Deep learning-based quantification of osteonecrosis using magnetic resonance images in Gaucher disease.

Gaucher disease is one of the most common lysosomal storage disorders. Osteonecrosis is a principal clinical manifestation of Gaucher disease and often leads to joint collapse and fractures. T1-weighted (T1w) modality in MRI is widely used to monitor bone involvement in Gaucher disease and to diagnose osteonecrosis. However, objective and quantitative methods for characterizing osteonecrosis are still limited. In this work, we present a deep learning-based quantification approach for the segmentation of osteonecrosis and the extraction of characteristic parameters. We first constructed two independent U-net models to segment the osteonecrosis and bone marrow unaffected by osteonecrosis (UBM) in spine and femur respectively, based on T1w images from patients in the UK national Gaucherite study database. We manually delineated parcellation maps including osteonecrosis and UBM from 364 T1w images (176 for spine, 188 for femur) as the training datasets, and the trained models were subsequently applied to all the 917 T1w images in the database. To quantify the segmentation, we calculated morphological parameters including the volume of osteonecrosis, the volume of UBM, and the fraction of total marrow occupied by osteonecrosis. Then, we examined the correlation between calculated features and the bone marrow burden score for marrow infiltration of the corresponding image, and no strong correlation was found. In addition, we analyzed the influence of splenectomy and the interval between the age at first symptom and the age of onset of treatment on the quantitative measurements of osteonecrosis. The results are consistent with previous studies, showing that prior splenectomy is closely associated with the fractional volume of osteonecrosis, and there is a positive relationship between the duration of untreated disease and the quantifications of osteonecrosis. We propose this technique as an efficient and reliable tool for assessing the extent of osteonecrosis in MR images of patients and improving prediction of clinically important adverse events.

The Two Substrate Reduction Therapies for Type 1 Gaucher Disease Are Not Equivalent. Comment on Hughes et al. Switching between Enzyme Replacement Therapies and Substrate Reduction Therapies in Patients with Gaucher Disease: Data from the Gaucher Outcome Survey (GOS). J. Clin. Med. 2022, 11, 5158.

In their paper, Hughes et al. [...].

Gaucher disease protects against tuberculosis.

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. Gaucher and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19th century.

Upregulation of non-canonical and canonical inflammasome genes associates with pathological features in Krabbe disease and related disorders.

Infantile Krabbe disease is a rapidly progressive and fatal disorder of myelin, caused by inherited deficiency of the lysosomal enzyme ß-galactocerebrosidase. Affected children lose their motor skills and other faculties; uncontrolled seizures are a frequent terminal event. Overexpression of the sphingolipid metabolite psychosine is a pathogenic factor, but does not fully account for the pleiotropic manifestations and there is a clear need to investigate additional pathological mechanisms. We examined innate immunity, caspase-11 and associated inflammatory pathways in twitcher mice, an authentic model of Krabbe disease. Combined use of molecular tools, RNAscope in situ hybridization and immunohistochemical staining established that the expression of pro-inflammatory non-canonical caspase-11, canonical caspase-1, gasdermin D and cognate genes is induced in nervous tissue. Early onset and progressive upregulation of these genes accompany demyelination and gliosis and although the molecules are scant in healthy tissue, abundance of the respective translation products is greatly increased in diseased animals. Caspase-11 is found in reactive microglia/macrophages as well as astrocytes but caspase-1 and gasdermin D are restricted to reactive microglia/macrophages. The inflammasome signature is not unique to Krabbe disease; to varying degrees, this signature is also prominent in other lysosomal diseases, Sandhoff and Niemann-Pick Type-C1, and the lysolecithin toxin model of focal demyelination. Given the potent inflammatory response here identified in Krabbe disease and the other neurodegenerative disorders studied, a broad induction of inflammasomes is likely to be a dominant factor in the pathogenesis, and thus represents a platform for therapeutic exploration.

Radiographic Cortical Thickness Index Predicts Fragility Fracture in Gaucher Disease.

