The mutations responsible for most lysosomal storage diseases (LSDs) have been largely elucidated; however, the molecular pathways through which the storage material causes cellular and organ pathology are largely unknown. Recent studies have underlined the importance of inflammation, apoptosis, alteration in signal transduction and transport for some of the lysosomal disorders. Almost all LSDs show a broad clinical spectrum with respect to severity of symptoms, progression and age of onset. This can be explained, in part, by the residual enzyme activity associated with the particular alleles the patient carries. This correlation is loose, however, and does not allow prediction of the clinical course on an individual basis. Other as yet unknown genetic and epigenetic factors influence the disease phenotype substantially.

Introduction

Receiving the Nobel prize in 1974 for his discovery of lysosomes, Christian de Duve stated that "with more than 20 distinct congenital lysosomal enzyme deficiencies identified, this mysterious chapter of pathology has been largely elucidated" [1]. Although appropriate at that time, this statement does not reflect the current view of the pathophysiology of lysosomal storage diseases (LSDs). In all LSDs, the central pathophysiological question is how the storage material affects the metabolism of a cell and subsequently leads to organ pathology and clinical symptoms. The pathophysiology of a disorder is determined by the chemical nature of the storage compound, the extent of storage, the kinetics of accumulation and the type and spectrum of storage cells [2]. For example, the abundance of cerebrosides and gangliosides in the nervous system explains why sphingolipid storage disorders are characterized by severe neurological symptoms. Likewise, the glycogen content of muscle provides an explanation as to why myopathy dominates in the pathology of Pompe disease, in which glycogen breakdown is defective. The diversity of pathology and clinical phenotypes requires that different pathophysiological mechanisms must be elucidated for each specific disorder.

Cellular pathophysiology

In some related disorders, such as the sphingolipidoses, pathogenic mechanisms may, at least partially, be shared [3, 4]. In these disorders, inflammation, apoptosis and alterations in signal transduction and transport are all involved in pathogenesis. Mice deficient in the β-subunit of β-hexosaminidase are a model of the GM2-gangliosidosis, Sandhoff disease. The animals develop severe neurological symptoms at the age of 3–4 months. The development of symptoms correlates with the activation of microglia and an increased frequency of apoptotic neurones [4]. Transplantation of the affected mice with bone marrow from a normal donor leads to a substantial reduction in the number of apoptotic neurones, which is accompanied by an increased lifespan. Surprisingly, however, transplantation does not result in delivery of β-hexosaminidase to the brain or to a reduction in GM2-ganglioside storage, suggesting that neuronal storage, per se, is not responsible for apoptosis. However, bone marrow transplantation markedly reduces the activation of microglia and, consequently, the production of cytokines and tumour necrosis factor-α (TNFα). This suggests that the initial neuronal damage results in microglial phagocytosis. Subsequent accumulation of GM2-ganglioside in microglia causes activation accompanied by secretion of various cytokines, which promotes apoptosis. This example demonstrates that pathogenic mechanisms cannot simply be explained by cellular alterations of a single cell type but are rather the result of a complex interplay between different cells.

Microglial activation also occurs in a number of other LSDs, such as Krabbe disease [3], metachromatic leukodystrophy [5] and Gaucher disease [6]. In twitcher mice – a model of Krabbe disease – microglial activation and increased levels of interleukins and TNFα also correlate with the frequency of apoptotic cells. Thus, the role of microglial activation in the pathophysiology of the mouse model of Sandhoff disease may also apply to other lipidoses. Similarly, in Gaucher disease, the accumulation of glucosylceramide in macrophages results in their activation [6]. Interleukins 1β, 6 and 10 and TNFα are elevated in the serum of patients with Gaucher disease and have been implicated in the development of osteopenia and gammopathies in type I Gaucher disease [7, 8]. Interestingly, splenomegaly and hepatomegaly in patients with Gaucher disease cannot be explained by lipid accumulation, which accounts for only about 2% of the tissue mass in the up to 25-fold enlarged organs [9]. It is possible that secretion of mitogenic factors by activated macrophages is the dominant factor causing an increase in the number of cells in the liver and spleen.

