The expanding boundaries of sphingolipid lysosomal storage diseases; insights from Niemann-Pick disease type C.

Lysosomal storage diseases are inborn errors of metabolism that arise due to loss of function mutations in genes encoding lysosomal enzymes, protein co-factors or lysosomal membrane proteins. As a consequence of the genetic defect, lysosomal function is impaired and substrates build up in the lysosome leading to 'storage'. A sub group of these disorders are the sphingolipidoses in which sphingolipids accumulate in the lysosome. In this review, I will discuss how the study of these rare lysosomal disorders reveals unanticipated links to other rare and common human diseases using Niemann-Pick disease type C as an example.

Gene Therapy of Sphingolipid Metabolic Disorders.

Sphingolipidoses are defined as a group of rare hereditary diseases resulting from mutations in the genes encoding lysosomal enzymes. This group of lysosomal storage diseases includes more than 10 genetic disorders, including GM1-gangliosidosis, Tay-Sachs disease, Sandhoff disease, the AB variant of GM2-gangliosidosis, Fabry disease, Gaucher disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease, Farber disease, etc. Enzyme deficiency results in accumulation of sphingolipids in various cell types, and the nervous system is also usually affected. There are currently no known effective methods for the treatment of sphingolipidoses; however, gene therapy seems to be a promising therapeutic variant for this group of diseases. In this review, we discuss gene therapy approaches for sphingolipidoses that are currently being investigated in clinical trials, among which adeno-associated viral vector-based approaches and transplantation of hematopoietic stem cells genetically modified with lentiviral vectors seem to be the most effective.

Zebra-Sphinx: Modeling Sphingolipidoses in Zebrafish.

Sphingolipidoses are inborn errors of metabolism due to the pathogenic mutation of genes that encode for lysosomal enzymes, transporters, or enzyme cofactors that participate in the sphingolipid catabolism. They represent a subgroup of lysosomal storage diseases characterized by the gradual lysosomal accumulation of the substrate(s) of the defective proteins. The clinical presentation of patients affected by sphingolipid storage disorders ranges from a mild progression for some juvenile- or adult-onset forms to severe/fatal infantile forms. Despite significant therapeutic achievements, novel strategies are required at basic, clinical, and translational levels to improve patient outcomes. On these bases, the development of in vivo models is crucial for a better understanding of the pathogenesis of sphingolipidoses and for the development of efficacious therapeutic strategies. The teleost zebrafish (Danio rerio) has emerged as a useful platform to model several human genetic diseases owing to the high grade of genome conservation between human and zebrafish, combined with precise genome editing and the ease of manipulation. In addition, lipidomic studies have allowed the identification in zebrafish of all of the main classes of lipids present in mammals, supporting the possibility to model diseases of the lipidic metabolism in this animal species with the advantage of using mammalian lipid databases for data processing. This review highlights the use of zebrafish as an innovative model system to gain novel insights into the pathogenesis of sphingolipidoses, with possible implications for the identification of more efficacious therapeutic approaches.

Rare Diseases in Glycosphingolipid Metabolism.

Sphingolipidoses is a cluster of genetic rare disorders regarding glycosphingolipid metabolism, classified as lysosomal storage disorders (LSD). Here, we focus on eight inheritable diseases, including GM1 gangliosidosis, GM2 gangliosidosis, Fabry disease, Gaucher's disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease A and B, and Farber disease. Mostly, pathogenic mutations in the key enzyme are loss-function, resulting in accumulation of substrates and deficiency of products. Thus, cellular overload of substrates causes lipotoxicity, which is deleterious to cellular and organ function. In the terms of clinical manifestations in sphingolipidoses, multiple systems and organs, especially central nervous system (CNS) are usually affected. As for diagnosis strategy, enzymatic activity assay and genetic sequencing are helpful. Up till now, limited treatment approaches have approved for treating sphingolipidoses, with some potential strategies for further evaluation. In general, enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and molecular chaperones are feasible choices for enzyme deficiency disorders, but these therapies are limited to relieve CNS lesions and symptoms due to prevention from blood-brain barrier. Other possible treatments such as gene therapy, bone marrow transplantation (BMT), and hematopoietic stem cell transplantation (HSCT) need further evaluation.

Multiple Sulfatase Deficiency: A Disease Comprising Mucopolysaccharidosis, Sphingolipidosis, and More Caused by a Defect in Posttranslational Modification.

