GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

Endogenous, non-reducing end glycosaminoglycan biomarkers for the mucopolysaccharidoses: Accurate diagnosis and elimination of false positive newborn screening results.

The mucopolysaccharidoses (MPS) are a family of inborn errors of metabolism resulting from a deficiency in a lysosomal hydrolase responsible for the degradation of glycosaminoglycans (GAG). From a biochemical standpoint, excessive urinary excretion of GAG has afforded first-tier laboratory investigations for diagnosis whereas newborn screening programs employ lysosomal hydrolase measurements. Given false positives are not uncommon, second-tier diagnostic testing relies on lysosomal hydrolase measurements following elevated urinary GAG, and newborn screening results are often corroborated with GAG determinations. Molecular genetics requires acknowledgement, as identifying pathogenic variants in the hydrolase genes confirms the diagnosis and allows cascade testing for families, but genetic variants of uncertain significance complicate this paradigm. Initiating cellular, tissue and organ damage that leads to an MPS phenotype is undoubtedly the accumulation of partially degraded GAG, and with mass spectrometry technologies now readily available in the biochemical genetics' laboratory, the ability to properly measure these GAG fragments has been realized. The most common approach involves bacterial lyase/hydrolase digestion of the long chain GAG polymers into their disaccharide units that can be measured by mass spectrometry. Another, less well-known method, the endogenous, non-reducing end method, does not require depolymerization of GAG but rather relies on the mass spectrometric measurement of the naturally produced oligosaccharides that arise from the enzyme deficiency. All MPS can be identified by this one method, and evidence to date shows it to be the only GAG analysis method that gives no false positives when employed as a first-tier laboratory diagnostic test and second-tier newborn screening test.

Spinal muscular atrophy-like phenotype in a mouse model of acid ceramidase deficiency.

Mutations in ASAH1 have been linked to two allegedly distinct disorders: Farber disease (FD) and spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME). We have previously reported FD-like phenotypes in mice harboring a single amino acid substitution in acid ceramidase (ACDase), P361R, known to be pathogenic in humans (P361R-Farber). Here we describe a mouse model with an SMA-PME-like phenotype (P361R-SMA). P361R-SMA mice live 2-3-times longer than P361R-Farber mice and have different phenotypes including progressive ataxia and bladder dysfunction, which suggests neurological dysfunction. We found profound demyelination, loss of axons, and altered sphingolipid levels in P361R-SMA spinal cords; severe pathology was restricted to the white matter. Our model can serve as a tool to study the pathological effects of ACDase deficiency on the central nervous system and to evaluate potential therapies for SMA-PME.

Elevation of gangliosides in four brain regions from Parkinson's disease patients with a GBA mutation.

A number of genetic risk factors have been identified over the past decade for Parkinson's Disease (PD), with variants in GBA prominent among them. GBA encodes the lysosomal enzyme that degrades the glycosphingolipid, glucosylceramide (GlcCer), with the activity of this enzyme defective in Gaucher disease. Based on the ill-defined relationship between glycosphingolipid metabolism and PD, we now analyze levels of various lipids by liquid chromatography/electrospray ionization-tandem mass spectrometry in four brain regions from age- and sex-matched patient samples, including idiopathic PD, PD patients with a GBA mutation and compare both to control brains (n = 21 for each group) obtained from individuals who died from a cause unrelated to PD. Of all the glycerolipids, sterols, and (glyco)sphingolipids (251 lipids in total), the only lipid class which showed significant differences were the gangliosides (sialic acid-containing complex glycosphingolipids), which were elevated in 3 of the 4 PD-GBA brain regions. There was no clear correlation between levels of individual gangliosides and the genetic variant in Gaucher disease [9 samples of severe (neuronopathic), 4 samples of mild (non-neuronopathic) GBA variants, and 8 samples with low pathogenicity variants which have a higher risk for development of PD]. Most brain regions, i.e. occipital cortex, cingulate gyrus, and striatum, did not show a statistically significant elevation of GlcCer in PD-GBA. Only one region, the middle temporal gyrus, showed a small, but significant elevation in GlcCer concentration in PD-GBA. We conclude that changes in ganglioside, but not in GlcCer levels, may contribute to the association between PD and GBA mutations.

