An increase in ER stress and unfolded protein response in iPSCs-derived neuronal cells from neuronopathic Gaucher disease patients.

Gaucher disease (GD) is a lysosomal storage disorder caused by a mutation in the GBA1 gene, responsible for encoding the enzyme Glucocerebrosidase (GCase). Although neuronal death and neuroinflammation have been observed in the brains of individuals with neuronopathic Gaucher disease (nGD), the exact mechanism underlying neurodegeneration in nGD remains unclear. In this study, we used two induced pluripotent stem cells (iPSCs)-derived neuronal cell lines acquired from two type-3 GD patients (GD3-1 and GD3-2) to investigate the mechanisms underlying nGD by biochemical analyses. These iPSCs-derived neuronal cells from GD3-1 and GD3-2 exhibit an impairment in endoplasmic reticulum (ER) calcium homeostasis and an increase in unfolded protein response markers (BiP and CHOP), indicating the presence of ER stress in nGD. A significant increase in the BAX/BCL-2 ratio and an increase in Annexin V-positive cells demonstrate a notable increase in apoptotic cell death in GD iPSCs-derived neurons, suggesting downstream signaling after an increase in the unfolded protein response. Our study involves the establishment of iPSCs-derived neuronal models for GD and proposes a possible mechanism underlying nGD. This mechanism involves the activation of ER stress and the unfolded protein response, ultimately leading to apoptotic cell death in neurons.

GBA1 inactivation in oligodendrocytes affects myelination and induces neurodegenerative hallmarks and lipid dyshomeostasis in mice.

BACKGROUND: Mutations in the ß-glucocerebrosidase (GBA1) gene do cause the lysosomal storage Gaucher disease (GD) and are among the most frequent genetic risk factors for Parkinson's disease (PD). So far, studies on both neuronopathic GD and PD primarily focused on neuronal manifestations, besides the evaluation of microglial and astrocyte implication. White matter alterations were described in the central nervous system of paediatric type 1 GD patients and were suggested to sustain or even play a role in the PD process, although the contribution of oligodendrocytes has been so far scarcely investigated. METHODS: We exploited a system to study the induction of central myelination in vitro, consisting of Oli-neu cells treated with dibutyryl-cAMP, in order to evaluate the expression levels and function of ß-glucocerebrosidase during oligodendrocyte differentiation. Conduritol-B-epoxide, a ß-glucocerebrosidase irreversible inhibitor was used to dissect the impact of ß-glucocerebrosidase inactivation in the process of myelination, lysosomal degradation and α-synuclein accumulation in vitro. Moreover, to study the role of ß-glucocerebrosidase in the white matter in vivo, we developed a novel mouse transgenic line in which ß-glucocerebrosidase function is abolished in myelinating glia, by crossing the Cnp1-cre mouse line with a line bearing loxP sequences flanking Gba1 exons 9-11, encoding for ß-glucocerebrosidase catalytic domain. Immunofluorescence, western blot and lipidomic analyses were performed in brain samples from wild-type and knockout animals in order to assess the impact of genetic inactivation of ß-glucocerebrosidase on myelination and on the onset of early neurodegenerative hallmarks, together with differentiation analysis in primary oligodendrocyte cultures. RESULTS: Here we show that ß-glucocerebrosidase inactivation in oligodendrocytes induces lysosomal dysfunction and inhibits myelination in vitro. Moreover, oligodendrocyte-specific ß-glucocerebrosidase loss-of-function was sufficient to induce in vivo demyelination and early neurodegenerative hallmarks, including axonal degeneration, α-synuclein accumulation and astrogliosis, together with brain lipid dyshomeostasis and functional impairment. CONCLUSIONS: Our study sheds light on the contribution of oligodendrocytes in GBA1-related diseases and supports the need for better characterizing oligodendrocytes as actors playing a role in neurodegenerative diseases, also pointing at them as potential novel targets to set a brake to disease progression.

Lysosomal storage, impaired autophagy and innate immunity in Gaucher and Parkinson's diseases: insights for drug discovery.

