Direct targeting of wild-type glucocerebrosidase by antipsychotic quetiapine improves pathogenic phenotypes in Parkinson's disease models.

Current treatments for Parkinson's disease (PD) provide only symptomatic relief, with no disease-modifying therapies identified to date. Repurposing FDA-approved drugs to treat PD could significantly shorten the time needed for and reduce the costs of drug development compared with conventional approaches. We developed an efficient strategy to screen for modulators of ß-glucocerebrosidase (GCase), a lysosomal enzyme that exhibits decreased activity in patients with PD, leading to accumulation of the substrate glucosylceramide and oxidized dopamine and α-synuclein, which contribute to PD pathogenesis. Using a GCase fluorescent probe and affinity-based fluorescence polarization assay, we screened 1280 structurally diverse, bioactive, and cell-permeable FDA-approved drugs and found that the antipsychotic quetiapine bound GCase with high affinity. Moreover, quetiapine treatment of induced pluripotent stem cell-derived (iPSC-derived) dopaminergic neurons from patients carrying mutations in GBA1 or LRRK2 led to increased wild-type GCase protein levels and activity and partially lowered accumulation of oxidized dopamine, glucosylceramide, and α-synuclein. Similarly, quetiapine led to activation of wild-type GCase and reduction of α-synuclein in a GBA mutant mouse model (Gba1D409V/+ mice). Together, these results suggest that repurposing quetiapine as a modulator of GCase may be beneficial for patients with PD exhibiting decreased GCase activity.

Ambroxol increases glucocerebrosidase (GCase) activity and restores GCase translocation in primary patient-derived macrophages in Gaucher disease and Parkinsonism.

Mutations in the glucocerebrosidase gene (GBA) encoding the lysosomal enzyme glucocerebrosidase (GCase) cause Gaucher disease (GD) and are the most commonly known genetic risk factor for Parkinson disease (PD). Ambroxol is one of the most effective pharmacological chaperones of GCase. Fourteen GD patients, six PD patients with mutations in the GBA gene (GBA-PD), and thirty controls were enrolled. GCase activity and hexosylsphingosine (HexSph) concentration were measured in dried blood and macrophage spots using liquid chromatography coupled with tandem mass spectrometry. The effect of ambroxol on GCase translocation to lysosomes was assessed using confocal microscopy. The results showed that ambroxol treatment significantly increased GCase activity in cultured macrophages derived from patient blood monocytic cell (PBMC) of GD (by 3.3-fold) and GBA-PD patients (by 3.5-fold) compared to untreated cells (p < 0.0001 and p < 0.0001, respectively) four days after cultivation. Ambroxol treatment significantly reduced HexSph concentration in GD (by 2.1-fold) and GBA-PD patients (by 1.6-fold) (p < 0.0001 and p < 0.0001, respectively). GD macrophage treatment resulted in increased GCase level and increased enzyme colocalization with the lysosomal marker LAMP2. The possible binding modes of ambroxol to mutant GCase carrying N370S amino acid substitution at pH 4.7 were examined using molecular docking and molecular dynamics simulations. The ambroxol position characterized by minimal binding free energy was observed in close vicinity to the residue, at position 370. Taken together, these data showed that PBMC-derived macrophages could be used for assessing ambroxol therapy response for GD patients and also for GBA-PD patients.

Chemical inhibition of ß-glucocerebrosidase does not affect phagocytosis and early containment of Leishmania by murine macrophages.

