C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid ß-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/-) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/- mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Glucosylceramidase Maintains Influenza Virus Infection by Regulating Endocytosis.

Influenza virus is an RNA virus encapsulated in a lipid bilayer derived from the host cell plasma membrane. Previous studies showed that influenza virus infection depends on cellular lipids, including the sphingolipids sphingomyelin and sphingosine. Here we examined the role of a third sphingolipid, glucosylceramide, in influenza virus infection following clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR-Cas9)-mediated knockout (KO) of its metabolizing enzyme glucosylceramidase (GBA). After confirming GBA knockout of HEK 293 and A549 cells by both Western blotting and lipid mass spectrometry, we observed diminished infection in both KO cell lines by a PR8 (H1N1) green fluorescent protein (GFP) reporter virus. We further showed that the reduction in infection correlated with impaired influenza virus trafficking to late endosomes and hence with fusion and entry. To examine whether GBA is required for other enveloped viruses, we compared the results seen with entry mediated by the glycoproteins of Ebola virus, influenza virus, vesicular stomatitis virus (VSV), and measles virus in GBA knockout cells. Entry inhibition was relatively robust for Ebola virus and influenza virus, modest for VSV, and mild for measles virus, suggesting a greater role for viruses that enter cells by fusing with late endosomes. As the virus studies suggested a general role for GBA along the endocytic pathway, we tested that hypothesis and found that trafficking of epidermal growth factor (EGF) to late endosomes and degradation of its receptor were impaired in GBA knockout cells. Collectively, our findings suggest that GBA is critically important for endocytic trafficking of viruses as well as of cellular cargos, including growth factor receptors. Modulation of glucosylceramide levels may therefore represent a novel accompaniment to strategies to antagonize "late-penetrating" viruses, including influenza virus.IMPORTANCE Influenza virus is the pathogen responsible for the second largest pandemic in human history. A better understanding of how influenza virus enters host cells may lead to the development of more-efficacious therapies against emerging strains of the virus. Here we show that the glycosphingolipid metabolizing enzyme glucosylceramidase is required for optimal influenza virus trafficking to late endosomes and for consequent fusion, entry, and infection. We also provide evidence that promotion of influenza virus entry by glucosylceramidase extends to other endosome-entering viruses and is due to a general requirement for this enzyme, and hence for optimal levels of glucosylceramide, for efficient trafficking of endogenous cargos, such as the epidermal growth factor (EGF) receptor, along the endocytic pathway. This work therefore has implications for the basic process of endocytosis as well as for pathogenic processes, including virus entry and Gaucher disease.

Intracellular metabolite ß-glucosylceramide is an endogenous Mincle ligand possessing immunostimulatory activity.

Sensing and reacting to tissue damage is a fundamental function of immune systems. Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. Notably, Mincle also recognizes glycolipid ligands on pathogens. To elucidate endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as ß-glucosylceramide (GlcCer), which is a ubiquitous intracellular metabolite. Synthetic ß-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated in Mincle-deficient cells. Sterile inflammation induced by excessive cell death in the thymus was exacerbated by hematopoietic-specific deletion of degrading enzyme of ß-GlcCer (ß-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1-deficient dendritic cells (DCs) in which ß-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas these responses were compromised in DCs from GBA1 × Mincle double-deficient mice. These results suggest that ß-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity.

Clinical and neuropathological differences between Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies - current issues and future directions.

Lewy body diseases share clinical, pathological, genetic and biochemical signatures, and are regarded as a highly heterogeneous group of neurodegenerative disorders. Inclusive of Parkinson's disease (PD), Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB), controversy still exists as to whether they should be considered as separate disease entities or as part of the same disease continuum. Here we discuss emerging knowledge relating to both clinical, and neuropathological differences and consider current biomarker strategies as we try to improve our diagnostic capabilities and design of disease modifying therapeutics for this group of debilitating neurodegenerative disorders. This article is part of the Special Issue "Synuclein".

Glucocerebrosidase modulates cognitive and motor activities in murine models of Parkinson's disease.

