Retinal Remodeling and Metabolic Alterations in Human AMD

Abstract

Age-related macular degeneration (AMD) is a progressive retinal degeneration resulting in central visual field loss, ultimately causing debilitating blindness. AMD affects 18% of Americans from 65 to 74, 30% older than 74 years of age and is the leading cause of severe vision loss and blindness in Western populations. While many genetic and environmental risk factors are known for AMD, we currently know less about the mechanisms mediating disease progression. The pathways and mechanisms through which genetic and non-genetic risk factors modulate development of AMD pathogenesis remain largely unexplored. Moreover, current treatment for AMD is palliative and limited to wet/exudative forms. Retina is a complex, heterocellular tissue and most retinal cell classes are impacted or altered in AMD. Defining disease and stage-specific cytoarchitectural and metabolic responses in AMD is critical for highlighting targets for intervention. The goal of this article is to illustrate cell types impacted in AMD and demonstrate the implications of those changes, likely beginning in the retinal pigment epithelium (RPE), for remodeling of the the neural retina. Tracking heterocellular responses in disease progression is best achieved with computational molecular phenotyping (CMP), a tool that enables acquisition of a small molecule fingerprint for every cell in the retina. CMP uncovered critical cellular and molecular pathologies (remodeling and reprogramming) in progressive retinal degenerations such as retinitis pigmentosa (RP). We now applied these approaches to normal human and AMD tissues mapping progression of cellular and molecular changes in AMD retinas, including late-stage forms of the disease.

Keywords: Müller cell; age-related macular degeneration (AMD); computational molecular phenotyping (CMP); neural remodeling; photoreceptor; retina; retinal pigment epithelium (RPE); retinal remodeling.

Figure 1

Normal aged, peripheral human retina. (A) γ.G.E → rgb mapping of human peripheral retina from a 78 year old male with no diagnosis of AMD. Normal topology and stratification of the retina is observed, but the earliest indications of potential pathology are present in the bricking of the retinal pigment epithelium (RPE), a common finding in aged, human retina. Also, note the existence of a small druse, labeled with asterisk. (B) τ.Q.E → rgb mapping revealing Müller glial populations in yellow. Scale bar = 40 μM.

Figure 2

Human parafoveal retina from a 76 year old patient with advanced AMD showing taurine labeling in the RPE internal to the vascular choroid (Ch). Normal levels of taurine in the RPE are typically high and uniform. As RPE cells experience cell stress, they adopt a “bricked” or non-uniform appearance suggesting that they are becoming uncoupled. While this pathology is present to a limited degree in ostensibly normal aged human retina, it becomes much more dramatic in regions of obvious AMD related pathology. The white spots in the RPE likely indicate regions of lipid accumulation and could represent lipofuscin/melanolipofuscin granules. Also of note, are the accumulations of intensely taurine positive deposits underneath the RPE and above the photoreceptor outer segments (arrows). These deposits are not apical processes of the RPE as they have much higher concentrations of taurine than do the RPE cells. Scale bar = 40 μM.

Figure 3

Peripheral retina from a 71 year old AMD patient demonstrating small and mid-size drusen (arrows), and RPE bricking particularly underneath large druse. (A) γ.G.E → rgb. (B) Post-mortem ex-vivo OCT data demonstrating correlates with histology in (A,C,D) with inset demonstrating higher magnification views of RPE histology shown in rectangle. (C) Taurine labeling demonstrating RPE bricking. (D) Glutamate labeling demonstrating RPE bricking and altered cone photoreceptors (boxes) revealing cones with low glutamate concentrations. Scale bar = 80 μM.

Figure 4

(A) RPE from a 75 year old male with no history of AMD. (B) A 76 year old male with AMD and (C) mature male Papio anubis. The RPE is shown in each column labeled for taurine (τ), glutamine (Q), glutathione (J), GABA (γ), CRALBP and glutamate (E). This image most notably demonstrates the variability in taurine content of the RPE cells compared with normal human and non-human primate but also demonstrates the lack of staining in GABA of the RPE indicating that any pigment granules in the RPE do not alter or influence small molecule epitope detection. Also, note that CRALBP is upregulated over the normal human in AMD and that a subset of the taurine deposits differentially contains glutamate as well. Scale bar = 80 μM.

