Biochemistry of Brain

By Sudhir Kumar

Biochemistry of Brain is a collection of articles dealing with the developments in the biochemistry of the brain. This book gives a comprehensive and critical discussion of important developments in studies concerning the above subject. This text discusses the structure, function, and metabolism of glycosphingolipids, which are related to the study of sphingolipid storage diseases. Inborn defects of metabolism are found in Gaucher's and Fabry's disease, which are characterized by lipid accumulation in the brain. Another paper reviews the chemical and genetics of critically lysosomal hydrolase deficiencies that can cause the storage of sphingolipids. This book then explains the role of myelin basic protein in lipids in vivo that the weak bonding of the protein is not a major component of myelin stability. Another paper discusses the procedures for isolating subfractions of myelin and myelin-related membranes, with some attention given on the alterations in the subfractionation of myelin in pathological hypomyelinating and demyelinating conditions. Another article discusses the biochemical and enzymatic composition of lysosomes and the biosynthesis, intracellular transport, storage, and the degradation of lysosomal constituents. This collection of papers will benefit scientists doing research in microbiology, microchemistry, molecular genetics, and neurochemistry.

• Language: English
• Publisher: Elsevier Science
• Release date: Oct 22, 2013
• ISBN: 9781483153599

Book preview

KUMAR

STRUCTURE, FUNCTION AND METABOLISM OF GLYCOSPHINGOLIPIDS

YOGESH C. AWASTHI and SATISH K. SRIVASTAVA,     Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550

Publisher Summary

This chapter discusses the chemical structure, physical functions, and metabolism of glycosphingolipids. Neutral glycosphingolipids based on glucocerebrosides are in higher concentrations in non-neuronal tissue than in the neuronal tissue. Galactocerebroside and sulfatides constitute a significant portion of brain glycosphingolipid, especially in myelin sheath and white matter. Cerebrosides, sphingomyelin, and sulfatide form a significant portion of the lipids of myelin sheath for which several structural models have been proposed, showing the arrangement hydrophobic and hydrophilic groups of constituent lipids and proteins. The chapter further discusses the role of gangliosides in the transmission of nerve impulses. Both gangliosides and neutral glycosphingolipids have antigenic properties; however, the latter are known to be more effective in raising antibodies. Metabolism of sphingolipids was primarily generated by attempts to understand the biochemistry and genetics of inborn errors of metabolism in which one or more glycosphingolipids are stored. Both neutral and sialic acid-containing oligoglycosyl ceramides are degraded by a stepwise removal of terminal sugar residues leading finally to the ceramide. The last sialic acid residue of gangliosides is not cleaved by neuraminidase until it becomes the terminal moiety as a result of the cleavage of other monosaccharides.

CONTENTS

Introduction

Structure and Nomenclature of Sphingosine and Related Bases

Classification of Sphingolipids

Chemical Structures and Occurrence

Isolation of Glycosphingolipids

Biosynthesis of Glycosphingolipids

Catabolism of Glycosphingolipids

Physiological Functions of Glycosphingolipids

INTRODUCTION

The widely-accepted term sphingolipid is derived from the aliphatic base sphingosine which is present in the structural framework of all these compounds. The isolation of sphingosine from hydrolysates of brain lipids was reported by Thudichum (1882, 1901) who assigned to it the empirical formula C16H35NO2. The molecular formula was corrected to C18H37NO2, by Klenk in 1929 but it was not until the 1950′s that the full structure of sphingosine was elucidated (Carter & Humiston, 1951) and confirmed by its total synthesis (Shapiro & Segal, 1954; Shapiro et al., 1958). The sudden spurt of interest in the chemistry of sphingosine and related lipids since then is primarily due to interest in the sphingolipid storage diseases which are probably the best understood congenital storage disorders of the nervous system.

STRUCTURE AND NOMENCLATURE OF SPHINGOSINE AND RELATED BASES

Sphingosine is the major naturally occurring base present in sphingolipids. Carter and Humiston (1951) determined its structure (Table I) to be (D+) erythro-1, 3-dihydroxy-2-amino-4-transoctadecene. Minor constituents related to sphingosine that have also been isolated from brain tissue include sphingosines with chain lengths either longer or shorter than C18, and branched-chain sphingosines and bases with more than one double bond or more than two hydroxyl groups. The fully-saturated analogue of sphingosine, dihydrosphingosine, is also almost invariably present along with sphingosine. The names and structures of some of the more frequently-occurring sphingosine bases are given in Table 1.

TABLE I

Structure and Nomenclature of Sphingosine Bases

In the present system of nomenclature the C18 saturated base, dihydrosphingosine, is tentatively designated as sphinganine. According to this nomenclature, sphingosine is 4-sphingenine (the prefix 4 indicates the position of the double bond and phytosphingosine is termed 4-hydroxysphinganine. Homologues of C-18 are designated by an appropriate prefix (Table 1).

The primary amino group at C-2 in sphingosine is always N-acylated in sphingolipids, whereas the primary hydroxyl group at position 1 is either esterified or glycosylated. The N-acylated derivative of sphingosine, ceramide is the precursor of most of the sphingolipids and it has been isolated in the free state from neuronal and several other tissues (Gatt, 1963; Martensson, 1969; Samuelsson, 1969). Although various fatty acids have been detected in ceramide, the C20-C24 fatty acids predominate in neutral glycosphingolipids and sphingomyelin, whereas stearic acid is the major component of gangliosides.

CLASSIFICATION OF SPHINGOLIPIDS

The classification of sphingolipids is primarily based on the substituent groups attached to the hydroxyl group at C-1 of sphingosine or its derivatives. In phosphosphingolipids this hydroxyl group is esterified in a phosphate diester, as with phosphoryl choline in sphingomyelin, whereas in the glycosphingolipids the C-1 hydroxyl group is directly glycosylated by mono-, di-, or oligosaccharides. The glycosphingolipids acquire an anionic nature if the oligosaccharide moiety has acidic groups, as in sulfatide (galactose 3-sulphate) or in the gangliosides which contain sialic acid. Gangliosides are an important group of water-soluble acidic sphingolipids containing 3 or more hexose units attached to the C-1 hydroxyl of ceramide together with one or more sialic acid residues.

Sphingolipids are present in virtually all mammalian tissues and fluids although they are generally less abundant than the glycerides and cholesterol. They were once considered to be confined to the membranes of eukaryotic cells and to be absent from bacteria. Recent studies, however, have shown their occurrence in some of these organisms. In extra-neuronal tissues, the sphingolipids are believed to be localized mainly in plasma membrane and to contribute to the surface properties and to specific membrane functions of the cell.

