Bidirectional regulation of glycoprotein nonmetastatic melanoma protein B by β-glucocerebrosidase deficiency in GBA1 isogenic dopaminergic neurons from a patient with Gaucher disease and parkinsonism

Abstract

Variants in GBA1 are common genetic risk factors for several synucleinopathies. The increased risk has been attributed to the toxic effects of misfolded glucocerebrosidase (GCase) (gain-of-function), and the accumulation of lipid substrates due to reduced enzyme activity (loss-of-function). To delineate GBA1 pathogenicity, an iPSC line was generated from a patient with both type 1 Gaucher disease (GBA1: N370S/N370S; p.N409S/p.N409S) and Parkinson disease (PD). From this line, we created a reverted wild-type (WT) line and a GBA1 knockout (KO) line to eliminate misfolded GCase and intensify lipid accumulation. N370S/N370S and KO dopaminergic neurons (DANs) exhibited decreasing GCase levels and progressive accumulation of lipid substrates compared to WT DANs. Notably, the expression of GPNMB, whose levels correlate with PD risk, was upregulated by the mild lipid accumulation in N370S/N370S DANs but disrupted in KO DANs. These findings refine the loss-of-function mechanism by associating PD risk levels of GPNMB with lipid levels specific to GBA1 risk variants.

Keywords: GBA1; GCase; GPNMB; Gaucher disease; Parkinson disease; iPSC.

Figure 1:. Genetic background of HT809.

A. The distribution of insertion-deletion (indel), loss-of-function (LoF), and missense SNVs in HT809 and KOLF2.1J. B.C. PD and AD polygenic risk scores for HT809 and KOLF2.1J.

Figure 2:. Engineering HT809 to monitor DAN differentiation and to enable LysoIP.

A. Insertion of tdTomato into the endogenous TH locus. B. DAN differentiation protocol optimized for HT809. C. DAN differentiation efficiency evaluated by FACS. D. Immunostaining TH and FOXA2 in tdTomato+ cells. Scale bar, 35 μm. E, F. RNA-seq comparing HT809 iPSCs and day 75 DANs. G. LysoIP and MitoIP strategy in HT809. H. Enrichment of lysosomal proteins in LysoIP samples. Lysosomal proteins are indicated with blue color. I. Localization of TMEM192-GFP-3xHA to lysosomes in DANs. Scale bar, 10 μm; insert scale bar, 2 μm. J. Characterization of lysosomes purified from DANs by Western blotting using the Automated Western Blot JESS System.

Figure 3:. GBA1 isogenic iPSC lines generated using CRISPR-Cas9 and piggyBac transposase.

A. Schematic representation of GBA1 and GBAP1. B. gRNA specifically targets GBA1 to generate the KO line. C. Editing outcome in the KO line revealed by short-read amplicon sequencing. D. Reversion to the WT line using CRISPR-Cas9 and piggyBac transposase. E. Editing outcome in the WT line revealed by short-read amplicon sequencing. F. Specific editing of GBA1 as demonstrated by PacBio HiFi long-read amplicon sequencing.

Figure 4:. GCase characterization in GBA1 isogenic lines.

A. GCase protein levels in isogenic iPSCs and DANs. B. GCase glycosylation analysis with Endo H and PNGase F digestion in WT and N370S/N370S iPSCs and DANs. C. GCase activity levels in isogenic iPSCs and DANs. D. GCase localization in isogenic DANs. Scale bar, 5 μm; insert scale bar, 1 μm. E. Levels of LIMP2, prosaposin, and saposin C in isogenic DANs. F. Lysosomal GCase activity in isogenic DANs measured using LysoFQ-GBA. Scale bar, 10 μm. Intensities measured in the presence of GCase inhibitor CBE represent the fluorescence of TMEM192-GFP-3xHA in isogenic DANs.

Figure 5:. Levels of GlcCer and GlcSph in HT809 isogenic DANs.

GlcCer and GlcSph quantification by SFC-MS/MS in GBA1 isogenic DANs. Phosphate (Pi) amounts were determined in all samples for data normalization.

Figure 6:. Distinctive GPNMB phenotypes in N370S/N370S and KO DANs.

A,B. Volcano plots comparing KO and WT lysosomes (A), and N370S/N370S and WT lysosomes (B). C. GPNMB structure predicted by ALPHAFOLD. D. GPNMB levels in DANs. E. Characterization of GPNMB antibody specificity using GPNMB KO DANs. F. Expression of HiBiT-GPNMB-V5 in DANs. G. Enrichment of GPNMB fragment B4 in lysosomes. H. GPNMB glycosylation analysis with E4D7P. I. GPNMB expression in isogenic DANs after treatment with recombinant GCase.

Figure 7:. Working model illustrating the levels of GPNMB in GBA1 isogenic DANs.

In WT DANs a portion of GPNMB is processed to a 12-kDa fragment that is enriched in lysosomes. Levels of GPNMB, including both the full-length protein and the 12-kDa fragment, are elevated in N370S/N370S DANs but reduced in KO DANs.