Lipofuscin: The “Wear and Tear” Pigment
Sabrina S. Seehafer
David A. Pearce
Many substances fluoresce spontaneously (autofluoresce, i.e., emit light of a particular wavelength) when illuminated by light of a different wavelength. Pathologists are well used to observing autofluorescence (AF) in certain cells, such as macrophages, when viewing specimens with a cobalt blue light filter in the microscope. More recently, ophthalmologists have become accustomed to visualizing AF in images of the fundus. This book reviews the basic science and clinical knowledge regarding fundus AF in the human eye, a phenomenon that was first noted when fluorescein angiographic imaging of the eye was introduced and has become more clearly evident with the development of scanning laser ophthalmoscopy. AF is thought to be due to lipofuscin present in cells of the retina, especially retinal pigment epithelial cells. This first chapter serves as an introduction to the biochemistry and mechanisms behind the accumulation of lipofuscin and other autofluorescent storage material in tissues, with a particular focus on the central nervous system (CNS).
Lipofuscin, commonly referred as the “wear and tear” pigment, is an autofluorescent storage material that accumulates as a result of cell senescence. Lipofuscin has also been termed lipopigment (LP), autofluorescent storage material, yellow-brown material, and aging pigment. Although all cells accumulate lipofuscin, it is seen in the highest quantity in tissues or cells that are postmitotic, such as neurons, retina, and muscle. However, aging is not the only phenomenon associated with accumulation of autofluorescent storage material. Autofluorescent LPs have also been shown to accumulate as a result of pathological conditions, in which case the autofluorescent storage material is known as ceroid. Such conditions include the pediatric neurodegenerative disorders called neuronal ceroid lipofuscinoses (NCLs). This distinction between ceroid (AF material that accumulates in disease) and lipofuscin (AF material that accumulates as a result of aging), however, is not generally used in ophthalmology. Both ceroid and lipofuscin have been shown to primarily accumulate in the lysosome; however, they have also been shown to accumulate in vesicles, in the cytoplasm, and in the perikaryon of neurons. Table 1.1 lists different disease states and aging pathologies reported to involve an accumulation of lipofuscin/ceroid. The mechanisms and biochemistry of lipofuscin will be primarily discussed in the context of LP accumulation in all tissue types; in the case of ceroid, the focus will be on the CNS.
All types of autofluorescent LPs were originally described as autofluorescent material in postmortem tissue. All autofluorescent storage materials are not identical. The LPs are often defined based on their fluorescent spectral properties, i.e., the wavelength of light use to excite the intrinsic fluorophore (excitation) and the light emitted as a result of this initial excitation (emission). Lipofuscin has a yellow-brown appearance and a wide range of spectral properties, with excitation wavelengths of 320-460 nm and emission wavelengths of 460-630 nm (1). A detailed
description of the spectral characteristics of retinal lipofuscin can be found in Chapter 2. The most marked signal for the autofluorescent storage material is seen under far-ultraviolet excitation. Ceroid from NCLs has excitation and emission wavelengths similar to those of lipofuscin, with an excitation maximum of 460 nm and an emission maximum of 539 nm (2). The range of excitation and emission wavelengths for both lipofuscin and ceroid reflects the different methods of measurement used, different types of tissue studied, and different corrections for spectrum. It is important to use age-matched controls in studies of LP biology to distinguish between the accumulation of lipofuscin (the result of aging) and LPs (the result of different diseases).
description of the spectral characteristics of retinal lipofuscin can be found in Chapter 2. The most marked signal for the autofluorescent storage material is seen under far-ultraviolet excitation. Ceroid from NCLs has excitation and emission wavelengths similar to those of lipofuscin, with an excitation maximum of 460 nm and an emission maximum of 539 nm (2). The range of excitation and emission wavelengths for both lipofuscin and ceroid reflects the different methods of measurement used, different types of tissue studied, and different corrections for spectrum. It is important to use age-matched controls in studies of LP biology to distinguish between the accumulation of lipofuscin (the result of aging) and LPs (the result of different diseases).
