Lysosomal Lipid Storage Diseases

Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.Lysosomal lipid storage diseases are a group of inherited catabolic disorders in which typically large amounts of complex lipids accumulate in cells and tissues. Macromolecules such as complex lipids and oligosaccharides are constitutively degraded in the acidic compartments of the cell, the endosomes, and lysosomes, into their building blocks. The resulting catabolites are exported to the cytosol and reused in cellular metabolism. When lysosomal function is impaired because of a defect in a catabolic step, degradation cannot proceed normally and undegraded compounds accumulate. Lysosomal lipid storage diseases comprise mainly the sphingolipidoses, Niemann-Pick type C disease (NPC), and Wolman disease, including the less severe form of this disease, called cholesteryl ester storage. NPC is a complex lipid storage disease mainly characterized by the accumulation of unesterified cholesterol in the late endosomal/lysosomal compartment (Bi and Liao 2010). The sphingolipidoses are caused by defects in genes encoding proteins involved in the lysosomal degradation of sphingolipids (Kolter and Sandhoff 2006). First reports on these diseases were given more than a century ago. Already in 1881, Warren Tay described the clinical symptoms of a disease, which is today called Tay-Sachs disease (Tay 1881). After Christian de Duve discovered the lysosome in 1955 (de Duve 2005), Henri-Géry Hers established the first correlation between an enzyme deficiency and a lysosomal storage disorder (Pompe’s disease) in 1963 (Hers 1963). In the following decades, the enzymes and cofactors deficient in the sphingolipidoses have been identified. Though lysosomal lipid storage diseases have been known for a long time, treatment is only available for a few mild forms of the diseases, such as the adult forms of Gaucher disease (Barton et al. 1991). For several lysosomal storage diseases, therapies like enzyme replacement or bone marrow transplantation are in the clinical trial stage (Platt and Lachmann 2009). For a long time, lysosomal diseases have been considered a problem of superabundance (storage) in which the storage material can slowly spread to other cellular membranes, impairing their function. More recently, it came into focus that massive storage prevents lysosomal functions such as nutrition delivery through the endolysosomal system, leading to a state of cellular starvation. In mouse models of both GM1 and GM2 gangliosidoses iron is progressively depleted in brain tissue. Administration of iron prolonged survival in the diseased mice by up to 38% (Jeyakumar et al. 2009).     

Role of membranes in disease

The membranes of mammalian cells are composed of an ordered array of lipids and proteins, the latter containing carbohydrate residues directed towards the exterior and important in the interaction of cells with each other and with external proteins. This external (plasma) membrane and other more simple membranes within the cell are damaged in all diseases which compromise the integrity of the cell. However, in many cases, chemical or functional changes in these membranes are central to the pathogenesis of the disease. These processes are illustrated, and a classification of membrane-related diseases is proposed. This includes: receptor-related diseases such as type II familial hypercholesterolaemia, Grave's disease, some lysosomal storage diseases and some forms of diabetes and obesity; structural instability as manifested by red cell abnormalities and multiple sclerosis; changes in lipid state as in muscular dystrophy and multiple sclerosis; altered permeability or transport as in cystic fibrosis, diseases associated with specific transport defects, and the action of many bacterial toxins, and abnormality of the cytoskeleton-membrane interface as in Chediak-Higashi disease and some diseases associated with red cell abnormalities. Different mechanisms can contribute to the membrane disorder in a single disease state and many of these are described to illustrate this diversity.     

Binding of the small-molecule kinase inhibitor ruxolitinib to membranes does not disturb membrane integrity

Ruxolitinib is a small-molecule protein kinase inhibitor, which is used as a therapeutic agent against several diseases. Due to its anti-inflammatory impact, ruxolitinib has also been considered recently for usage in the treatment of Covid-19. While the specific effects of ruxolitinib on Janus kinases (JAK) is comparatively well investigated, its (unspecific) impact on membranes has not been studied in detail so far. Therefore, we characterized the interaction of this drug with lipid membranes employing different biophysical approaches. Ruxolitinib incorporates into the glycerol region of lipid membranes causing an increase in disorder of the lipid chains. This binding, however, has only marginal influence on the structure and integrity of membranes as found by leakage and permeation assays.     