CHEMICAL STRUCTURES AND OCCURRENCE

Structural studies of glycosphingolipids were mainly carried out in order to characterize the lipids accumulated in the brain and/or other tissues in inborn errors of metabolism such as Gaucher’s and Fabry’s disease. These diseases will be discussed in detail later in this volume. The structures of some glycosphingolipids are shown in Tables 2 and 3.

TABLE II

Structure of Some of the Neutral Sphingolipids

Cer = ceramide. glu = glucose. gal = galactose. N-Acgal = N-acetyl galactosamine.

TABLE III

Structure of Major Gangliosides and their Nomenclature

NANA = N-acetylneuraminic acid. Other abbreviations are same as in Table II.

+Svennerholm (1964)

*Kuhn & Weigandt (1963)

**Klenk & Gielen (1960)

Neutral Glycosphingolipids and Sulfatide

The first sugar residue attached to ceramide is usually glucose or galactose giving rise to the simple members of the series, glucocerebroside and galactocerebroside, respectively. More complex oligosaccharides are formed by the addition of galactose or N-acetylgalactosamine residues to the first sugar unit. In sulfatide, the C-3 hydroxyl group of galactosyl ceramide is esterified with sulphuric acid (Yamakawa et al., 1962; Stoffyn & Stoffyn, 1963, 1963a; Stoffyn, 1966).

The stereochemical configurations of different sphingolipids derived from glucocerebroside usually follow a similar pattern (Table 2) (Yamakawa et al., 1965; Makita & Yamakawa, 1963; Makita et al., 1966). The sugar moieties exist in pyranoside form and the glycosidic linkages are normally in the β-configuration. Forssman hapten (Makita et al., 1966), a derivative of glucocerebroside, is a well-known exception in which the linkage between the C-1 hydroxyl group of terminal N-acetyl galactosamine and the C-3 hydroxyl of galactose is in the α anomeric configuration (Ando & Yamakawa 1970; Siddiqui & Hakamori, 1971; Stellner et al., 1973).

Sphingosine is the major component in all neutral glycosphingolipids and accounts for more than 90% of the basic fraction. Among the other bases, C18-dihydrosphingosine occurs in brain while phytosphingosine occurs in kidney as a minor constituent (Carter & Hirchberg, 1968; Karlsson, 1964; Karlsson & Martensson, 1968; Michalec & Kolman, 1966). Galactocerebroside consists predominantly of C18-sphingosine. The fatty acid compositions of neutral glycosphingolipids are somewhat similar to those of other sphingolipids. Predominantly, C18 to C24 saturated acids are attached to the free amino group of sphingosine (Martensson, 1966; Suomi & Agranoff, 1965; Miras et al., 1966; Yamakawa, 1966). Galactosylceramide, which is the major glycosphingolipid of human brain has a relatively high content of α-hydroxy fatty acids which comprise about 50% of the total fatty acids (Svennerholm & Stallberg-Stenhagen, 1968). Depending on the nature of the fatty acid attached to sphingosine, the brain galactosylceramide is designated as cerasin (having normal fatty acids) or pherosin (having α-hydroxy fatty acids). Kidney dihexosylceramide and monohexosyl ceramide also have substantial amounts of α-hydroxy fatty acids. Most of the other glycosphingolipids have only trace amounts of these fatty acids.

Neutral glycosphingolipids derived from galactocerebroside predominate in neuronal tissue. Galactocerebroside and sulfatides constitute a significant portion of brain glycosphingolipid, especially in myelin sheath and white matter where cerebrosides and sulfatide make up some 25% of the total lipids compared to only 7% in gray matter.

Neutral glycosphingolipids based on glucocerebroside are in higher concentrations in non-neuronal tissue. Glucosyl ceramide (glucocerebroside) is the major sphingolipid constituent of plasma although the concentration of galactosyl ceramide is higher in liver and spleen. In the kidney and in the red cell membrane tetrahexosylceramides derived from glucocerebroside predominate (Yamakawa et al., 1965).

Gangliosides

The currently accepted name ganglioside was applied by Klenk (1941, 1942) to the lipid material that he isolated from the brain of a Tay-Sachs patient. Because of their structural and corresponding unwieldly nomenclature, several shorthand notations for gangliosides have been suggested (Svennerholm, 1964; Kuhn & Weigandt, 1963; Klenk & Gielen, 1960; Penick et al., 1966). Table 3 gives the structures and shorthand nomenclature of some of the major gangliosides.

Gangliosides contain one or more residues of sialic acid attached to the same or different sugar moieties of the oligosaccharide chain. Sialic acid is the accepted name for compounds derived from neuraminic acid (5-amino-3, 5 dideoxy D-glycero-D-galacto-nonulosonic acid) (Fig. 1). In sialic acid, the amino group may be N-acetylated, as in the case of N-acetyl neuraminic acid (NANA), or N-glycolylated as in the case of N-glycolyl neuraminic acid (NGNA). Human brain gangliosides contain mostly NANA, whereas NGNA occurs as a minor constituent in sheep, pig and bovine brains (Yu & Leeden, 1970).

Fig. 1 Structure of neuraminic acid

Left-hand side: open form. Right-hand side: ring form

In NANA, R = CH3

In NGNA, R = CH2OH

In most of the gangliosides, the major bases are C18 and C20 sphingosines which occur in approximately equal proportions (Carter et al., 1947; Sambosivarao & McCluer, 1964) together with minor amounts of their dihydroanalogues. The main fatty acid component of the gangliosides is stearic acid. However, other saturated fatty acids also occur. Lactosyl ceramide (Cer-glu-gal) appears to be the precursor of all gangliosides which usually also contain N-acetylgalactosamine.

Gangliosides (mainly GM1, GD1a, GD1b and GT1) are concentrated in the gray matter of the brain where they constitute some 5% of the total lipid (Klenk, 1941, 1942; Lapetina et al., 1967; Weigandt, 1967) whereas they comprise only 0.6% of the total lipids of white matter (Leeden & Yu, 1973). They are also present in small amounts in spleen, erythrocytes, liver, kidney and spinal fluid.