In addition to Sandhoff disease, increased apoptosis has been demonstrated in a number of other LSDs, such as Krabbe, Gaucher, Niemann–Pick type C and Pompe diseases, mucopolysaccharidosis type VI and GM1-gangliosidosis [10–14]. Whether this is due to the activation of a common apoptotic pathway has not been elucidated. In contrast, apoptosis is inhibited in Niemann–Pick type A disease, which is caused by sphingomyelinase deficiency [15].

A pathological hallmark of GM2-gangliosidosis in humans, as well as in mice, is the development of meganeurites and additional spines on neurones, which is one reason for the severe neurological symptoms [16]. In the developing human brain, the appearance of GM2-ganglioside in the various layers of the cortex parallels dendritogenesis [17]. GM2-ganglioside is therefore considered to be part of the developmental programme involved in dendritogenesis. The accumulation of GM2-ganglioside in the brains of affected patients may result in an inability to terminate this developmental programme, leading to continued dendritogenesis and the development of meganeurites and spines. Contact of these spines with other dendrites can lead to abnormal spreading of signals within neuronal networks, which is likely to contribute to the neurological symptoms.

In some of the lysosomal disorders, toxic metabolites appear to play a major pathogenic role, rather than the storage material itself. Examples of these disorders are Krabbe disease, metachromatic leukodystrophy and Gaucher disease [18–20]. Besides galactosylceramide – which is the major storage compound in Krabbe disease and in its animal model, the twitcher mouse – patients and mice have substantially increased levels of the lysolipid galactosylsphingosine, also called psychosine. The structural difference between psychosine and galactosylceramide is the loss of the fatty acid chain attached to the amino group of the sphingosine backbone. Similarly, increased lysosulphatide and glucosylsphingosine concentrations have also been shown to occur in metachromatic leukodystrophy and Gaucher disease, respectively [19, 20]. Lysolipids are biologically very active compounds [21]. They are potent inhibitors of protein kinase C, interfere with the integrity of membranes and lead to activation of inducible nitric oxide synthase [21, 22]. Psychosine stimulates Ca²⁺ release from the endoplasmic reticulum [23] and binds with high affinity to a septahelical membrane receptor, termed TDAG 8 [24]. This receptor is coupled to G proteins, eliciting a cyclic AMP (cAMP) response. High concentrations of psychosine in the brains of patients with Krabbe disease may therefore produce constantly stimulated cAMP-dependent pathways, which is likely to be of pathophysiological relevance. Therefore, the interference of lysosphingolipids with protein kinase A, protein kinase C and Ca²⁺-mediated signal transduction pathways suggests a major pathophysiological role for these lipids in various disorders. This is supported by the fact that glucosylsphingosine is found in higher levels in the brains of neurologically affected patients with type I and type II Gaucher disease [25].

Effects of different mutations on pathophysiology

In the past two decades our knowledge about LSDs has grown considerably. Sequences of genes encoding lysosomal enzymes have been elucidated and resulted in the identification of many of the mutations responsible for the enzyme deficiencies in the various disorders. Today, from the genetic point of view, most LSDs are well understood; however, the biochemical mechanisms by which a mutation causes a deficiency have only partially been resolved. The molecular consequences of nonsense mutations, deletions, insertions and duplications that cause premature termination and/or frame shifts are mostly obvious, as they frequently lead to absent or truncated proteins, which results in a complete loss of functional enzyme. Several splice-site mutations have been identified in lysosomal disorders. In some instances, these mutations do not allow the generation of functional mRNA [35]. Frequently, however, the mutations are leaky, such that a small percentage of transcripts is still correctly spliced and translated, producing reduced amounts of normal enzyme. This kind of mutation, therefore, usually leads to attenuated forms of disease [36].