Mutations in GBA1, the gene encoding glucocerebrosidase, are associated with an enhanced risk of developing synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies. A higher prevalence and increased severity of motor and non-motor symptoms is observed in PD patients harboring mutant GBA1 alleles, suggesting a link between the gene or gene product and disease development. Interestingly, PD patients without mutations in GBA1 also exhibit lower levels of glucocerebrosidase activity in the central nervous system (CNS), implicating this lysosomal enzyme in disease pathogenesis. Here, we investigated whether modulation of glucocerebrosidase activity in murine models of synucleinopathy (expressing wild type Gba1) affected α-synuclein accumulation and behavioral phenotypes. Partial inhibition of glucocerebrosidase activity in PrP-A53T-SNCA mice using the covalent inhibitor conduritol-B-epoxide induced a profound increase in soluble α-synuclein in the CNS and exacerbated cognitive and motor deficits. Conversely, augmenting glucocerebrosidase activity in the Thy1-SNCA mouse model of PD delayed the progression of synucleinopathy. Adeno-associated virus-mediated expression of glucocerebrosidase in the Thy1-SNCA mouse striatum led to decrease in the levels of the proteinase K-resistant fraction of α-synuclein, amelioration of behavioral aberrations and protection from loss of striatal dopaminergic markers. These data indicate that increasing glucocerebrosidase activity can influence α-synuclein homeostasis, thereby reducing the progression of synucleinopathies. This study provides robust in vivo evidence that augmentation of CNS glucocerebrosidase activity is a potential therapeutic strategy for PD, regardless of the mutation status of GBA1.

Gaucher-Associated Parkinsonism.

Gaucher disease is associated with Parkinson's disease (PD) by mutations in glucocerebrosidase (GCase). The gene encoding GCase, glucosidase beta acid (GBA), is an important risk factor for PD. Findings from large studies have shown that patients with PD have an increased frequency of mutations in GBA and that GBA mutation carriers exhibit diverse parkinsonian phenotypes and Lewy body pathology. Although the mechanism for this association remains elusive, some hypotheses have been proposed to explain it, including gain of function caused by GBA mutations, which increases α-synuclein (α-syn) aggregation, loss of function due to lysosomal enzyme deficiency, which affects α-syn clearance, and even a bidirectional feedback loop, but each of these hypotheses has its limitations. It is also worth noting that many findings have implicated the interaction between α-syn and GCase, indicating the essential role of the interaction in the pathogenesis of GBA-associated parkinsonism. Therefore, the current review focuses on α-syn and GCase, and it provides some new thoughts that may be helpful for understanding the α-syn-GCase interaction and unraveling the exact mechanism underlying GBA-associated parkinsonism.

Gaucher disease: haematological presentations and complications.

Gaucher disease (GD) is an autosomal recessive lysosomal storage disease, caused by deficiency of the enzyme glucocerebrosidase, required for the degradation of glycosphingolipids. Clinical manifestations include hepatosplenomegaly, thrombocytopenia, bone disease and a bleeding diathesis, frequently resulting in presentation to haematologists. Historically managed by splenectomy, transfusions and orthopaedic surgery, the development of specific therapy in the form of intravenous enzyme replacement therapy in the 1990s has resulted in dramatic improvements in haematological and visceral disease. Recognition of complications, including multiple myeloma and Parkinson disease, has challenged the traditional macrophage-centric view of the pathophysiology of this disorder. The pathways by which enzyme deficiency results in the clinical manifestations of this disorder are poorly understood; altered inflammatory cytokine profiles, bioactive sphingolipid derivatives and alterations in the bone marrow microenvironment have been implicated. Further elucidating these pathways will serve to advance our understanding not only of GD, but of associated disorders.

The significance of GBA for Parkinson's disease.

From the first descriptions of Parkinson's disease (PD) and Gaucher's disease (GD) in the nineteenth century, it took more than 100 years to discover the link between the GBA gene and Parkinsonism. The observation that mutations in the GBA gene represent the most common genetic risk factor for PD so far only came into focus because of astute clinical observation of Gaucher patients and their families. In this review, we (i) outline how GBA was identified as a genetic risk factor for Parkinsonism, (ii) present clinical characteristics of GBA-associated Parkinsonism, (iii) discuss possible mechanisms of the underlying pathogenesis in GBA-associated Parkinsonism, and (iv) provide an outlook on potentially new areas of research and treatment that arise from this important discovery.

The association between lysosomal protein glucocerebrosidase and Parkinson's disease.