The long and the short of Huntington's disease: how the sphingolipid profile is shifted in the caudate of advanced clinical cases.

Huntington's disease is a devastating neurodegenerative disorder that onsets in late adulthood as progressive and terminal cognitive, psychiatric and motor deficits. The disease is genetic, triggered by a CAG repeat (polyQ) expansion mutation in the Huntingtin gene and resultant huntingtin protein. Although the mutant huntingtin protein is ubiquitously expressed, the striatum degenerates early and consistently in the disease. The polyQ mutation at the N-terminus of the huntingtin protein alters its natural interactions with neural phospholipids in vitro, suggesting that the specific lipid composition of brain regions could influence their vulnerability to interference by mutant huntingtin; however, this has not yet been demonstrated in vivo. Sphingolipids are critical cell signalling molecules, second messengers and membrane components. Despite evidence of sphingolipid disturbance in Huntington's mouse and cell models, there is limited knowledge of how these lipids are affected in human brain tissue. Using post-mortem brain tissue from five brain regions implicated in Huntington's disease (control n = 13, Huntington's n = 13), this study aimed to identify where and how sphingolipid species are affected in the brain of clinically advanced Huntington's cases. Sphingolipids were extracted from the tissue and analysed using targeted mass spectrometry analysis; proteins were analysed by western blot. The caudate, putamen and cerebellum had distinct sphingolipid changes in Huntington's brain whilst the white and grey frontal cortex were spared. The caudate of Huntington's patients had a shifted sphingolipid profile, favouring long (C13-C21) over very-long-chain (C22-C26) ceramides, sphingomyelins and lactosylceramides. Ceramide synthase 1, which synthesizes the long-chain sphingolipids, had a reduced expression in Huntington's caudate, correlating positively with a younger age at death and a longer CAG repeat length of the Huntington's patients. The expression of ceramide synthase 2, which synthesizes very-long-chain sphingolipids, was not different in Huntington's brain. However, there was evidence of possible post-translational modifications in the Huntington's patients only. Post-translational modifications to ceramide synthase 2 may be driving the distinctive sphingolipid profile shifts of the caudate in advanced Huntington's disease. This shift in the sphingolipid profile is also found in the most severely affected brain regions of several other neurodegenerative conditions and may be an important feature of region-specific cell dysfunction in neurodegenerative disease.

Impaired neural differentiation of MPS IIIA patient induced pluripotent stem cell-derived neural progenitor cells.

Mucopolysaccharidosis type IIIA (MPS IIIA) is characterised by a progressive neurological decline leading to early death. It is caused by bi-allelic loss-of-function mutations in SGSH encoding sulphamidase, a lysosomal enzyme required for heparan sulphate glycosaminoglycan (HS GAG) degradation, that results in the progressive build-up of HS GAGs in multiple tissues most notably the central nervous system (CNS). Skin fibroblasts from two MPS IIIA patients who presented with an intermediate and a severe clinical phenotype, respectively, were reprogrammed into induced pluripotent stem cells (iPSCs). The intermediate MPS IIIA iPSCs were then differentiated into neural progenitor cells (NPCs) and subsequently neurons. The patient derived fibroblasts, iPSCs, NPCs and neurons all displayed hallmark biochemical characteristics of MPS IIIA including reduced sulphamidase activity and increased accumulation of an MPS IIIA HS GAG biomarker. Proliferation of MPS IIIA iPSC-derived NPCs was reduced compared to control, but could be partially rescued by reintroducing functional sulphamidase enzyme, or by doubling the concentration of the mitogen fibroblast growth factor 2 (FGF2). Whilst both control heparin, and MPS IIIA HS GAGs had a similar binding affinity for FGF2, only the latter inhibited FGF signalling, suggesting accumulated MPS IIIA HS GAGs disrupt the FGF2:FGF2 receptor:HS signalling complex. Neuronal differentiation of MPS IIIA iPSC-derived NPCs was associated with a reduction in the expression of neuronal cell marker genes ßIII-TUBULIN, NF-H and NSE, revealing reduced neurogenesis compared to control. A similar result was achieved by adding MPS IIIA HS GAGs to the culture medium during neuronal differentiation of control iPSC-derived NPCs. This study demonstrates the generation of MPS IIIA iPSCs, and NPCs, the latter of which display reduced proliferation and neurogenic capacity. Reduced NPC proliferation can be explained by a model in which soluble MPS IIIA HS GAGs compete with cell surface HS for FGF2 binding. The mechanism driving reduced neurogenesis remains to be determined but appears downstream of MPS IIIA HS GAG accumulation.

