Comparative study of enriched dopaminergic neurons from siblings with Gaucher disease discordant for parkinsonism

Abstract

Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.

Keywords: GBA1; Gaucher disease; Parkinson disease; dopaminergic neuron differentiation; iPSC; neomycin resistance; neurodegeneration.

Figure 1. Characterization of patient-derived iPSC lines

a Summary of clinical features of iPSC donors. b Brightfield images of iPSCs (Scale bars, 10 µm) c The expression of pluripotency marker OCT3/4 in all iPSC lines demonstrated by Western blotting. d Quantification of the expression of pluripotency markers Tra1–60 and Nanog in all iPSC lines with flow cytometry.

Figure 2. Generation and characterization of TH-neo edited iPSC lines.

a CRISPR-mediated knock-in of neo in the TH locus. b Brightfield images of TH-neo iPSC lines (Scale bar, 10 µm). c Normal karyotypes of TH-neo iPSC lines. d Immunocytochemistry (ICC) of TH+ cells in HT711 and HT711 TH-neo at day 37 of differentiation (Scale bar, 50 µm; insert scale bar, 25 µm). e,f Quantification of TH+ cells using flow cytometry in HT711 and HT711 TH-neo at day 37 of differentiation (t-test, n=4 independent differentiations).

Figure 3. neo expression is limited to TH+ neurons.

a The expression of neo is limited in TH+ neurons as demonstrated with ICC. b Quantification of colocalization. No correlation is detected in HT711 (t-test, ***, p<0.001, n=3 independent differentiations). c Geneticin eliminates both HT711 and HT711 TH-neo neural progenitor cells (n=3 independent differentiations). d Dose dependent survival of HT711 TH-neo DA neurons following geneticin treatment (ANOVA, **p=0.01, ***p<0.001, n=3 independent differentiations).

Figure 4. Enrichment of TH+ neurons with geneticin treatment.

a Confocal image of TH+ DA neurons with and without geneticin treatment. Note the reduction of the nuclei without accompanying TH staining. b Quantification of TH+ DA neurons in ICC (1-way ANOVA, ***p<0.001, n=5 fields of view from 2 independent differentiations). c, d Western blot analysis of TH expression. Data is normalized to cells without geneticin treatment (1-way ANOVA, **p <0.01, n=3 independent differentiations). e Flow analysis of TH+ cells. (t-test, *p<0.05, n=4 independent differentiations).

Figure 5. Cellular proteomics comparing DA neurons

A Volcano plot showing differentially expressed proteins in HT711 TH-neo DA neurons with and without geneticin selection. DA neuron markers enriched by geneticin treatment include TH, DDC and ALDH1A1 (n=1 independent differentiation). b UMAP clustering of HT707, HT708, and HT711 DA neurons shows improved comparability after geneticin selection.

Fig 6.. Phenotyping high-purity DA neurons.

a,b Western blotting analyzing enriched DA neurons from HT707 TH-neo, HT708 TH-neo, and HT711 TH-neo as well as those from the parental lines. c Lipidomics analysis of enriched DA neurons. d A network of lysosomal hydrolases that is down-regulated in DA neurons from both HT707 TH-neo and HT708 TH-neo. e A network of molecular chaperones that is upregulated in HT708 TH-neo DA neurons compared to HT707 TH-neo. f Top 20 enriched GO term pathways comparing HT708 TH-neo and HT707 TH-neo DA neurons.