Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of alpha-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced alpha-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.

Gaucher disease is an autosomal recessive disorder caused by deficiency of the enzyme glucocerebrosidase. Although it is a monogenic disease, there is vast phenotypic heterogeneity, even among patients with the same genotype. MicroRNAs (miRNAs) are small non-coding RNAs involved in many biological processes and diseases. To determine whether miRNAs can affect glucocerebrosidase activity, we performed a screen of 875 different miRNA mimics. The screen was performed using Gaucher fibroblasts, and glucocerebrosidase activity was used as the initial outcome parameter. We found several miRNAs that either up- or down-regulated glucocerebrosidase activity. In follow-up assays, we confirmed that one specific miRNA (miR-127-5p) down-regulated both glucocerebrosidase activity and protein levels by down-regulation of LIMP-2, the receptor involved in proper trafficking of glucocerebrosidase from the endoplasmic reticulum to the lysosome. A conditioned media assay demonstrated that cells treated with this miRNA secreted glucocerebrosidase into the extracellular environment, supporting impaired LIMP-2 function. Two other miRNAs, miR-16-5p and miR-195-5p, were found to up-regulate glucocerebrosidase activity by greater than 40% and to enhance expression and protein levels of the enzyme. In conclusion, we show that miRNAs can alter glucocerebrosidase activity in patient cells, indicating that miRNAs can potentially act as modifiers in Gaucher disease.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.

computerized tomography (HRCT) mainly in type I GD adults. So, this study will determine the clinical spectrum of severity of lung involvement in Egyptian children mainly type 3 GD, the radiological changes,assess clinical significance of these findings and their response to ERT. The study included 34 GD children diagnosed by enzyme assay and genotype and are on ERT, imiglucerase 30-60 IU/kg/2 weeks. 31 GD type 3 children (29L444P/L444P, 2 D409H/D409H) and 3 GD type I (1 R359Q/R359Q and 2 with unknown genotype) were followed over 7 years. Their pulmonary status was clinically and radiologically assessed by chest x-ray and HRCT and its response to ERT determined. Clinical variables and radiological findings were statistically correlated. Patients’ median age is 8.6 years and mean duration of ERT is 6.5 years. Twenty (59%) type 3 L4444P/L444P GD children, 13 males and 7 females had chest symptoms with 14 having recurrent chest infections, dyspnea and wheezing, 4 requiring intensive care admission and 1 died of severe pulmonary involvement. Children showed statistically significant higher prevalence of chest symptoms at younger age (p= 0.04). The chest xray and HRCT findings included coarse interstitial pulmonary thickening of variable severity in 26/34(76.5%) and in 90% of the symptomatic group. Other changes included bronchiectasis, gound glass veiling, hyperinflation, mediastinal lymphadenopathy, military infiltration, consolidation, atelectasis and air trapping. In 12/14 asymptomatic GD children findings varied from interstitial thickening to ground glass veiling and bronchiectactic changes. 3 children were splenectomised without worsening of their chest condition. In 13 symptomatic children there was dramatic clinical response to ERT but radiological findings were stationary. All L444P/L444P children showed moderate to severe neurological involvement, 4 had epilepsy, 8 mesenteric lymphadenopathy, 3 severe bone involvement, 2 severe skeletal deformities, aortic and mitral calcifications in D409H/D409H patients. Nearly all children with recurrent infections and significant chest findings have at least one other severe system involvement. Our study showed heterogeneity in clinical presentation and response of lung involvement to ERT in type 3 GD children. 76.5% had variable degrees of interstitial lung disease and nearly all children without clinical lung involvement had abnormal radiological findings. Chest symptoms showed improvement in 65%. Lung involvement can be among the determinants of severity of type 3 GD in children.

Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.

The presynaptic protein α-synuclein (α-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between α-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that α-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the α-syn C-terminal residues, 118–137. This α-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 μm in the presence of 25 to 100 mm NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for α-syn, as does the inhibitor conduritol-β-epoxide-bound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the α-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.