Experience with the Urinary Tetrasaccharide Metabolite for Pompe Disease in the Diagnostic Laboratory.

Following clinical indications, the laboratory diagnosis of the inherited metabolic myopathy, Pompe disease (PD), typically begins with demonstrating a reduction in acid alpha-glucosidase (GAA), the enzyme required for lysosomal glycogen degradation. Although simple in concept, a major challenge is defining reference intervals, as even carriers can have reduced GAA, and pseudodeficiencies complicate interpretation. Here, we developed a mass spectrometric assay for quantification of a urinary glycogen metabolite (tetrasaccharide) and reported on its utility as a confirmatory test for PD in a diagnostic laboratory. Using two age-related reference intervals, eight returned tetrasaccharide concentrations above the calculated reference interval but did not have PD, highlighting non-specificity. However, retrospective analysis revealed elevated tetrasaccharide in seven infantile-onset (IOPD) cases and sixteen late-onset (LOPD) cases, and normal concentrations in one heterozygote. Prospective tetrasaccharide analysis in nine individuals with reduced GAA confirmed IOPD in one, LOPD in six and identified two heterozygotes. Using this metabolite as a biomarker of therapeutic response was not overly informative; although most patients showed an initial drop following therapy initiation, tetrasaccharide concentrations fluctuated considerably and remained above reference intervals in all patients. While useful as a confirmation of PD, its utility as a biomarker for monitoring treatment warrants further investigation.

Systemic scAAV9.U1a.hSGSH Delivery Corrects Brain Biochemistry in Mucopolysaccharidosis Type IIIA at Early and Later Stages of Disease.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a single gene (SGSH) childhood onset neurodegenerative disease for which gene therapy is in clinical trial. Theoretically, the transfer of a working gene should enable functional expression of the defective protein and rescue the phenotype when administered before the onset of irreversible disease. Recombinant adeno-associated virus (AAV) is being used as a vehicle for a number of gene therapy applications and the neurotropism of serotype 9 affords utility for monogenetic neurological disorders. To assess the efficacy of restoring the underlying biochemistry in the MPS IIIA brain, tail vein injections of self-complementary AAV9 human N-sulfoglucosamine sulfohydrolase (scAAV9.U1A.hSGSH) at 3 × 1013 vg/kg were administered to 6- and 16-week-old MPS IIIA mice. Heparan sulfate (HS) and GM2 and GM3 gangliosides were cleared from the cortex, hippocampus and subcortex with residual storage remaining in the brain stem and cerebellum. SGSH activity increased in the brain of the MPS IIIA-treated mice, but remained significantly reduced compared with wild-type. Motor activity as assessed in an open-field arena, and gait length, improved in MPS IIIA mice treated at both 6 and 16 weeks of age. However, functional assessment of cognition in the water cross-maze test, as well as gait width, normalized in mice treated at 6 weeks of age only, with mice treated at 16 weeks performing similar to untreated MPS IIIA mice. Astrogliosis was reduced in mice treated at 6 and 16 weeks of age compared to untreated MPS IIIA mice. These results demonstrate that the gene product is actively clearing primary HS and secondary ganglioside accumulation in MPS IIIA mice, but in older mice, neurocognitive impairments remain. This is likely due to secondary downstream consequences of HS affecting neurological functions that are not reversible upon substrate clearance.