Impairment of autophagic-lysosomal pathways is increasingly being implicated in Parkinson's disease (PD). GBA1 mutations cause the lysosomal storage disorder Gaucher disease (GD) and are the commonest known genetic risk factor for PD. GBA1 mutations have been shown to cause autophagic-lysosomal impairment. Defective autophagic degradation of unwanted cellular constituents is associated with several pathologies, including loss of normal protein homeostasis, particularly of α-synuclein, and innate immune dysfunction. The latter is observed both peripherally and centrally in PD and GD. Here, we will discuss the mechanistic links between autophagy and immune dysregulation, and the possible role of these pathologies in communication between the gut and brain in these disorders. Recent work in a fly model of neuronopathic GD (nGD) revealed intestinal autophagic defects leading to gastrointestinal dysfunction and immune activation. Rapamycin treatment partially reversed the autophagic block and reduced immune activity, in association with increased survival and improved locomotor performance. Alterations in the gut microbiome are a critical driver of neuroinflammation, and studies have revealed that eradication of the microbiome in nGD fly and mouse models of PD ameliorate brain inflammation. Following these observations, lysosomal-autophagic pathways, innate immune signalling and microbiome dysbiosis are discussed as potential therapeutic targets in PD and GD. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Lysosomal membrane integrity in fibroblasts derived from patients with Gaucher disease.

Gaucher disease (GD) is a recessively inherited lysosomal storage disorder characterized by a deficiency of lysosomal glucocerebrosidase (GBA1). This deficiency results in the accumulation of its substrate, glucosylceramide (GlcCer), within lysosomes. Here, we investigated lysosomal abnormalities in fibroblasts derived from patients with GD. It is noteworthy that the cellular distribution of lysosomes and lysosomal proteolytic activity remained largely unaffected in GD fibroblasts. However, we found that lysosomal membranes of GD fibroblasts were susceptible to damage when exposed to a lysosomotropic agent. Moreover, the susceptibility of lysosomal membranes to a lysosomotropic agent could be partly restored by exogenous expression of wild-type GBA1. Here, we report that the lysosomal membrane integrity is altered in GD fibroblasts, but lysosomal distribution and proteolytic activity is not significantly altered.Key words: glucosylceramide, lysosome, Gaucher disease, lysosomotropic agent.

Proteomics analysis of the brain from a Gaucher disease mouse identifies pathological pathways including a possible role for transglutaminase 1.

Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid ß-glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra-peritoneal injection of conduritol B-epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non-targeted, mass spectrometry-based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE-injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up-regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE-injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology.

Autophagic dysfunction and gut microbiota dysbiosis cause chronic immune activation in a Drosophila model of Gaucher disease.

Mutations in the GBA1 gene cause the lysosomal storage disorder Gaucher disease (GD) and are the greatest known genetic risk factors for Parkinson's disease (PD). Communication between the gut and brain and immune dysregulation are increasingly being implicated in neurodegenerative disorders such as PD. Here, we show that flies lacking the Gba1b gene, the main fly orthologue of GBA1, display widespread NF-kB signalling activation, including gut inflammation, and brain glial activation. We also demonstrate intestinal autophagic defects, gut dysfunction, and microbiome dysbiosis. Remarkably, modulating the microbiome of Gba1b knockout flies, by raising them under germ-free conditions, partially ameliorates lifespan, locomotor and immune phenotypes. Moreover, we show that modulation of the immune deficiency (IMD) pathway is detrimental to the survival of Gba1 deficient flies. We also reveal that direct stimulation of autophagy by rapamycin treatment achieves similar benefits to germ-free conditions independent of gut bacterial load. Consistent with this, we show that pharmacologically blocking autophagosomal-lysosomal fusion, mimicking the autophagy defects of Gba1 depleted cells, is sufficient to stimulate intestinal immune activation. Overall, our data elucidate a mechanism whereby an altered microbiome, coupled with defects in autophagy, drive chronic activation of NF-kB signaling in a Gba1 loss-of-function model. It also highlights that elimination of the microbiota or stimulation of autophagy to remove immune mediators, rather than prolonged immunosuppression, may represent effective therapeutic avenues for GBA1-associated disorders.

Animal Models for the Study of Gaucher Disease.

