A novel allosteric GCase modulator prevents Tau accumulation in GBA1WT and GBA1L444P/L444P cellular models

Abstract

A slow decline in the autophagy-lysosomal pathway is a hallmark of the normal aging brain. Yet, an acceleration of this cellular function may propel neurodegenerative events. In fact, mutations in genes associated with the autophagy-lysosomal pathway can lead to Parkinson's disease. Also, amyloidogenic protein deposition is observed in lysosomal storage disorders, which are caused by genetic mutations representing risk factors for Parkinson's disease. For example, Gaucher's disease GBA1 mutations leading to defects in lysosomal sphingolipid metabolism cause α-synuclein accumulation. We observed that increased lysosomal Tau accumulation is found in human dermal fibroblasts engineered for inducible Tau expression. Inhibition of the GBA1 product GCase augmented Tau-dependent lysosomal stress and Tau accumulation. Here, we show increased Tau seed-induced Tau accumulation in Gaucher's fibroblasts carrying GBA1 mutations when compared to normal fibroblasts. Pharmacological enhancement of GCase reversed this effect, notably, also in normal fibroblasts. This suggests that boosting GCase activity may represent a therapeutic strategy to slow down aging-dependent lysosomal deficits and brain protein deposition.

Fig. 1

(A) Molecular structure of the GCase pharmacological chaperone GT-02216. (B) SPR dose-response for GT-02216 binding to immobilized human GCase protein monitored at acidic (pH 5.0) and neutral (pH 7.4) conditions. (C) SPR binding properties determined at the indicated pH values.

Fig. 2

(A) Basal GCase activity in wild-type and mutant GBA1 fibroblasts (mean ± SD normalized on GBA1^WT(XY), n = 6–48). Ordinary 1way ANOVA (p < .0001) and Dunnet’s multiple tests against GBA1^WT(XY), **** p < .0001. (B) Effect of 4-days treatment with 12.5 µmol/L GT-02216 on GCase activity on fibroblast lines (mean ± SD normalized on the respective basal activity, n = 5–42). Ordinary 1way ANOVA (p < .0001) and Šidák’s multiple tests against the respective basal activity, **** p < .0001. (C) GT-02216 dose-response on GBA1^{L444P/L444P(I)}α fibroblasts (mean ± sem normalized on vehicle, n = 14) or (D) on GBA1^{WT (XY)} fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC₅₀ = 2.4 µmol/L on GBA1^{L444P/L444P(I)}α (1.3–10.5 µmol/L 95% confidentiality interval, top 1.8–2.4 µmol/L, R² = 0.71) and 1.5 µmol/L on GBA1^WT(XY) (1.3–1.8 µmol/L 95% confidentiality interval, top 1.9-2.0 µmol/L, R² = 0.95).

Fig. 3

(A) Quantification of total HexCer in GBA1^{L444P/L444P(I)}α fibroblasts treated with vehicle or CBE (mean ± SD normalized on ctrl, n = 3). Unpaired t test, *** p 0.0007. (B) GBA1^{L444P/L444P(I)}α fibroblasts treated for 10 days with the indicated amount of GT-02216 (mean ± SD normalized on vehicle, n = 3). Ordinary 1way ANOVA (p < .0001) and Dunnett’s multiple tests against vehicle. ** p < .01, *** p < .001, **** p < .0001. (C) Quantification of total HexCer in GBA1^{L444P/L444P(I)}α compared to GBA1^WT(XX) fibroblasts (mean ± SD normalized on GBA1^WT(XX), n = 3). Unpaired t test, *** p 0.0006. (D) As in B. for GBA1^WT(XX) fibroblasts. Ordinary 1way ANOVA (p 0.0002) and Dunnett’s multiple tests against vehicle. ** p < .01, *** p < .001, **** p < .0001.