ISOLATION OF GLYCOSPHINGOLIPIDS

Gangliosides are soluble in water and form high molecular weight micelles, whereas the solubility of neutral glycosphingolipids in water increases from that of the insoluble cerebrosides with increasing chain length of the oligosaccharide portion of the molecule. Details of procedures for extraction of total lipids and separation of neutral and anionic sphingolipids are documented in reviews by Rouser et al. (1967) and Skipsky (1975). Commensurate with the scope of the present chapter, we will briefly outline the principles of isolation and characterization of glycosphingolipids. The ground tissue, homogenate, or acetone powder, is extracted with a mixture of chloroform and methanol. Although solvent mixtures containing different proportions of chloroform have been used (Booth, 1962; Svennerholm, 1963), the original extraction procedure of Folch et al. (1957) using chloroform-methanol, 2:1, appears to be most effective. The lipids are then partitioned using the Folch procedure (Folch et al., 1957) or modified Folch procedures (Suzuki, 1965; Radin, 1969), when gangliosides separate in the upper layer of aqueous methanol containing KCl. The lower layer of chloroform-methanol contains neutral glycosphingolipids together with phospholipids, glycerides, fatty acids, steroids and their esters. Separation of the glycosphingolipids from the chloroform-methanol fraction can be achieved by thin layer chromatography, or column chromatography using silicic acid or ion exchange resins. In column chromatography neutral lipids are eluted first with chloroform followed by the glycosphingolipids with acetone-methanol, 9:1, while the phospholipids are retained on the column and can be eluted only with methanol. Sequential elution of different classes of lipids from chromatographic columns of various adsorbants using gradually increasing polar solvents for successive elutions is a widely used technique for the separation not only of glycosphingolipids but for almost all lipid classes. Excellent detailed review articles are available in the literature (Rouser et al., 1967; Sweeley, 1969).

Thin layer chromatography (TLC) is probably the most extensively used technique for qualitative and quantitative analyses of both neutral glycosphingolipids and the anionic gangliosides. The lipids are extracted from the tissue and partitioned by the Folch procedure which separates the gangliosides in the aqueous phase and the neutral glycosphingolipids in the organic solvent phase. The neutral glycosphingolipids can then be directly isolated from phospholipids and the neutral lipids by TLC. Using TLC plates containing 80% silica gel and 20% magnesium silicate and a two-step development in acetone-pyridine-chloroform water (40 : 60 : 5 : 4) followed by ethyl ether-pyridine-ethanol and 2N NH4OH (65 : 30 : 8 : 2) Skipsky et al. (1967) have separated these lipids in one step. Gangliosides are largely retained in the aqueous phase during partitioning in the Folch procedure but because of the formation of water-insoluble complexes of calcium and gangliosides, some gangliosides are retained in the chloroform layer (Berh & Lehn, 1973). A modified procedure described by Carter & Kanfer (1975) allows a complete extraction of gangliosides in the aqueous phase. Gangliosides form micelles of very high molecular weight in water solution, and column chromatography over ion-exchange resins of florisil is often performed to remove phospholipids and other contaminants before the sample is ready for analytical or preparative TLC. Contaminating phospholipids may be cleaved by mild alkaline hydrolysis in the crude ganglioside extract at this stage. Acid hydrolysis may be employed to remove sialic acid residues when the asialo derivatives are to be isolated.

Radioactive Labeling of Glycosphingolipids

Radioactively-labeled gangliosides and neutral glycosphingolipids are used as natural substrates for the assay of specific sphingoglycosidases as discussed later. For radioactive labeling of sphingolipids, two major approaches have been used. In the first, radioactively-labeled [¹⁴C] precursors are injected into experimental animals from which the labeled products are subsequently isolated. [¹⁴C] sialic acid—and [¹⁴C] N-acetylgalactosomine—labeled GM2 ganglioside and several other sphingolipids have been labeled by this technique (Kolodny et al., 1970). The other approach consists of enzymatic oxidation of terminal galactose or N-acetyl galactosamine residue by galactose oxidase followed by reduction with tritiated sodium borohydride. Galactosyl ceramide, lactosyl ceramide and GM2 ganglioside labeled at terminal galactose residue have been prepared in this way (Radin et al., 1969; Suzuki & Suzuki, 1972). Syntheses of glycosphingolipids radioactively-labeled at the sphingosine (Iwamori et al., 1975) and fatty acid moieties (Mapes et al., 1973) have also been achieved.

BIOSYNTHESIS OF GLYCOSPHINGOLIPIDS

Sphingosines

The biosynthesis of dihydrosphingosine or its analogues can proceed via condensation of the fatty aldehyde (e.g. palmitoyl aldehyde) with serine linked in the form of a Schiff’s base to pyridoxal phosphate (Brady & Koval, 1958; Brady et al., 1958; Brady, 1969). Manganese ions are required for this reaction (Fig. 2A). Stoffel et al. (1968) however have isolated enzymes from several tissues which seem to favor an alternate biosynthetic pathway as shown in Fig. 2B. This pathway does not require aldehyde as the intermediate and proceeds directly with fatty acyl CoA. Formation of sphingosine from dihydrosphingosine is not well understood.

Figs. 2A & 2B Biosynthesis of sphingosine

Neutral Glycosphingolipids and Sulfatide

Addition of fatty acid and hexose to sphingosine leading to the biosynthesis of cerebrosides can proceed either through the addition of the hexose to ceramide or through the direct addition of the sugar residue to sphingosine followed by acylation of the free-NH2 group of sphingosine (Fig. 3).

Fig. 3 Biosynthesis of sulfatide

The biosynthesis of sulfatide, or 3-sulfate ester of galactosyl ceramide, occurs by sulfatation of the 3-hydroxyl group of galactose by active sulfate, or 3′-phosphoadenosine-5-phosphosulfate (Balasubramanian & Bachhawat, 1965; McKahnn et al., 1965; Cumar et al., 1968), as shown in Fig. 3. Stepwise addition of sugar residues to cerebrosides via respective uridine diphosphate (UDP)-derivatives gives rise to ceramide oligosaccharides. Lactosyl ceramide formed by the addition of galactose to glucocerebroside via UDP-galactose is a key intermediate as it is the precursor for many of the ceramide oligosaccharides and gangliosides (Fig. 4). These reactions are catalyzed by glycosyl transferases present in Golgi apparatus.

