David N. Palmer

Storage of saposins A and D in infantile neuronal ceroid-lipofuscinosis

We have isolated storage cytosomes from brain tissue of patients with infantile neuronal ceroid‐lipofuscinosis. The purified storage bodies were subjected to compositional analysis which revealed a high content of proteins, accounting for 43% of dry weight. Saposins A and D, also known as sphingolipid activator proteins (SAPs), were shown to constitute a major portion of the accumulated protein using gel electrophoresis and sequence analysis. This is the first time that saposins have been found to be stored in any form of neuronal ceroid‐lipofuscinosis.

Separation of some neutral lipids by normal-phase high-performance liquid chromatography on a cyanopropyl column: Ubiquinone, dolichol, and cholesterol levels in sheep liver

The normal-phase high-performance liquid chromatographic separation of neutral lipids into molecular classes was carried out on a cyanopropyl (CN) column eluted with isopropanol in hexane. Cholesteryl, retinyl, and dolichyl esters, triglycerides and vitamin E, ubiquinone, dolichol, phytol, and cholesterol eluted as separate peaks with 0.05% isopropanol in hexane. Cholesterol, retinol, diglyceride, and monoglyceride eluted as separate peaks with 0.75% isopropanol in hexane. These separations could not be achieved on a silica gel column. The method was used to assay sheep liver ubiquinone, dolichol, and cholesterol levels, that were determined as 77, 108 and 1864 micrograms/g wet wt, respectively.

The analytical approach to the nature of lipofuscin (age pigment)

Analytical studies of three lipopigments show that much can be achieved. Lipopigment from ovine ceroid-lipofuscinosis is composed of discrete protein and lipid molecules in orderly arrays and lipid peroxidation is not involved in its formation. Subunit c of mitochondrial ATP synthase accounts for approximately 50% of accumulated material and is specific to the disease process in this and other forms of the disease. Lipofuscin from bovine heart was mostly soluble and also contained discrete proteins, lipids and metals. Equine thyroid lipofuscin was less soluble but also had a relatively high protein content, probably derived from thyroglobulin. Although sugar could not be measured quantitatively, staining reactions and elemental analyses suggested it could also be a significant component. Some may be present as derivatives in the form of advanced glycation products. It is proposed that protein, the dominant molecular species present, is the important constituent in lipofuscinogenesis rather than lipid peroxidation. Whereas this latter may play some part in the maturation of lipofuscin, this has not been shown experimentally and is not likely to be the initiating mechanism.

Bovine ceroid-lipofuscinosis (Batten's disease): The major component stored is the DCCD-reactive proteolipid, subunit c, of mitochondrial ATP synthase

The ceroid-lipofuscinoses (Battens disease) are a group of recessively inherited lysosomal storage diseases of children and animals in which there is intracellular accumulation of a fluorescent lipopigment in a wide variety of cells. Lipopigment bodies isolated from pancreas, liver, kidney and brain tissue from a heifer affected with ceroid-lipofuscinosis contained between 55 and 62% protein. A dominant component comigrated on LDS-PAGE with the major low molecular weight protein stored in ovine ceroid-lipofuscinosis. It was identified by amino acid sequence and mass spectroscopy as the full subunit c of mitochondrial ATP synthase, normally found only in the inner mitochondrial membrane, where it is estimated to account for 2–4% of the membrane protein. In pancreatic lipopigment it accounted for at least 40% of the total lipopigment mass and this storage was considered specific to the disease. No other mitochondrial proteins were found in storage bodies. These results are similar to those found in studies on the ovine and the late infantile and juvenile human forms of the disease. It is concluded that bovine ceroid-lipofuscinosis is also a proteolipid proteinosis in which subunit c of mitochondrial ATP synthase is specifically stored in lysosome derived organelles.

Phospholipid fatty acids in brains of normal sheep and sheep with ceroid-lipofuscinosis.