Gaucher disease is a lysosomal storage disease in which a genetic deficiency in ß-glucocerebrosidase leads to the accumulation of glycosphingolipids in lysosomes. Macrophages are amongst the cells most severely affected in Gaucher disease patients. One phenotype associated with Gaucher macrophages is the impaired capacity to fight bacterial infections. Here, we investigate whether inhibition of ß-glucocerebrosidase activity affects the capacity of macrophages to phagocytose and act on the early containment of human pathogens of the genus Leishmania. Towards our aim, we performed in vitro infection assays on macrophages derived from the bone marrow of C57BL/6 mice. To mimic Gaucher disease, macrophages were incubated with the ß-glucocerebrosidase inhibitor, conduritol B epoxide (CBE), prior to contact with Leishmania. This treatment guaranteed that ß-glucocerebrosidase was fully inhibited during the contact of macrophages with Leishmania, its enzymatic activity being progressively recovered along the 48 h that followed removal of the inhibitor. Infections were performed with L. amazonensis, L. infantum, or L. major, so as to explore potential species-specific responses in the context of ß-glucocerebrosidase inactivation. Parameters of infection, recorded immediately after phagocytosis, as well as 24 and 48 h later, revealed no noticeable differences in the infection parameters of CBE-treated macrophages relative to non-treated controls. We conclude that blocking ß-glucocerebrosidase activity during contact with Leishmania does not interfere with the phagocytic capacity of macrophages and the early onset of leishmanicidal responses.

New Era in disease modification in Parkinson's disease: Review of genetically targeted therapeutics.

Disease modification remains a major unmet need in Parkinson's disease (PD) therapeutics. Despite multiple attempts, not a single study has yet been successful, perhaps due to our incomplete understanding of the underlying disease mechanisms. Genetic and epidemiologic studies of the last decade have substantially increased our comprehension of the etiology of PD. Once considered a pure sporadic disease, the discovery of familial mutations provided the initial paradigm shift and it is now widely accepted that PD has a substantial genetic component. These genetic discoveries have allowed the development of novel therapeutics aimed at halting or slowing the underlying disease process, rather than just ameliorating symptoms. Here, we discuss the latest advances in therapeutics based on three genetic discoveries (SNCA, LRRK2 and GBA) that are currently reaching the clinical arena and outline the challenges of therapeutic development of genetically targeted therapeutics.

Ambroxol effects in glucocerebrosidase and α-synuclein transgenic mice.

OBJECTIVE: Gaucher disease is caused by mutations in the glucocerebrosidase 1 gene that result in deficiency of the lysosomal enzyme glucocerebrosidase. Both homozygous and heterozygous glucocerebrosidase 1 mutations confer an increased risk for developing Parkinson disease. Current estimates indicate that 10 to 25% of Parkinson patients carry glucocerebrosidase 1 mutations. Ambroxol is a small molecule chaperone that has been shown to increase glucocerebrosidase activity in vitro. This study investigated the effect of ambroxol treatment on glucocerebrosidase activity and on α-synuclein and phosphorylated α-synuclein protein levels in mice. METHODS: Mice were treated with ambroxol for 12 days. After the treatment, glucocerebrosidase activity was measured in the mouse brain lysates. The brain lysates were also analyzed for α-synuclein and phosphorylated α-synuclein protein levels. RESULTS: Ambroxol treatment resulted in increased brain glucocerebrosidase activity in (1) wild-type mice, (2) transgenic mice expressing the heterozygous L444P mutation in the murine glucocerebrosidase 1 gene, and (3) transgenic mice overexpressing human α-synuclein. Furthermore, in the mice overexpressing human α-synuclein, ambroxol treatment decreased both α-synuclein and phosphorylated α-synuclein protein levels. INTERPRETATION: Our work supports the proposition that ambroxol should be further investigated as a potential novel disease-modifying therapy for treatment of Parkinson disease and neuronopathic Gaucher disease to increase glucocerebrosidase activity and decrease α-synuclein and phosphorylated α-synuclein protein levels. Ann Neurol 2016;80:766-775.

Cellular Uptake of Glucocerebrosidase in Gaucher Patients Receiving Enzyme Replacement Treatment.