The effects of missense mutations or small in-frame deletions are less apparent and need molecular biological characterization to understand the cause of the enzyme deficiency. Compared with the large number of missense mutations identified in lysosomal disorders, however, only a limited number have been investigated to elucidate the biochemical consequences for the respective enzyme. The deleterious effect of a missense mutation is obvious if it affects amino acid residues that are involved in the active centre [37–39] or that are important for proper folding. Active-site mutations may lead to a complete loss of enzyme activity, but may also result in alterations of enzyme kinetics [39]. Substitution of cysteine residues or charged residues involved in formation of intramolecular disulphide bonds or salt bridges, respectively, is likely to interfere with proper folding of the enzyme [40, 41]. Mutations affecting the active centre or intramolecular bonds, however, are in the minority.

In spite of the fact that structural information [38, 40] was expected to shed more light on the molecular consequences of amino acid substitutions, the effects of most missense mutations are still not obvious. Thus, it is difficult to understand why even very conservative amino acid substitutions can severely affect a lysosomal enzyme [41]. In many cases, amino acid substitutions cause misfolding of the enzyme, which results in retention of the mutant polypeptide in the endoplasmic reticulum and its subsequent proteasomal degradation [42–44]. It has been estimated that degradation associated with the endoplasmic reticulum is responsible for about 50% of protein defects in all genetic diseases and thus represents the most frequent cause of deficiencies [45]. Although this proportion has never been specifically calculated for LSDs, it is reasonable to assume that comparable numbers apply [43]. In many cases, retention of the misfolded enzyme in the endoplasmic reticulum is complete, such that no functional enzyme reaches the Golgi apparatus to be phosphorylated and sorted into the lysosome [40–42]. Therefore, these mutations frequently lead to a complete deficiency of the enzyme. Even if these mutant enzymes still retain some activity they will not be functional, as they are mislocated [39]. As with splice-site mutations, however, the process of endoplasmic quality control can be leaky. There are examples in which the majority of misfolded enzyme is retained in the endoplasmic reticulum, with a small fraction bypassing the quality control and being sorted to the lysosomes (5, 12). If this enzyme still has residual activity, it can result in an attenuated disease phenotype [46, 47].

For therapeutic reasons, an escape from the endoplasmic reticulum may also be induced by 'molecular chaperones' – small molecules such as competitive enzyme inhibitors. In Fabry disease, the R301Q-substituted enzyme is usually retained in the endoplasmic reticulum. Treatment with a competitive inhibitor, however, improves folding and increases lysosomal delivery of the enzyme, which can be therapeutically exploited [48] (Figure 1).

Figure 1

Concept of treatment by molecular chaperones. All panels show schematically the rough endoplasmic reticulum (RER), the Golgi apparatus and a lysosome within a cell. A single lysosomal enzyme is represented below each cell. (a) A correctly folded enzyme (more...)

Alternatively, amino acid substitutions allow for proper folding of the enzyme to pass the quality control of the endoplasmic reticulum. Arylsulphatase A deficiency causes meta-chromatic leukodystrophy, a lipid storage disease. The most frequent disease-associated allele in adult patients is characterized by a missense mutation causing substitution of proline 426 by leucine (P426L) [35]. The P426L substituted enzyme is active, reaches the Golgi apparatus, receives M6P residues and is correctly sorted to the lysosomes. Due to the amino acid substitution, however, the mutant protein is rapidly degraded by lysosomal proteases [49]. The half-life of the mutant enzyme, however, is still sufficient to provide a small amount of residual enzyme activity, explaining the juvenile or adult-onset forms of metachromatic leukodystrophy in patients carrying this allele. If the fibroblasts of patients expressing this mutant enzyme are treated with inhibitors of lysosomal proteases, the enzyme can be stabilized to yield low-normal activity values [49]. This demonstrates how a precise understanding of mutation effects can provide strategies for possible therapies.

To understand the molecular basis of enzyme deficiencies in even more detail requires not only the biochemical characterization but also the crystallization of mutant proteins. P426L-substituted arylsulphatase A is so far the only example of a lysosomal enzyme in which a mutant enzyme has been crystallized and the molecular consequences of the amino acid substitution revealed in detail [49]. A cleavage site for cathepsin L was found in close proximity to proline 426. In the normal enzyme, this cleavage site becomes inaccessible to cathepsin L, because arylsulphatase A octamerizes in the low pH environment of the lysosome. Thus, octamerization is a prerequisite for the intralysosomal stability of the enzyme.