Background Patients with Gaucher disease (GD) have a high risk of fragility fractures. Routine evaluation of bone involvement in these patients includes radiography and repeated dual-energy x-ray absorptiometry (DXA). However, osteonecrosis and bone fracture may affect the accuracy of DXA. Purpose To assess the utility of DXA and radiographic femoral cortical thickness measurements as predictors of fragility fracture in patients with GD with long-term follow-up (up to 30 years). Materials and Methods Patients with GD age 16 years and older with a detailed medical history, at least one bone image (DXA and/or radiographs), and minimum 2 years follow-up were retrospectively identified using three merged UK-based registries (Gaucherite study, enrollment 2015-2017; Clinical Bone Registry, enrollment 2003-2006; and Mortality Registry, enrollment 1993-2019). Cortical thickness index (CTI) and canal-to-calcar ratio (CCR) were measured by two independent observers, and inter- and intraobserver reliability was calculated. The fracture-predictive value of DXA, CTI, CCR, and cutoff values were calculated using receiver operating characteristic curves. Statistical differences were assessed using univariable and multivariable analysis. Results Bone imaging in 247 patients (123 men, 124 women; baseline median age, 39 years; IQR, 27-50 years) was reviewed. The median follow-up period was 11 years (IQR, 7-19 years; range, 2-30 years). Thirty-five patients had fractures before or at first bone imaging, 23 patients had fractures after first bone imaging, and 189 patients remained fracture-free. Inter- and intraobserver reproducibility for CTI/CCR measurements was substantial (range, 0.96-0.98). In the 212 patients with no baseline fracture, CTI (cutoff, ≤0.50) predicted future fractures with higher sensitivity and specificity (area under the receiver operating characteristic curve [AUC], 0.96; 95% CI: 0.84, 0.99; sensitivity, 92%; specificity, 96%) than DXA T-score at total hip (AUC, 0.78; 95% CI: 0.51, 0.91; sensitivity, 64%; specificity, 93%), femoral neck (AUC, 0.73; 95% CI: 0.50, 0.86; sensitivity, 64%; specificity, 73%), lumbar spine (AUC, 0.69; 95% CI: 0.49, 0.82; sensitivity, 57%; specificity, 63%), and forearm (AUC, 0.78; 95% CI: 0.59, 0.89; sensitivity, 70%; specificity, 70%). Conclusion Radiographic cortical thickness index of 0.50 or less was a reliable independent predictor of fracture risk in Gaucher disease. Clinical trial registration no. NCT03240653 © RSNA, 2022 Supplemental material is available for this article.

Long-term effects of eliglustat on skeletal manifestations in clinical trials of patients with Gaucher disease type 1.

PURPOSE: Most patients with Gaucher disease have progressive and often disabling skeletal manifestations. We examined the long-term effect of eliglustat treatment on bone outcomes in clinical trials in adults with Gaucher disease type 1. METHODS: Data from 4 completed phase 2 and 3 trials were evaluated in treatment-naïve patients or patients switching to eliglustat from enzyme replacement therapy (ERT). RESULTS: Overall, 319 of 393 (81%) eliglustat-treated patients remained in their trials until completion or commercial eliglustat became available. Mean eliglustat treatment duration ranged from 3.3 to 6.5 years. In treatment-naïve patients and ERT-switch patients, frequency and severity of bone pain decreased during eliglustat treatment. Mean lumbar spine T-scores shifted from abnormal to normal in treatment-naïve patients and remained in the healthy reference range or improved modestly in ERT-switch patients. Mean total bone marrow burden score shifted from marked-to-severe to moderate in treatment-naïve patients and remained moderate in ERT-switch patients. MIP-1ß (marker of active bone disease) was elevated at baseline and decreased to the healthy reference range in treatment-naïve patients and remained in the healthy reference range among ERT-switch patients. CONCLUSION: These findings confirm the long-term efficacy of eliglustat on skeletal complications of Gaucher disease in treatment-naïve and ERT-switch patients.

Venglustat combined with imiglucerase for neurological disease in adults with Gaucher disease type 3: the LEAP trial.