Chondroitin sulfate disaccharide is a specific and sensitive biomarker for mucopolysaccharidosis type IVA.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism characterized by a deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulphatase (GALNS). Consequently, partially degraded GAG, chondroitin 6-sulfate (CS) and keratan sulfate (KS), accumulate in the lysosomes of affected cells, primarily in cartilage resulting in skeletal disease. Excessive urinary excretion of these GAG is often used as the initial biochemical parameter to inform a laboratory diagnosis. Here we present the utility of a CS-disaccharide with a non-reducing 6-sulfated N-acetylgalactosamine residue (HNAc-UA (1S))-the enzyme's substrate-for the diagnosis and biochemical monitoring of MPS IVA patients. Following implementation of this method into the diagnostic laboratory, we identified one MPS IVA patient over 3 years of MPS urine screening, with no false positive results from 2050 urines tested. Uniquely, urinary concentrations of HNAc-UA (1S) are independent of age meaning that age-related reference ranges are not required. Urinary HNAc-UA (1S) was also able to identify two MPS IVA siblings who remained misdiagnosed with spondyloepiphyseal dysplasia for 5 years because of normal urinary GAG. HNAc-UA (1S) could also be used as a biomarker for monitoring response to enzyme replacement therapy (ERT) as there was a drop in urinary concentration following the administration of ERT in all 12 patients and concentrations correlated with urinary KS (R 2 = 0.92). In conclusion, HNAc-UA (1S) is a reliable, sensitive and specific biomarker for the diagnosis of MPS IVA and can be used to biochemically monitor the response to ERT.

Sphingolipid dyshomeostasis in the brain of the mouse model of mucopolysaccharidosis type IIIA.

Gangliosides are complex glycosphingolipids that are vital for proper brain development and function. Alterations in ganglioside metabolism are evident in neurological disorders including the inherited metabolic disease mucopolysaccharidosis type IIIA (MPS IIIA/Sanfilippo A syndrome). Here we sought to comprehensively analyse alterations in ganglioside metabolism within the brain of a naturally occurring MPS IIIA mouse model at early (one month) and late (six months of age) stages of disease progression, as well as the impact on related sphingolipids in the ganglioside metabolic pathway. The simple gangliosides GM2 and GM3 were elevated in the brain stem, cerebellum and sub-cortex of the MPS IIIA mouse at one month of age, but not in the cortex. By six months accumulation was significant throughout the brain, with GD2 gangliosides also elevated. Elevations in other sphingolipids were limited to the upstream synthetic precursors, ceramide and dihexosylceramide (DHC) species containing 18:0 and 20:0 acyl chains, likely due to the abundance of these fatty acids in the elevated gangliosides. In contrast, sphingomyelin, sulphatide and DHC containing a 24:1 fatty acid, were all decreased in the brain stem of the MPS IIIA mice, suggestive of alterations in myelination. These perturbations in sphingolipid metabolism could provide an avenue for therapeutic intervention by manipulation with specific drugs that target the production of these lipids.

Increased monohexosylceramide levels in the serum of established rheumatoid arthritis patients.

OBJECTIVES: To identify serum sphingolipids that could act as candidate biomarkers in RA. METHODS: We performed lipidomic analyses in the serum of 82 participants: 19 established RA patients, 18 untreated early RA patients, 13 untreated early arthritis patients not fulfilling the classification criteria for RA, 12 established SpA patients and 20 controls. We compared the lipid levels from the different patient groups with the control group through multiple-regression analyses controlling for age at diagnosis, gender and medication (cDMARDs and corticoids). RESULTS: Established RA patients had significantly increased levels of sphingosine, monohexosylceramide and ceramide compared with controls, when controlling for age and gender. Monohexosylceramide levels remained significantly increased when additionally controlling for medication. On the contrary, SpA patients had significantly decreased levels of ceramide, in both analyses. CONCLUSION: We observed a detectable increase in the levels of certain sphingolipids in the serum of established RA patients when compared with controls, in line with previous observations in the synovial fluid. Such findings provide further evidence that sphingolipids may play a key role in the pathophysiology of RA.

Expanding the clinical utility of glucosylsphingosine for Gaucher disease.