In Gaucher disease (GD), a relatively common sphingolipidosis, the mutant lysosomal enzyme acid ß-glucocerebrosidase (GCase), encoded by the GBA1 gene, fails to properly hydrolyze the sphingolipid glucosylceramide (GlcCer) in lysosomes, particularly of tissue macrophages. As a result, GlcCer accumulates, which, to a certain extent, is converted to its deacylated form, glucosylsphingosine (GlcSph), by lysosomal acid ceramidase. The inability of mutant GCase to degrade GlcSph further promotes its accumulation. The amount of mutant GCase in lysosomes depends on the amount of mutant ER enzyme that shuttles to them. In the case of many mutant GCase forms, the enzyme is largely misfolded in the ER. Only a fraction correctly folds and is subsequently trafficked to the lysosomes, while the rest of the misfolded mutant GCase protein undergoes ER-associated degradation (ERAD). The retention of misfolded mutant GCase in the ER induces ER stress, which evokes a stress response known as the unfolded protein response (UPR). GD is remarkably heterogeneous in clinical manifestation, including the variant without CNS involvement (type 1), and acute and subacute neuronopathic variants (types 2 and 3). The present review discusses animal models developed to study the molecular and cellular mechanisms underlying GD.

Establishment of MUi030-A: A human induced pluripotent stem cell line carrying homozygous L444P mutation in the GBA1 gene to study type-3 Gaucher disease.

Gaucher disease (GD) is a common lysosomal storage disease resulting from mutations in the glucocerebrosidase (GBA1) gene. This genetic disorder manifests with symptoms affecting multiple organs, yet the underlying mechanisms leading to pathology remain elusive. In this study, we successfully generated the MUi030-A human induced pluripotent stem cell (hiPSC) line using a non-integration method from a male type-3 GD patient with a homozygous c.1448T>C (L444P) mutation. These hiPSCs displayed a normal karyotype and pluripotency markers and the remarkable ability to differentiate into cells representing all three germ layers. This resourceful model holds significant promise for illuminating GD's underlying pathogenesis.

Pseudophosphatase STYXL1 depletion enhances glucocerebrosidase trafficking to lysosomes via ER stress.

Pseudophosphatases are catalytically inactive but share sequence and structural similarities with classical phosphatases. STYXL1 is a pseudophosphatase that belongs to the family of dual-specificity phosphatases and is known to regulate stress granule formation, neurite formation and apoptosis in different cell types. However, the role of STYXL1 in regulating cellular trafficking or the lysosome function has not been elucidated. Here, we show that the knockdown of STYXL1 enhances the trafficking of ß-glucocerebrosidase (ß-GC) and its lysosomal activity in HeLa cells. Importantly, the STYXL1-depleted cells display enhanced distribution of endoplasmic reticulum (ER), late endosome and lysosome compartments. Further, knockdown of STYXL1 causes the nuclear translocation of unfolded protein response (UPR) and lysosomal biogenesis transcription factors. However, the upregulated ß-GC activity in the lysosomes is independent of TFEB/TFE3 nuclear localization in STYXL1 knockdown cells. The treatment of STYXL1 knockdown cells with 4-PBA (ER stress attenuator) significantly reduces the ß-GC activity equivalent to control cells but not additive with thapsigargin, an ER stress activator. Additionally, STYXL1-depleted cells show the enhanced contact of lysosomes with ER, possibly via increased UPR. The depletion of STYXL1 in human primary fibroblasts derived from Gaucher patients showed moderately enhanced lysosomal enzyme activity. Overall, these studies illustrated the unique role of pseudophosphatase STYXL1 in modulating the lysosome function both in normal and lysosome-storage disorder cell types. Thus, designing small molecules against STYXL1 possibly can restore the lysosome activity by enhancing ER stress in Gaucher disease.

Neuronopathic Gaucher disease models reveal defects in cell growth promoted by Hippo pathway activation.

Gaucher Disease (GD), the most common lysosomal disorder, arises from mutations in the GBA1 gene and is characterized by a wide spectrum of phenotypes, ranging from mild hematological and visceral involvement to severe neurological disease. Neuronopathic patients display dramatic neuronal loss and increased neuroinflammation, whose molecular basis are still unclear. Using a combination of Drosophila dGBA1b loss-of-function models and GD patient-derived iPSCs differentiated towards neuronal precursors and mature neurons we showed that different GD- tissues and neuronal cells display an impairment of growth mechanisms with an increased cell death and reduced proliferation. These phenotypes are coupled with the downregulation of several Hippo transcriptional targets, mainly involved in cells and tissue growth, and YAP exclusion from nuclei. Interestingly, Hippo knock-down in the GBA-KO flies rescues the proliferative defect, suggesting that targeting the Hippo pathway can be a promising therapeutic approach to neuronopathic GD.

