) being the leading cause of irreversible vision loss in adults in the United States. Histopathological and clinical studies have shown that AMD and a subset of inherited macular dystrophies (MDs) share extensive phenotypic and clinical similarities (1-4). Specifically, AMD and related MDs have a cumulative etiology with adult onset of signs and symptoms and similar disease presentation including drusen formation, extracellular matrix (ECM) protein accumulation, thickening of Bruch's membrane, development of choroidal neovascularization, retinal pigment epithelium (RPE) atrophy, and ultimately the loss of vision (1-5). Although, the major pathological manifestations in these maculopathies are localized to the RPE-ECM interface in the retina, the multifactorial nature of these diseases, including the involvement of multiple retinal cell layers (photoreceptors, RPE, and choriocapillaris) (3, 6-9) and environmental risk factors (e.g., cigarette smoke) (10), has complicated the pursuit of t...

Properties of purified F1-ATPase from Escherichia coli mutant strain AN484 (uncD412) have been studied in an attempt to understand why the amino acid substitution in the beta-subunit of this enzyme causes a tenfold reduction from normal MgATP hydrolysis rate. In most properties that were studied, uncD412 F1-ATPase resembled normal E. coli F1-ATPase. Both enzymes were found to contain a total of six adenine-nucleotide-binding sites, of which three were found to be non-exchangeable and three were exchangeable (catalytic) sites. Binding of the non-hydrolysable substrate analogue adenosine 5'-[beta gamma-imido]triphosphate (p[NH]ppA) to the three exchangeable sites showed apparent negative co-operativity. The binding affinities for p[NH]ppA, and also ADP, at the exchangeable sites were similar in the two enzymes. Both enzymes were inhibited by efrapeptin, aurovertin and p[NH]ppA, and were inactivated by dicyclohexylcarbodi-imide, 4-chloro-7-nitrobenzofurazan and p-fluorosulphonyl-benzoyl-5'-adenosine. Km values for CaATP and MgATP were similar in the two enzymes. uncD412 F1-ATPase was abnormally unstable at high pH, and dissociated into subunits readily with consequent loss of activity. The reason for the impairment of catalysis in uncD412 F1-ATPase cannot be stated with certainty from these studies. However we discuss the possibility that the mutation interrupts subunit interaction, thereby causing a partial impairment in the site-site co-operativity which is required for 'promotion' of catalysis in this enzyme.

Morgan and colleagues demonstrated that the RPE cell mosaic can be resolved in the living human eye non-invasively by imaging the short-wavelength autofluorescence using an adaptive optics (AO) ophthalmoscope. This method, based on the assumption that all subjects have the same longitudinal chromatic aberration (LCA) correction, has proved difficult to use in diseased eyes, and in particular those affected by age-related macular degeneration (AMD). In this work, we improve Morgan's method by accounting for chromatic aberration variations by optimizing the confocal aperture axial and transverse placement through an automated iterative maximization of image intensity. The increase in image intensity after algorithmic aperture placement varied depending upon patient and aperture position prior to optimization but increases as large as a factor of 10 were observed. When using a confocal aperture of 3.4 Airy disks in diameter, images were obtained using retinal radiant exposures of less than 2.44 J/cm 2 , which is ~22 times below the current ANSI maximum permissible exposure. RPE cell morphologies that were strikingly similar to those seen in postmortem histological studies were observed in AMD eyes, even in areas where the pattern of fluorescence appeared normal in commercial fundus autofluorescence (FAF) images. This new method can be used to study RPE morphology in AMD and other diseases, providing a powerful tool for understanding disease pathogenesis and progression, and offering a new means to assess the efficacy of treatments designed to restore RPE health. L. Dunaief, J. Z. Baffi, and J. Ambati, "DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration," Nature 471(7338), 325-330 (2011).

PurposeTo characterize in vivo morphometry and multispectral autofluorescence of the retinal pigment epithelial (RPE) cell mosaic and its relationship to cone cell topography across the macula.MethodsRPE cell morphometrics were computed in regularly spaced regions of interest (ROIs) from contiguous short-wavelength autofluorescence (SWAF) and photoreceptor reflectance images collected across the macula in one eye of 10 normal participants (23–65 years) by using adaptive optics scanning light ophthalmoscopy (AOSLO). Infrared autofluorescence (IRAF) images of the RPE were collected with AOSLO in seven normal participants (22–65 years), with participant overlap, and compared to SWAF quantitatively and qualitatively.ResultsRPE cell statistics could be analyzed in 84% of SWAF ROIs. RPE cell density consistently decreased with eccentricity from the fovea (participant mean ± SD: 6026 ± 1590 cells/mm2 at fovea; 4552 ± 1370 cells/mm2 and 3757 ± 1290 cells/mm2 at 3.5 mm temporally and nasally, respectively). Mean cone-to-RPE cell ratio decreased rapidly from 16.6 at the foveal center to <5 by 1 mm. IRAF revealed cells in six of seven participants, in agreement with SWAF RPE cell size and location. Differences in cell fluorescent structure, contrast, and visibility beneath vasculature were observed between modalities.ConclusionsImprovements in AOSLO autofluorescence imaging permit efficient visualization of RPE cells with safe light exposures, allowing individual characterization of RPE cell morphometry that is variable between participants. The normative dataset and analysis of RPE cell IRAF and SWAF herein are essential for understanding microscopic characteristics of cell fluorescence and may assist in interpreting disease progression in RPE cells.