Fig. 4 Possible routes of ganglioside biosynthesis

abbreviations:

Cer = ceramide

glc = glucose

gal = galactose

galNAc = N-acetylgalactosamine

UDP-glc = uridine diphosphate glucose

UDP-gal = uridine diphosphate galactose

UDP-galNAc = uridine diphosphate N-acetylgalactosamine

CMP = cytidine monophosphate

NANA = N-acetylneuraminic acid

Gangliosides

The biosynthesis of gangliosides is similar to that of neutral glycosphingolipids. The addition of NANA to ceramide oligosaccharides is catalysed by sialyl transferase using the active nucleotide derivative, cytidine monophosphate-N-acetyl-neuraminicacid (CMP-NANA). Glycosyl transferases leading to the synthesis of gangliosides from ceramide have been located in embryonic chicken brain (Basu et al., 1968; Kaufman et al., 1968; Steigerwald et al., 1975). Although the exact sequences are not without controversy, Fig. 4 outlines biosynthetic pathways for different gangliosides.

CATABOLISM OF GLYCOSPHINGOLIPIDS

As indicated earlier, interest in the chemistry and metabolism of sphingolipids was primarily generated by attempts to understand the biochemistry and genetics of inborn errors of metabolism in which one or more glycosphingolipids are stored. Once the nature of the stored lipid was established the metabolic lesion leading to its accumulation was sought. It is now known that the accumulation of a particular sphingolipid results in most cases from the deficiency of one of a battery of lysosomal acid hydrolases which sequentially cleave off the monosaccharide units from sphingolipid oligosaccharides. Both neutral and sialic acid-containing oligoglycosyl ceramides are degraded by stepwise removal of terminal sugar residues leading finally to the ceramide, which is either re-utilized for biosynthesis or is broken down to sphingosine and fatty acid. When the enzyme that cleaves the terminal group of a particular sphingolipid is missing, that lipid is stored in lysosomes.

Initial steps in the degradation of gangliosides involve the removal of neuraminic acid residues. Both tri- and di-sialo-gangliosides are converted to mono-sialogangliosides by nonspecific neuraminidases. Unlike the acid hydrolases, neuraminidases are not localized only in lysosomes, but have also been found in soluble fractions (Leibovitz & Gatt, 1968; Ohman et al., 1970). The last sialic acid residue of gangliosides is not cleaved by neuraminidase until it becomes the terminal moiety as a result of the cleavage of other monosaccharides. Thus, the sialic acid of GM1 ganglioside is not cleaved by neuraminidase until the galactose and N-acetyl galactosamine residues are cleaved. Similarly, the removal of N-acetyl galactosamine is an essential prerequisite for the hydrolysis of sialic acid from GM2 ganglioside. This explains why GM1 and GM2 gangliosides are stored in generalized GM1 gangliosidosis and Tay-Sachs or Sandhoff’s disease, respectively, rather than their asialo derivatives. Kolodny et al. (1971) have reported a mammalian neuraminidase preparation that cleaves the sialic acid from GM2 gangliosides prior to removal of N-acetyl galactosamine. The physiological role of this enzyme cannot be specifically defined at present, however. Figure 5 summarizes the catabolic pathways of glycosphingolipids.

Fig. 5 Catabolism of gangliosides

Abbreviations:

Cer = ceramide

glu = glucose

gal = galactose

galNAc = N-acetylgalactosamine

NANA = N-acetylneuraminic acid

Turnover of Glycosphingolipids

Studies have been carried out to determine the relative rates of degradation of various sphingolipids in vivo. Turnover rates of gangliosides have been shown to be more rapid than those of other glycosphingolipids in the rat brain, with a half life of 10 to 24 days depending on the nature of the precursor used (Burton, 1967). Suzuki (1967) has shown that gangliosides in rat brain show a slow turnover rate for the first 10 days after birth. The rate increases rapidly for the next 8 days and thereafter declines with the onset of maturation. The rapid turnover rate of gangliosides in rat brain corresponds to the periods of optimum myelination. In another study it has been shown (Suzuki, 1970) that GM1 ganglioside of rat brain has a higher turnover rate in the whole brain homogenate compared with that of isolated myelin fraction. Apart from gangliosides the turnover rates of other glycosphingolipids of brain have been studied. Using ³⁵S-sulfate as a precursor for sulfatides Davison & Gregson (1966) have demonstrated that the turnover rate of sulfatide in myelin fraction is very slow after the onset of maturation. When ³⁵S-sulfate was injected into the brain of 12-day-old rats there was no noticeable decay in the myelin sulfatides over a period of 76 days. The sulfatides of other subcellular fractions of brain, on the other hand, had a shorter half life.

Pritchard (1966) has shown that sulfatides in brain stem metabolise more rapidly than sulfatides of other regions of brain. While the remarkable metabolic stability of myelin sheath is well established, its constituent lipids do undergo a slow turnover. Cerebrosides and sulfatides are the most stable lipids of myelin and their half lives in rat brain myelin have been shown to be well over one year (Smith, 1967).

Enzymes of Sphingolipid Catabolism

Korey & Stein (1963) reported a gangliosidase from rat and human brain capable of partially degrading the complex ganglioside mixture to unidentified products. Sandhoff et al. (1964) demonstrated the enzymatic degradation of tetraglycosylceramide to ceramide by enzyme preparations from mammalian brain and kidney tissues and identified the tri-, di- and monoglycosyl ceramide intermediates. Svennerholm (1967) demonstrated the conversion of ganglioside GT1b to GM3 by preparations from neonatal human brain. In the past decade, the enzymes of glycosphingolipid catabolism listed in Fig. 5 have been defined and will be discussed in detail later. Most of these are membrane bound enzymes and are usually referred to as the group of lysosomal glycosphingolipid hydrolases. It is possible that these enzymes responsible for the stepwise degradation of brain glycosphingolipids, exist together as a multienzyme complex and are oriented on the membrane in such a way that the degradation product of each of the enzymes is directly passed on to the enzyme catalyzing the next step of degradation. Such an arrangement would bring about the sequential degradation of sphingolipids smoothly without any unnecessary buildup of the intermediate products. Low levels of all the intermediate catabolites in mature human brain (Suzuki & Chen, 1967) seem to favor this hypothesis.