Interaction with ERp57 is required for progranulin protection against Type 2 Gaucher disease.

Gaucher disease (GD), one of the most common lysosomal storage diseases, is caused by GBA1 mutations resulting in defective glucocerebrosidase (GCase) and consequent accumulation of its substrates ß-glucosylceramide (ß-GlcCer). We reported progranulin (PGRN), a secretary growth factor-like molecule and an intracellular lysosomal protein was a crucial co-factor of GCase. PGRN binds to GCase and recruits Heat Shock Protein 70 (Hsp70) to GCase through its C-terminal Granulin (Grn) E domain, termed as ND7. In addition, both PGRN and ND7 are therapeutic against GD. Herein we found that both PGRN and its derived ND7 still displayed significant protective effects against GD in Hsp70 deficient cells. To delineate the molecular mechanisms underlying PGRN's Hsp70-independent regulation of GD, we performed a biochemical co-purification and mass spectrometry with His-tagged PGRN and His-tagged ND7 in Hsp70 deficient cells, which led to the identification of ERp57, also referred to as protein disulfide isomerase A3 (PDIA3), as a protein that binds to both PGRN and ND7. Within type 2 neuropathic GD patient fibroblasts L444P, bearing GBA1 L444P mutation, deletion of ERp57 largely abolished the therapeutic effects of PGRN and ND7, as manifested by loss of effects on lysosomal storage, GCase activity, and ß-GlcCer accumulation. Additionally, recombinant ERp57 effectively restored the therapeutic effects of PGRN and ND7 in ERp57 knockout L444P fibroblasts. Collectively, this study reports ERp57 as a previously unrecognized binding partner of PGRN that contributes to PGRN regulation of GD.

A 20-Year Longitudinal Study of Plasma Chitotriosidase Activity in Treated Gaucher Disease Type 1 and 3 Patients-A Qualitative and Quantitative Approach.

Chitotriosidase is an enzyme produced and secreted in large amounts by activated macrophages, especially macrophages loaded with phagocytozed glycosphingolipid in Gaucher disease. Macrophages phagocytose decayed blood cells that contain a lot of sphingolipid-rich cell membranes. In Gaucher disease, due to a deficit in beta-glucocerebrosidase activity, the phagocytozed substrate glucocerebroside cannot undergo further catabolism. In such a situation, macrophages secrete chitotriosidase in proportion to the degree of overload. Gaucher disease (GD) is a recessively inherited disorder resulting in storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. It is directly caused by the deficiency of beta-glucocerebrosidase (GBA) activity. Chitotriosidase has been measured systematically each year in the same group of 49 patients with type 1 and 3 GD for over 20 years. Our analysis showed that chitotriosidase is very sensitive biomarker to enzyme replacement therapy (ERT). The response to treatment introduction is of an almost immediate nature, lowering pathologically high chitotriosidase levels by a factor of 2 in a time scale of 8 months, on average. Long term enzyme replacement therapy (ERT) brings chitotriosidase activity close to reference values. Finally, reducing the dose of ERT quickly boosts chitotriosidase activity, but restoring the initial dose of treatment brings chitotriosidase level of activity back onto the decreasing time trajectory.

Acid ceramidase involved in pathogenic cascade leading to accumulation of α-synuclein in iPSC model of GBA1-associated Parkinson's disease.

Bi-allelic mutations in GBA1, the gene that encodes ß-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson's disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD-DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD-DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

Characterization of peripheral blood T follicular helper (TFH) cells in patients with type 1 Gaucher disease and carriers.