The crystal structure of the P426L-substituted arylsulphatase A reveals that the amino acid substitution interferes with the capacity of the enzyme to octamerize at low pH [49]. The reason for enzyme deficiency due to the P426L subsitution is therefore the inability to octamerize intralysosomally and to protect the enzyme from attack by cathepsin L.

Factors influencing the clinical heterogeneity of LSDs

Most of the LSDs show a wide spectrum of clinical phenotypes (e.g. [50, 51]. In many disorders, severe early-onset forms of disease are distinguished from attenuated intermediate and late-onset forms. This classification suggests the existence of distinctive clinical entities. The clinical spectrum, however, represents a continuum with respect to severity, progression and age of onset. The identification of mutations in the various disorders was accompanied by a hope that clear genotype–phenotype correlations would be found, ideally allowing prediction of the clinical course of disease in individual patients. Investigations of genotype–phenotype correlations, however, have only partially revealed the molecular basis of the clinical heterogeneity of disease [35, 52–54]. In many disorders, the clinical phenotype is loosely related to the amount of residual enzyme activity associated with the particular mutation.

One of the first diseases in which a genotype–phenotype correlation was identified was metachromatic leukodystrophy [35, 52]. In this disease, two defective alleles are particularly frequent, each representing about 25% of alleles among European patients. One allele is characterized by a splice donor-site mutation, which results in a complete loss of mRNA and, therefore, no enzyme is synthesized. This allele represents a null mutation. The other frequent allele is the above-mentioned P426L allele, which still allows for the expression of low enzyme activity. When the distribution of these alleles was investigated in patients with metachromatic leukodystrophy displaying clinical phenotypes of varying severity, a genotype–phenotype correlation became apparent. Homozygosity for null alleles was always associated with the most severe late-infantile form of disease; heterozygosity for a null allele and an allele with residual enzyme activity mitigated the course to the intermediate juvenile form; and homozygosity for alleles with residual enzyme activity was associated with the attenuated, mostly adult, form of metachromatic leukodystrophy [35].

Correlations with alleles allowing for the expression of low residual enzyme activity with attenuated clinical forms of disease are found in a number of lysosomal disorders, including Gaucher disease [53]. However, it must be emphasized that genotype–phenotype correlations become apparent only when large numbers of patients are studied [35, 54]. The variations between individual patients with an identical genotype can be enormous, even within the same family [54]. Therefore, in none of the LSDs does the genotype of a patient allow the prediction of the clinical course for the individual. The correlation between residual enzyme activity and attenuation of clinical phenotype has not only been found at the genetic level, but was also confirmed biochemically for metachromatic leukodystrophy and Tay–Sachs disease (52). The methods applied in this study were sophisticated and cannot be applied as routine diagnostic procedures. For this reason, it must be emphasized that enzyme activities that are determined for diagnostic purposes in, for example, leukocyte homogenates, do not enable even an approximate estimate of residual enzyme activity. Residual enzyme activities leading to attenuated forms of disease are in the range of 2–8% of normal activity. In routine assays, it is impossible to quantify enzyme activities reliably within this low range (Figure 2).

Figure 2

Correlation of enzyme activity and lipid-degrading capacity in fibroblasts of patients with Tay–Sachs disease. Tay–Sachs disease is a lipid storage disorder caused by deficiency of hexosaminidase A. Patients store GM2-ganglioside and suffer (more...)