Fig. 4

(A) GCase activity in doxycycline-inducible Tau-mCherry human fibroblasts with the genotype GBA^WT(XX) or GBA1^{L444P/L444P(I)}α (mean ± SD normalized on GBA^WT(XX), n = 7). Mann-Whitney test, *** p 0.0006. (B) GT-02216 dose-response on Tau- GBA1^{L444P/L444P(I)}α fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC₅₀ = 1.1 µmol/L (0.7–1.6 µmol/L 95% confidentiality interval, top 1.5–1.6 µmol/L, R² = 0.79). (C) Basal and GT-02166-rescued GCase activity is not affected by the induction of Tau-mCherry expression with doxycycline in Tau-GBA1^WT(XX) fibroblasts (mean ± SD normalized on basal ctrl, n = 3). 2way ANOVA (p < .0001 for treatment, ns for Tau expression) and Šidák’s multiple tests against the respective ctrl, **** p < .0001. (D) GT-02216 dose-response on Tau-GBA1^WT(XX) fibroblasts (mean ± sem normalized on vehicle, n = 8) treated for 4 days. Non-linear fit with four parameters log(agonist); EC₅₀ = 1.0 µmol/L (0.7–2.3 µmol/L 95% confidentiality interval, top 1.4–1.6 µmol/L, R² = 0.78).

Fig. 5

(A) Quantification of Tau Puncta (TP) in Tau-GBA1^WT(XX) and Tau-GBA1^{L444P/L444P(I)}α fibroblasts at basal conditions (mean ± sem normalized on Tau-GBA^WT(XX), n = 55–60). Mann-Whitney test, *** p 0.0004. (B) Quantification of Tau puncta in Tau-GBA1^{L444P/L444P(I)}α fibroblasts treated overnight in the absence (ctrl) or presence (seeds) of Alzheimer’s brain-derived Tau seeds (mean ± sem normalized on ctrl, n = 45). Mann-Whitney test, **** p 4 × 10^− 11. A representative image of ctrl or Tau seeds conditions are shown (Tau in magenta, nuclei stained with DAPI in blue). (C) GCase activity in Tau-GBA1^{L444P/L444P(I)}α fibroblasts treated overnight in the absence or presence of Tau seeds (mean ± sem normalized on ctrl, n = 3). Mann-Whitney test, not significant. (D) as in B. for Tau-GBA1^WT(XX) fibroblasts (n = 45). Mann-Whitney test, **** p 0.00001. (E) as in C. for Tau-GBA1^WT(XX) fibroblasts (n = 6–9). Mann-Whitney test, not significant. (F) Dose-dependent reduction of Tau Puncta in Tau- GBA1^{L444P/L444P(I)}α fibroblasts or (G) in GBA1^WT(XX) fibroblasts treated for 4 days with the indicated amount of GT-02216 (mean ± sem normalized on vehicle, no seed ctrl, n = 15–45). 2way ANOVA (p < .0001 for GT-02216 concentration and seeds) and Šídák’s multiple tests against vehicle, **** p < .0001.

Fig. 6

(A) Pearson’s Correlation Coefficients (PCC) for Tau-mCherry on LAMP1-positive DOs in Tau-GBA1^{L444P/L444P(I)}α fibroblasts treated for 4 days with the indicated GT-02216 concentration and overnight in the presence or absence of Tau seeds (mean ± sem, n = 100 cells). (B) As in A. for Tau-GBA1 ^WT(XX) fibroblasts. (C) Number of LAMP1-positive DOs per Tau-GBA1^{L444P/L444P(I)}α fibroblast treated as in A. (mean ± sem, n = 20 cells). A representative image of the three conditions (ctrl, Tau seeds, Tau seeds and 12.5 µmol/L GT-02216 are shown (Tau in magenta, LAMP1 one in yellow, nuclei stained with DAPI in blue). (D) As in C. for Tau-GBA1^WT(XX) fibroblasts. Ordinary 1way ANOVA (p < .0001, except for p < .01 in D.) and Dunnett’s multiple tests against the respective controls (light grey bars), * p < .05, **** p < .0001. (E) Number of LysoTracker-positive DOs per Tau-GBA1^{L444P/L444P(I)}α fibroblast treated as in A. (mean ± sem, n = 20 cells). (F) As in E. for Tau-GBA1^WT(XX) fibroblasts. Ordinary 1way ANOVA (p < .0001 for E; p 0.0024 for F) and Dunnett’s multiple tests against the respective controls (light grey bars), * p < .05, ** p < .01, **** p < .0001.

Fig. 7

Viability of rat hippocampal primary neurons treated for 2 days with GT-02216 at the indicated concentration or with BDNF before being challenged with 5 µmol/L Tau oligomers for 1d. Cell viability was assessed with the MTT assay (mean ± SD, n = 3–4 wells). Ordinary 1way ANOVA (p < .0001) and Dunnett’s multiple tests against the control, **** p < .0001.