Figure 5

(A) Parafoveal retina from a 71 year old male patient, 2 h 41 min post mortem with a diagnosis of AMD labeled for rhodopsin demonstrating extensive rod opsin delocalization in the rod photoreceptors down around the inner segments and cell bodies. Asterisks denote small possible subretinal drusenoid desposits (SDD) or aberrant apical processes of RPE deforming the tips of the outer segments of photoreceptors. Notably, no rhodopsin buildup is occurring underneath the RPE as occurs in many retinitis pigmentosa (RP) diseases. (B) Demonstrating a slightly more oblique section of rhodopsin labeling in a normal parafoveal non-human primate retina revealing no rhodopsin delocalization. Scale bar = 8 μM.

Figure 6

Parafoveal retina from a 76 year old patient with AMD demonstrating CRALBP.rg-opsin.τ → rgb in (A), taurine labeling in (B), glutamate labeling in (C) and cone opsin in (D). This image composite shows RPE bricking as well as cone opsin bounded taurine and glutamate rich deposits underneath the RPE. Cone opsin delocalization is also shown in (A,D). Scale bar = 80 μM.

Figure 7

Graphs demonstrating glutathione concentration in the inner segments of cone photoreceptors over retinal eccentricity in four example patients, normal, early-AMD, early wet-AMD, with neovascularization present, but prior to large scale bleeds in the retina and late wet-AMD with prevalent evidence of resolved retinal bleeding. Normal glutathione concentration is very low, but becomes dramatically variable in central retina of wet-AMD. Each plot represents data from a single individual (n = 1) of normal, early, dry-AMD, early wet-AMD and late we-AMD.

Figure 8

Two retinas from 75 year old patients with early dry-AMD in (A–C) with J.Q.DAPI → rgb in (A), glutathione labeling in (B), glutamine labeling in (C), and mid to late stage wet-AMD in (D–F) with γ.G.E → rgb shown in (D), glutathione in (E) and glutamine in (F). Boxes (higher magnification in G,H) show Müller glia that has dramatically elevated glutathione and glutamine signals in isolated or groups of glia. In the case of wet-AMD (D–F), the ONL is dramatically thinner than in the early dry-AMD retina (A–C) and appears much more distinct because of this loss of photoreceptor cell nucleii. (G,H) Show increased magnification views of the boxes in (B,C,E,F) for glutathione and glutamine channels respectively. Scale bar = 80 μM. Asterisks demonstrate drusen (*).

Figure 9

Peripheral retina from late stage dry AMD patient with drusen (*) demonstrating hypertrophy and metabolic alterations in Müller glial cells. (A) Shows γ.G.E → rgb and (B) shows τ.Q.E → rgb. The inset rectangles demonstrate magnified regions shown in (C,D) revealing GABAergic labeling of aberrant GABAergic processes (arrows) outside the normal lamination of the IPL in (C) and glycinergic labeling of a misplaced glycinergic amacrine cell in (D) demonstrating clear plasticity and remodeling in inhibitory neuronal classes. Note, in the post-mortem state, GABA and glycine increase in the Müller cells. Scale bar = 200 μM in (A,B). Scale bar = 40 μM in (C,D).

Figure 10

Early stage dry AMD patient with very small drusen showing in τ.Q.J → rgb labeling, (A) and τ.Q.J → rgb overlay on top of transmission electron microscopy (TEM) imagery in (B) with pure TEM imagery in (C). Black arrows demonstrate RPE cells with varying taurine, glutamine and glutathione concentrations. Asterisks demonstrate cone photoreceptors with altered metabolic signatures showing decreases in taurine, and elevations in glutamine. Vertical white arrows denote the presence of a small druse. Scale bar = 8 μM.