Gaucher disease type 3 is a chronic neuronopathic disorder with wide-ranging effects, including hepatosplenomegaly, anaemia, thrombocytopenia, skeletal disease and diverse neurological manifestations. Biallelic mutations in GBA1 reduce lysosomal acid ß-glucosidase activity, and its substrates, glucosylceramide and glucosylsphingosine, accumulate. Enzyme replacement therapy and substrate reduction therapy ameliorate systemic features of Gaucher disease, but no therapies are approved for neurological manifestations. Venglustat is an investigational, brain-penetrant, glucosylceramide synthase inhibitor with potential to improve the disease by rebalancing influx of glucosylceramide with impaired lysosomal recycling. The Phase 2, open-label LEAP trial (NCT02843035) evaluated orally administered venglustat 15 mg once-daily in combination with maintenance dose of imiglucerase enzyme replacement therapy during 1 year of treatment in 11 adults with Gaucher disease type 3. Primary endpoints were venglustat safety and tolerability and change in concentration of glucosylceramide and glucosylsphingosine in CSF from baseline to Weeks 26 and 52. Secondary endpoints included change in plasma concentrations of glucosylceramide and glucosylsphingosine, venglustat pharmacokinetics in plasma and CSF, neurologic function, infiltrative lung disease and systemic disease parameters. Exploratory endpoints included changes in brain volume assessed with volumetric MRI using tensor-based morphometry, and resting functional MRI analysis of regional brain activity and connectivity between resting state networks. Mean (SD) plasma venglustat AUC0-24 on Day 1 was 851 (282) ng•h/ml; Cmax of 58.1 (26.4) ng/ml was achieved at a median tmax 2.00 h. After once-daily venglustat, plasma concentrations (4 h post-dose) were higher compared with Day 1, indicating ∼2-fold accumulation. One participant (Patient 9) had low-to-undetectable venglustat exposure at Weeks 26 and 52. Based on mean plasma and CSF venglustat concentrations (excluding Patient 9), steady state appeared to be reached on or before Week 4. Mean (SD) venglustat concentration at Week 52 was 114 (65.8) ng/ml in plasma and 6.14 (3.44) ng/ml in CSF. After 1 year of treatment, median (inter-quartile range) glucosylceramide decreased 78% (72, 84) in plasma and 81% (77, 83) in CSF; median (inter-quartile range) glucosylsphingosine decreased 56% (41, 60) in plasma and 70% (46, 76) in CSF. Ataxia improved slightly in nine patients: mean (SD, range) total modified Scale for Assessment and Rating of Ataxia score decreased from 2.68 [1.54 (0.0 to 5.5)] at baseline to 1.55 [1.88 (0.0 to 5.0)] at Week 52 [mean change: -1.14 (95% CI: -2.06 to -0.21)]. Whole brain volume increased slightly in patients with venglustat exposure and biomarker reduction in CSF (306.7 ± 4253.3 mm3) and declined markedly in Patient 9 (-13894.8 mm3). Functional MRI indicated stronger connectivity at Weeks 26 and 52 relative to baseline between a broadly distributed set of brain regions in patients with venglustat exposure and biomarker reduction but not Patient 9, although neurocognition, assessed by Vineland II, deteriorated in all domains over time, which illustrates disease progression despite the intervention. There were no deaths, serious adverse events or discontinuations. In adults with Gaucher disease type 3 receiving imiglucerase, addition of once-daily venglustat showed acceptable safety and tolerability and preliminary evidence of clinical stability with intriguing but intrinsically inconsistent signals in selected biomarkers, which need to be validated and confirmed in future research.

Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts. We describe an attenuated juvenile form of VPS33A-related syndrome-mucopolysaccharidosis plus in a man who is homozygous for a hitherto unknown missense mutation (NM_022916.4: c.599 G>C; NP_075067.2:p. Arg200Pro) in a conserved region of the VPS33A gene. Urinary glycosaminoglycan (GAG) analysis revealed increased heparan, dermatan sulphates, and hyaluronic acid. We showed decreased abundance of VPS33A in patient derived fibroblasts and provided evidence that the p.Arg200Pro mutation leads to destablization of the protein and proteasomal degradation. As in the infantile form of mucopolysaccharidosis plus, the endocytic compartment in the fibroblasts also expanded-a phenomenon accompanied by increased endolysosomal acidification and impaired intracellular glycosphingolipid trafficking. Experimental treatment of the patient's cultured fibroblasts with the proteasome inhibitor, bortezomib, or exposure to an inhibitor of glucosylceramide synthesis, eliglustat, improved glycosphingolipid trafficking. To our knowledge this is the first report of an attenuated juvenile form of VPS33A insufficiency characterized by appreciable residual endosomal-lysosomal trafficking and a milder mucopolysaccharidosis plus than the disease in infants. Our findings expand the proof of concept of redeploying clinically approved drugs for therapeutic exploitation in patients with juvenile as well as infantile forms of mucopolysaccharidosis plus disease.