Multiple sulfatase deficiency (MSD, MIM #272200) is an ultra-rare disease comprising pathophysiology and clinical features of mucopolysaccharidosis, sphingolipidosis and other sulfatase deficiencies. MSD is caused by impaired posttranslational activation of sulfatases through the formylglycine generating enzyme (FGE) encoded by the sulfatase modifying factor 1 (SUMF1) gene, which is mutated in MSD. FGE is a highly conserved, non-redundant ER protein that activates all cellular sulfatases by oxidizing a conserved cysteine in the active site of sulfatases that is necessary for full catalytic activity. SUMF1 mutations result in unstable, degradation-prone FGE that demonstrates reduced or absent catalytic activity, leading to decreased activity of all sulfatases. As the majority of sulfatases are localized to the lysosome, loss of sulfatase activity induces lysosomal storage of glycosaminoglycans and sulfatides and subsequent cellular pathology. MSD patients combine clinical features of all single sulfatase deficiencies in a systemic disease. Disease severity classifications distinguish cases based on age of onset and disease progression. A genotype- phenotype correlation has been proposed, biomarkers like excreted storage material and residual sulfatase activities do not correlate well with disease severity. The diagnosis of MSD is based on reduced sulfatase activities and detection of mutations in SUMF1. No therapy exists for MSD yet. This review summarizes the unique FGE/ sulfatase physiology, pathophysiology and clinical aspects in patients and their care and outlines future perspectives in MSD.

Emerging mechanisms of drug-induced phospholipidosis.

Drug-induced phospholipidosis is a lysosomal storage disorder characterized by excessive accumulation of phospholipids. Its cellular mechanism is still not well understood, but it is known that cationic amphiphilic drugs can induce it. These drugs have a hydrophilic amine head group that can be protonated in the endolysosomal compartment. As cationic amphiphiles, they are trapped in lysosomes, where they interfere with negatively charged intralysosomal vesicles, the major platforms of cellular sphingolipid degradation. Metabolic principles observed in sphingolipid and phospholipid catabolism and inherited sphingolipidoses are of great importance for lysosomal function and physiological lipid turnover at large. Therefore, we also propose intralysosomal vesicles as major platforms for degradation of lipids and phospholipids reaching them by intracellular pathways like autophagy and endocytosis. Phospholipids are catabolized as components of vesicle surfaces by protonated, positively charged phospholipases, electrostatically attracted to the negatively charged vesicles. Model experiments suggest that progressively accumulating cationic amphiphilic drugs inserting into the vesicle membrane with their hydrophobic molecular moieties disturb and attenuate the main mechanism of lipid degradation as discussed here. By compensating the negative surface charge, cationic enzymes are released from the surface of vesicles and proteolytically degraded, triggering a progressive lipid storage and the formation of inactive lamellar bodies.

Advances in Sphingolipidoses: CRISPR-Cas9 Editing as an Option for Modelling and Therapy.

Sphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood-brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.

In Silico Analysis of Missense Mutations as a First Step in Functional Studies: Examples from Two Sphingolipidoses.

In order to delineate a better approach to functional studies, we have selected 23 missense mutations distributed in different domains of two lysosomal enzymes, to be studied by in silico analysis. In silico analysis of mutations relies on computational modeling to predict their effects. Various computational platforms are currently available to check the probable causality of mutations encountered in patients at the protein and at the RNA levels. In this work we used four different platforms freely available online (Protein Variation Effect Analyzer- PROVEAN, PolyPhen-2, Swiss-model Expert Protein Analysis System-ExPASy, and SNAP2) to check amino acid substitutions and their effect at the protein level. The existence of functional studies, regarding the amino acid substitutions, led to the selection of the distinct protein mutants. Functional data were used to compare the results obtained with different bioinformatics tools. With the advent of next-generation sequencing, it is not feasible to carry out functional tests in all the variants detected. In silico analysis seems to be useful for the delineation of which mutants are worth studying through functional studies. Therefore, prediction of the mutation impact at the protein level, applying computational analysis, confers the means to rapidly provide a prognosis value to genotyping results, making it potentially valuable for patient care as well as research purposes. The present work points to the need to carry out functional studies in mutations that might look neutral. Moreover, it should be noted that single nucleotide polymorphisms (SNPs), occurring in coding and non-coding regions, may lead to RNA alterations and should be systematically verified. Functional studies can gain from a preliminary multi-step approach, such as the one proposed here.