BACKGROUND: In recent years, mutations in glucocerebrosidase gene (GBA), which encodes the lysosomal enzyme glucocerebrosidase (GCase) deficient in Gaucher disease (GD), were found to be the most widespread genetic for the development of Parkinson disease. AIM: In this work, we investigated the possibility of a biological linkage between GCase and alpha-synuclein. MATERIALS AND METHODS: siRNA was used to knockdown the GBA, then the related proteins such as alpha-synuclein were detected, additionally, the mutations of GBA were also detected. We also provide evidence that a mouse model of Gaucher disease (GBAD409H/D409H) to detect the gene types of GBA. RESULTS: The results showed functional knockdown (KD) of GBA in neuroblastoma cells culture causes a significant accumulation of alpha-synuclein and alpha-synuclein-mediated neurotoxicity. Furthermore, KD of GBA in rat primary neurons expressing the A53T mutation of alpha-synuclein, decreases cell viability. In addition, we observed that overexpression of several GBA mutants (N370S, L444P, D409H, D409V) significantly raised human alpha-syn levels of vector control. Glucosylceramide (GlcCer), the GCase substrate, influenced formation of purified a-syn by stabilizing soluble oligomeric intermediates. We also provide evidence that a mouse model of Gaucher disease (GBAD409H/D409H) exhibited alpha-syn aggregates in substantia nigra, cortex and hippocampus regions. ELISA analysis showed a significant rise in membrane-associated α-syn and western blot analysis showed that two forms of alpha-syn oligomers were present in brain homogenates from the hippocampus D409H mice. CONCLUSIONS: These studies support the contention that both WT and mutant GBA can cause Parkinson disease-like alpha-synuclein pathology.

Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease.

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba-/-) mouse model. Gba-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.

The role of glucocerebrosidase mutations in Parkinson disease and Lewy body disorders.

Mutations in the gene encoding glucocerebrosidase (GBA), the enzyme deficient in the lysosomal storage disorder Gaucher disease, are associated with the development of Parkinson disease and other Lewy body disorders. In fact, GBA variants are currently the most common genetic risk factor associated with parkinsonism, and identified subjects with Parkinson disease are more than five times more likely to carry mutations in GBA. The mechanisms underlying this association are not known, but proposed theories include enhanced protein aggregation, alterations in lipid levels, and autophagy-lysosomal dysfunction promoting the retention of undegraded proteins. We review the genetic studies linking GBA to parkinsonism, as well as several of the mechanisms postulated to explain the association of GBA mutations and the synucleinopathies, which demonstrate how studies of a rare mendelian disease may provide insights into our understanding of a common complex disorder.

The pathophysiology of GD - current understanding and rationale for existing and emerging therapeutic approaches.

Gaucher disease is a genetic disorder of sphingolipid metabolism resulting from dysfunction of the lysosomal membrane-associated glycoprotein glucocerebrosidase (GBA) and resulting in intracellular accumulation of glucosylceramide and other glycolipids. Although the gene defect and relevant biochemical pathways have been defined, the mechanisms by which substrate accumulation causes disease manifestations are not well understood. The direct effects of a build up of substrate laden cells may account for some aspects of disease but the overall pathology is likely to be more complex with effects of stored material on a variety of intra and extra cellular functions. In this article we review the GBA gene and its protein product, with associated defects, lipid metabolism and storage, enzyme misfolding and endoplasmic reticulum stress, calcium homeostasis, oxidative stress and autophagy and at each point examine how therapies that are currently available, in clinical development or at earlier stages of basic research might address the pathological mechanisms.

The underrecognized progressive nature of N370S Gaucher disease and assessment of cancer risk in 403 patients.

Mutations in GBA1 gene that encodes lysosomal glucocerebrosidase result in Type 1 Gaucher Disease (GD), the commonest lysosomal storage disorder; the most prevalent disease mutation is N370S. We investigated the heterogeneity and natural course of N370S GD in 403 patients. Demographic, clinical, and genetic characteristics of GD at presentation were examined in a cross-sectional study. In addition, the relative risk (RR) of cancer in patients compared with age-, sex-, and ethnic-group adjusted national rates of cancer was determined. Of the 403 patients, 54% of patients were homozygous (N370S/N370S) and 46% were compound heterozygous for the N370S mutation (N370S/other). The majority of N370S/N370S patients displayed a phenotype characterized by late onset, predominantly skeletal disease, whereas the majority of N370S/other patients displayed early onset, predominantly visceral/hematologic disease, P < 0.0001. There was a striking increase in lifetime risk of multiple myeloma in the entire cohort (RR 25, 95% CI 9.17-54.40), mostly confined to N370S homozygous patients. The risk of other hematologic malignancies (RR 3.45, 95% CI 1.49-6.79), and overall cancer risk (RR 1.80, 95% CI 1.32-2.40) was increased. Homozygous N370S GD leads to adult-onset progressive skeletal disease with relative sparing of the viscera, a strikingly high risk of multiple myeloma, and an increased risk of other cancers. High incidence of gammopathy suggests an important role of the adaptive immune system in the development of GD. Adult patients with GD should be monitored for skeletal disease and cancers including multiple myeloma.