BACKGROUND: Gaucher disease (GD) is the most common autosomal recessive lipid storage disease. In this study, the changes in TFH cells and IL-4 and IL-21 cytokines in blood samples of GD patients, carriers and healthy volunteers were investigated. METHODS: Two pretreatment type 1 GD patients, 20 currently treated type 1 GD patients, 6 carriers, and 27 healthy volunteers were enrolled in the study. TFH cell (CD45RA-CD4+CXCR5+) number, phenotype (PD1, ICOS expression), and cytokine production (IL-21, IL-4) were assessed via flow cytometric assays. RESULTS: No significant differences were found between the groups with respect to the number, frequency and PD1 or ICOS expression of TFH cells between healthy controls, patients and carriers. However, IL-4+ TFH cells were significantly reduced both in percent and number in the treated GD patients compared with healthy controls (p < 0.05). Interestingly, the IL-21+ TFH cell number was increased in treated GD patients. When TFH cells were examined based on CXCR3 expression, the frequency of the PD1+Th17-Th2-like fraction (CXCR3-) was found to be significantly increased in treated GD patients. CONCLUSION: To our knowledge, this is the first study to assess TFH cells in GD patients, and to show that the production of IL-4 and IL-21 by TFH cells and their subsets may be altered in type 1 GD patients.

a-Synuclein and lipids in erythrocytes of Gaucher disease carriers and patients before and after enzyme replacement therapy.

It is well established that patients with Gaucher disease, as well as carriers of the disease have an increased risk for developing Parkinson's disease. A plethora of evidence suggests that disturbed α-Synuclein homeostasis is the link between Gaucher disease and Parkinson's disease. The pathogenic mechanism linking these entities is still a topic of debate and both gain- and loss-of-function theories have been put forward, which however are not mutually exclusive. In the present study we expanded our previous studies to include not only Gaucher disease patients but also Gaucher disease carriers and Gaucher disease patients following Enzyme Replacement Therapy. In these groups we investigated α-Synuclein in red blood cell membranes in association with lipid abnormalities described in Gaucher disease. These included glucosylceramide and its species, glucosylsphingosine, glucosylcholesterol and plasmalogens. Increased oligomerization of α-Synuclein in red blood cell membranes was observed not only in Gaucher disease patients but also in carriers of the disease. There were no qualitative differences in the lipids identified in the groups studied. However, significant quantitative differences compared to controls were observed in Gaucher disease patients but not in Gaucher disease carriers. Enzyme Replacement Therapy reversed the biochemical defects and normalized α-Synuclein homeostasis, providing for the first time evidence in human subjects that such homeostatic dysregulation is reversible. Further studies investigating α-Synuclein status during the differentiation of erythroid progenitors could provide new data on the pathogenic mechanism of α-Synuclein oligomerization in this system.

Inhibitors of Glucosylceramide Synthase.

Glucosylceramide synthase can be targeted by high affinity small molecular weight inhibitors for the study of glycosphingolipid metabolism and function or for the treatment of glycosphingolipid storage disorders, including Gaucher and Fabry disease. This work is exemplified by the discovery and development of eliglustat tartrate, the first stand-alone small chemical entity approved for the treatment of Gaucher disease type 1. The development of inhibitors of glucosylceramide synthase that have utility for either research or clinical purposes begins with a testing funnel for screening candidate inhibitors for activity against this enzyme and for activity in lowering the content of glucosylceramide in intact cells. Two common assays for glucosylceramide synthase, one enzyme based and another cell based, are the focus of this chapter.

Brain-Derived Neurotrophic Factor (BDNF) Is Associated with Platelet Activity and Bleeding Tendency in Patients with Gaucher Disease.

Bleeding tendency, a prominent feature of patients with Gaucher disease (GD), is associated with abnormal platelet function. Brain-derived neurotrophic factor (BDNF) is a protein with neuroprotective potential stored in alpha granules of circulating platelets. Here we studied BDNF levels in 50 patients with type I GD (GD1) and their correlation with platelet activity and bleeding tendency. Flow cytometry was used to test unstimulated and stimulated measurement of platelet surface-activated expression of αIIbß3 integrin, P-selectin and lysosomal-associated membrane protein (LAMP3/CD63). Serum and plasma BDNF levels were quantified using ELISA. The bleeding history was recorded by a bleeding questionnaire. Serum BDNF levels were positively correlated with platelet count and moderately correlated with unstimulated and stimulated platelet P-selectin expression. Patients with more than one bleeding manifestation were shown to have lower serum BDNF levels, albeit similar platelet count. Plasma BDNF levels were significantly elevated in splenectomized patients and showed a moderate positive correlation with stimulated platelet CD63 expression. These observations demonstrate the first association between BDNF levels in the peripheral blood with platelet dysfunction and increased bleeding manifestation. The role of measuring serum BDNF for assessing platelet alpha degranulation defects and bleeding risk in patients with GD and the general population needs further study.