Acid ceramidase and the treatment of ceramide diseases: The expanding role of enzyme replacement therapy.

Ceramides are a diverse group of sphingolipids that play important roles in many biological processes. Acid ceramidase (AC) is one key enzyme that regulates ceramide metabolism. Early research on AC focused on the fact that it is the enzyme deficient in the rare genetic disorder, Farber Lipogranulomatosis. Recent research has revealed that deficiency of the same enzyme is responsible for a rare form of spinal muscular atrophy associated with myoclonic epilepsy (SMA-PME). Due to their diverse role in biology, accumulation of ceramides also has been implicated in the pathobiology of many other common diseases, including infectious lung diseases, diabetes, cancers and others. This has revealed the potential of AC as a therapy for many of these diseases. This review will focus on the biology of AC and the potential role of this enzyme in the treatment of human disease.

Fluid levity of the cell: Role of membrane lipid architecture in genetic sphingolipidoses.

Sphingolipidoses arise from inherited loss of function of key enzymes regulating the sphingolipid (SL) metabolism and the accumulation of large quantities of these lipids in affected cells. Most frequently, toxicity is manifested in the nervous system, where survival and function of neurons and glial cells are most affected. Although detailed information is available on neuroglial alterations during terminal stages of the disease, the initial pathogenic mechanisms triggering neuropathology are largely unclear. Because they are key components of biological membranes, changes in the local concentration of SLs are likely to impact the organization of membrane domains and functions. This Commentary proposes that SL toxicity involves initial defects in the integrity of lipid domains, membrane fluidity, and membrane bending, leading to membrane deformation and deregulation of cell signaling and function. Understanding how SLs alter membrane architecture may provide breakthroughs for more efficient treatment of sphingolipidoses. © 2016 Wiley Periodicals, Inc.

Demographic characteristics and distribution of lysosomal storage disorder subtypes in Eastern China.

Lysosomal storage disorders (LSDs) are a group of >50 different types of inherited metabolic disorders that result from defects in the lysosome. The aim of this study was to investigate the distribution and demographic characteristics of the different subtypes of LSDs in Eastern China. From 2006 to 2012, 376 out of 1331 clinically suspected patients were diagnosed with 17 different subtypes of LSDs at our hospital. Mucopolysaccharidoses (MPS) were the most common group of LSDs (50.5%), followed by sphingolipidoses (25.4%) and Pompe disease (19.8%). Mucolipidosis type II/III accounted for the remaining 4% of diagnosed LSDs. MPS II was the most common form of MPS, comprising 47.4% of all MPS cases diagnosed, followed by MPS IVA (26.8%) and MPS I (16.3%). Gaucher disease and Niemann-Pick disease type A/B were the two most common forms of sphingolipidoses. There was a large variation in the time between disease onset and eventual diagnosis, from 0.3 years in infantile-onset Pompe disease to 30 years in Fabry disease, highlighting timely and accurate diagnosis of LSDs as the main challenge in China.

The consequences of genetic and pharmacologic reduction in sphingolipid synthesis.

A new therapy based on substrate synthesis reduction in sphingolipidoses is showing promise. The consequences of decreasing sphingolipid synthesis depend on the level at which synthetic blockage occurs and on the extent of the blockage. Complete synthetic blockage may be lethal if it includes all sphingolipids, such as in a global knockout of serine palmitoyltransferase. Partial inhibition of sphingolipid synthetic pathways is usually benign and may have beneficial effects in a number of lysosomal diseases and in more common pathologies, as seen in animal models for atherosclerosis, polycystic kidney disease, diabetes, and asthma. Studies of various forms of sphingolipid synthesis reduction serve to highlight not only the cellular role of these lipids but also the potential risks and therapeutic benefits of pharmacological agents to be used in therapy for human diseases.

The genetics of sphingolipid hydrolases and sphingolipid storage diseases.

The relationship of sphingolipids with human disease first arose from the study of sphingolipid storage diseases over 50 years ago. Most of these disorders are due to inherited deficiencies of specific sphingolipid hydrolases, although a small number also result from defects in sphingolipid transport or activator proteins. Due to the primary protein deficiencies sphingolipids and other macromolecules accumulate in cells and tissues of affected patients, leading to a diverse presentation of clinical abnormalities. Over 25 sphingolipid storage diseases have been described to date. Most of the genes have been isolated, disease-causing mutations have been identified, the recombinant proteins have been produced and characterized, and animal models exist for most of the human diseases. Since most sphingolipid hydrolases are enriched within the endosomal/lysosomal system, macromolecules first accumulate within these compartments. However, these abnormalities rapidly spread to other compartments and cause a wide range of cellular dysfunction. This review focuses on the genetics of sphingolipid storage diseases and related hydrolytic enzymes with an emphasis on the relationship between genetic mutations and human disease.