Gaucher disease (GD) is an inherited metabolic disorder characterised by impaired catabolism of the glycosphingolipid, glucosylceramide. The deacetylated derivative, glucosylsphingosine (GluSph, lyso-Gb1) has materialised as a biomarker for GD. Further appraisal of the clinical utility of GluSph is required in terms of its prognostic power to inform disease course and pre-symptomatic testing. In this study, we show that plasma GluSph concentrations are significantly higher in GD patients with neuronopathic disease compared with non-neuronopathic disease, even in the neonatal period. A neonate diagnosed at 1 day of age (homozygous for N370S) due to an affected older sibling, returned GluSph of 70 nmol/L compared with 1070-2620 nmol/L for four neuronopathic patients diagnosed <20 days of age. Given this result shows promise for newborn screening, we developed a rapid, simple, and robust assay for GluSph in dried filter paper blood spots (DBS) and were able to detect 23 GD patients from 220 unaffected individuals. Neuronopathic GD patients also had significantly higher DBS concentrations of GluSph than their non-neuronopathic counterparts. We went on to measure GluSph in tissue extracts prepared from chorionic villus sampling and confirmed concentrations were undetectable in unaffected tissue but elevated in GD tissue demonstrating utility in the prenatal setting. Additionally, GluSph is a pharmacodynamic biomarker, revealing a precipitous drop following initiation of enzyme replacement therapy. In conclusion, GluSph is a reliable and specific biomarker for GD and shows promise for prenatal diagnosis and DBS screening programmes.

Collection of cerebrospinal fluid from murine lateral ventricles for biomarker determination in mucopolysaccharidosis type IIIA.

BACKGROUND: Cerebrospinal fluid (CSF) collection is currently the only feasible means to obtain biological fluid for the quantitative determination of biomarkers that may reflect disease activity within the brain. Studies in mouse models of human neurological disease benefit from ascertainment and subsequent analysis of brain tissue that is not afforded in human patients. The CSF provides a translational forum, however, due to the practical constraints presented by the mouse's small size, CSF is often ignored in experimental mouse models. NEW METHOD: Here we report a method for the controlled, precise and predictable collection of 10 µL of CSF from the lateral ventricles of adult mice using stereotaxic equipment and a micro-syringe pump. RESULTS: Collected CSF was clear and manifested the consistency of water and moreover, quantification of a disease-specific biomarker for the neurodegenerative disorder, mucopolysaccharidosis type IIIA (MPS IIIA) was possible in this small volume of CSF. In the naturally occurring mouse model of MPS IIIA, that faithfully recapitulates the human form of the disease, this biomarker was present at concentrations of >100 pmol/mL and undetectable in wild-type mice. COMPARISON WITH EXISTING METHOD: Advantages of this method over the most commonly used cisterna magna collection technique include increased CSF sample volume (10 µL) and reduced blood contamination. CONCLUSION: The ability to collect CSF from mouse models of neurological disease enables a forum for translating research outcomes in experimental models to the human equivalent in which CSF collection is also possible.

Evaluation of biomarkers for Sanfilippo syndrome.

Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III) is a childhood metabolic disorder marked by neuropathology arising due to impaired heparan sulphate (HS) catabolism. Consequently, partially degraded HS accumulates in the lysosomes of affected cells and is excreted in the urine. The measurement of HS in urine has long been considered a biomarker of Sanfilippo syndrome although it is largely non-specific. Using blood, urine and CSF collected from a cohort of Sanfilippo patients we investigated the utility of primary and secondary biomarkers to inform on disease activity. These included enzyme activity, specific oligosaccharides with non-reducing end residues reflective of the enzyme deficiency, and gangliosides. The diagnostic oligosaccharides - a HS disaccharide and tetrasaccharide - were elevated in the urine, plasma and CSF of all MPS IIIA and IIIB patients, respectively. There was no correlation between the concentrations in any of the matrices suggesting they reflect specific tissues and not overall disease burden. Enzyme activity did not inform on disease severity, with no measurable activity in CSF and activity approaching normal in MPS IIIA plasma. The concentration of gangliosides, GM2 and GM3, were significantly higher in the CSF of all MPS III subjects when compared to controls and correlated with the age of onset of first symptoms. Given that these gangliosides reflect delayed brain development they may be useful measures of disease burden, within the limitations of the clinical surrogates. Observation of these biochemical measurements in MPS III patients enrolled in clinical trials may determine whether they represent true pharmacodynamics biomarkers.

Disease and subtype specific signatures enable precise diagnosis of the mucopolysaccharidoses.