Fig. 6 Catabolism of ceramide trihexoside, sulfatide and sphingomyelins

Abbreviations:

Cer = ceramide

glu = glucose

gal = galactose

PC = phosphoryl choline

Lysosomal glycosphingolipid hydrolases have several common features. All of them are glycoproteins, have acidic pH optima, and many including hexosaminidase, β-galactosidase, α-galactosidase, aryl sulfatase and sphingomyelinase are known to exist in several isozymic forms. In various sphingolipidoses, one or more isozymes of the particular enzyme are deficient. It is known that in GM2 gangliosidosis (Tay-Sachs disease) only hexosaminidase A is missing (Okada & O’Brien, 1969) while in Sanhoff’s disease, which is a variant of GM2 gangliosidosis (variant O) both A and B isozymes are missing (Sandhoff et al., 1968). In metachromatic leukodystrophy (MLD) usually the major isozyme, aryl sulfatase A is missing. However, in multiple sulfatase deficiency, a variant of MLD, all the three known isozymes of aryl sulfatase are deficient (Austin et al., 1965). Similar observations have been made in Neimann-Pick disease (Callahan et al., 1975), Fabry’s disease (Beutler & Kuhl, 1972) and several other sphingolipidoses which will be discussed in detail later.

During the past few years, a great deal of interesting work has led to a better understanding of the genetic relationship between variants of GM2 gangliosidosis and GM1 gangliosidosis. Biochemical genetics of the two variants of GM2 gangliosidosis (Tay-Sachs and Sandhoff’s disease) have been studied in great detail (Robinson & Sterling, 1968; Srivastava & Beutler, 1973, 1974; Srivastava et al., 1974; 1974a, 1975, 1976; Tallman et al., 1974) and the genetic relationship between these two inborn disorders is probably the best understood of all the sphingolipidoses. It has been demonstrated that hexosaminidase A is (αβ)3 and hexosaminidase B is (ββ)3 where α and β are polypeptide chains of about 18,000 molecular weight and are coded by different genes (Srivastava et al., 1976). This model explains the genetic origin of Sandhoff’s disease (where only hexosaminidase A is missing) by a single gene mutation. Also, since cross-reacting material has been shown to be present in Tay-Sachs and Sandhoff’s diseases (Srivastava et al., 1976a and Srivastava and Ansari, 1978) a single structural gene mutation has been suggested for both these congenital disorders (Srivastava & Beutler, 1974; Srivastava et al., 1976 and 1976a and Srivastava and Ansari, 1978). Existence of several other isozymes of hexosaminidase and their relationship to hexosaminidase A and B is also explained by this model. Norden et al., (1974) have shown that GM1 β-galactosidases A and B have at least one subunit in common. Recently, aryl sulfatase A has been purified to homogeneity (Stevens et al., 1975) and further studies may reveal the nature of relationships between the various isozymes of aryl sulfatase. The interrelationship between the isozymes of β-galactosidases, aryl sulfatases sphingomyelinases, α-galactosidases and other glycosphingolipid hydrolases, is currently being studied and progress in this direction is described in detail later. These studies are vital to the exploration of possible therapeutic approaches, including enzyme replacement, to these congenital abnormalities. The presence of a structurally altered non-catalytic enzyme protein in some of the sphingolipidoses, e.g. MM1 gangliodisosis and Tay-Sachs disease has been demonstrated by the presence of cross-reacting material (CRM) against the antibodies of the normal enzyme. Structural studies with these proteins could shed further light on the exact nature of genetic events leading to these abnormalities. It has been speculated that Krabbe’s disease in different cases does not necessarily arise out of the same structural mutation (Suzuki & Suzuki, 1974). O’Brien (1975) has suggested that GM1 ganglioside-β-galactosidase is a heterocatalytic protein having different active sites against different groups of natural substrates and that mutations at different sites are responsible for different variants of GM1 gangliosidosis. Such hypotheses could be verified only if the homogeneous CRM from the tissues of these patients is available for structural and kinetic studies. Recently, several enzymes of glycosphingolipid metabolism have been shown to require a heat-stable factor (Li & Li, 1976) for effective degradation of their natural substrates. This heat-stable factor has been identified as a glycoprotein having a molecular weight of about 21,000. It would also be interesting to study the structural and kinetic properties of this factor from the tissues of patients having various sphingolipidoses. Involvement of this factor, or other cofactors in the interrelationship of closely related sphingolipidoses such as the three distinct disorders involving glycosphingolipid β-galactosidases, could be a real possibility.

PHYSIOLOGICAL FUNCTIONS OF GLYCOSPHINGOLIPIDS

Sphingolipids generally, including sphingomyelin and the glycosphingolipids, are localized primarily on mammalian membranes, in which they may be assumed to be of structural importance. Cerebrosides, sphingomyelin and sulfatide form a significant portion of the lipids of myelin sheath for which, on the basis of lipid composition and electron microscopic studies, several structural models (Finean, 1968; Vandenheuvel, 1965) have been proposed showing the arrangement hydrophobic and hydrophilic groups of constituent lipids and proteins. A role for gangliosides in the transmission of nerve impulses at the synapse has been suggested in view of their localization at the nerve ending and of their anionic nature and their interactions with acetyl choline, serotonin, dopamine, norepinephrine and histamine, have been postulated (Dekirmenjian et al., 1969). Both gangliosides and neutral glycosphingolipids have antigenic properties, although the latter are known to be more effective in raising antibodies. Forssman hapten and several other neutral glycosphingolipids including lactosyl and galactosyl ceramides (Rapport et al., 1958) are known to be antigenic, while the antigenic determinants of the ABO and Lewis blood group systems are believed to be carried by glycosphingolipids of the red cell membrane (Hakomori & Strycharz, 1968; Hakamori & Andrews, 1970).

ACKNOWLEDGEMENT

We gratefully acknowledge the support of DHEW Grants GM 21655, EY 02260 and The National Foundation—March of Dimes.

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METABOLIC DISORDERS IN SPHINGOLIPIDOSES

SATISH K. SRIVASTAVA and YOGESH C. AWASTHI,     Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77550

Publisher Summary

This chapter provides an overview of the metabolic disorders in sphingolipidoses. Lipid storage diseases attracted considerable attention after the concept of lysosomes was introduced. Following the chemical identification of stored sphingolipids in sphingolipidoses, the enzymes involved in their degradation were investigated. These sphingolipid glycosidases were shown to be exozymes that hydrolyze the glycosidic bond of the terminal sugar only. Recent studies have been aimed at the detection of heterozygotes having intermediate levels of the enzyme, at prenatal diagnosis of the enzyme deficiency, and at an understanding of the mutational events that produce the deficiency. To understand the biochemical genetics of sphingolipidoses, investigators have purified the enzymes involved in the catabolism of sphingolipids from normal human tissues—such as placenta, liver, brain, and kidney—and have studied their kinetic, structural, and immunological properties. The distribution of sphingo myelinase and the deficiency of this enzyme in the lysosome or the cytosol, or both, play an important role in the pathophysiology of the Niemann–Pick disease. The various forms of the Niemann-Pick disease are genetically heterogeneous, which in some cases are caused by an alteration in the subcellular localization of sphingomyelinase. Splenomegaly, hepatomegaly, mild hypochromic anemia, leukopenia, and thrombocytopenia are the main clinical symptoms of Gaucher’s disease. This disease is transmitted as an autosomal recessive trait.