Altered expression and distribution of cathepsins in neuronopathic forms of Gaucher disease and in other sphingolipidoses.

The neuronopathic forms of the human inherited metabolic disorder, Gaucher disease (GD), are characterized by severe neuronal loss, astrogliosis and microglial proliferation, but the cellular and molecular pathways causing these changes are not known. Recently, a mouse model of neuronopathic GD was generated in which glucocerebrosidase deficiency is limited to neural and glial progenitor cells. We now show significant changes in the levels and in the distribution of cathepsins in the brain of this mouse model. Cathepsin mRNA expression was significantly elevated by up to approximately 10-fold, with the time-course of the increase correlating with the progression of disease severity. Cathepsin activity and protein levels were also elevated. Significant changes in cathepsin D distribution in the brain were detected, with cathepsin D elevated in areas where neuronal loss, astrogliosis and microgliosis were observed, such as in layer V of the cerebral cortex, the lateral globus pallidus and in various nuclei in the thalamus, brain regions known to be affected in the disease. Cathepsin D elevation was greatest in microglia and also noticeable in astrocytes. The distribution of cathepsin D was altered in neurons in a manner consistent with its release from the lysosome to the cytosol. Remarkably, ibubrofen treatment significantly reduced cathepsin D mRNA levels in the cortex of Gaucher mice. Finally, cathepsin levels were also altered in mouse models of a number of other sphingolipidoses. Our findings suggest the involvement of cathepsins in the neuropathology of neuronal forms of GD and of other lysosomal storage diseases, and are consistent with a crucial role for reactive microglia in neuronal degeneration in these diseases.

My journey into the world of sphingolipids and sphingolipidoses.

Analysis of lipid storage in postmortem brains of patients with amaurotic idiocy led to the recognition of five lysosomal ganglioside storage diseases and identification of their inherited metabolic blocks. Purification of lysosomal acid sphingomyelinase and ceramidase and analysis of their gene structures were the prerequisites for the clarification of Niemann-Pick and Farber disease. For lipid catabolism, intraendosomal vesicles are formed during the endocytotic pathway. They are subjected to lipid sorting processes and were identified as luminal platforms for cellular lipid and membrane degradation. Lipid binding glycoproteins solubilize lipids from these cholesterol poor membranes and present them to water-soluble hydrolases for digestion. Biosynthesis and intracellular trafficking of lysosomal hydrolases (hexosaminidases, acid sphingomyelinase and ceramidase) and lipid binding and transfer proteins (GM2 activator, saposins) were analyzed to identify the molecular and metabolic basis of several sphingolipidoses. Studies on the biosynthesis of glycosphingolipids yielded the scheme of Combinatorial Ganglioside Biosynthesis involving promiscuous glycosyltransferases. Their defects in mutagenized mice impair brain development and function.

Genetic defects in the sphingolipid degradation pathway and their effects on microglia in neurodegenerative disease.

Sphingolipids, which function as plasma membrane lipids and signaling molecules, are highly enriched in neuronal and myelin membranes in the nervous system. They are degraded in lysosomes by a defined sequence of enzymatic steps. In the related group of disorders, the sphingolipidoses, mutations in the genes that encode the individual degradative enzymes cause lysosomal accumulation of sphingolipids and often result in severe neurodegenerative disease. Here we review the information indicating that microglia, which actively clear sphingolipid-rich membranes in the brain during development and homeostasis, are directly affected by these mutations and promote neurodegeneration in the sphingolipidoses. We also identify parallels between the sphingolipidoses and more common forms of neurodegeneration, which both exhibit evidence of defective sphingolipid clearance in the nervous system.

[Lysosomal storage diseases]. / Lysosomale Speicherkrankheiten.

Lysosomal storage diseases are a heterogeneous group of disorders caused by lysosomal enzyme dysfunction. Individually they are very rare, but this group as a whole has a prevalence of more than 1:8,000 live births. While severe phenotypes are easily diagnosed this can be a real challenge with attenuated forms. Because musculoskeletal complaints are frequently the first reason for the patient to seek medical advice, the rheumatologist plays a key role for the early recognition of these diseases. Since several of these can be treated very effectively by enzyme replacement, a timely diagnosis and start of therapy are essential to avoid irreversible organ damage and poor quality of life. Therefore, each clinical rheumatologist should be aware of the cardinal symptoms of lysosomal storage diseases.

