Chapter 24

I. LYSOSOMAL BIOLOGY

II. LYSOSOMAL STORAGE DISEASES (LSDs)

III. PATHOGENESIS

IV. CLINICAL SIGNS

V. DIAGNOSIS

VI. THERAPY

A. ERT

B. BMT

C. Gene Therapy

REFERENCES

I. LYSOSOMAL BIOLOGY

In 1955, de Duve et al. named the cytoplasmic particles that contain a series of hydrolytic enzymes lytic bodies, or “lysosomes.” These organelles have a single lipoprotein membrane and contain several dozen different acid hydrolase enzymes (Holtzman, 1989), which typically catalyze catabolic reactions A-B + H₂O →A-H + B-OH, optimally at acid pH. Lysosomes and their “housekeeping” enzymes degrade many substrates that are found in all nucleated mammalian cells. Deficiencies of these enzymes lead to lysosomal accumulation of their substrates, thereby causing lysosomal storage disease (LSDs), many of which have been discovered and characterized in domestic animals.

In normal cells, most lysosomal hydrolases are synthesized as preproenzymes on rough endoplasmic reticulum (ER) ribosomes. Through a signal-recognition particle complex, the enzymes are translocated into the lumen of the ER where high mannose oligosaccharides are added (Fig. 24-1; reviewed in Kornfeld and Sly [2001]). These oligosaccharides are trimmed, and the glycoprotein moves to the Golgi apparatus where further shortening occurs. Further posttranslational modification results from the action of two enzymes that add a mannose 6-phosphate (M6P) marker. Deficiency in activity of these transferases can result in unique forms of LSD (e.g., mucolipidosis II in domestic shorthair cats). The M6P moiety can be recognized by two similar integral membrane glycoprotein receptors, which transfer the enzyme to the lysosome. These two receptors are (1) small and cation dependent for binding and (2) large and cation independent, which in some species also bind insulin-like growth factor II. Both receptors appear responsible for the transport of the enzymes from the Golgi apparatus via clathrin-coated vesicles to the prelysosomal/endosomal compartment. Once the lysosomal enzymes dissociate, the receptors recycle to the Golgi apparatus.

A proportion of the M6P modified enzyme in the Golgi may also leave the cell via secretory granules (Fig. 24-1). The secreted enzymes can then move from the extracellular space into the circulation. Different enzymes appear to be secreted from cells in varied amounts (Dobrenis et al., 1994). Thus, the level of activity in serum of any particular enzyme is related to how much is secreted and its stability at plasma pH. Secreted enzymes can ultimately reach the lysosome of other cells because the cation-independent receptor is present in the plasma membrane on many cells (Distler et al., 1979; Kaplan et al., 1977; Natowicz et al., 1979). Thus, enzymes that connect with this receptor can be internalized and transferred to lysosomes. This pathway provides the mechanism for therapy for lysosomal storage diseases discussed later.

Although posttranslational glycosylation is common to most lysosomal enzymes, other modifications or activator proteins are necessary for the function of a subset of the hydrolases. For example, the lysosomal sulfatases (17 in humans; 14 in rodents) undergo an additional posttranslational modification by sulfatase modifying factor 1 (SUMF-1), which converts a cysteine residue into C (alpha)-formylglycine (FGly) at the active site (Dierks et al., 2005; Preusser-Kunze et al., 2005). The absence of this conversion results in multiple sulfatase deficiency. A second factor, SUMF-2, which is also part of this system, apparently down-regulates SUMF-1 activity (Zito et al., 2005). The degradation of sphingolipids with short hydrophilic head groups requires sphingolipid activator proteins (SAPs), which are small, nonenzymatic glycoproteins (reviewed in Sandhoff et al., 2001). Deficiency in activity of SAPs is also known to cause lysosomal storage diseases.

