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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Niemann-Pick type C disease: The atypical sphingolipidosis.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder resulting from mutations in either the NPC1 (95%) or NPC2 (5%) genes. NPC typically presents in childhood with visceral lipid accumulation and complex progressive neurodegeneration characterized by cerebellar ataxia, dysphagia, and dementia, resulting in a shortened lifespan. While cholesterol is widely acknowledged as the principal storage lipid in NPC, multiple species of sphingolipids accumulate as well. This accumulation of sphingolipids led to the initial assumption that NPC disease was caused by a deficiency in a sphingolipid catabolism enzyme, similar to sphingomyelinase deficiencies with which it shares a family name. It took about half a century to determine that NPC was in fact caused by a cholesterol trafficking defect, and still as we approach a century after the initial identification of the disease, the mechanisms by which sphingolipids accumulate remain poorly understood. Here we focus on the defects of sphingolipid catabolism in the endolysosomal compartment and how they contribute to the biology and pathology observed in NPC disease. This review highlights the need for further work on understanding and possibly developing treatments to correct the accumulation of sphingolipids in addition to cholesterol in this currently untreatable disease.

Recent advances and novel treatments for sphingolipidoses.

Sphingolipidoses are genetically inherited diseases in which genetic mutations lead to functional deficiencies in the enzymes needed for lysosomal degradation of sphingolipid substrates. As a consequence, nondegradable lipids enrich in the lysosomes and lead to fatal pathological phenotypes in affected individuals. In this review, different drug-based treatment strategies including enzyme replacement therapy and substrate reduction therapy are discussed. A special focus is on the concept of pharmacological chaperones, one of which recently acquired clinical approval within the EU. On the basis of the different limitations for each approach, possible future directions of research are discussed.

Characterization of Drosophila Saposin-related mutants as a model for lysosomal sphingolipid storage diseases.

Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT), this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling events.

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.

Thirteen year retrospective review of the spectrum of inborn errors of metabolism presenting in a tertiary center in Saudi Arabia.

BACKGROUND: Inborn errors of metabolism (IEMs) are individually rare; however, they are collectively common. More than 600 human diseases caused by inborn errors of metabolism are now recognized, and this number is constantly increasing as new concepts and techniques become available for identifying biochemical phenotypes. The aim of this study was to determine the type and distribution of IEMs in patients presenting to a tertiary care center in Saudi Arabia. METHOD: We conducted a retrospective review of children diagnosed with IEMs presenting to the Pediatric Department of King Abdulaziz Medical City in Riyadh, Saudi Arabia over a 13-year period. RESULTS: Over the 13- year period of this retrospective cohort, the total number of live births reached 110,601. A total of 187 patients were diagnosed with IEMs, representing a incidence of 169 in 100,000 births (1:591). Of these, 121 patients (64.7 %) were identified to have small molecule diseases and 66 (35.3 %) to have large molecule diseases. Organic acidemias were the most common small molecule IEMs, while lysosomal storage disorders (LSD) were the most common large molecule diseases. Sphingolipidosis were the most common LSD. CONCLUSION: Our study confirms the previous results of the high rate of IEMs in Saudi Arabia and urges the health care strategists in the country to devise a long-term strategic plan, including an IEM national registry and a high school carrier screening program, for the prevention of such disorders. In addition, we identified 43 novel mutations that were not described previously, which will help in the molecular diagnosis of these disorders.

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.

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.

Mitochondrial Respiration Controls Lysosomal Function during Inflammatory T Cell Responses.

The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4(+) T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation, and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward proinflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD(+) levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify strategies for intervention in mitochondrial-related diseases.

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.