Photoredox-based late-stage functionalization in SAR study for in vivo potent glucosylceramide synthase inhibitor.

Glucosylceramide synthase (GCS) has drawn much attention as an attractive protein target in the disease pathways of Parkinson's Disease (PD) and lysosomal storage disorders, such as Gaucher's Disease (GD). In previous our study, T-036 and its analogue, 2a, were discovered as novel GCS inhibitors. To further improve activity of this chemical series, SAR was investigated on the fused pyridyl ring core of 2a by employing a photoredox reaction that significantly reduced synthetic demand. Herein, we successfully applied the decarboxylation C-H alkylation photoredox reaction to introduce a wide variety of substituents at the 6-position of the fused pyridine core scaffold. This quick SAR acquisition facilitated the swift identification of the potent GCS inhibitors 2b (IC50 = 5.9 nM) and 2g (IC50 = 3.6 nM). Moreover, 2b exhibited superior in vivo potency to that of our previously reported lead compound, T-036.

Galactosyl- and glucosylsphingosine induce lysosomal membrane permeabilization and cell death in cancer cells.

Isomeric lysosphingolipids, galactosylsphingosine (GalSph) and glucosylsphingosine (GlcSph), are present in only minute levels in healthy cells. Due to defects in their lysosomal hydrolysis, they accumulate at high levels and cause cytotoxicity in patients with Krabbe and Gaucher diseases, respectively. Here, we show that GalSph and GlcSph induce lysosomal membrane permeabilization, a hallmark of lysosome-dependent cell death, in human breast cancer cells (MCF7) and primary fibroblasts. Supporting lysosomal leakage as a causative event in lysosphingolipid-induced cytotoxicity, treatment of MCF7 cells with lysosome-stabilizing cholesterol prevented GalSph- and GlcSph-induced cell death almost completely. In line with this, fibroblasts from a patient with Niemann-Pick type C disease, which is caused by defective lysosomal cholesterol efflux, were significantly less sensitive to lysosphingolipid-induced lysosomal leakage and cell death. Prompted by the data showing that MCF7 cells with acquired resistance to lysosome-destabilizing cationic amphiphilic drugs (CADs) were partially resistant to the cell death induced by GalSph and GlcSph, we compared these cell death pathways with each other. Like CADs, GalSph and GlcSph activated the cyclic AMP (cAMP) signalling pathway, and cAMP-inducing forskolin sensitized cells to cell death induced by low concentrations of lysosphingolipids. Contrary to CADs, lysosphingolipid-induced cell death was independent of lysosomal Ca2+ efflux through P2X purinerigic receptor 4. These data reveal GalSph and GlcSph as lysosome-destabilizing lipids, whose putative use in cancer therapy should be further investigated. Furthermore, the data supports the development of lysosome stabilizing drugs for the treatment of Krabbe and Gaucher diseases and possibly other sphingolipidoses.

Secondary Mitochondrial Dysfunction as a Cause of Neurodegenerative Dysfunction in Lysosomal Storage Diseases and an Overview of Potential Therapies.

Mitochondrial dysfunction has been recognised a major contributory factor to the pathophysiology of a number of lysosomal storage disorders (LSDs). The cause of mitochondrial dysfunction in LSDs is as yet uncertain, but appears to be triggered by a number of different factors, although oxidative stress and impaired mitophagy appear to be common inhibitory mechanisms shared amongst this group of disorders, including Gaucher's disease, Niemann-Pick disease, type C, and mucopolysaccharidosis. Many LSDs resulting from defects in lysosomal hydrolase activity show neurodegeneration, which remains challenging to treat. Currently available curative therapies are not sufficient to meet patients' needs. In view of the documented evidence of mitochondrial dysfunction in the neurodegeneration of LSDs, along with the reciprocal interaction between the mitochondrion and the lysosome, novel therapeutic strategies that target the impairment in both of these organelles could be considered in the clinical management of the long-term neurodegenerative complications of these diseases. The purpose of this review is to outline the putative mechanisms that may be responsible for the reported mitochondrial dysfunction in LSDs and to discuss the new potential therapeutic developments.