Thirty-year clinical outcomes after haematopoietic stem cell transplantation in neuronopathic Gaucher disease.

BACKGROUND: Neuronopathic Gaucher Disease (nGD) describes the condition of a subgroup of patients with the Lysosomal Storage Disorder (LSD), Gaucher disease with involvement of the central nervous system (CNS) which results from inherited deficiency of ß-glucosylceramidase. Although systemic manifestations of disease are now corrected by augmentation with macrophage-targeted therapeutic enzyme (enzyme replacement therapy, ERT), neurological disease progresses unpredictably as a result of failure of therapeutic enzyme to cross the blood-brain barrier (BBB). Without therapy, the systemic and neurological effects of the disease progress and shorten life: investigators, principally in Sweden and the UK, pioneered bone marrow transplantation (BMT; Haematopoietic Stem Cell Transplantation HSCT) to supply healthy marrow-derived macrophages and other cells, to correct the peripheral disease. Here we report the first long-term follow-up (over 20 years in all cases) of nine patients in the UK and Sweden who underwent HSCT in the 1970s and 1980s. This retrospective, multicentre observational study was undertaken to determine whether there are neurological features of Gaucher disease that can be corrected by HSCT and the extent to which deterioration continues after the procedure. Since intravenous administration of ERT is approved for patients with the neuronopathic disease and ameliorates many of the important systemic manifestations but fails to correct the neurological features, we also consider the current therapeutic positioning of HSCT in this disorder. RESULTS: In the nine patients here reported, neurological disease continued to progress after transplantation, manifesting as seizures, cerebellar disease and abnormalities of tone and reflexes. CONCLUSIONS: Although neurological disease progressed in this cohort of patients, there may be a future role for HSCT in the treatment of nGD. The procedure has the unique advantage of providing a life-long source of normally functioning macrophages in the bone marrow, and possibly other sites, after a single administration. HSCT moreover, clearly ameliorates systemic disease and this may be advantageous-especially where sustained provision of high-cost ERT cannot be guaranteed. Given the remaining unmet needs of patients with neuronopathic Gaucher disease and the greatly improved safety profile of the transplant procedure, HSCT could be considered to provide permanent correction of systemic disease, including bone disease not ameliorated by ERT, when combined with emerging therapies directed at the neurological manifestations of disease; this could include ex-vivo gene therapy approaches.

A novel brain-penetrant oral UGT8 inhibitor decreases in vivo galactosphingolipid biosynthesis in murine Krabbe disease.

Krabbe disease is a rare, inherited neurodegenerative disease due to impaired lysosomal ß-galactosylceramidase (GALC) activity and formation of neurotoxic ß-galactosylsphingosine ('psychosine'). We investigated substrate reduction therapy with a novel brain-penetrant inhibitor of galactosylceramide biosynthesis, RA 5557, in twitcher mice that lack GALC activity and model Krabbe disease. This thienopyridine derivative selectively inhibits uridine diphosphate-galactose glycosyltransferase 8 (UGT8), the final step in the generation of galactosylceramides which are precursors of sulphatide and, in the pathological lysosome, the immediate source of psychosine. Administration of RA 5557, reduced pathologically elevated psychosine concentrations (72-86%) in the midbrain and cerebral cortex in twitcher mice: the inhibitor decreased galactosylceramides by about 70% in midbrain and cerebral cortex in mutant and wild type animals. Exposure to the inhibitor significantly decreased several characteristic inflammatory response markers without causing apparent toxicity to myelin-producing cells in wild type and mutant mice; transcript abundance of oligodendrocyte markers MBP (myelin basic protein) and murine UGT8 was unchanged. Administration of the inhibitor before conception and during several breeding cycles to mice did not impair fertility and gave rise to healthy offspring. Nevertheless, given the unchanged lifespan, it appears that GALC has critical functions in the nervous system beyond the hydrolysis of galactosylceramide and galactosylsphingosine. Our findings support further therapeutic exploration of orally active UGT8 inhibitors in Krabbe disease and related galactosphingolipid disorders. The potent thienopyridine derivative with effective target engagement here studied appears to have an acceptable safety profile in vivo; judicious dose optimization will be needed to ensure efficacious clinical translation.