CONTENTS

Introduction

Biochemical Genetics of Sphingolipid Storage Disorders

GM1 Gangliosidosis

Krabbe’s Disease

Lactosylceramidosis

Tay-Sachs and Sandhoff’s Diseases

Fabry’s Disease.

Metachromatic Leukodystrophy

Niemann-Pick Disease

Gaucher’s Disease

INTRODUCTION

The purpose of this chapter is to review critically lysosomal hydrolase deficiencies leading to the storage of sphingolipids. Although various sphingolipidoses have been known for several decades, the biochemical and genetic studies of these congenital disorders have, in general, only been carried out after the identification of the relevant stored lipids in the tissues of patients. Aghion (1934) first identified the material that accumulates in patients of Gaucher’s disease as glucocerebroside, a finding that was confirmed a few years later by Halliday et al. (1940) and in 1935, Klenk demonstrated the storage of sphingomyelin in Niemann-Pick disease. In the following years, the lipids that are stored in Tay-Sachs disease and various other sphingolipidoses have been identified.

Lipid storage diseases attracted considerable attention after the concept of lysosomes was introduced by de Duve et al. in 1955, and it soon became apparent that the lipids stored in these diseases are concentrated in the lysosomes where the various hydrolases responsible for the degradation of sphingolipids are localized. Following the chemical identification of stored sphingolipids in sphingolipidoses, the enzymes involved in their degradation were investigated. These sphingolipid glycosidases were shown to be exozymes which hydrolyze the glycosidic bond of the terminal sugar only. This led to the development of the concept of sequential hydrolysis of glycosphingolipids. Recent studies have been aimed at the detection of heterozygotes having intermediate levels of the enzyme, at prenatal diagnosis of the enzyme deficiency and at an understanding of the mutational events that produce the deficiency. In order to understand the biochemical genetics of sphingolipidoses, investigators have purified the enzymes involved in the catabolism of sphingolipids, from normal human tissues, such as placenta, liver, brain and kidney and have studied their kinetic, structural and immunological properties.

Figure 1 describes the catabolism of glycosphingolipids and the primary enzymatic defects leading to different sphingolipidoses. Table 1 summarizes the enzyme defects, storage material and clinical symptoms in various sphingolipid storage disorders.

TABLE 1

ENZYME DEFICIENCY, STORED MATERIAL AND CLINICAL SYMPTOMS IN SPHINGOLIPIDOSES

Fig. 1 Metabolic blocks in Sphingolipidoses

BIOCHEMICAL GENETICS OF SPHINGOLIPID STORAGE DISORDERS

β-D-Galactosidase Deficiencies

Three clinically distinct genetic disorders, GM1 gangliosidosis, Krabbe’s disease or globoid cell leukodystrophy and lactosyl ceramidosis have been recognized as involving deficiencies of specific sphingolipid β-galactosidases.

GM1-Gangliosidosis

Landing et al. (1964) were the first to recognize the so-called psuedo-Hurler disease (Craig et al., 1959; Sanfilippo et al., 1962) or Tay-Sachs disease with visceral involvement (Norman et al., 1959) as a specific entity, which they called familial neurovisceral lipidosis. The present name GM1 gangliosidosis was assigned to this disease (O’Brien, 1965) when the major stored lipid material was characterized as GM1 ganglioside (Gonatas & Gonatas, 1965; Leeden et al., 1965; O’Brien et al., 1965). Suzuki (1968) demonstrated that besides GM1 ganglioside, keratan sulfate-like mucopolysaccharide was also accumulated in the viscera, van Hoof and Hers (1969) demonstrated a characteristic lysosomal hypertrophy in these patients along with the deficiency of GM1 β-galactosidase. The deficiency of GM1 β-galactosidase, which cleaves the terminal galactose from GM1 ganglioside (Okada & O’Brien, 1968), leads to accumulation of GM1 ganglioside predominantly within the central nervous system. GM1 β-galactosidase acts on 4-methyl umbelliferyl β-D-galactoside and this activity is also absent in GM1 gangliosidosis patients. GM1 gangliosidosis is shown to be transmitted as an autosomal recessive trait.

Variants of GM1 Gangliosidosis

Two major clinical subtypes of GM1 gangliosidosis have been delineated. In GM1 gangliosidosis Type 1, also referred to as generalized gangliosidosis, the clinical symptoms usually appear within the first six months of life. These symptoms include a rapid deterioration of the central nervous system, severe bone abnormalities and hepa to splenomegaly. Some of the clinical features such as bone deficiency, facial dysmorphy, macroglossia, distended abdomen, and sometimes hirsutism are similar to those of Hurler’s syndrome. In generalized GM1 gangliosidosis the lungs are usually rich in foam cells. Only a few cases have been reported with corneal opacity. Some of the other symptoms e.g. lysosomes in neurons appear as membranous cytoplasmic bodies are similar to those of Tay-Sachs disease. (Terry & Weiss, 1963). As in Hurler’s syndrome, Zebra bodies are present in neurons. Other symptoms include psychomotor regression and hypotony in the first stages; later spastic quadriparesis, clonic movements, sometimes convulsion; amaurosis with degeneration of the macula; and in some cases, a cherry-red spot. Death usually occurs by two years of age.

In GM1 gangliosidosis Type II (Juvenile GM1 gangliosidosis) the symptoms which appear by one or two years of age are relatively mild. The central nervous system deterioration is less rapid, bony abnormalities are minimal and hepatomegaly is usually absent. At the final stage, the children lie immobile in a state of decerebrate rigidity and death occurs between 3 and 10 years of age (Derry, et al., 1968; O’Brien et al., 1972). In addition to these two types, several subtypes of GM1 gangliosidosis have been described. A subtype of generalized GM1 gangliosidosis having minimal bony deformities has been reported (Feldges et al., 1973) as have variants with clinical similarity to Type II but with late onset and prolonged survival (Goldberg et al., 1971; Loonen et al., 1974; Lowden et al., 1974; Wenger et al., 1974).

