A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism

Abstract

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 α-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 α-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.

Significance statement: Because 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 α-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 α-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.

Keywords: dopaminergic neurons; glucocerebrosidase; induced pluripotent stem cells; parkinsonism; pharmacological chaperone; α-synuclein.

Figure 1.

Synthesis of NCGC00241607. The synthesis of NCGC00241607 (NCGC607) was executed according to the scheme above. A synopsis of the procedure used in each transformation is given along with the synthetic yield and ¹H NMR, ¹³C NMR, and HRMS data characterizing NCGC607.

Figure 2.

NCGC607 enhances GCase activity and facilitates its translocation to lysosomes in Gaucher macrophages. A, GCase activity in the presence of different concentrations of NCGC607 measured in iMacs from a control and iPSC line GD1–1 (N370S/N370S). Graph represents two independent experiments. RFU, Relative fluorescence units. B, Western blot analysis of GCase in iMacs from two subjects with GD1 (N370S/N370S) and one with Type 2 with and without NCGC607 (3 μm for 6 d). Control iMacs (C) are shown on the right. β-Actin is used as the loading control. Quantification of the results as the ratio of GCase to β-actin appears in the graph below. C, GlcCer levels were measured using HPLC/MS/MS in control and iMacs from the iPSC lines indicated. Graph represents two independent experiments (each experiment in triplicate). Statistically significant differences between patient and control values: *p = 0.03; **p = 0.0092 and p = 0.0046; ***p = 0.0005. D, Immunofluorescence study of iMacs treated with NCG607 (3 μm) for 6 d and costained for GCase (green), lysosomal marker Lamp2 (red), and nuclear marker DAPI (blue). Z-stack images were acquired using a Zeiss 510 confocal microscope (63× magnification). Scale bars, 5 μm. E, Pearson's coefficient was quantified reflecting the degree of colocalization between GCase and Lamp2 using Imaris software. Each data point represents the mean value of the cells in a field.

Figure 3.

Differentiation of iDA neurons from GD or GD-PD iPSCs. A, Each iPSC line was differentiated into rosettes (first panel), NPCs (second panel), and then iDA neurons and analyzed by immunofluorescence staining during the different stages of differentiation. Stains include antibodies to Sox1 (red), Nestin/FoxA2/TuJ1 (green/red/pink), Nestin/Map2 (red/green), and Map2/TH (red/green). B, RT-PCR analysis of PIRX3, FOXA2, AADC, and HPRT (control) gene expression levels in NPCs and iDA neurons from four of the lines. C, GCase activity (percentage of control) was measured in NPCs from the iPSC lines indicated. Graph represents data from two independent experiments in quadruplicate. ***p = 0.0003 and p = 0.00022. D, Western blot analysis of GCase in NPCs, using the GCase-specific irreversible inhibitor MDW933. Tuj1 and β-actin were used as loading controls. E, Western blot analysis of TH levels in iDA neurons after 100 d in culture. A total of 10 μg of protein was loaded per lane.

Figure 4.

Dopamine release and uptake in iPSC-derived GD iDA neurons. A, DA and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured in iDA neurons. Data represent two independent experiments in triplicates: ***p = 0.0038; ***p = 0.0016; *p = 0.043. B, Dopamine uptake in iDA neurons in the presence and absence of 10 and 20 μm GBR12909, used as a specific DAT inhibitor. Graph represents data from three independent experiments in pentaplicates: *p = 0.043; ***p = 0.0042. C, E, Immunofluorescence analyses of GD1, GD2, or GD1-PD iDA neurons after 100 d in culture costained for TH (green), DAT1 (red) (C), or VMAT2 (red) (E). Insets, Single-channel DAT1 or VMAT2. Scale bars, 5 μm. D, F, RT-PCR analysis of DAT1 in NPCs and iDA neurons. Graphs represent two independent experiments. G, Action potential firing in GD2 iDA neurons. Ga, Gb, Whole-cell recordings of spikes evoked by a range of current injections. Gc, Family of current traces evoked by voltage steps covering a range of potentials (5 mV increments). Note the presence of transient outward current characteristic of potassium currents in native DA neurons. Red tracing represents current evoked by step to 0 mV. Inset, Fast inward Na⁺-mediated component.