BACKGROUND: Enzyme replacement therapy (ERT) is currently the standard treatment for patients with Gaucher disease type I (GD1), but the pharmacokinetics have hardly been studied. This study aimed to quantify in vivo enzyme activity in peripheral leukocytes from patients receiving long-term treatment with imiglucerase or velaglucerase for GD1, and set out to assess the process of enzymatic uptake by peripheral leukocytes. METHODS: A prospective semi-experimental study was conducted. Four time points for blood withdrawal were planned per patient to quantify the intra-leukocyte enzymatic activity. In order to assess the uptake process, the rate of enzyme uptake by leukocytes (Rupt) and the rate of enzyme disappearance from the plasma (Rdis) were estimated. RESULTS: Eight GD1 patients were included. Intra-leukocyte activity was 24.31 mU/mL [standard deviation (SD) 6.32 mU/mL; coefficient of variation (CV) 25.96 %] at baseline and 27.14 mU/mL (SD  6.96 mU/mL; CV 25.65 %) at 15 min post-perfusion. The relationships with the administered dose were linear. The Rupt value was 37.73 mU/mL/min [95 % confidence interval (CI) 25.63-49.84] and showed a linear correlation with the administered enzyme dose (p < 0.05), and the Rdis value was 189.43 mU/mL/min (95 % CI 80.31-298.55) and also showed a linear correlation with the dose (p < 0.05). CONCLUSION: This was the first in vivo study to quantify the accumulated enzymatic activity in patients receiving ERT for GD1. It showed that intra-leukocyte activity at baseline and at 15 min post-perfusion could be used as a possible marker for therapeutic individualization in patients receiving ERT for GD1.

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease.

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.

Pilot study using ambroxol as a pharmacological chaperone in type 1 Gaucher disease.

The purpose of this pilot was to assess the tolerability and efficacy of ambroxol as a pharmacological chaperone in patients with symptomatic, type 1 Gaucher disease who present with measurable disease parameters but are not receiving enzyme replacement therapy (ERT) in order to provide proof of concept and/or ascertain the suitability of ambroxol for a larger clinical trial. The Israeli Ministry of Health Form 29c was employed to prescribe ambroxol for off-label use. Twelve patients were dispensed 2 capsules of 75 mg of ambroxol daily for 6 months. There were 8 females (66.7%). Mean age at entry was 41.1 (range: 24-63) years. Mean body weight at entry was 66.4 (range: 46.5-100) kg. One patient withdrew because of a hypersensitivity reaction, one because of elective splenectomy. No patient experienced clinically relevant deterioration in disease parameters measured. One patient achieved a robust response relative to baseline: +16.2% hemoglobin; +32.9% platelets; -2.8% liver volume; and -14.4% spleen volume. Three patients, including the above one, elected to continue on ambroxol for a further 6 months: hemoglobin levels and liver volumes were relatively stable, but platelet counts further increased in the above patient (+52.6% from baseline) and spleen volumes decreased further in all three patients (-6.4%, -18.6%, and -23.4% from baseline). Thus, ambroxol may be a safe option for Gaucher disease patients with potential disease-specific efficacy and should be expanded into a clinical trial using higher doses and placebo-controlled design.

Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase ß-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients.

Selective action of the iminosugar isofagomine, a pharmacological chaperone for mutant forms of acid-beta-glucosidase.

Gaucher disease is a lysosomal glycolipid storage disorder characterized by defects in acid-beta-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. We recently demonstrated that isofagomine (IFG), an iminosugar that binds to the active site of GlcCerase, enhances the folding, transport and activity of the N370S mutant form of GlcCerase. In this study we compared the effects of IFG on a number of other glucosidases and glucosyltransferases. We report that IFG has little or no inhibitory activity towards intestinal disaccharidase enzymes, ER alpha-glucosidase II or glucosylceramide synthase at concentrations previously shown to enhance N370S GlcCerase folding and trafficking in Gaucher fibroblasts. Furthermore, treatment of wild type fibroblasts with high doses of IFG did not alter the processing of newly synthesized N-linked oligosaccharides. These findings support further evaluation of IFG as a potential therapeutic agent in the treatment of some forms of Gaucher disease.

The iminosugar isofagomine increases the activity of N370S mutant acid beta-glucosidase in Gaucher fibroblasts by several mechanisms.