PURPOSE: Expanding treatments for the mucopolysaccharidoses-a family of genetic disorders-place unprecedented demands for accurate, timely diagnosis because best outcomes are seen with early initiation of appropriate therapies. Here we sought to improve the diagnostic odyssey by measuring specific glycosaminoglycan fragments with terminal residues complicit with the genetic defect resulting in precise diagnosis rather than the usual first-line, ambiguous total glycosaminoglycan determinations that return poor diagnostic yield. METHODS: A derivatizing reagent was added to urine aliquots (0.5 µmol creatinine) before separation of the glycosaminoglycan fragments by liquid chromatography and quantification with electrospray ionization-tandem mass spectrometry using multiple reaction monitoring for 10 targeted fragments plus the internal standard. RESULTS: All 93 mucopolysaccharidosis patients were correctly identified as 1 of 10 subtypes from a total of 723 de-identified subjects-blinded to diagnosis-based on the presence of specific "signature" glycosaminoglycan fragments. Employing reference intervals calculated from 630 unaffected urines, with 99% confidence intervals, provided a laboratory test with 100% specificity and sensitivity. CONCLUSION: This novel urine assay allows diagnosis of 10 mucopolysaccharidosis subtypes in a single test. The precise quantification of unique glycosaminoglycan fragments also enables longitudinal biochemical monitoring following therapeutic interventions.

Maternal overnutrition by hypercaloric diets programs hypothalamic mitochondrial fusion and metabolic dysfunction in rat male offspring.

BACKGROUND: Maternal overnutrition including pre-pregnancy, pregnancy and lactation promotes a lipotoxic insult leading to metabolic dysfunction in offspring. Diet-induced obesity models (DIO) show that changes in hypothalamic mitochondria fusion and fission dynamics modulate metabolic dysfunction. Using three selective diet formula including a High fat diet (HFD), Cafeteria (CAF) and High Sugar Diet (HSD), we hypothesized that maternal diets exposure program leads to selective changes in hypothalamic mitochondria fusion and fission dynamics in male offspring leading to metabolic dysfunction which is exacerbated by a second exposure after weaning. METHODS: We exposed female Wistar rats to nutritional programming including Chow, HFD, CAF, or HSD for 9 weeks (pre-mating, mating, pregnancy and lactation) or to the same diets to offspring after weaning. We determined body weight, food intake and metabolic parameters in the offspring from 21 to 60 days old. Hypothalamus was dissected at 60 days old to determine mitochondria-ER interaction markers by mRNA expression and western blot and morphology by transmission electron microscopy (TEM). Mitochondrial-ER function was analyzed by confocal microscopy using hypothalamic cell line mHypoA-CLU192. RESULTS: Maternal programming by HFD and CAF leads to failure in glucose, leptin and insulin sensitivity and fat accumulation. Additionally, HFD and CAF programming promote mitochondrial fusion by increasing the expression of MFN2 and decreasing DRP1, respectively. Further, TEM analysis confirms that CAF exposure after programing leads to an increase in mitochondria fusion and enhanced mitochondrial-ER interaction, which partially correlates with metabolic dysfunction and fat accumulation in the HFD and CAF groups. Finally, we identified that lipotoxic palmitic acid stimulus in hypothalamic cells increases Ca2+ overload into mitochondria matrix leading to mitochondrial dysfunction. CONCLUSIONS: We concluded that maternal programming by HFD induces hypothalamic mitochondria fusion, metabolic dysfunction and fat accumulation in male offspring, which is exacerbated by HFD or CAF exposure after weaning, potentially due to mitochondria calcium overflux.

Mass spectrometry-directed structure elucidation and total synthesis of ultra-long chain (O-acyl)-ω-hydroxy fatty acids.

The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.

Glycosaminoglycan fragments as a measure of disease burden in the mucopolysaccharidosis type I mouse.