L-Arginine Ameliorates Defective Autophagy in GM2 Gangliosidoses by mTOR Modulation.

AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by ß-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy.

In-depth phenotyping for clinical stratification of Gaucher disease.

BACKGROUND: The Gaucher Investigative Therapy Evaluation is a national clinical cohort of 250 patients aged 5-87 years with Gaucher disease in the United Kingdom-an ultra-rare genetic disorder. To inform clinical decision-making and improve pathophysiological understanding, we characterized the course of Gaucher disease and explored the influence of costly innovative medication and other interventions. Retrospective and prospective clinical, laboratory and radiological information including molecular analysis of the GBA1 gene and comprising > 2500 variables were collected systematically into a relational database with banking of collated biological samples in a central bioresource. Data for deep phenotyping and life-quality evaluation, including skeletal, visceral, haematological and neurological manifestations were recorded for a median of 17.3 years; the skeletal and neurological manifestations are the main focus of this study. RESULTS: At baseline, 223 of the 250 patients were classified as type 1 Gaucher disease. Skeletal manifestations occurred in most patients in the cohort (131 of 201 specifically reported bone pain). Symptomatic osteonecrosis and fragility fractures occurred respectively in 76 and 37 of all 250 patients and the first osseous events occurred significantly earlier in those with neuronopathic disease. Intensive phenotyping in a subgroup of 40 patients originally considered to have only systemic features, revealed neurological involvement in 18: two had Parkinson disease and 16 had clinical signs compatible with neuronopathic Gaucher disease-indicating a greater than expected prevalence of neurological features. Analysis of longitudinal real-world data enabled Gaucher disease to be stratified with respect to advanced therapies and splenectomy. Splenectomy was associated with an increased hazard of fragility fractures, in addition to osteonecrosis and orthopaedic surgery; there were marked gender differences in fracture risk over time since splenectomy. Skeletal disease was a heavy burden of illness, especially where access to specific therapy was delayed and in patients requiring orthopaedic surgery. CONCLUSION: Gaucher disease has been explored using real-world data obtained in an era of therapeutic transformation. Introduction of advanced therapies and repeated longitudinal measures enabled this heterogeneous condition to be stratified into obvious clinical endotypes. The study reveals diverse and changing phenotypic manifestations with systemic, skeletal and neurological disease as inter-related sources of disability.

Expression of Ripk1 and DAM genes correlates with severity and progression of Krabbe disease.

Krabbe disease, an inherited leukodystrophy, is a sphingolipidosis caused by deficiency of ß-galactocerebrosidase: it is characterized by myelin loss, and pathological activation of macrophage/microglia and astrocytes. To define driving pathogenic factors, we explored the expression repertoire of candidate neuroinflammatory genes: upregulation of receptor interacting protein kinase 1 (Ripk1) and disease-associated microglia (DAM) genes, including Cst7 and Ch25h, correlated with severity of Krabbe disease genetically modelled in the twitcher mouse. Upregulation of Ripk1 in Iba1/Mac2-positive microglia/macrophage associated with the pathognomic hypertrophic/globoid phenotype of this disease. Widespread accumulation of ubiquitinin1 in white and grey matter co-localised with p62. In Sandhoff disease, another sphingolipid disorder, neuroinflammation, accumulation of p62 and increased Ripk1 expression was observed. The upregulated DAM genes and macrophage/microglia expression of Ripk1 in the authentic model of Krabbe disease strongly resemble those reported in Alzheimer disease associating with disturbed autophagosomal/lysosomal homeostasis. Activation of this shared molecular repertoire, suggests the potential for therapeutic interdiction at a common activation step, irrespective of proximal causation. To clarify the role of Ripk1 in the pathogenesis of Krabbe disease, we first explored the contribution of its kinase function, by intercrossing twitcher and the K45A kinase-dead Ripk1 mouse and breeding to homozygosity. Genetic ablation of Ripk1 kinase activity neither altered the neuropathological features nor the survival of twitcher mice. We conclude that Ripk1 kinase-dependent inflammatory and degenerative capabilities play no instrumental role in Krabbe disease; however, putative kinase-independent functions of Ripk1 remain formally to be explored in its molecular pathogenesis.