Gaucher disease is a lysosomal storage disorder caused by deficiency in lysosomal acid beta-glucosidase (GlcCerase), the enzyme responsible for the catabolism of glucosylceramide. One of the most prevalent disease-causing mutations, N370S, results in an enzyme with lower catalytic activity and impaired exit from the endoplasmic reticulum. Here, we report that the iminosugar isofagomine (IFG), an active-site inhibitor, increases GlcCerase activity 3.0 +/- 0.6-fold in N370S fibroblasts by several mechanisms. A major effect of IFG is to facilitate the folding and transport of newly synthesized GlcCerase in the endoplasmic reticulum, thereby increasing the lysosomal pool of the enzyme. In addition, N370S GlcCerase synthesized in the presence of IFG exhibits a shift in pH optimum from 6.4 to 5.2 and altered sensitivity to SDS. Although IFG fully inhibits GlcCerase in the lysosome in an in situ assay, washout of the drug leads to partial recovery of GlcCerase activity within 4 h and full recovery by 24 h. These findings provide support for the possible use of active-site inhibitors in the treatment of some forms of Gaucher disease.

Acid beta-glucosidase: intrinsic fluorescence and conformational changes induced by phospholipids and saposin C.

Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.

Galactocerebroside and not glucocerebroside or ceramide stimulate epidermal beta-glucocerebrosidase activity.

Glycosphingolipids including glucocerebroside (GluCer) and galactocerebroside (GalCer) have been recognized as bioreguratory lipids by our group and others. In addition, our recent study demonstrated that GalCer corrects dry skin conditions in humans. The processing of stratum corneum lipids, which occurs when beta-glucocerebrosidase (beta-GluCer'ase) changes GluCer to ceramide (Cer), is required to form the epidermal permeability barrier. We herein investigated the effects of GluCer, GalCer and Cer on the processing of GluCer to Cer by assaying epidermal beta-GluCer'ase in mice (155%, P < 0.01) when compared to vehicle treated controls, while neither GluCer nor Cer had this effect. Studies using inhibitors of beta-GluCer'ase or beta-galactosidase and measuring the optimum pH of the enzyme verified that GalCer specifically activated beta-GluCer'ase. We confirmed that GalCer significantly increased beta-GluCer'ase activity in the outer epidermal fraction (172%, P < 0.01) and that the activation of beta-GluCer'ase is not due to a direct activating effect of GalCer on the enzyme. Furthermore, the induction of beta-GluCer'ase activity by GalCer was also observed in cultured normal human deratinocytes (123%, P < 0.01). Finally, acylceramide content in stratum corneum was increased in mice treated with GalCer (194%, P < 0.0005). These results indicate that GalCer appears to affect the Cer construct in the stratum corneum by the activation of beta-GluCer'ase, which ultimately contribute to an enhancement of barrier formation.

Complete restoration of glucocerebrosidase deficiency in Gaucher fibroblasts using a bicistronic MDR retrovirus and a new selection strategy.

Retrovirus-mediated gene transfer is currently the most common method for the application of genetic therapy to cancer and many inherited and acquired disorders. Here we report the generation of an amphotropic producer cell line (CA2) that synthesizes viral particles carrying a bicistronic cassette in which the selectable MDR1 cDNA encoding P-glycoprotein (P-gp) a multidrug efflux pump, and the human glucocerebrosidase (GC) gene are transcriptionally fused. Transduction of human Gaucher fibroblasts with this recombinant virus allowed coordinate expression of P-gp and GC. Treatment of the transduced fibroblasts with various cytotoxic substrates of P-gp selected for cells with increased expression of GC, which paralleled the stringency of drug selection. Thus, selection of the genetically modified Gaucher fibroblasts in 1 microgram/ml colchicine raised their GC activity levels from nearly undetectable to those present in WI-38 normal human fibroblasts, correcting the enzyme deficiency present in Gaucher cells. Moreover, by simultaneously inhibiting the P-gp pump, it was possible to use much lower concentrations of colchicine to select for high-level expression of MDR1 and GC. Thus, selection with colchicine at 5 ng/ml in combination with the P-gp inhibitors verapamil or PSC 833 produced a complete correction of the GC deficiency in the CA2-transduced fibroblasts. These combination regimens, already in clinical use for the treatment of multidrug-resistant malignancies, may prove useful in gene therapy trials when utilized for high level selection of a nonselectable gene such as glucocerebrosidase when transcriptionally fused to the MDR1 gene.