Glycosaminoglycan (GAG) catabolism involves endo-hydrolysis of polysaccharides followed by the sequential removal of the non-reducing end residue from the resulting oligosaccharides by exo-enzymes. In the inherited metabolic disorder, mucopolysaccharidosis type I (MPS I), a deficiency in the exo-enzyme, α-l-iduronidase, prevents removal of α-l-iduronic acid residues from the non-reducing end of the GAGs, heparan sulphate (HS) and dermatan sulphate (DS). The excretion of partially degraded HS and DS in urine of MPS I patients has long been recognized, but the question of whether they do indeed reflect GAG load in a particular tissue has not been addressed - an important issue in the context of biomarkers for assessment of disease burden in MPS I. Therefore, we measured specific low molecular weight HS and DS oligosaccharides with terminal α-l-iduronic acid residues, in the brain, liver, kidney, serum and urine, and correlated these findings with total GAG in the MPS I mouse model. Six oligosaccharides were identified in the urine, ranging from di- to pentasaccharides. Of these, five were observed in the kidney, four in the liver and brain, with the three most abundant in urine also seen in serum. These oligosaccharides accounted for just 0.1-2% of total GAG, with a disaccharide showing the best correlation with total GAG. The oligosaccharides and total GAG were most abundant in the liver, with the least observed in the brain. The concentration of oligosaccharides as a percentage of total GAG in urine was similar to that observed in the kidney, and both revealed a similar ratio of HS:DS, suggesting that the oligosaccharide storage pattern in urine is a reflection of that in the kidney. Serum, liver and brain had a similar ratio of HS:DS, which was lower to that seen in the urine and kidney. The distribution of oligosaccharides when ranked from most to least abundant, was also the same between serum, liver and brain suggesting that serum more closely reflects the oligosaccharides of the brain and liver and may therefore be a more informative measurement of disease burden than urine. The accumulation of HS and DS oligosaccharides was observed in the brain as early as one month of age, with the disaccharide showing a continuous increase with age. This demonstrates the progressive nature of the disease and as such this disaccharide could prove to be a useful biomarker to measure disease burden in MPS I.

Reduced cerebral vascularization in experimental neuronopathic Gaucher disease.

The glycosphingolipidosis, Gaucher disease, in which a range of neurological manifestations occur, results from a deficiency of acid ß-glucocerebrosidase, with subsequent accumulation of ß-glucocerebroside, its upstream substrates, and the non-acylated congener ß-glucosylsphingosine. However, the mechanisms by which end-organ dysfunction arise are poorly understood. Here, we report strikingly diminished cerebral microvascular density in a murine model of disease, and provide a detailed analysis of the accompanying cerebral glycosphingolipidome in these animals, with marked elevations of ß-glucosylsphingosine. Further in vitro studies confirmed a concentration-dependent impairment of endothelial cytokinesis upon exposure to quasi-pathological concentrations of ß-glucosylsphingosine. These findings support a premise for pathogenic disruption of cerebral angiogenesis as an end-organ effect, with potential for therapeutic modulation in neuronopathic Gaucher disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Subregional brain distribution of simple and complex glycosphingolipids in the mucopolysaccharidosis type I (Hurler syndrome) mouse: impact of diet.

Gangliosides are the most complex oligosaccharide-containing glycosphingolipids defined by the presence of sialic acid and although present in all tissues, predominate in the brain. Considering their importance in neural development, it is unsurprising that ganglioside metabolism is altered in neurodegenerative diseases. The severe form of mucopolysaccharidosis type I, Hurler syndrome (HS), is characterised by progressive loss of neuronal function through largely undefined mechanisms. Here, we sought to interrogate brain gangliosides in a murine model of HS and further, assessed whether dietary modulation of lipid metabolism effected correction of the metabolic abnormalities. The simple gangliosides, GM2 , GM3 , GD2 and GD3 were elevated in the five subregions examined - brain stem, cerebellum, cortex, hippocampus, subcortex - in HS mice as early as 2 months of age compared with their wild type counterparts. Their elevation persisted at 6 months of age, imparting protracted neurological development as these simple gangliosides have usually subsided by this stage of brain development. Their immediate synthetic precursor, lactosylceramide, was also elevated, suggesting that their increase arises at this metabolic intermediary, as dihydroceramide, ceramide and monohexosylceramide were unaffected. Dietary linoleic acid supplementation significantly reduced GM2 and GM3 , and furthermore, improved exploratory behaviour as assessed by the open field test, highlighting the possibility of further exploring dietary intervention as a therapeutic consideration.