MEET QPS AT AD/PD™ 2024; March 5-9, Lisbon, Portugal

GBA-D409V-KI mice for in vivo and in vitro drug screening

QPS Neuropharmacology

Mutations in the human GBA (glucosylceramidase-β) gene are associated with lowered glucosylceramidase-β (GCase) activity and are shown to be relevant for the development of Gaucher disease. The enzyme is further highly discussed as therapeutic target in Parkinson`s disease research. The GBA-D409V-KI mouse model expresses the mutant D427V GBA protein which corresponds to the D409V mutation in the mature human GBA protein. Biochemical analysis of these mice for GCase activity in the liver and brain showed severely reduced GCase activity levels in homozygous GBA-D409V-KI mice and moderately, but also highly significant reduced GCase activity levels in heterozygous GBA-D409V-KI mice (Figure 1A and B). Evaluation of soluble and insoluble α-synuclein levels in the brain of GBA-D409V-KI mice showed significantly increased α-synuclein levels in the soluble brain fraction of homozygous GBA-D409V-KI mice compared to wild type littermates. α-synuclein levels in the insoluble brain fraction did not change (data not shown).

To test new drugs in a corresponding in vitro model, mouse embryonic fibroblasts of E14 GBA-D409V-KI mice were evaluated for GCase activity, showing a strong reduction of enzyme activity in homozygous GBA-D409V-KI mice. Isofagomine was able to significantly increase GCase activity in heterozygous mice compared to vehicle controls (VC, Figure 2).

GBA-D409V-KI mice and MEFs of these mice are thus a valuable tool to evaluate new compounds targeting GCAse activity.

Figure 1

Figure 1. GCase activity in the liver and brain of GBA-D409V-KI mice with age. GCase activity as µM/h in liver (A) and brain (B) of homo- (tm/tm) and heterozygous (wt/tm) GBA-D409V-KI mice as well as age-matched wild type littermates (wt/wt) at 4, 6, 8 and 12 months of age; n=12 per group. Two-way ANOVA with Bonferroni`s post hoc test; mean + SEM; ***p<0.001.

Figure 2

Figure 2. GCase activity in GBA-D409V-KI mouse embryonic fibroblasts (MEFs) treated with vehicle (VC) or Isofagomine (IFG). Mouse embryonic fibroblasts were isolated from homo- (tm/tm) and heterozygous (wt/tm) GBA-D409V-KI E14 embryos as well as age-matched wild type littermates (wt/wt). Cells were cultivated in 96-well plates and treated with either vehicle (0.1 % DMSO) or 20 µM isofagomine for 7 days. Thereafter, cells were subjected to either an adapted on-cell 4-MUG or crystal violet assay. Data are given as relative fluorescent units (RFU) of 4-MUG assay normalized to optical density (OD) values derived from crystal violet assay; n=5 per group. Two-way ANOVA with Bonferroni`s post hoc test; mean + SEM; *p<0.05; **p<0.01.

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