The ceroid-lipofuscinoses are a group of inherited diseases of humans and animals characterised by brain atrophy and the storage of a fluorescent lipopigment. Brain grey matter phospholipid fatty acids of diseased sheep are compared with those of normal sheep. Phosphatidylethanolamine of diseased sheep contains more 18:1(n-9) and less 22:6(n-3) than normal and their phosphatidylcholine less 16:0. Other differences are minor. All differences are in the same direction as those reported for the infantile form of human ceroid-lipofuscinosis, but are smaller. Normal sheep grey matter phosphatidylinositol contains 8.5% 20:4(n-6) and 24.6% 22:6(n-3), in contrast to 28.5 and 4.6%, respectively, in humans. The other sheep phospholipids have similar fatty acid profiles to those from humans. Apart from low levels of 20:3(n-9) and 22:3(n-9) they contain no additional non-essential fatty acid derived species. No sign of essential fatty acid deficiency occurs in either diseased or normal sheep. It is concluded that sheep must conserve their restricted essential fatty acid supply for structural functions, and that an abnormality in fatty acid metabolism is not primarily involved in the pathogenesis of ceroid-lipofuscinosis. The results also call into question the primary role of peroxidation of polyunsaturated fatty acids in lipopigment formation in this disease.

Ceroid, lipofuscin and the ceroid-lipofuscinoses (Batten Disease)

ConclusionThe similar histochemical, fluorescent and ultrastructural appearance of some lipofuscins and the pigment of Batten disease imply some underlying common chemical feature. The similar chloroform—methanol solubility of the protein subunit c in Batten disease and the specific proteins accumulated in thyroid lipofuscin imply that disordered or delayed catabolism of hydrophobic protein may be that common feature.

Analysis of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis

Ovine ceroid-lipofuscinosis is an inherited neurodegenerative disorder characterised by the accumulation of storage cytosomes in brain and visceral organs. Phosphorylated dolichol-containing compounds, largely in the form of dolichyl pyrophosphoryl oligosaccharides, have been shown to constitute 1-2% of the dry weight of storage cytosomes isolated from brain and pancreas, and 0.5 and 0.1% respectively of storage cytosomes isolated from liver and kidney. The carbohydrate portion of these glyconjugates in storage cytosomes isolated from brain, pancreas and liver consisted of a series of oligosaccharides of composition Man2-9GlcNAc2, with Man5-8GlcNAc2 predominating. The concentrations of dolichyl pyrophosphoryl oligosaccharides in storage cytosomes from ovine ceroid-lipofuscinosis are much higher than has been reported for endoplasmic reticulum, their normal functional location.

Lipofuscin and abnormalities in colloid in the equine thyroid gland in relation to age

Lipofuscin accumulation and other histological changes in thyroid tissue, previously reported to be age-related, were studied in 31 horses aged up to 35 years. The number of lipofuscin granules relative to thyrocytes increased from birth to 5 years of age. There was a wide individual variation in the number of lipofuscin granules in thyrocytes in mature horses, but this was not directly related to age. Several abnormalities were identified in thyroid colloid. The prevalence of spherites, lipofuscin granules, nucleated cells and shreds of colloid increased with age, but the prevalence of calcium oxalate crystals, erythrocytes, basophilic zones and solid fragments of colloid did not. In horses younger than 7 years, particularly large lipofuscin granules were found in thyrocytes of a small proportion of follicles which also contained abnormal colloid. Such follicles became more common in older horses without being accompanied by large lipofuscin granules. No correlation was found between granule numbers and frequency of colloid abnormalities. These results cast doubt on the traditional assumption that lipofuscin is indigestible cellular residue, since there was little evidence for excretion of granules. It is postulated that lipofuscin in this tissue may be a normal stage in lysosomal catabolism.

Lysosomal storage of a mitochondrial protein in Batten’s disease (ceroid lipofuscinosis)

The neuronal ceroid lipofuscinoses are a group of recessively inherited lysosomal storage disease of children and animals. Clinical signs include blindness, seizures and dementia, culminating in premature death. There are three main forms of human ceroid lipofuscinosis (Batten’s disease). These are the infantile, late infantile and juvenile forms, distinguished from each other by the age of onset and the clinical course of the disease. Other variant types and an adult form (Kuf’s disease) have also been reported (Rider and Rider, 1988; Boustany et al., 1988; Wisniewski et al., 1988; Dyken, 1988; Eto et al., 1988; Lake, 1984). Collectively they are probably the most common lysosomal storage diseases. The incidence has been estimated as high as 1 in 12 500 (Rider and Rider, 1988). Ceroid lipofuscinoses also occur in a number of animals. In particular, a flock of sheep with the disease has been maintained and studied as a model of the human diseases (Jolly et al., 1980, Jolly et al., 1982; Graydon and Jolly, 1984; Mayhew et al., 1985). The clinical course of ovine ceroid lipofuscinosis most closely resembles the juvenile form of the human disease.