[Metachromatic leucodystrophy. Clinical, biological, and therapeutic aspects]. / La leucodystrophie métachromatique Aspects clinique, biologique et thérapeutique.

Scholz's disease or metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a deficiency in arylsulfatase A (ARSA: EC 3.1.6.8). This enzyme is responsible for the degradation of sulfatides commonly called cerebroside-3-sulfate or 3-O-sulfogalactosylcéramide in galactocérébroside and sulfate. The success of hydrolysis of these sphingolipids by ARSA necessarily depends on the presence of saposine B forms a complex with the substrate. The pathological accumulation of sulfatides in the nervous system (myelin, neurons and glial cells) results most often neurological, mental retardation, nervous disorders, blindness. The metachromatic granules accumulated in the central nervous system and peripheral compounds are highly toxic. These are at high levels in the urine of patients affected by the MLD. Arylsulfatase A activity is collapsed in these patients. Unfortunately, the value of enzyme activity is not a predictor of clinical severity of the neuropathology. In contrast, the study of the gene that codes for the ARSA is seen as a way to diagnose the simplest and most reliable of the disease to avoid misdiagnosis due to the presence of pseudodeficit. The conventional therapeutic approaches are essentially symptomatic. They were made in order to restore the enzyme activity of arylsulfatase A and prevent the progression of the pathological accumulation of sulfatides and consequently reduce morbidity associated with MLD.

The sphingolipid salvage pathway in ceramide metabolism and signaling.

Sphingolipids are important components of eukaryotic cells, many of which function as bioactive signaling molecules. Of these, ceramide is a central metabolite and plays key roles in a variety of cellular responses, including regulation of cell growth, viability, differentiation, and senescence. Ceramide is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups. Sphingosine serves as the product of sphingolipid catabolism, and it is mostly salvaged through reacylation, resulting in the generation of ceramide or its derivatives. This recycling of sphingosine is termed the "salvage pathway", and recent evidence points to important roles for this pathway in ceramide metabolism and function. A number of enzymes are involved in the salvage pathway, and these include sphingomyelinases, cerebrosidases, ceramidases, and ceramide synthases. Recent studies suggest that the salvage pathway is not only subject to regulation, but it also modulates the formation of ceramide and subsequent ceramide-dependent cellular signals. This review focuses on the salvage pathway in ceramide metabolism, its regulation, its experimental analysis, and emerging biological functions.

Inhibition of the IGF-1-PI3K-Akt-mTORC2 pathway in lipid rafts increases neuronal vulnerability in a genetic lysosomal glycosphingolipidosis.

Glycosphingolipid (GSL) accumulation is implicated in the neuropathology of several lysosomal conditions, such as Krabbe disease, and may also contribute to neuronal and glial dysfunction in adult-onset conditions such as Parkinson's disease, Alzheimer's disease and multiple sclerosis. GSLs accumulate in cellular membranes and disrupt their structure; however, how membrane disruption leads to cellular dysfunction remains unknown. Using authentic cellular and animal models for Krabbe disease, we provide a mechanism explaining the inactivation of lipid raft (LR)-associated IGF-1-PI3K-Akt-mTORC2, a pathway of crucial importance for neuronal function and survival. We show that psychosine, the GSL that accumulates in Krabbe disease, leads to a dose-dependent LR-mediated inhibition of this pathway by uncoupling IGF-1 receptor phosphorylation from downstream Akt activation. This occurs by interfering with the recruitment of PI3K and mTORC2 to LRs. Akt inhibition can be reversed by sustained IGF-1 stimulation, but only during a time window before psychosine accumulation reaches a threshold level. Our study shows a previously unknown connection between LR-dependent regulation of mTORC2 activity at the cell surface and a genetic neurodegenerative disease. Our results show that LR disruption by psychosine desensitizes cells to extracellular growth factors by inhibiting signal transmission from the plasma membrane to intracellular compartments. This mechanism serves also as a mechanistic model to understand how alterations of the membrane architecture by the progressive accumulation of lipids undermines cell function, with potential implications in other genetic sphingolipidoses and adult neurodegenerative conditions. This article has an associated First Person interview with the first author of the paper.