Lysosomes degrade large complex substrates that have been taken into a cell by endocytosis or autophagy (the degradation/turnover of a cell’s own molecules). The endosome containing the substrates fuses with a primary lysosome, producing a secondary lysosome, which contains the mixture of hydrolases and substrates. Degradation of most substrates occurs by the activity of a cascade of hydrolases, each step requiring the action of the previous hydrolase to modify the substrate, thereby permitting catabolism to proceed to the next enzyme step in the pathway. If one step in the process fails, further degradation ceases. For example, the glycosaminoglycans (GAGs), formerly known as mucopolysaccharides, are long molecules of repeating subunits and are, as part of proteoglycans, a component of the ground substance of the extracellular matrix. Figure 24-2 illustrates the series of hydrolases that are responsible for the sequential stepwise degradation of one of the glycosaminoglycans, dermatan sulfate. Each of the enzymes in this pathway has been described as deficient in a domestic animal causing different mucopolysaccharidosis.

II. LYSOSOMAL STORAGE DISEASES (LSDs)

The LSDs are defined as a group of individually rare genetic disorders of cellular catabolism involving the lysosome. The earliest detailed clinical reports of an LSD were in humans by Tay (1881) and Sachs (1887). Eight decades later, the stored material in “Tay-Sachs disease” was defined as GM2 ganglioside (Svennerholm, 1962); 7 years later, the enzyme that is deficient in activity (beta-hexosaminidase A) was identified (Okada and O’Brien, 1969; Sandhoff, 1969). Isolation and sequencing of the cDNA coding for the alpha subunit of beta-hexosaminidase A was reported 15 years later (Korneluk et al., 1986; Myerowitz and Proia, 1984) and was quickly followed by the identification of the first of more than 50 mutations responsible for Tay-Sachs disease (Myerowitz and Hogikyan, 1986, 1987) and sequencing of the entire gene (Proia and Soravia, 1987). Similar rapid progress has been made in identifying and characterizing the molecular bases of all lysosomal diseases since the 1980s.

LSDs are inherited as autosomal recessive traits (except MPS II, which is X-linked) and result from mutations in the coding sequence of one of the acid hydrolases located in the lysosome. Point mutations, deletions, insertions, and other alterations in sequence may occur anywhere along the length of DNA coding the enzyme protein. Each individual alteration will produce a unique change in the protein affecting structure, stability, and function. Thus, these genetic abnormalities result in the reduction or elimination of the catalytic activity of the particular enzyme. This, in turn, results in the accumulation within the lysosome of the substrate of that enzyme (Fig. 24-3), hence the name LSD. In many LSDs, the reduction in the amount of product of the metabolic pathway does not appear to produce disease. However, the storage of cholesterol in Niemann-Pick type C disease may result in a downstream deficiency of neurosteroids (Griffin et al., 2004; Mellon et al., 2004). LSDs are classified by the primary substrates that accumulate and are defined by the individual enzyme that is deficient in activity. For example, the mucopolysaccharidoses (MPSs) are a group of diseases resulting from defective catabolism of GAGs (previously mucopolysaccharides). Each of the MPSs is caused by impaired function of one of 12 enzymes required for normal GAG degradation. In humans, these disorders were initially defined by clinical phenotype then by the particular GAGs (heparan, dermatan, chondroitin, keratan sulfates) present in the patient’s urine. Now, in addition to defining the diseases by the specific enzyme deficiency, many are subdivided by the particular mutation in the coding sequence of the gene responsible for the defect.

