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.
ABSTRACT
Mutations in GBA1, the gene encoding glucocerebrosidase, are common genetic risk factors for Parkinson disease (PD). While the mechanism underlying this relationship is unclear, patients with GBA1-associated PD often have an earlier onset and faster progression than idiopathic PD. Previously, we modeled GBA1-associated PD by crossing gba haploinsufficient mice with mice overexpressing a human mutant α-synuclein transgene (SNCAA53T), observing an earlier demise, shorter life span and faster symptom progression, although behavioral testing was not performed. To assess whether gba+/-//SNCAA53T mice exhibit a prodromal behavioral phenotype, we studied three cardinal PD features: olfactory discrimination, memory dysfunction, and motor function. The longitudinal performance of gba+/-//SNCAA53T (n = 8), SNCAA53T (n = 9), gba+/- (n = 10) and wildtype (n = 6) mice was evaluated between ages 8 and 23 months using the buried pellet test, novel object recognition test and the beam walk. Fifteen-month-old gba+/-//SNCAA53T mice showed more olfactory and motor deficits than wildtype mice. However, differences between gba+/-//SNCAA53T and SNCAA53T mice generally did not reach statistical significance, possibly due to small sample sizes. Furthermore, while gba haploinsufficiency leads to a more rapid demise, this might not result in an earlier prodromal stage, and other factors, including aging, oxidative stress and epigenetics, may contribute to the more fulminant disease course.