Enzymology and Storage Material

GM1 gangliosidosis results from the deficiency of β-galactosidase which, as well as cleaving the natural substrate ganglioside GM1, cleaves synthetic substrates such as 4-methyl umbelliferyl-β-D-galactoside and p-nitrophenyl-β-D-galactoside (Ho et al., 1973; Norden & O’Brien, 1973). This enzyme can also cleave the β-galactosyl residues of glycoproteins (Norden et al., 1974) and glycosaminoglycans (MacBrin et al., 1969). It is interesting to note that the terminal galactose in these compounds is predominantly linked β(1–4) to acetylglycosamine, whereas in GM1 ganglioside the linkage is exclusively β(1–3) to acetylgalactosamine. However, it is now established that the biological role of GM1 ganglioside β-galactosidose involves the hydrolysis of galactosyl residues of glycoproteins and glycosaminoglycans in addition to the cleavage of GM1 ganglioside. The absence of this enzyme may therefore lead not only to generalized GM1 gangliosidosis, but also to storage of partially degraded glycoproteins or desulfated keratan sulfate (Tsay & Dawson, 1973; Wolfe et al., 1974) in vesceral organs. These compounds are, in fact, reported to be stored in GM1 gangliosidosis (Suzuki et al., 1968; Wolfe et al., 1970; Brunngraber et al., 1973; Tsay & Dawson, 1973; and Wolfe et al., 1974). The bony deformities in GM1 gangliosidosis probably result from disturbed connective tissue metabolism due to glycosaminoglycan and glycoprotein accumulation.

At least two isozymes of GM1 ganglioside β-galactosidase have been characterized in human tissues. GM1 ganglioside β-galactosidase A has been purified to homogeneity and the B isozyme has been partially purified (Norden et al., 1974) from human liver. Besides these two isozymes, β-galactosidase C, having a neutral pH optimum, has also been isolated from soluble fraction. This enzyme, however, does not cleave GM1 ganglioside (O’Brien, 1975).

GM1 ganglioside β-galactosidases A and B both have a pH optimum of 4.5, both cleave natural as well as artificial substrates and both are glycoproteins. Antibodies raised in rabbits against homogeneous preparation of the A isozyme cross-react with the B isozyme (Norden et al., 1974). indicating that both proteins have, at least in part, a common genetic origin. The A isozyme has been shown to have a single subunit of about 78,000 molecular weight, whereas the molecular weight of B is several fold higher. It is suggested that the B isozyme is a polymer of the A isozyme (O’Brien, 1975).

The homogeneous GM1 β-galactosidase A has been investigated for its catalytic activity toward various natural and artificial substrates. Besides GM1 ganglioside, the enzyme has some catalytic activity toward the galactosidic bonds of asialofetuin, lactosyl ceramide and galactosyl ceramide (Norden et al., 1974). However, antibodies raised against this enzyme did not precipitate any galactosyl ceramide β-galactosidase activity and only about 14% of the lactosyl ceramide β-galactosidase activity from crude liver homogenate, whereas the entire GM1 β-galactosidase activity was precipitated. This points toward the separate genetic origin of GM1 β-galactosidase from that of galactosyl and lactosyl ceramide β-galactosidase. However, the significant catalytic activity of homogeneous GM1 ganglioside β-galactosidase A toward galactosyl and lactosyl ceramides (Norden et al., 1974) remains unexplained.

In different variants of GM1 gangliosidosis, the cross-reacting material (CRM) has been shown to be present. It has been suggested (O’Brien, 1975) that GM1-β-galactosidase A has different catalytic activity sites for GM1 ganglioside, mucopolysaccharides and glycoproteins. It is the site of mutation which will determine the residual enzyme activity of the altered enzyme towards different compounds. If the altered enzyme has sufficient activity towards glycosaminoglycans and glycoproteins, the catabolism of these compounds would not be impaired. In this case the visceral involvement and bony deformities would either be absent or less severe. On the other hand, if the altered enzyme has impaired activity toward these compounds, severe bony deformities and visceral involvement would be expected. The variants of GM1 gangliosidosis, having intermediate characteristics between Type I and Type II, can be explained by this hypothesis (O’Brien, 1975) which also predicts heretofore undiscovered variants of the disease. This hypothesis, although very attractive, has yet to be substantiated by experimentation. The most logical way to test its validity would be to get homogeneous cross reacting material from several variants of GM1 gangliosidosis and to study their kinetic properties using different substrates.

Krabbe’s Disease

Krabbe (1916) described clinical and histological findings from two siblings who died of an acute infantile familial defuse sclerosis of the brain. He gave a detailed description of the globoid cells, which are unique histological characteristic of the disease. Suzuki and co-workers (Suzuki & Suzuki, 1970; 1971; Malone, 1970 and Suziki et al., 1971) have established the primary genetic defect in Krabbe’s disease, or globoid cell leukodystrophy, to be the deficiency of galactocerebroside β-galactosidase (galactosyl ceramide β-galactosyl hydrolase), a lysosomal hydrolase (Bowen & Radin, 1968).

The clinical manifestations of Krabbe’s disease are almost exclusively neurological. The onset of the disease is between three and six months after birth (Hagberg, 1963). It starts with vague hyperirritability, stiffness of limbs and episodes of fever. In the second stage, rapid and progressive mental and motor deterioration develops with marked hypertonicity and hyperactive tendon reflexes. In the final stages the patient is decerebrate, blind and has no contact with the surroundings. Clinical and electrophysiological signs of peripheral nerve involvement are almost always present (Dunn et al., 1969).

Morphological Changes

In Krabbe’s disease both the central and the peripheral nervous systems are involved. White matter of the brain is always primarily affected and morphological lesions of gray matter are usually minimal or mild. The most conspicuous findings are the presence of the unique globoid cells, profound lack of myelin and severe astrocytic gliosis.