N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing.

We have previously reported that the imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits glycolipid biosynthesis, in addition to its known activity as an inhibitor of the N-linked oligosaccharide processing enzyme alpha-glucosidase I. In an attempt to dissociate these two activities and identify an inhibitor which was more selective for the glycolipid biosynthetic pathway, several imino sugars have been N-alkylated and tested for inhibitory activity. The galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) was found to be a potent inhibitor of glycolipid biosynthesis but in contrast to NB-DNJ had no effect on the maturation of N-linked oligosaccharides or on lysosomal glucocerebrosidase. The effect of increasing N-alkyl chain length on glycolipid inhibition was investigated. Nonalkylated DGJ, the N-methyl and N-ethyl derivatives, were noninhibitory. However, N-propylation resulted in partial inhibition while the N-butyl and N-hexyl derivatives resulted in maximal inhibition. Increasing alkyl chain length also resulted in increased potency of glucosyltransferase inhibition. In an in vitro Gaucher's disease model NB-DGJ was as effective as NB-DNJ in preventing glycolipid storage and may represent a more selective potential therapeutic agent than NB-DNJ for the management of this and other glycosphingolipidoses.

beta-Glucocerebrosidase activity in murine epidermis: characterization and localization in relation to differentiation.

The intercellular lipids of the stratum corneum, which are highly enriched in ceramides, are critical for the mammalian epidermal permeability barrier. During the terminal stages of epidermal differentiation, the glucosylceramide content is dramatically reduced, while the content of free ceramides increases. To investigate whether beta-glucocerebrosidase (beta-GlcCer'ase) could be responsible for this change in lipid content, we characterized its activity in murine epidermis, compared enzyme activity to other murine tissues, and localized beta-GlcCer'ase activity within the epidermis. Epidermal extracts demonstrated linear 4-methylumbelliferyl-beta-D-glucose hydrolysis (to 3 h) with protein concentrations between 1 and 250 micrograms/ml. Whole epidermis contained comparable beta-glucosidase activity (9.1 +/- 0.4 nmol/min per mg DNA) to murine brain and liver, and 5-fold higher activity than spleen. Epidermal beta-glucosidase activity was stimulated greater than 15-fold by sodium taurocholate at pH 5.6, and inhibited at acidic pH (3.5-4.0). Bromoconduritol B epoxide (greater than or equal to 1.0 microM), inhibited epidermal enzyme activity by greater than 75%, while activity in brain, liver, and spleen was only inhibited by 6, 17, and 14%, respectively. Moreover, beta-GlcCer'ase mRNA expression in murine epidermis exceeded levels in liver, brain, and spleen. Finally, beta-GlcCer'ase activity was highest in the outer, more differentiated epidermal cell layers including the stratum corneum. In summary, mammalian epidermis contains an usually high percentage (approximately 75%) of beta-glucocerebrosidase activity, and the concentration of activity in the more differentiated cell layers may account for the replacement of glucosylceramide by ceramides in the outer epidermis.

Acid lability of the mutated glucosylceramide-beta-glucosidase in a lymphoid cell line from type 2 Gaucher disease.

Lymphoid cell lines from patients with infantile (type-2) and juvenile (type 3) Gaucher disease have been established by Epstein-Barr virus transformation and investigated and compared with the adult phenotype (type 1) with the view to enzymology. The enzymatic defect in glucosylceramide(GlcCer)-beta-glucosidase activity was more severe in type 2 and 3 than in type 1 cells. The mutant GlcCer-beta-glucosidase from our studied type 2 lymphoid cells was profoundly labile at pH 4.0 and 37 degrees C, whereas the residual GlcCer-beta-glucosidase from type 1 and type 3 were stable similar to the normal enzyme. In contrast to the distinct stability of the GlcCer-beta-glucosidases from the three phenotypes, the acid lability of the nonspecific membrane-bound beta-glucosidases from type 1, 2 and 3 were quite similar.