Identification of a feedback loop involving ß-glucosidase 2 and its product sphingosine sheds light on the molecular mechanisms in Gaucher disease.

The lysosomal acid ß-glucosidase GBA1 and the non-lysosomal ß-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of ß-glucosidase activity in general.

Delineating pathological pathways in a chemically induced mouse model of Gaucher disease.

Great interest has been shown in understanding the pathology of Gaucher disease (GD) due to the recently discovered genetic relationship with Parkinson's disease. For such studies, suitable animal models of GD are required. Chemical induction of GD by inhibition of acid ß-glucosidase (GCase) using the irreversible inhibitor conduritol B-epoxide (CBE) is particularly attractive, although few systematic studies examining the effect of CBE on the development of symptoms associated with neurological forms of GD have been performed. We now demonstrate a correlation between the amount of CBE injected into mice and levels of accumulation of the GD substrates, glucosylceramide and glucosylsphingosine, and show that disease pathology, indicated by altered levels of pathological markers, depends on both the levels of accumulated lipids and the time at which their accumulation begins. Gene array analysis shows a remarkable similarity in the gene expression profiles of CBE-treated mice and a genetic GD mouse model, the Gba(flox/flox) ;nestin-Cre mouse, with 120 of the 144 genes up-regulated in CBE-treated mice also up-regulated in Gba(flox/flox) ;nestin-Cre mice. We also demonstrate that various aspects of neuropathology and some behavioural abnormalities can be arrested upon cessation of CBE treatment during a specific time window. Together, our data demonstrate that injection of mice with CBE provides a rapid and relatively easy way to induce symptoms typical of neuronal forms of GD. This is particularly useful when examining the role of specific biochemical pathways in GD pathology, since CBE can be injected into mice defective in components of putative pathological pathways, alleviating the need for time-consuming crossing of mice. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

From sheep to mice to cells: tools for the study of the sphingolipidoses.

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.

Ablation of ceramide synthase 2 causes chronic oxidative stress due to disruption of the mitochondrial respiratory chain.

Ceramide is a key intermediate in the pathway of sphingolipid biosynthesis and is an important intracellular messenger. We recently generated a ceramide synthase 2 (CerS2) null mouse that cannot synthesize very long acyl chain (C22-C24) ceramides. This mouse displays severe and progressive hepatopathy. Significant changes were observed in the sphingolipid profile of CerS2 null mouse liver, including elevated C16-ceramide and sphinganine levels in liver and in isolated mitochondrial fractions. Because ceramide may be involved in reactive oxygen species (ROS) formation, we examined whether ROS generation was affected in CerS2 null mice. Levels of a number of anti-oxidant enzymes were elevated, as were lipid peroxidation, protein nitrosylation, and ROS. ROS were generated from mitochondria due to impaired complex IV activity. C16-ceramide, sphingosine, and sphinganine directly inhibited complex IV activity in isolated mitochondria and in mitoplasts, whereas other ceramide species, sphingomyelin, and diacylglycerol were without effect. A fluorescent analog of sphinganine accumulated in mitochondria. Heart mitochondria did not display a substantial alteration in the sphingolipid profile or in complex IV activity. We suggest that C16-ceramide and/or sphinganine induce ROS formation through the modulation of mitochondrial complex IV activity, resulting in chronic oxidative stress. These results are of relevance for understanding modulation of ROS signaling by sphingolipids.

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

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

Altered lysosome distribution is an early neuropathological event in neurological forms of Gaucher disease.

In the lysosomal storage disorder Gaucher disease (GD), glucosylceramide (GlcCer) accumulates due to the defective activity of glucocerebrosidase. A subset of GD patients develops neuropathology. We now show mislocalization of Limp2-positive puncta and a large reduction in the number of Lamp1-positive puncta, which are associated with impaired tubulin. These changes occur at an early stage in animal models of GD, prior to development of overt symptoms and considerably earlier than neuronal loss. Altered lysosomal localization and cytoskeleton disruption precede the neuroinflammatory pathways, axonal dystrophy and neuronal loss previously characterized in neuronal forms of GD.

Identification of a biomarker in cerebrospinal fluid for neuronopathic forms of Gaucher disease.

Gaucher disease, a recessive inherited metabolic disorder caused by defects in the gene encoding glucosylceramidase (GlcCerase), can be divided into three subtypes according to the appearance of symptoms associated with central nervous system involvement. We now identify a protein, glycoprotein non-metastatic B (GPNMB), that acts as an authentic marker of brain pathology in neurological forms of Gaucher disease. Using three independent techniques, including quantitative global proteomic analysis of cerebrospinal fluid (CSF) in samples from Gaucher disease patients that display neurological symptoms, we demonstrate a correlation between the severity of symptoms and GPNMB levels. Moreover, GPNMB levels in the CSF correlate with disease severity in a mouse model of Gaucher disease. GPNMB was also elevated in brain samples from patients with type 2 and 3 Gaucher disease. Our data suggest that GPNMB can be used as a marker to quantify neuropathology in Gaucher disease patients and as a marker of treatment efficacy once suitable treatments towards the neurological symptoms of Gaucher disease become available.

Distinct origins and genetic programs of head muscle satellite cells.

Adult skeletal muscle possesses a remarkable regenerative capacity, due to the presence of satellite cells, adult muscle stem cells. We used fate-mapping techniques in avian and mouse models to show that trunk (Pax3(+)) and cranial (MesP1(+)) skeletal muscle and satellite cells derive from separate genetic lineages. Similar lineage heterogeneity is seen within the head musculature and satellite cells, due to their shared, heterogenic embryonic origins. Lineage tracing experiments with Isl1Cre mice demonstrated the robust contribution of Isl1(+) cells to distinct jaw muscle-derived satellite cells. Transplantation of myofiber-associated, Isl1-derived satellite cells into damaged limb muscle contributed to muscle regeneration. In vitro experiments demonstrated the cardiogenic nature of cranial- but not trunk-derived satellite cells. Finally, overexpression of Isl1 in the branchiomeric muscles of chick embryos inhibited skeletal muscle differentiation in vitro and in vivo, suggesting that this gene plays a role in the specification of cardiovascular and skeletal muscle stem cell progenitors.