The activity of lactosyl ceramide β-galactosidase in Krabbe’s patients is disputed and the reports of Wenger et al., (1974) on the one hand, and of Suzuki & Suzuki (1974) on the other, are conflicting. This controversy will be discussed later. Although 4-methylumbelliferyl β-galactosidase activity is present in Krabbe’s disease, it has recently been shown (Suzuki & Suzuki, 1974a; Suzuki et al., 1974) to be qualitatively altered. Differences in isoelectricfocusing data (Suzuki & Suzuki, 1974a), have been pointed out between 4-methylumbelliferyl β-galactosidase of Krabbe’s patients and of normal subjects. More interesting is the observation that the enzyme shows significant alterations in different cases of Krabbe’s disease. Suzuki et al. (1974) have put forward a hypothesis that the structural mutations in different cases of Krabbe’s disease may not necessarily be at the same locus. Apart from the isoelectricfocusing data, however, there is no other experimental evidence to support this hypothesis. Recently, we have also investigated 4-methylumbelliferyl β-galactosidase in the liver and brain of a patient who died of Krabbe’s disease (Awasthi & Srivastava, 1978). The fractionation of β-galactosidase by column or thin layer isoelectricfocusing from the liver homogenates of the normal and Krabbe’s disease patients was significantly different.

Unlike GM1 gangliosidosis or Tay-Sachs disease, the biochemical diagnosis of Krabbe’s disease can be performed only by using the natural substrate, radioactively-labeled galactosyl ceramide, for enzyme assays. Recently, Gal et al. (1976) have reported the use of an artificial substrate to demonstrate the deficiency of galactosylceramide β-galactosidase in Krabbe’s disease.

The loss of galactosyl ceramide β-galactosidase activity in Krabbe’s disease is accompanied by the loss of activity towards monogalactosyl diglyceride and β-galactosyl sphingosine, suggesting that these three lipids are degraded by the same enzyme (Wenger et al., 1973). It may be galactosyl sphingosine, a cytotoxic compound, that is responsible for the loss of oligodendroglial cells. Krabbe’s disease is unique among sphingolipidoses in that there is no accumulation of the affected metabolite, galactosylceramide, probably because of the nearly total disappearance of oligodendroglial (myelin-forming) cells which normally synthesize glycolipid (Suzuki & Suzuki, 1973). If this hypothesis is correct, then an abnormal accumulation of galactosyl ceramide should occur in other tissues, especially in kidney, of patients with Krabbe’s disease. Suzuki (1971) demonstrated a 25% elevation of galactocerebroside content in kidneys of Krabbe’s patients compared with normal kidneys. However, a concommitent increase was also obtained in the glucocerebroside content and the lack of accumulation of galactosyl ceramide in neuronal tissue is as yet not clearly understood.

Lactosylceramidosis

This disease is characterized by the accumulation of lactosyl ceramide and the deficiency of lactosyl ceramide β-galactosidase in cells and tissues of the one known patient (Dawson et al., 1971). As this enzyme is present in both GM1 gangliosidosis (Dawson et al., 1971) and in most Krabbe’s patients (Suzuki & Suzuki, 1974), the existence of a distinct β-galactosidase for the cleavage of lactoslyl ceramide is indicated.

Suzuki and Suzuki (1974) reported that the activities of lactosyl ceramide β-galactosidase were normal in livers of patients with Krabbe’s disease whereas the activity in the livers of GM1 gangliosidosis patients, was only about 12% of the normal value. In contrast, Wenger et al. (1974) reported that the brain and liver tissues of GMJ gangliosidosis patients had normal lactosyl ceramide β-galactosidase activity. Moreover, they demonstrated an almost total lack of lactosyl ceramide β-galactosidase in brain, liver and fibroblasts of the Krabbe’s patients. These contradictory findings attracted considerable attention and Tanaka & Suzuki (1975) appear to have solved this controversy. These authors have demonstrated that normally there are two distinct lactosyl ceramide β-galactosidases having separate genetic origins. One of these enzymes, lactosyl ceramide β-galactosidase I (Lac-cer I), is identical with the enzyme missing in Krabbe’s disease (galactosyl ceramidase), while the other enzyme, lactosyl ceramide β-galactosidase II (Lac-cer II), is closely related to nonspecific 4-methylumbelliferyl β-galactosidase or GM1 β-galactosidase. Therefore, the Type I is missing in Krabbe’s disease while Type II is absent in GM1 gangliosidosis. According to these authors, the normal brain contains mostly the Type I enzyme while normal liver contains Type II enzyme. They showed that Type I is significantly activated by crude or pure taurocholate and by oleic acid, but only slightly by chloride ions. Type II, on the other hand, is activated by only crude taurocholate and not by pure taurocholate. For Type II, chloride was a more effective activator than for Type I, while the effect of oleate was less profound as an activator on Type I than on Type II. Tanaka & Suzuki (1975) showed that the assay system of Wenger et al. (1974), which contained pure taurocholate and oleic acid, determined Type I enzyme exclusively whereas the assay system of Suzuki, et al., which had crude taurocholate and no oleic acid, determined the Type II enzyme almost exclusively. Although the conflicting data of the two groups have been satisfactorily explained fundamental questions have arisen concerning the validity of earlier results, the primary question being: What is the exact relationship between these three catalytic activities whose deficiency causes Krabbe’s disease, GM1 gangliosidosis and lactosyl ceramidosis, respectively? If lactosyl ceramide β-galactosidase has a close functional relationship with Krabbe’s enzyme on the one hand, and with GM1 ganglioside β-galactosidase on the other, then what is the relationship between Krabbe’s enzyme and GM1 gangliosidosis enzyme? The very existence of lactosyl ceramide β-galactosidase as a distinct enzyme from GM1 ganglioside and galactosyl ceramide β-galactosidase is in question. Recently, on reexamination of the only case of the so-called lactosyl ceramidosis, Wenger et al. (1975) have discovered both Lac-cer I and Lac-cer II activity in the patient’s fibroblasts and these authors contend that the patient probably suffered from Niemann-Pick disease which has a wide variety of phenotypic variants, often difficult to diagnose. Thus at present, barring possible future discovery of a lactosyl ceramide β-galactosidase deficiency, lactosyl ceramidosis should not be accommodated in the list of clearly defined inborn errors of sphingolipid metabolism.

TAY-SACHS AND SANDHOFF’S DISEASES

In 1881, a British ophthalmologist, Warren Tay, described a condition in an infant of Jewish ancestry characterized by severe mental regression and a curious cherry-red spot in the fundus (Tay, 1881). Several years later, an American neurologist, Bernard Sachs, reported an infant who exhibited similar clinical symptoms and ancestry (Sachs, 1887). In 1942 Klenk & Schumann discovered an accumulation of ganglioside in the brain of Tay-Sachs disease patients and while the genetic nature of this condition was recognized early the primary pathological cause remained unknown until the accumulated lipid was fractionated and