Hematopoietic stem cell transplantation leads to biochemical and functional correction in two mouse models of acid ceramidase deficiency.

Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME) are ultra-rare lysosomal storage disorders caused by deficient acid ceramidase (ACDase) activity. Although both conditions are caused by mutations in the ASAH1 gene, clinical presentations differ considerably. FD patients usually die in childhood, while SMA-PME patients can live until adulthood.

LysoGb3 quantification facilitates phenotypic categorization of Fabry disease patients: Insights gained by a novel MS/MS method.

Background: Fabry disease (FD) is an X-linked lysosomal storage disease resulting from pathogenic variants in the GLA gene coding α-galactosidase A (AGAL) and cleaving terminal alpha-linked galactose. Globotriaosylceramide (Gb3) is the predominantly accumulated sphingolipid. Gb3, deacylated-Gb3 (lysoGb3), and methylated-Gb3 (metGb3) have been suggested as FD biomarkers.

Simultaneous deletion of ORMDL1 and ORMDL3 proteins disrupts immune cell homeostasis.

ORMDL3 is a prominent member of a family of highly conserved endoplasmic reticulum resident proteins, ORMs (ORM1 and ORM2) in yeast, dORMDL in and ORMDLs (ORMDL1, ORMDL2, and ORMDL3) in mammals. ORMDL3 mediates feedback inhibition of sphingolipid synthesis. Expression levels of ORMDL3 are associated with the development of inflammatory and autoimmune diseases including asthma, systemic lupus erythematosus, type 1 diabetes mellitus and others.

Simultaneous reduction of all ORMDL proteins decreases the threshold of mast cell activation.

In mammals, the ORMDL family of evolutionarily conserved sphingolipid regulators consists of three highly homologous members, ORMDL1, ORMDL2 and ORMDL3. ORMDL3 gene has been associated with childhood-onset asthma and other inflammatory diseases in which mast cells play an important role. We previously described increased IgE-mediated activation of mast cells with simultaneous deletions of ORMDL2 and ORMDL3 proteins.

Skin inflammation and impaired adipogenesis in a mouse model of acid ceramidase deficiency.

Acid ceramidase catalyzes the degradation of ceramide into sphingosine and a free fatty acid. Acid ceramidase deficiency results in lipid accumulation in many tissues and leads to the development of Farber disease (FD). Typical manifestations of classical FD include formation of subcutaneous nodules and joint contractures as well as the development of a hoarse voice.

Loading of cell cultures with cholesterol-dextran particles as a new functional test for Niemann-Pick type C disease.

Deuterium-labeled cholesterol-dextran particles (d4-CholDex), prepared by co-precipitation, were internalized by cultured human skin fibroblasts and HEK293 cells. Subcellular particles from d4-CholDex-treated HEK293 cells were fractionated on iodixanol gradients. More than 60% of d4-cholesterol (d4-UC) in the gradient co-fractionated with lysosomal markers and NPC1.

Lipidomic profiling of human serum enables detection of pancreatic cancer.

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth.

Crosstalk between ORMDL3, serine palmitoyltransferase, and 5-lipoxygenase in the sphingolipid and eicosanoid metabolic pathways.

Leukotrienes (LTs) and sphingolipids are critical lipid mediators participating in numerous cellular signal transduction events and developing various disorders, such as bronchial hyperactivity leading to asthma. Enzymatic reactions initiating production of these lipid mediators involve 5-lipoxygenase (5-LO)-mediated conversion of arachidonic acid to LTs and serine palmitoyltransferase (SPT)-mediated de novo synthesis of sphingolipids. Previous studies have shown that endoplasmic reticulum membrane protein ORM1-like protein 3 (ORMDL3) inhibits the activity of SPT and subsequent sphingolipid synthesis.

ORMDL2 Deficiency Potentiates the ORMDL3-Dependent Changes in Mast Cell Signaling.

The systemic anaphylactic reaction is a life-threatening allergic response initiated by activated mast cells. Sphingolipids are an essential player in the development and attenuation of this response. synthesis of sphingolipids in mammalian cells is inhibited by the family of three ORMDL proteins (ORMDL1, 2, and 3).

A new role for dolichol isoform profile in the diagnostics of CDG disorders.

Dolichol is a membrane lipid which carries monosaccharides and glycans for N-linked protein glycosylation occurring in the endoplasmic reticulum. Recently, some types of congenital disorders of glycosylation (CDG) have been described as consequences of defects in dolichol biosynthesis and metabolism, yet these types of CDG are not detectable by standard screening methods. The aim of this project was to evaluate the potential of dolichol as a biomarker of CDG.

Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations.

Background: Niemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms.

Lipidomic characterization of exosomes isolated from human plasma using various mass spectrometry techniques.

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides.