Figure 5.

Small-molecule NCGC607 increases GCase activity and translocation to lysosomes. A, Western blot analysis of GCase in untreated and NCGC6070-treated iDA neurons using green fluorescent MDW933. Tuj1 was used as the loading control. Imiglucerase (recombinant GCase) was loaded as a positive control. B, Graph represents quantification of the blots in A. C, GCase activity (percentage control) in iDA neurons in the presence and absence of NCGC607 (3 μm). Graph represents data from three independent experiments in quadruplicate: **p = 0.005; *p = 0.02; **p = 0.0025. D, Immunofluorescence study of iDA neurons before and after treatment with NCGC607 (3 μm) for 21 d. Cells were costained for GCase (green), Lamp2 (red), and DAPI (blue). Z-stack images were acquired using a Zeiss 510 confocal microscope (63× magnification). Insets, Higher magnification of the areas outlined in the images. Scale bars, 5 μm. E, The degree of colocalization reflected in Pearson's coefficient in areas of colocalization using Imaris software. Each data point represents the mean value of at least five cells per field.

Figure 6.

NCGC607 reduces GlcSph and GlcCer accumulation in GD iDA neurons. A, Levels of different species of glucosylceramide in iDA neurons measured by mass spectrometry. B, GlcSph and GalSph measured in iDA neurons using HPLC/MS/MS. The portion from GalSph is shown in black. C, GlcSph levels. *p = 0.013, p = 0.047, p = 0.034, and p = 0.031for GD1–1, GD1–2, GD1-PD2, and GD2, respectively. **p = 0.0034 for GD1-PD1. D, GlcCer levels. *p = 0.045, p = 0.035, p = 0.025, and p = 0.02 for GD1–1, GD1–2, GD1-PD1, and GD1-PD2, respectively, in iDA neurons after treatment with 3 μm NCGC607 for 21 d. **p = 0.0016 for GD2 in iDA neurons after treatment with 3 μm NCGC607 for 21 d. Graph represents the mean of two independent experiments (n = 6). E, Immunofluorescence staining with antibodies to GlcCer (green), Lamp2 (red), and Map2 (pink) in iDA neurons in the presence and absence of NCGC607. Nuclei are counterstained with DAPI. Insets, Higher magnification of the areas outlined in the images. Insets stained in pink, Single-channel views of Map2. Scale bars, 5 μm.

Figure 7.

α-Syn levels in iDA neurons are reduced after NCGC607 treatment. A, Immunofluorescence analysis of iDA neurons stained with antibody to TH (green) and α-syn (red). Scale bars, 5 μm. B, Western blot analysis of α-syn levels in iDA neurons after 150 d in culture and treated with 3 μm NCGC607 for 21 d. Tuj1 was used as the loading control. C, Quantification of the α-syn levels in iDA neurons in the presence and absence of NCGC607. Statistically significant differences between the untreated and treated cells: ***p = 0.00017; p = 0.0005; p = 0.0002. ^###Significant differences between the patient iDA neurons compared with control (n = 2). D, mRNA analysis of α-syn levels in iDA neurons after 100 d in culture. HPRT was used as internal control. Graph represents the mean of two independent experiments. E, iDA neurons were costained for α-syn (green), Lamp2 (red), and TH (purple) in the absence (left) and presence (right) of NCGC607 (3 μm, 21 d). Insets, Higher magnification of the areas outlined in the images. Z-stack images were acquired using a Zeiss 510 confocal microscope (63× magnification). Scale bars, 5 μm. F, Colocalization between Lamp2 and α-syn was measured for 50 individual cells and Pearson's coefficient quantified in ROI volume, using Imaris software.