Roles for tumor necrosis factor receptor p55 and sphingomyelinase in repairing the cutaneous permeability barrier.

Epidermal TNF expression increases in response to cutaneous permeability barrier disruption and wound healing. TNF signaling is mediated by acid and neutral sphingomyelinases (A- and N-SMase), which generate ceramide, an important regulator of proliferation, differentiation, and apoptosis. In the epidermis, ceramide is known to be an integral part of the extracellular stratum corneum (SC) lipid bilayers that constitute the permeability barrier of the skin. We show here that topical application of TNF after experimental injury to the SC of hairless mice (hr(-/-)) enhances barrier repair. In TNF receptor p55-deficient (TNF-R55-deficient) mice (hr(+/+)), cutaneous barrier repair was delayed compared with wild-type (hr(+/+)) or TNF-R75-deficient (hr(+/+)) animals. After barrier disruption in hairless (hr(-/-)) and wild-type (hr(+/+)), but not in TNF-R55-deficient (hr(+/+)) mice, the enzymatic activities of both A-SMase and N-SMase were significantly enhanced. Stimulation of SMase activities was accompanied by an increase in C(24)-ceramide levels. Most A-SMase activity in hairless mice (hr(-/-)) was found in the outer epidermal cell layers and colocalized in the lamellar bodies with A-SMase and sphingomyelin. Reduction of epidermal A-SMase activity by the inhibitor imipramine resulted in delayed permeability barrier repair after SC injury. Together, these results suggest that TNF-R55 signaling pathways contribute to cutaneous permeability barrier repair through SMase-mediated generation of ceramide.

Expression of human glucocerebrosidase in murine macrophages: identification of efficient retroviral vectors.

Gaucher's disease is an autosomal recessive disorder characterized by a functional deficiency in beta-glucocerebrosidase enzymatic activity and the resultant accumulation of the glycolipid glucocerebroside in macrophages. Due to the nature of the affected cells, Gaucher's disease is an excellent candidate for gene therapy of hematopoietic stem cells and autologous bone marrow transplantation of transduced cells using retroviral vectors containing the glucocerebrosidase (GC) gene. In order to identify a retroviral vector capable of high levels of expression of the GC gene in macrophages, we have used the murine myeloid leukemia cell line, M1, a cell line that can be differentiated with interleukin-6 (IL-6) from blasts to macrophages. Two vectors use the Moloney murine leukemia virus (MoMLV) enhancer/promoter (LG vector) or the myeloproliferative sarcoma virus (MPSV) enhancer/MoMLV promoter (MG vector), both located in the viral long-terminal repeat (LTR); the third vector uses the phosphoglycerate kinase (PGK) promoter located internally in the vector (PG vector). The amphotropic PA317 and GP+am12 packaging cell lines were used as virus producer cells, and the GP+am12 cell line demonstrated higher titers, higher levels of GC protein expression, and specific GC enzymatic activity as well as higher transduction efficiencies for all three vectors. The LG retroviral vector was the most efficient in transducing the M1 cells. On average, higher levels of RNA and protein expression were seen in the M1 clones transduced with the LG vector, and these levels increased after differentiation. Thus, the LG retroviral vector in which the expression of the GC gene is driven by the MoMLV LTR enhancer/promoter is the best vector of the three studied for future studies for gene therapy of Gaucher's disease and other hematopoietic disorders that involve macrophages.

The link between the GBA gene and parkinsonism.

Mutations in the glucocerebrosidase (GBA) gene, which encodes the lysosomal enzyme that is deficient in Gaucher's disease, are important and common risk factors for Parkinson's disease and related disorders. This association was first recognised in the clinic, where parkinsonism was noted, albeit rarely, in patients with Gaucher's disease and more frequently in relatives who were obligate carriers. Subsequently, findings from large studies showed that patients with Parkinson's disease and associated Lewy body disorders had an increased frequency of GBA mutations when compared with control individuals. Patients with GBA-associated parkinsonism exhibit varying parkinsonian phenotypes but tend to have an earlier age of onset and more associated cognitive changes than patients with parkinsonism without GBA mutations. Hypotheses proposed to explain this association include a gain-of-function due to mutations in glucocerebrosidase that promotes α-synuclein aggregation; substrate accumulation due to enzymatic loss-of-function, which affects α-synuclein processing and clearance; and a bidirectional feedback loop. Identification of the pathological mechanisms underlying GBA-associated parkinsonism will improve our understanding of the genetics, pathophysiology, and treatment for both rare and common neurological diseases.