Chronic visceral acid sphingomyelinase deficiency (Niemann-Pick disease type B) in 16 Polish patients: long-term follow-up.

Background: Acid sphingomyelinase deficiency (ASMD), due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, is divided into infantile neurovisceral ASMD (Niemann-Pick type A), chronic neurovisceral ASMD (intermediate form, Niemann-Pick type A/B) and chronic visceral ASMD (Niemann-Pick type B). We conducted a long-term observational, single-center study including 16 patients with chronic visceral ASMD.

Results: 12 patients were diagnosed in childhood and 4 others in adulthood, the oldest at the age of 50.

Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B.

Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs.

Conserved roles of C. elegans and human MANFs in sulfatide binding and cytoprotection.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER) protein that can be secreted and protects dopamine neurons and cardiomyocytes from ER stress and apoptosis. The mechanism of action of extracellular MANF has long been elusive. From a genetic screen for mutants with abnormal ER stress response, we identified the gene Y54G2A.

Neural cells generated from human induced pluripotent stem cells as a model of CNS involvement in mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPSII) is a rare X-linked lysosomal storage disorder caused by mutations in the iduronate-2-sulfatase (IDS) gene (IDS, Xq28). MPSII is characterized by skeletal deformities, hearing loss, airway obstruction, hepatosplenomegaly, cardiac valvular disease, and progressive neurological impairment. At the cellular level, IDS deficiency leads to lysosomal storage of glycosaminoglycans (GAGs), dominated by accumulation of dermatan and heparan sulfates.

N-butyldeoxynojirimycin delays motor deficits, cerebellar microgliosis, and Purkinje cell loss in a mouse model of mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease exhibiting progressive intellectual disability, motor impairment, and premature death. There is currently no cure or corrective treatment. The disease results from mutations in the gene encoding mucolipin-1, a transient receptor potential channel believed to play a key role in lysosomal calcium egress.

Quantitation of plasmatic lysosphingomyelin and lysosphingomyelin-509 for differential screening of Niemann-Pick A/B and C diseases.

Acid sphingomyelinase deficiency (ASMd, Niemann-Pick disease A/B) and Niemann-Pick type C disease (NPC) share core clinical symptoms. Initial diagnostic discrimination of these two rare lysosomal storage diseases is thus difficult. As sphingomyelin accumulates in ASMd as well as NPC, lysosphingomyelin (sphingosylphosphorylcholine) and its m/z 509 analog were suggested as biomarkers for both diseases.

Tandem Mass Spectrometry of Sphingolipids: Applications for Diagnosis of Sphingolipidoses.

In recent years, mass spectrometry (MS) has become the dominant technology in lipidomic analysis. It is widely used in diagnosis and research of lipid metabolism disorders including those characterized by impairment of lysosomal functions and storage of nondegraded-degraded substrates. These rare diseases, which include sphingolipidoses, have severe and often fatal clinical consequences.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration.

Fabry disease: renal sphingolipid distribution in the α-Gal A knockout mouse model by mass spectrometric and immunohistochemical imaging.

Fabry disease is an X-linked lysosomal storage disease due to deficient α-galactosidase A (α-Gal A) activity and the resultant lysosomal accumulation of globotriaosylceramide (Gb3) and related lipids primarily in blood vessels, kidney, heart, and other organs. The renal distribution of stored glycolipid species in the α-Gal A knockout mouse model was compared to that in mice to assess relative distribution and absolute amounts of accumulated sphingolipid isoforms. Twenty isoforms of five sphingolipid groups were visualized by mass spectrometry imaging (MSI), and their distribution was compared with immunohistochemical (IHC) staining of Gb3, the major stored glycosphingolipid in consecutive tissue sections.

Direct tandem mass spectrometric profiling of sulfatides in dry urinary samples for screening of metachromatic leukodystrophy.

Background: Prediagnostic steps in suspected metachromatic leukodystrophy (MLD) rely on clinical chemical methods other than enzyme assays. We report a new diagnostic method which evaluates changes in the spectrum of molecular types of sulfatides (3-O-sulfogalactosyl ceramides) in MLD urine.

Methods: The procedure allows isolation of urinary sulfatides by solid-phase extraction on DEAE-cellulose membranes, transportation of a dry membrane followed by elution and tandem mass spectrometry (MS/MS) analysis in the clinical laboratory.

Rapid isolation of lysosomal membranes from cultured cells.

We present a simple method for enrichment of lysosomal membranes from HEK293 and HeLa cell lines taking advantage of selective disruption of lysosomes by methionine methyl ester. Organelle concentrate from postnuclear supernatant was treated with 20 mmol/l methionine methyl ester for 45 min to lyse the lysosomes. Subsequently, lysosomal membranes were resolved on a step sucrose gradient.