Different mutations of the same gene may produce similar diseases or somewhat varied degrees of disease as is seen in humans with MPS IH (Hurler) and MPS IS (Scheie), with and without CNS disease, respectively (Neufeld and Meunzer, 2001), and in cats with MPS VI (Crawley et al., 1998). Affected individuals can be either homozygous for the same mutation in both alleles (typical of most LSDs in animals) or heteroallelic (having one mutation in the allele on one chromosome and a different mutation in the allele on the other chromosome [Crawley et al., 1998], common in humans with LSDs). In addition, if the substrates being stored in different diseases have similar pathological effects, defects in different lysosomal enzymes may produce similar diseases, as has been described in humans and animals with MPS III A-D (Aronovich et al., 2001; Bhaumik et al., 1999; Ellinwood et al., 2003; Fischer et al., 1998; Jones et al., 1998; Neufeld and Meunzer, 2001; Yogalingam et al., 2002). Furthermore, it is now recognized that the expression of lysosomal genes, similar to other inborn errors of metabolism, is also influenced by other (modifying) genes and the environment, which explains the phenotypic variation in animals homozygous for the same mutation. Finally, the clinical features and disease course of animals with all types of LSDs closely resemble their human counterparts.

Animals of several species were diagnosed clinically and pathologically as having an LSD before recognizing that the group of diseases were caused by deficiencies in hydrolase activity. Because of the distinctive central and peripheral nervous system lesions, the first of these diseases to be described was globoid cell leukodystrophy in Cairn and West highland white terriers (Fankhauser et al., 1963). These two related dog breeds (primarily a color variation) are now known to have the same mutation in the gene coding for galactosylceramidase (Victoria et al., 1996), which apparently originated in the 19th century from an ancestor common to these two breeds that diverged around the beginning of the 20th century. The first definitive discovery of an enzyme deficiency in a nonhuman mammal was GM1 gangliosidosis in a Siamese cat by Baker and colleagues in 1971 (Baker et al., 1971). Since then, naturally occurring LSDs defined by a deficiency in lysosomal enzyme activity have been recognized in cats, cattle, dogs, goats, mice, pigs, rats, horses, sheep, and two avian species, emus and flamingos (Table 24-1).

Additional storage diseases do not involve lysosomal enzymes and, thus, are not strictly LSDs, but some have been included in Table 24-1. These include glycogen storage disease IV Niemann-Pick disease C, and ceroid lipofuscinoses. Many mouse models of LSDs have been created by gene knockout technology, but have not been included in Table 24-1. In creating murine knockouts, the phenotype has ranged from essentially the same as in humans, to no disease, to being fatal soon after birth. New knockout models of LSDs will continue to be created in mice to learn more about the pathogenesis of these debilitating disorders and to evaluate therapy. However, companion animals appear often to be better disease homologues and are important to translating novel therapies to humans.

In LSDs, the continued presentation of substrates to the cell and their lack of degradation result in their storage and swelling of the lysosomes. By electron microscopy, lysosomes within the cytoplasm can be seen as membrane-bound inclusions containing the stored substrate (Fig. 24-4). As the lysosomes become larger, they can be seen with light microscopy (Figure 24-5). However, in some LSDs, the accumulated substrate may be lost during tissue processing, leaving empty vacuolar artifacts. The accumulation of the primary substrate for a particular enzyme pathway may also interfere with other lysosomal hydrolases necessary for different catabolic pathways (Kint et al., 1973), thereby leading to the secondary accumulation of additional substrates. As more substrates accumulate, the lysosomes occupy more of the cytoplasm (Fig. 24-6). This increase in the number and size of lysosomes may obscure the other cellular organelles and may deform the nuclear outline. As the process continues, the affected cells enlarge, which is one cause of organomegaly. Just as with the CNS, cartilage, and bone, pathophysiology is probably not solely related to the increase in the cell, tissue, or organ size. The storage of GAGs within the mitral heart valve causes the normally fusiform cells to become rounded (Fig. 24-6). This, in turn, causes the valve leaflet and cordae tendinea to become thick (Fig. 24-7), interfering with normal valve function and producing mitral regurgitation. Similarly, storage within the cells of the cornea (Figure 24-8) results in reflection and refraction of light, producing the cloudiness observed grossly and by ophthalmoscopy (Fig. 24-9). However, in the cornea there is also an abnormality in collagen biosynthesis resulting in larger fibrils that are more widely spaced than normal (Alroy et al., 1999), and the cornea of the MPS VI cat, rather than being thicker because of increased cell size, is thinner than normal (Aguirre et al., 1992).

