Biology Cell and Physiology, Biochemistry,•tinal3 rchapteR

Figure 3.4  Autofluorescence imaging of patient with Stargardt’s macular dystrophy showing central atrophy (dark area) with surrounding increased fluorescence spots of lipofuscin accumulation.

Stargardt’s disease and lipofuscin

Stargardt’s disease, a macular degeneration of juvenile onset, is caused by mutations in both alleles of the ABCA4 (formerly known as ABCR) gene. ABCA4 is a transporter protein called ATP-binding cassette transporter and is expressed in the discs of the outer segments of the rods and cones. In the visual cycle, the all-trans retinal formed in the lumen of the disc interacts with phosphatidylamine to form NRPE. The NRPE is flipped across the disc membrane by ABCA4 to be reduced to all-trans retinol in the cytosol of the rods. It acts as a flippase for NRPE. So, a loss of ABCA4 function leads to accumulation of NRPE and all-trans retinal in the lumen of the discs, with a consequent increase in the formation of lipofuscin (Figure 3.4). Pathologically, Stargardt’s disease is associated with accumulation of excessive lipofuscin in the RPE, accounting for the dark choroid on fundus fluorescein angiography. Work with a mouse model of Stargardt’s disease, the ABCA4 knockout mouse, has shown that the abundant A2E levels that accompany a loss of ABCA4 activity predispose to RPE atrophy. Short-wavelength visible light adversely affects this disease due to the RPE cell damage caused by the light-assisted generation of photoxidation of A2E.

Age-related macular degeneration and lipofuscin

Lipofuscin in the RPE has been implicated in the pathogenesis of atrophic age-related macular degeneration. In addition to the causes of atrophy mentioned above, photo-oxidation products of A2E may interact with complement at cell surface and activate the alternate pathway of the complement system in the pathogenesis of age-related macular degeneration. The interplay of photo-oxidation of lipofuscin, genetic susceptibility, and environmental factors may play a role in inducing low-grade inflammation in the pathogenesis of age-related macular degeneration. The accumulation of lipofuscin is also shown to activate retinoic acid receptor genes and vascular endothelial growth factor expression that increases the risk of choroidal neovascularization.

MATRIX BIOLOGY

STRUCTURAL COMPOSITION OF THE BRUCH’S MEMBRANE

The BM is a thin (2–4 m) connective tissue interposed between the metabolically active RPE and its source of nutrition, the choriocapillaris.

This pentalaminar structure is classically described as consisting of the basement membrane of the RPE, an inner collagenous zone (ICZ), a fenestrated elastic layer, an outer collagenous zone (OCZ), and the basement membrane of the endothelium of the choriocapillaris.

MACROSCOPIC CHANGES OF THE BRUCH’S MEMBRANE

The fundamental age-related change of BM is increased thickness: thickness is reported to increase by 135% in ten decades. The maximum thickness occurs in the substrata of the OCZ. Disease risk is associated with age-related thickness of the BM. A prolonged choroidal filling phase during fluorescein angiography signals the presence of diffuse BM thickening. In addition, serial measurement of the thickness of BM may be a useful prognostic parameter in longitudinal studies of agerelated macular degeneration.

CELL BIOLOGY OF BRUCH’S MEMBRANE

The three anatomical changes that occur in BM with age are the progressive accumulation of debris, lipid deposition, and alteration of the ECM. The deposition of periodic acid–Schiff-positive granular, vesicular, and filamentary deposits in the ICZ of BM has been identified as early as the first decade of life. As age advances, the debris accumulates and contaminates all the collagenous layer of the BM, and is seen on both sides of the elastic lamina, thus forming the bulk of the age-related debris in the collagenous layers, especially in the OCZ. Depending on their proximity to the RPE, deposits may represent incompletely digested waste material emanating from a dysfunctional RPE, an inappropriately directed immune response, or altered remodeling of the membrane.

The proteins identified in the drusen/BM include both locally derived (neural retina, RPE, and choroid) and extracellular nonocular components. The oxidative modifications of some of these components may also be the primary catalyst in drusen formation. The complement system is now known to play a role in the pathogenesis of age-related macular degeneration.

LIPID ACCUMULATION

An age-related exponential accumulation of lipids occurs in the BM. The predominant lipids in the BM consist of phospholipids and fatty acids. About 50% of the phospholipids are phosphatidylcholine, suggesting that the lipids in the BM are more likely of a cellular origin (a potential source being the photoreceptor outer-segment membranes) than from plasma. However, studies using filipin histochemistry and hot-stage polarizing microscopy revealed that lipid content of BM consists of both esterified and unesterified cholesterol, suggesting a vascular origin akin to atherosclerosis.

MATRIX DYSREGULATION

Collagen synthesis increases with age in BM. Newsome et al. found an age-related increase in collagen I in the BM with age.11 Other collagens noted in the BM include types III, IV, and V. In addition, atypical banding periodicity has been observed in the collagen produced. The ICZ consists more of 640A type collagen while the OCZ contains more 1000A type collagen with age. The meshes formed by the tightly interwoven collagen fibers in the ICZ also become irregular and coarse with age. The long spacing collagen is a material with periodicity ranging between 100 and 140 nm found mainly in the OCZ and which extends as intercapillary pegs to areas where choriocapillaris have undergone age-related atrophy. The increased insoluble collagen may contribute to debris accumulation and serve as a depot for lipoproteins, growth factors, and cytokines.

The amount of noncollagen protein in BM also increases significantly with age, as suggested by an increase in deposition of noncollagen