Mitochondrial alterations of retinal pigment epithelium in age-related macular degeneration

Abstract

Mitochondrial dysfunctions have been implicated in the pathophysiology of several age-related diseases including age-related macular degeneration (AMD), a progressive neurodegenerative disease affecting primarily the retinal pigment epithelium (RPE). The aims of our electron microscopic and morphometric studies were to reveal qualitative and quantitative alterations of mitochondria in human RPE from AMD and from age- and sex-matched controls. With increasing age a significant decrease in number and area of mitochondria, as well as loss of cristae and matrix density were found in both AMD and control specimens. These decreases were significantly greater in AMD than in normal aging. Alterations of mitochondria were accompanied by proliferation of peroxisomes and lipofuscin granules in both AMD and control specimens, although the difference between groups was significant only for peroxisomes. Unexpectedly, morphometric data showed that the RPE alterations seen in AMD may also develop in normal aging, 10–15 years after appearing in AMD patients. These findings suggest that (i) the severity of mitochondrial and peroxisomal alterations are different between AMD and normal aging, and (ii) the timing of damage to RPE may be critical for the development of AMD. We conclude that besides the well-documented age-related changes in mitochondrial DNA, alterations of mitochondrial membranes may also play a role in the pathogenesis of AMD. These membranes could be a new target for treatment of AMD and other age-related diseases.

Introduction

Age-related macular degeneration (AMD) is a progressive neurodegenerative disease of the central retinal area (macula lutea), and it represents the most common cause of legal blindness in industrialized countries [26], [45]. Epidemiologic studies from several countries also showed dramatic increases in prevalence and in severity of AMD with age. For example, early retinal alterations were detected in 6–15% of patients in the sixth decade and 30–60% in their eighth decade of which 8.5% were blind [75]. Clinically, early AMD (dry form), synonymously known as age-related maculopathy, is characterized by the presence of small, yellowish deposits (drusen) under the retinal pigment epithelium (RPE), accompanied with either loss or focal accumulation of melanin pigment. Although the affected subjects usually have good visual acuity, they often complain of worsened quality of vision [51]. Progression of early AMD may lead to (i) the atrophic form in which the choriocapillaries and RPE-photoreceptor complex atrophies at the macular area or (ii) exudative or wet form in which exudation and hemorrhage into the subretinal space occurs, finally terminating with chorioretinal scar formation and neovascularisation. Both atrophic and exudative forms are associated with severe impairment of visual functions [11].

AMD is a multifactorial disease that affects primarily the RPE, a specialized glial tissue that provides metabolic support to both photoreceptor cells and the thin connective tissue layer (Bruch's membrane) interposed between the RPE and choriocapillaries [12]. The current pathophysiologic concept on AMD assigns a primary role to the age-related, cumulative oxidative damage to the RPE due to an imbalance between generation and elimination of reactive oxygen species (ROS) [7], [19], [77]. Specifically, lipofuscin, a byproduct of photoreceptor outer segment turnover, has been hypothesized to be the primary source of ROS responsible for both cellular and extracellular matrix alterations in AMD [36], [39], [78]. Lipofuscin is a heterogeneous material composed of a mixture of lipids, particularly lipid peroxides, proteins, and different fluorescent compounds derived mainly from vitamin A. It is frequently referred to as “age-pigment” because it accumulates in an age-dependent manner. Accumulation of lipofuscin, other lipid peroxides, and potentially toxic substances may dramatically influence the RPE physiology. In vitro it greatly reduces the phagocytic capacity, lysosomal enzyme activities and antioxidant potential of human RPE [63], [69]. N-Retinylidene-N-retinylethanolamine (A2E), the major autofluorescent component of lipofuscin, does not alter early steps of phagocytosis in cultured human RPE, but it inhibits the complete digestion of phospholipids [23]. Moreover, the autophagic sequestration of cytoplasmic material, i.e., turnover, is also markedly reduced after A2E loading [9]. In vivo studies support the in vitro effects of lipofuscin on RPE metabolism. A transgenic mouse model with impaired processing of phagocytized photoreceptor outer segments accumulates lipofuscin and manifests several clinical and histological features of AMD [58]. A2E also specifically targets cytochrome c oxidase [62], [70], and it causes caspase activation and RPE cell apoptosis [39], [78]. Thus lipofuscin is thought to be responsible for oxidative damage to RPE resulting in impaired metabolism and apoptosis characteristic for late AMD [19].

Mitochondria play a central role in aging and in the pathogenesis of age-related neurodegenerative diseases [47]. Earlier studies revealed several abnormalities of mitochondrial DNA (mtDNA) and subsequent disorders of respiratory enzyme complexes accompanied by reduced energy production, generation of excessive ROS, and activation of the apoptosis pathway [67]. However, recent studies suggest that mitochondrial dysfunctions may also include several other processes in which compositional and structural alterations of mitochondrial membranes (mtMEM) are primarily involved [38], [72]. Alterations in membrane lipid composition, such as decreases in cardiolipin content or changes in the omega-3:omega-6 ratio, impair the electron transport chain and energy production [55], ion channel and Ca²⁺ homeostasis of the mitochondria and of the cell [16], [28], [61], [68]. These alterations can also impair carnitine-mediated lipid transport, mitochondrial lipid metabolism [5], [79], cholesterol biosynthesis [6], [14], [31], [54], activity of pyruvate dehydrogenase complex [18], [60], and finally, cause activation of the apoptosis pathway [71].

Mutations of mtDNA and decreases in RPE cell number during aging and AMD have been described [3], [4], [17], [49]; however, neither alterations of mtMEM in aging nor a role in AMD have been elucidated. In the present paper, detailed electron microscopy of human RPE cells from aged and AMD specimens are described. Based on morphometric data, we found that (i) progressive deterioration of mtMEM with aging occurred in association with peroxisome proliferation and accumulation of lipofuscin in the RPE, and (ii) alterations of the RPE mtMEM and proliferation of peroxisomes were significantly more severe in AMD compared to normal aging.