In many LSDs, the CNS contains swollen neurons (Fig. 24-10) with lysosomes that contain lamellar substrate (Fig. 24-11). The pathogenesis of the CNS lesions includes the development of meganeurites and neurite sprouting, which appear correlated to alterations in ganglioside metabolism (Purpura and Baker, 1977, 1978; Purpura et al., 1978; Siegel and Walkley, 1994; Walkley, 1988; Walkley et al., 1988, 1990, 1991). Gangliosides, whether stored as a primary substrate (in G_M1 and G_M2 gangliosidosis) or secondarily (in MPS I and III), appear to stimulate the development of neurite sprouts with synapses. The presence of new neurites and their synapses apparently plays a role in the CNS dysfunction of these diseases (Walkley, 2003).

Non-CNS clinical signs associated with lysosomal storage disorders include failure to thrive, growth retardation (Fig. 24-12), umbilical hernia, corneal clouding, hepatosplenomegaly, cardiac murmurs, renal dysfunction, and skeletal abnormalities including facial dysmorphia and vertebral, rib, and long bone deformities (Fig. 24-13). The MPS disorders, in general, have more organ systems affected than the other diseases. The age of onset and severity of clinical signs are usually relatively consistent for a particular disease in animals; however, some variability can exist even in a family having the same disease-causing mutation. In research colonies of dogs and cats with LSDs kept in a relatively consistent environment, the explanation for variation in clinical signs rests with the variable genetic background (modifying genes) on which the mutation is expressed.

Most LSDs are manifest within a few months after birth, with some evident at birth or before weaning and fewer with adult onset (canine MPS IIIA and B). In severely affected animals, death often occurs at birth or before weaning.

A pedigree analysis should be performed as part of the history to determine information about the inbreeding of the parents and the presence of other family members with similar clinical signs or that died early. As most LSDs are inherited as autosomal recessive traits, parents are often related and are carriers (heterozygotes) but are clinically (phenotypically) normal. On average, one-fourth of the offspring of heterozygous parents are affected, two-thirds of unaffected offspring are carriers, and other relatives may also be affected (Fig. 24-14).

Abnormal metabolites may be found in urine and their presence points toward specific metabolic pathways that warrant further evaluation. A metabolic screen of urine (Fig. 24-15) (Giger and Jezyk, 1992; Jezyk et al., 1982) for GAGs is a relatively simple and inexpensive approach to identify the mucopolysaccharidoses and some cases of gangliosidosis (toluidine blue or MPS spot test; Fig. 24-16). Thin layer chromatography of urinary oligosaccharides is helpful to identify mannosidosis. Urine samples to be evaluated should be kept refrigerated or frozen and sent to an appropriate laboratory.¹

A final diagnosis for LSDs requires the demonstration of a particular enzyme deficiency by either determining the lack of enzyme activity or a disease-causing mutation in an enzyme gene; these tests do not only identify affected animals but are also helpful in identifying carriers. Enzyme assays using artificial substrates can usually be performed on serum, white blood cells, cultured fibroblasts, or liver. Generally, there is a profound deficiency in activity of the enzyme, making the diagnosis straightforward. In addition, the activities of other lysosomal enzymes in the cells or tissues are frequently higher than normal. The biochemical status of the clinically normal parents should be evaluated when possible. In an autosomal recessive disease, heterozygous parents are expected to have half-normal activity of the enzyme in question because each parent carries one normal and one mutant allele. Although in a population, heterozygotes (carriers) have on average half-normal activity, there is overlap between the ranges for enzyme activity values from normal and obligate heterozygous animals (Fig. 24-17). Thus, accurate determination of an asymptomatic individual as normal or a carrier may be difficult with an enzyme assay alone but can best be achieved by molecular DNA tests for the specific mutation in those diseases and families where the mutation has been identified.²

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