Residual enzyme activity is only one of the determinants of clinical outcome in many LSDs. The influence of other factors is substantial. These, so far unidentified factors, appear to be genetic as well as epigenetic. Clinical variability in Sandhoff and Pompe diseases suggests the existence of genetic factors [36, 55–57]. Sandhoff disease is caused by mutations in the gene for the β-subunit of β-hexosaminidase, which leads to the accumulation of GM2-ganglioside. Four siblings of a Canadian family were identified who were heterozygous for a null allele and an allele containing a mutation close to a splice acceptor site [36]. The latter is associated with low residual enzyme activity, as the normal splice site is used in a small percentage of transcripts. One of the siblings developed a very attenuated form of Sandhoff disease in his 50s, whereas the other siblings, aged 55–61 years, were still presymptomatic. A single copy of this splice-site allele allows for an attenuated form of disease or even an asymptomatic state in these individuals. The same mutation was independently found in a Japanese patient who suffered from a far more severe juvenile form of the disease [55]. Surprisingly, this patient was homozygous for the splice-site allele. A possible explanation could be that minor variations in genes coding for components of the splicing machinery lead to a more efficient use of the correct splicing site in the Canadian patients than in the Japanese patient. A similar situation occurs in Pompe disease, where a D645E missense mutation in the α-glucosidase gene causes divergent phenotypes in Chinese and African–American patients [56, 57]. This strongly suggests that differences in the genetic background account for the clinical outcome of the disease in patients from different ethnic groups.

The most frequent allele causing nonneuronopathic type I Gaucher disease is characterized by substitution of asparagine 370 by serine (N370S) in the glucocerebrosidase gene. This allele codes for an enzyme with 10–20% of normal activity [54]. When more than 200 patients homozygous for this allele were examined, an enormous heterogeneity became apparent [54]. The age of onset of disease in this group of patients varied between early childhood and senescence. In fact, calculation of allele frequencies and comparison with the prevalence of patients revealed that about two-thirds of the N370S homozygotes remain asymptomatic [58]. A particular case study of Gaucher disease revealed the importance of epigenetic factors for clinical variability in this disorder. N370S homozygous monozygotic twin sisters were recently identified [59]. They did not marry, lived together during their entire lives and both died at 84 years of age. Only one of the sisters, however, developed type I Gaucher disease, whereas the other twin remained asymptomatic. These monozygotic siblings demonstrate that epigenetic factors, perhaps infections, play a substantial role in the phenotypic variability of lysosomal disease.

Some diseases vary not only with respect to severity and age of onset but also with respect to organ involvement. Type I Gaucher disease is primarily a disease of macrophages, affecting the bone marrow, spleen, liver and lungs, but is not associated with CNS involvement [51]. In contrast, type II Gaucher disease has, in addition, severe nervous system involvement [51]. The degree of residual enzyme activity may explain these considerable differences in phenotype. The pool of glucosylceramide that must be degraded in a particular cell is derived from endogeneous synthesis and/or exogenous sources. The latter are particularly important for phagocytosing macrophages. Thus, in the case of a severe enzyme deficiency, the amount of glucosylceramide synthesized in neurones is sufficient to cause storage in these cells. In the case of sufficiently high residual glucocerebrosidase activity, the endogenously synthesized lipid in neurones is degraded. The lipid load of macrophages, however, is much higher, as among other debris they phagocytose aged erythrocytes, which contain glucosylceramide. Storage in type I Gaucher disease therefore occurs in macrophages but not in neurones [53]. A comparable situation is also found in Niemann–Pick disease. Here, the Δ608 single amino acid deletion in the sphingomyelinase gene allows for the expression of some residual enzyme activity. The presence of at least one copy of this allele appears to prevent the development of the neuronopathic Niemann–Pick type A disease, but attenuates the course to type B disease, with no or little neuronal involvement [60, 61].

Conclusions

The cellular pathophysiology of LSDs is a consequence of the underlying mutation and the toxic effects of the accumulating compounds. Mutations causing a complete loss of enzyme activity result mostly in severe disease of early onset. In contrast, alleles that still allow the expression of low amounts of residual enzyme activity are frequently associated with attentuated forms of disease. These forms, in particular, show substantial variability even among siblings. The factors causing this variability are unknown. Among the LSDs, lipidoses have been most intensively studied with respect to pathogenic mechanisms. In these disorders, microglial activation and/or apoptosis frequently play an important role in pathogenesis. In some disorders, toxic lysolipids accumulate. Calcium release from the endoplasmic reticulum is altered in some lipidoses and may account for alterations in calcium-mediated signal transduction. As the genetics of most disorders is now well understood, future research in the area of LSDs will focus on cellular pathophysiology and the development of therapies.

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