Ito cells in lysosomal storage disorders. An ultrastructural study

An ultrastructural study was performed in a series of liver biopsies from patients with various lysosomal storage diseases to evaluate the extent of lysosomal hypertrophy and hyperplasia in Ito cells (ICs). In previous studies this has been considered to be absent or only rudimentary. Lysosomal storage was recognized by the presence of storage cytosomes surrounded by limiting membranes and by the appearance of their content which was identical to that in other hepatic storage lysosomes. Storage was found in sphingomyelinase deficiency (Niemann-Pick disease types A, B), in Wolman's disease, GM1 gangliosidosis, mucopolysaccharidosis and in multiple sulphatase deficiency. In type C Niemann-Pick disease it was virtually absent with the exception of cases with prominent hepatic symptomatology. Storage was of variable degree and was accompanied by a decrease in the physiological fat content (cytoplasmic lipid droplets). The degree to which ICs were affected correlated only with the extent to which nonspecific fibroblasts were involved in the specimens studied and thus seems to reflect storage in the fibroblastic population.     

Basement membranes and human disease

In 1990, the role of basement membranes in human disease was established by the identification of COL4A5 mutations in Alport’s syndrome. Since then, the number of diseases caused by mutations in basement membrane components has steadily increased as has our understanding of the roles of basement membranes in organ development and function. However, many questions remain as to the molecular and cellular consequences of these mutations and the way in which they lead to the observed disease phenotypes. Despite this, exciting progress has recently been made with potential treatment options for some of these so far incurable diseases.     

Substrate deprivation therapy: a new hope for patients suffering from neuronopathic forms of inherited lysosomal storage diseases

Lysosomal storage diseases are a group of disorders caused by defects in enzymes responsible for degradation of particular compounds in lysosomes. In most cases, these diseases are fatal, and until recently no treatment was available. Introduction of enzyme replacement therapy was a breakthrough in the treatment of some of the diseases. However, while this therapy is effective in reduction of many somatic symptoms, its efficacy in the treatment of the central nervous system is negligible, if any, mainly because of problems with crossing the blood-brain-barrier by intravenously administered enzyme molecules. On the other hand, there are many lysosomal storage diseases in which the central nervous system is affected. Results of very recent studies indicate that in at least some cases, another type of therapy, called substrate deprivation therapy (or substrate reduction therapy) may be effective in the treatment of neuronopathic forms of lysosomal storage diseases. This therapy, based on inhibition of synthesis of the compounds that cannot be degraded in cells of the patients, has been shown to be effective in several animal models of various diseases, and recent reports demonstrate its efficacy in the treatment of patients suffering from Niemann-Pick C disease and Sanfilippo disease.     

Storage solutions: treating lysosomal disorders of the brain

Many neurodegenerative diseases are characterized by the accumulation of undegradable molecules in cells or at extracellular sites in the brain. One such family of diseases is the lysosomal storage disorders, which result from defects in various aspects of lysosomal function. Until recently, there was little prospect of treating storage diseases involving the CNS. However, recent progress has been made in understanding these conditions and in translating the findings into experimental therapies. We review the developments in this field and discuss the similarities in pathological features between these diseases and some more common neurodegenerative disorders.     

Dissecting desaturation: plants prove advantageous

Fatty acid desaturases play important roles in controlling the physical properties o f membranes and in the synthesis of signal molecules such as prostaglandins and pheromones. Most desaturases are membrane proteins that have been recalcitrant to characterization by conventional biochemical methods. Only one enzyme o f this class has been characterized from animals or fungi. In this context, plants have proved to be useful sources of experimental materials. Substantial progress has been made in characterizing and manipulating nine classes of desaturases that control the fatty acid composition o f both plant membranes and plant storage lipids, which account for approximately -30% of the calories in the human diet.     

