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Lipofuscin: The “Wear and Tear” Pigment

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).
TABLE 1.1 Occurrences of LPs (Lipofuscin/Ceroid)
Aging Best disease

Age-related macular degenerationStargardt disease

Neuronal ceroid lipofuscinoses Maternal inherited diabetes and deafness

Mucolipodosis IV Choroideremia

MPS III Sanfilippo disease Osteopetrosis with neuronal storage disease

Alzheimer disease Adult-onset glycogen storage disease type 2

Retinitis pigmentosa Macular ABCA4 disease

Cone and cone-rod dystrophy Wilson disease

X-linked retinoschisis Crohn disease

Leber congenital amaurosis Choroidal tumors

Pattern dystrophy

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.
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
Lipofuscin Ceroid

Location Primarily lysosome Primarily lysosome

Spectral properties (nm)

Excitation 320-460 320-460

Emission 460-630 460-630

Storage components

Proteins heterogeneous mix AβPP subunit C mitochondria ATPase saposins A & D, AβPP

Lipids triglycerides, cholesterol phospholipids, free fatty acidsphosphorylated dolichols, phospholipids neutral lipids

Carbohydrates heterogeneous mix dolichol-linked oligosaccharides

Metals Fe, Cu, Al, Zn, Mn, Ca predominantly Fe

Staining characteristics

Sudan black B Yes Yes

Periodic Schiff base Yes Yes

Lectin concanavalin A concanavalin A, agglutinin

Ultrastructures GRODS GRODs, fingerprint, curvilinear

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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