Human glucocerebrosidase mediates formation of xylosyl-cholesterol by ß-xylosidase and transxylosidase reactions.

Deficiency of glucocerebrosidase (GBA), a lysosomal ß-glucosidase, causes Gaucher disease. The enzyme hydrolyzes ß-glucosidic substrates and transglucosylates cholesterol to cholesterol-ß-glucoside. Here we show that recombinant human GBA also cleaves ß-xylosides and transxylosylates cholesterol. The xylosyl-cholesterol formed acts as an acceptor for the subsequent formation of di-xylosyl-cholesterol. Common mutant forms of GBA from patients with Gaucher disease with reduced ß-glucosidase activity were similarly impaired in ß-xylosidase, transglucosidase, and transxylosidase activities, except for a slightly reduced xylosidase/glucosidase activity ratio of N370S GBA and a slightly reduced transglucosylation/glucosidase activity ratio of D409H GBA. XylChol was found to be reduced in spleen from patients with Gaucher disease. The origin of newly identified XylChol in mouse and human tissues was investigated. Cultured human cells exposed to exogenous ß-xylosides generated XylChol in a manner dependent on active lysosomal GBA but not the cytosol-facing ß-glucosidase GBA2. We later sought an endogenous ß-xyloside acting as donor in transxylosylation reactions, identifying xylosylated ceramide (XylCer) in cells and tissues that serve as donor in the formation of XylChol. UDP-glucosylceramide synthase (GCS) was unable to synthesize XylChol but could catalyze the formation of XylCer. Thus, food-derived ß-D-xyloside and XylCer are potential donors for the GBA-mediated formation of XylChol in cells. The enzyme GCS produces XylCer at a low rate. Our findings point to further catalytic versatility of GBA and prompt a systematic exploration of the distribution and role of xylosylated lipids.

Improving the quantitative classification of Erlenmeyer flask deformities.

The Erlenmeyer flask deformity is a common skeletal modeling deformity, but current classification systems are binary and may restrict its utility as a predictor of associated skeletal conditions. A quantifiable 3-point system of severity classification could improve its predictive potential in disease. Ratios were derived from volumes of regions of interests drawn in 50 Gaucher's disease patients. ROIs were drawn from the distal physis to 2 cm proximal, 2 cm to 4 cm, and 4 cm to 6 cm. Width was also measured at each of these boundaries. Two readers rated these 100 femurs using a 3-point scale of severity classification. Weighted kappa indicated reliability and one-way analysis of variance characterized ratio differences across the severity scale. Accuracy analyses allowed determination of clinical cutoffs for each ratio. Pearson's correlations assessed the associations of volume and width with a shape-based concavity metric of the femur. The volume ratio incorporating the metaphyseal region from 0 to 2 cm and the diametaphyseal region at 4-6 cm was most accurate at distinguishing femurs on the 3-point scale. Receiver operating characteristic curves for this ratio indicated areas of 0.95 to distinguish normal and mild femurs and 0.93 to distinguish mild and severe femurs. Volume was moderately associated with the degree of femur concavity. The proposed volume ratio method is an objective, proficient method at distinguishing severities of the Erlenmeyer flask deformity with the potential for automation. This may have application across diseases associated with the deformity and deficient osteoclast-mediated modeling of growing bone.

Lysosomal Diseases and Neuropsychiatry: Opportunities to Rebalance the Mind.