A secretion granule membrane protein (GRAMP 92) is found in non-granule membranes including those of the endocytic pathway.

GRAMP 92, a secretion granule-associated membrane protein, has been identified in exocrine and endocrine storage granule membranes using a monoclonal antibody against rat parotid secretion granule membranes. This integral membrane glycoprotein has a M(r) of 92,000 in pancreatic zymogen granule membranes, and is slightly smaller in endocrine granule membranes. In both cases, deglycosylation produces core proteins of M(r) 52,000, that have identical peptide fingerprints. Unlike the slightly smaller zymogen granule membrane glycoprotein GP-2, GRAMP 92 does not appear to be bound to the membrane by a glycophosphatidyl inositol anchor, is not found on the plasma membrane and is not released into the secretion. Within acinar cells, low levels of antigen are observed immunocytochemically over the membranes of most granules. Antigen is highly concentrated on small vesicles that are closely apposed to (and possibly interact with) granules. As well, antigen is localized to organelles in the Golgi and basolateral regions that are part of the endocytic pathway. In hepatocytes a glycoprotein similar if not identical to GRAMP 92 marks the endocytic pathway including lysosomes. These findings indicate that GRAMP 92 is a widely distributed endocytic component and suggest that cells specialized for regulated secretion may adapt such components for storage granule function. Granule-associated GRAMP 92-rich membranes may link the exocytotic and endocytic pathways.     

Future perspectives for glycolipid research in medicine

Medical interest in glycolipids has been mainly directed to the rare and complex glycosphingolipid storage disorders that are principally caused by unitary deficiencies of lysosomal acid hydrolases. However, glycolipids are critical components of cell membranes and occur within newly described membrane domains known as lipid rafts. Glycolipids are components of important antigen systems and membrane receptors; they participate in intracellular signalling mechanisms and may be presented to the immune system in the context of the novel CD1 molecules present on T lymphocytes. A knowledge of their mechanism of action in the control of cell growth and survival as well as developmental pathways is likely to shed light on the pathogenesis of the glycosphingolipid storage disorders as well as the role of lipid second messengers in controlling cell mobility and in the mobilization of intracellular calcium stores (a biological role widely postulated particularly for the lysosphingolipid metabolite sphingosine 1-phosphate). Other sphingolipid metabolites such as ceramide 1-phosphate may be involved in apoptotic responses and in phagocytosis and synaptic vesicle formation. The extraordinary pharmaceutical success of enzymatic complementation for Gaucher's disease using macrophage-targeted human glucocerebrosidase has focused further commercial interest in other glycolipid storage diseases: the cost of targeted enzyme therapy and its failure to restore lysosomal enzymatic deficiencies in the brain has also stimulated interest in the concept of substrate reduction therapy using diffusible inhibitory molecules. Successful clinical trials of the iminosugar N-butyldeoxynojirimycin in type 1 Gaucher's disease prove the principle of substrate reduction therapy and have attracted attention to this therapeutic method. They will also foster important further experiments into the use of glycolipid synthesis inhibitors for the severe neuronopathic glycosphingolipidoses, for which no definitive treatment is otherwise available. Future glycolipid research in medicine will be directed to experiments that shed light on the role of sphingolipids in signalling pathways, and in the comprehensive characterization and their secretory products in relation to the molecular pathogenesis of the storage disorders; experiments of use to improve the efficiency of complementing enzymatic delivery to the lysosomal compartment of storage cells are also needed. Further systematic screening for inhibitory compounds with specific actions in the pathways of glycosphingolipid biosynthesis will undoubtedly lead to clinical trials in the neuronopathic storage disorders and to wider applications in the fields of immunity and cancer biology.     

Pathogenesis of lipid storage diseases

Lipidoses are rare genetic disorders characterized by defects of the digestive system that impair the way the body uses dietary fat. When the body is unable to properly digest fats, lipids such as cholesterol, sphingolipids or glycolipids may accumulate in body tissues in abnormal amounts. It has been also suggested that molecular mechanisms leading to development of human diseases, including obesity, diabetes type II and atherosclerosis, consist of impaired transport and storage of lipids, as well as disturbed structure and function of lipid membrane microdomains. In this review we discuss probable mechanisms, including role of lipid membrane microdomains, which may participate in pathogenesis of lipid storage diseases such as Niemann-Pick type A/B and type C, as well as Gaucher type I diseases.     

