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Gaucher's disease is the most prevalent lysosomal storage disorder in humans and results from an autosomally inherited deficiency of the enzyme glucocerebrosidase (beta-D-glucosyl-N-acylsphingosine glucohydrolase), which is responsible for degrading the sphingolipid glucocerebroside. An animal model for Gaucher's disease would be important for investigating its phenotypic diversity and pathogenesis and for evaluating therapeutic approaches. A naturally occurring canine model has been reported but not propagated. Attempts to mimic the disease in animals by inhibiting glucocerebrosidase have been inadequate. Here we generate an animal model for Gaucher's disease by creating a null allele in embryonic stem cells through gene targeting and using these genetically modified cells to establish a mouse strain carrying the mutation. Mice homozygous for this mutation have less than 4% of normal glucocerebrosidase activity, die within twenty-four hours of birth and store glucocerebroside in lysosomes of cells of the reticuloendothelial system.

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 ␣-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 ␣-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. Key words: ␣-synuclein; dopaminergic neurons; glucocerebrosidase; induced pluripotent stem cells; parkinsonism; pharmacological chaperone Significance StatementBecause GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of ␣-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced ␣-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Communicated by Mark Paalman Gaucher disease, the most common lysosomal storage disorder, results from the inherited deficiency of the enzyme glucocerebrosidase. Three clinical types are recognized: type 1, nonneuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. Type 2 Gaucher disease, the rarest type, is progressive and fatal. We have performed molecular analyses of a cohort of 31 patients with type 2 Gaucher disease. The cases studied included fetuses presenting prenatally with hydrops fetalis, infants with the collodion baby phenotype, and infants diagnosed after several months of life. All 62 mutant glucocerebrosidase (GBA) alleles were identified. Thirtythree different mutant alleles were found, including point mutations, splice junction mutations, deletions, fusion alleles and recombinant alleles. Eleven novel mutations were identified in these patients: R131L, H255Q, R285H, S196P, H311R, c.330delA, V398F, F259L, c.533delC, Y304C and A190E. Mutation L444P was found on 25 patient alleles. Southern blots and direct sequencing demonstrated that mutation L444P occurred alone on 9 alleles, with E326K on one allele and as part of a recombinant allele on 15 alleles. There were no homozygotes for point mutation L444P. The recombinant alleles that included L444P resulted from either reciprocal recombination or gene conversion with the nearby glucocerebrosidase pseudogene, and seven different sites of recombination were identified. Homozygosity for a recombinant allele was associated with early lethality. We have also summarized the literature describing mutations associated with type 2 disease, and list 50 different mutations. This report constitutes the most comprehensive molecular study to date of type 2 Gaucher disease, and it demonstrates that there is significant phenotypic and genotypic heterogeneity among patients with type 2 Gaucher disease. Hum Mutat 15:181-188, 2000.