TABLE 1.1 Occurrences of LPs (Lipofuscin/Ceroid) | ||||||||||||||||||||||
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Other properties examined in characterizing LPs are histochemical staining techniques, such as differential dyes, lectin binding, and ultrastructure analysis. Tissue sections for both NCLs and aging brains have been shown to stain with periodic acid Schiff, a carbohydrate stain, and Sudan black, a lipid stain (3, 4, 5). Lectin histochemistry has been shown to distinguish between ceroid in NCLs and lipofuscin in the aging brain, with both LPs binding concanavalin A, but only ceroid in NCL brain tissue binding to agglutinin (6).
Electron microscopy (EM) has also been carried out on LPs to determine their ultrastructure. It has been shown by EM that lipofuscin-loaded tissue has granular osmophilic deposits (GRODs) that appear as very densely packed vesicles with dark granules filling the entire vesicle (3,5). For ceroid from NCLs, EM has shown GRODS identical to lipofuscin (5). However, two unique ultrastructures are also found only in the ceroid: fingerprint and curvilinear profiles. Curvilinear profiles are vesicles that have an amorphous arrangement of lamellar structures forming C- or S-shaped forms. Fingerprint profiles also have lamellar structures in the vesicles; however, the arrangement is in swirling circles, similar to the skin on a fingertip, and has a more dense arrangement of the lamellar structures.
Although considerable work has been done to characterize lipofuscin/ceroid at the microscopic level, the basic components of lipofuscin and its pathological counterpart remain to be determined. Various studies have shown that lipofuscin is composed of 19% to 51% lipids and 30% to 58% proteins (reviewed in Ref. 7). Further examination of lipofuscin has shown that the lipid component consists of triglycerides, cholesterol, phospholipids, and free fatty acids. The protein component is a heterogeneous mixture of proteins, with only one identified component: amyloid β-precursor protein (AβPP) (8). The carbohydrate component of lipofuscin is also a heterogeneous mixture (9). Iron, copper, aluminum, zinc, calcium, and magnesium account for approximately
2% of the lipofuscin components (10). Retinal specific lipofuscin has been shown to have a very particular composition (11,12) (see Chapter 2). One study of lipofuscin isolated from retinal pigment epithelium (RPE) demonstrated that the components were highly damaged by peroxidation and glucoxidation (13). These components were specifically damaged at lysine and cysteine adducts, such as malondialdehyde (MDAs) and 4- hydroxynonenal (HNE). Moreover, the same study identified advanced glycation end products (AGEs). This and other studies suggest that the components of lipofuscin are highly modified by oxidative stress.
2% of the lipofuscin components (10). Retinal specific lipofuscin has been shown to have a very particular composition (11,12) (see Chapter 2). One study of lipofuscin isolated from retinal pigment epithelium (RPE) demonstrated that the components were highly damaged by peroxidation and glucoxidation (13). These components were specifically damaged at lysine and cysteine adducts, such as malondialdehyde (MDAs) and 4- hydroxynonenal (HNE). Moreover, the same study identified advanced glycation end products (AGEs). This and other studies suggest that the components of lipofuscin are highly modified by oxidative stress.
For ceroid in the NCLs, except for the infantile variant, it has been shown that the primary protein component (50%) is the subunit c of mitochondrial ATPase. In the infantile variant of NCL, the primary component is composed of sphingolipid activating proteins (saposins/SAPs) A and D (14, 15, 16, 17, 18). In other NCL variants, ceroid contains SAPs A and D, but not to the extent of subunit c accumulation. Other identified components include AβPP, dolichol pyrophosphate-linked oligosacharides, lipid-linked oligosaccharides, and metals (primarily iron) (16,19, 20, 21). Although all LP components vary in terms of the types of autofluorescent storage material and tissue, they all appear to be composed of undegraded or partially degraded proteins.