Lyophilized membranes as a test system for analyzing the properties of neuromediator receptors and activity of neurotropic agents

The effect of lyophilization and prolonged storage of rat brain membrane preparations on the properties of receptors of cholinergic and adrenergic neuromediator systems has been studied by the radioreceptor assay. Lyophilized membrane preparations have been shown to be highly stable as compared with fresh membranes and retain their binding properties during storage for 1 year.     

Accelerated fluid endocytosis and re-exocytosis by lysosomal storage disease fibroblasts

Fluid-phase endocytosis was significantly faster in three lines of human lysosomal storage disease fibroblasts (two I cells and one Sandhoff's) than in three normals. Re-exocytosis of fluid showed similar kinetics in all lines when release was expressed as a percentage of the radioactivity associated with the cells at the commencement of the measurement. This indicates a greater absolute exocytosis of radioactive sucrose from the storage disease cells. The data imply that the interactions of lysosomes with other cellular membranes are perturbed in the storage diseases.     

Biotechnical and other applications of nanoporous membranes

Recent advances mean that arrays of nearly uniform cylindrical, conical and pyramidal shaped pores can be produced in several types of substrates. Surface modification of nanopore surfaces can give unique mass transport characteristics that have recently been explored for biomolecule separation, detection and purification. Recent interest has focused on the use of nanoporous membranes for mass transfer diodes that act analogous to solid-state devices based on electron conduction. Asymmetric pores such as conical pores can show superior performance characteristics compared to traditional cylindrical pores in ion rectification. However, many phenomena for membranes with asymmetric pores still remain to be exploited in biomolecular separation, biosensing, microfluidics, logic gates, and energy harvesting and storage.     

Stop-codon read-through for patients affected by a lysosomal storage disorder

Lysosomal storage disorders are a group of inherited diseases that can result in severe and progressive pathology due to a specific lysosomal dysfunction. Current treatment strategies include bone-marrow transplantation, substrate reduction, chemical-chaperone and enzyme-replacement therapy. However, each of these treatments has its limitations. Enhanced stop-codon read-through is a potential alternative or adjunct therapeutic strategy for treating lysosomal-storage-disorder patients. Premature stop-codon mutations have been identified in a large cohort of patients with a lysosomal storage disorder, making stop-codon read-through a possible treatment for this disease. In lysosomal-storage-disorder cells (mucopolysaccharidosis type I, alpha-L-iduronidase deficient), preclinical studies have shown that gentamicin induced the read-through of premature stop codons, resulting in enzyme activity that reduced substrate storage.     

3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.     

Cellular models of Batten disease

The Neuronal Ceroid Lipofuscinoses (NCL), otherwise known as Batten disease, are a group of neurodegenerative diseases caused by mutations in 13 known genes. All except one NCL is autosomal recessive in inheritance, with similar aetiology and characterised by the accumulation of autofluorescent storage material in the lysosomes of cells. Age of onset and the rate of progression vary between the NCLs. They are collectively one of the most common lysosomal storage diseases, but the enigma remains of how genetically distinct diseases result in such remarkably similar pathogenesis. Much has been learnt from cellular studies about the function of the proteins encoded by the affected genes. Such research has utilised primitive unicellular models such as yeast and amoeba containing gene orthologues, cells derived from naturally occurring (sheep) and genetically engineered (mouse) animal models or patient-derived cells. Most recently, patient-derived induced pluripotent stem cell (iPSC) lines have been differentiated into neural cell-types to study molecular pathogenesis in the cells most profoundly affected by disease. Here, we review how cell models have informed much of the biochemical understanding of the NCLs and how more complex models are being used to further this understanding and potentially act as platforms for therapeutic efficacy studies in the future.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.