The brain is the physical organ of the mind but efforts to understand mental illness within a neurobiological context have hitherto been unavailing. Mental disorders (anxiety, depression, bipolar disorder, and schizophrenia) affect about one fifth of the population and present an almost endless societal challenge at the frontier of human sciences. Prodigious technological advances in functional neuroimaging and large-scale genetics have not yet delivered the prospect of refined molecular understanding of mental illness beyond early anatomical descriptions of brain metabolism. However, intensive clinical phenotyping and quantitative metabolic studies using sophisticated radio-ligands in positron-emission tomography, persistently favor the neurobiological approach. This Perspective pursues a familiar maxim in Medicine, aptly summarized in the words of Arthur Koestler: "Nature is generous in her senseless experiments on mankind." Hitherto, studies in neuropsychiatry have largely ignored rare genetic disorders but derangements of specific components within the cerebral laboratory offer rich opportunities for mechanistic exploration. Aberrant psychic behavior is characteristic of many inborn errors of metabolism and although each disorder represents a universe of its own, we are at a threshold for understanding, since contemporary genetics and cell biology furnish abundant materials to take on the perturbing enigma of mental derangement. A further development relates to orphan drugs with actions on defined molecular targets: these represent new ways to study the pathogenesis of psychiatric phenomena associated with rare diseases and in a manner not formerly possible. Here we introduce the frontier of schizophrenia and its strong association with late-onset Tay-Sachs disease as a paradigm to explore.

Decrease in Myelin-Associated Lipids Precedes Neuronal Loss and Glial Activation in the CNS of the Sandhoff Mouse as Determined by Metabolomics.

Sandhoff disease (SD) is a lysosomal disease caused by mutations in the gene coding for the ß subunit of ß-hexosaminidase, leading to deficiency in the enzymes ß-hexosaminidase (HEX) A and B. SD is characterised by an accumulation of gangliosides and related glycolipids, mainly in the central nervous system, and progressive neurodegeneration. The underlying cellular mechanisms leading to neurodegeneration and the contribution of inflammation in SD remain undefined. The aim of the present study was to measure global changes in metabolism over time that might reveal novel molecular pathways of disease. We used liquid chromatography-mass spectrometry and 1H Nuclear Magnetic Resonance spectroscopy to profile intact lipids and aqueous metabolites, respectively. We examined spinal cord and cerebrum from healthy and Hexb -/- mice, a mouse model of SD, at ages one, two, three and four months. We report decreased concentrations in lipids typical of the myelin sheath, galactosylceramides and plasmalogen-phosphatidylethanolamines, suggesting that reduced synthesis of myelin lipids is an early event in the development of disease pathology. Reduction in neuronal density is progressive, as demonstrated by decreased concentrations of N-acetylaspartate and amino acid neurotransmitters. Finally, microglial activation, indicated by increased amounts of myo-inositol correlates closely with the late symptomatic phases of the disease.

The lysosomal disease caused by mutant VPS33A.

A rare lysosomal disease resembling a mucopolysaccharidosis with unusual systemic features, including renal disease and platelet dysfunction, caused by the defect in a conserved region of the VPS33A gene on human chromosome 12q24.31, occurs in Yakuts-a nomadic Turkic ethnic group of Southern Siberia. VPS33A is a core component of the class C core vacuole/endosome tethering (CORVET) and the homotypic fusion and protein sorting (HOPS) complexes, which have essential functions in the endocytic pathway. Here we show that cultured fibroblasts from patients with this disorder have morphological changes: vacuolation with disordered endosomal/lysosomal compartments and-common to sphingolipid diseases-abnormal endocytic trafficking of lactosylceramide. Urine glycosaminoglycan studies revealed a pathological excess of sialylated conjugates as well as dermatan and heparan sulphate. Lipidomic screening showed elevated ß-D-galactosylsphingosine with unimpaired activity of cognate lysosomal hydrolases. The 3D crystal structure of human VPS33A predicts that replacement of arginine 498 by tryptophan will de-stabilize VPS33A folding. We observed that the missense mutation reduced the abundance of full-length VPS33A and other components of the HOPS and CORVET complexes. Treatment of HeLa cells stably expressing the mutant VPS33A with a proteasome inhibitor rescued the mutant protein from degradation. We propose that the disease is due to diminished intracellular abundance of intact VPS33A. Exposure of patient-derived fibroblasts to the clinically approved proteasome inhibitor, bortezomib, or inhibition of glucosylceramide synthesis with eliglustat, partially corrected the impaired lactosylceramide trafficking defect and immediately suggest therapeutic avenues to explore in this fatal orphan disease.