To date, very little is known about the fluorescent components (fluorophores) in most LPs that generate the spectral properties of the autofluorescent storage material. Some have hypothesized that the fluorophore is a single compound. However, the fluorescent signal may also be generated after interactions between several different nonfluorescent molecules. It has also been hypothesized that the fluorescence comes from lipid oxidation; however, some favor the hypothesis that modifications to the stored proteins result in the fluorescence. It is certain that the ranges of spectral properties reported for autofluorescent material make identification of a single fluorophore challenging. Isolation of lipofuscin/ceroid has also proven problematic, with spectral properties decreasing or attenuating during the isolation process. In vitro studies have shown that reactions between carbonyls and amino compounds that produce Schiff bases such as 1,4 dihydropyridine and 2-hydroxy-1,2-dihydropyrrol-3-ones demonstrate natural lipofuscin-like spectral properties (reviewed in Ref. 22). In retinal lipofuscin, it has been shown that the major blue absorbing fluorophore is pyridinium bisretinoid (A2E) (23,24) (see Chapter 2). Ceroid is similar to lipofuscin outside of the retina and currently has no identified fluorophore. Furthermore, it cannot be excluded that each type of ceroid or lipofuscin might have a specific fluorophore, or multiple fluorophores with overlapping spectral properties that result in the overall autofluorescent signal. Numerous studies have examined the biochemical properties of LPs (Table 1.2), but the larger question is, Why does this autofluorescent storage material accumulate?
MECHANISMS OF LIPOFUSCIN ACCUMULATION
Three different mechanisms have been proposed for accumulation of lipofuscin/ceroid: lysosomal dysfunction, autophagy, and cellular stress. The accumulation of LP at the lysosome implies an underlying lysosomal dysfunction that results in a buildup of lipofuscin/ceroid. Autophagy, the major degradation/recycling pathway, could be altered, leading to LP/ceroid deposition. Cellular stresses in the form of oxidative stress or starvation could have an impact on the cell physiology and result in lipofuscin/ceroid accumulation.
TABLE 1.2 Biochemical Properties of LPs | |||||||||||||||||||||||||||||||||||||||
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Lysosomal Dysfunction As a Cause for Lipofuscin Accumulation
A large percentage of lipofuscin/ceroid has been shown to accumulate in a specific organelle, the lysosome. There are two possible fundamental mechanisms that could result in accumulation of LP in the lysosomes: substrate accumulation due to mutation/dysfunction in enzymes, or an imbalance in lysosomal homeostasis resulting in an altered lysosomal environment changing multiple enzyme activities/functions. In the case of the NCLs, three variant diseases are caused by mutations in lysosomal enzymes: congenital, infantile, and late infantile NCL. These diseases are caused by mutations in the lysosomal enzymes cathepsin D, palmitoyl protein thioesterase 1, and tripeptyl protease, respectively (reviewed in Ref. 25). However, not all of the autofluorescent storage material can be accounted for by such specific enzymatic defects. Undefined ways to affect lysosomal enzymes, such as alterations in lysosomal homeostasis, are also likely. Other NCLs have defects in proteins that have not yet been assigned a definitive function. Juvenile NCL (JNCL) has a defect in the CLN3 protein, which resides in the lysosomal/late endosomal membrane (reviewed in Ref. 26). In fibroblasts from patients with JNCL, a decrease in lysosomal pH was observed. Most recently, the pH of lysosomes was shown to be regulated by a membrane channel protein (TRP-ML1). Defects in this protein lead to lysosomal accumulation of lipid deposits (27), so there may be a general mechanism at fault in the accumulation of lysosomal material related to the intralysosomal milieu. This shift in intralysosomal conditions may not be optimal for enzymatic activity. Suboptimal lysosomal enzyme activity could potentially underlie the accumulation of lipofuscin/ceroid (28). In support of this are studies demonstrating that administration of leupeptin, a general lysosome inhibitor, or chloroquine, an amine that raises lysosomal pH to an alkaline environment, in rats resulted in accumulation of lipofuscin-like autofluorescent storage material in brain tissue and hepatocytes (29, 30, 31, 32) (see also Chapter 4).
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