Figure 6.
Ambroxol Treatment Recovers GCase Activity and Increases GCase Protein
(A and D) The level of GCase protein was increased following ambroxol (ABX) treatment in heterozygous GBA1 mutant (Het1–3; N370S/wt) and iPD (PD1–3) neurons, but not in neurons derived from controls (C1–3; CTRL). For all three groups n = 3 cell lines.
(B) Ambroxol treatment significantly increased GCase activity in all three groups of neurons. Addition of the GCase inhibitor, conduritol-B-epoxide (CBE), showed that the contribution to the increase from non-lysosomal GCase (GBA2) was minimal. For all three groups n = 3 cell lines.
(C)The activity of hexosaminidase was not affected by ambroxol treatment. For all three groups n = 3 cell lines.
(E and F) Ambroxol treatment (60 μM) significantly decreased α-synuclein protein in GBA1 mutant (N370S/WT) neurons. The lower level of α-synuclein protein also occurred in treated WT and iPD neurons, but the decrease was not significant. For control and iPD groups n = 3 cell lines; for the GBA1 mutant group n = 4 cell lines.
(G) The α-synuclein mRNA level in all three groups of neurons was not affected by ambroxol treatment. For control and iPD groups n = 3 cell lines; for GBA1 mutant group n = 4 cell lines.
(H) There was no significant difference in the amount of α-synuclein released in the culture medium between treated and untreated cells of all three groups. For control and iPD groups n = 3 cell lines; for GBA1 mutant group n = 4 cell lines.
(I and J) Mature cathepsin D protein level was significantly increased following ambroxol treatment in GBA1 mutant (N370S/WT) human neurons. For all groups n = 3 cell lines.
(K and L) High level of α-synuclein protein (red) was seen in untreated in the GBA1 mutant (N370S/WT) neurons.
(M and N) The lower level of α-synuclein protein (red) was seen in the ambroxol-treated GBA1 mutant (N370S/WT) neurons. The neuronal marker β-III tubulin was stained green. Nuclei were stained with DAPI (blue).
Data are presented as means ± SEM. All comparisons were carried out with one-way ANOVA and Student's t test. ∗∗p < 0.01; ∗p < 0.05; NS, not significant (p > 0.05). Scale bars, 10 μm.