Figures & data
Figure 1. ShRNA-mediated knockdown of GBA leads to SNCA accumulation in SK-N-SH neuroblastoma cells. (A) Efficiency of GBA knockdown with shGBA #9847–1 and shGBA #9848–1 was assessed at 12, 24, and 48 h. (B) GBA protein expression in cells transfected with shRNA constructs was reduced at 12 and 24 h. (C) GBA activity was reduced in SK-N-SH neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. *P< 0.05 vs. normal; n = 3. (D, E) Increase in SNCA protein expression in GBA knockdown cells. (F, G) GBA and SNCA mRNA expression after GBA knockdown was assessed by quantitative RT-PCR. *P< 0.05, **P< 0.01 vs. normal; n = 3.
Figure 2. GBA knockdown inhibits autophagy in SK-N-SH neuroblastoma cells and rat cortical neurons. (A) Decreased LC3-II level in cells transfected with shGBA #9847–1 or shGBA #9848–1. (B) GBA protein expression was reduced in GBA knockdown cells. (C) Decrease in autophagic activity upon GBA knockdown, as assessed by the ratio of LC3-II/ACTB. **P< 0.01 compared to normal; n = 3. (D, E) Efficiency of GBA knockdown upon infection of neurons with different LV. LV-GFP-shGba #6 showed the greatest effect, causing a 50% reduction in GBA protein level. (F) GBA activity was reduced in cortical neurons infected with LV-GFP-shGba #6, an effect that was reversed by coinfection with full-length GBA (LV-GFP-shGba #6+HsGBA). *P< 0.05 vs. LV-GFP-sh-con group, #P< 0.05 vs. LV-GFP-shGba #6 group; n = 3. (G) Decreased LC3-II level upon GBA knockdown. Neurons treated with rapamycin (LV-GFP-shGba #6+Rapa; 40 nM for 6 h) served as a positive control. (H) GBA protein expression was reduced in cells infected with LV-GFP-shGba #6, an effect that was rescued by coinfection of full-length GBA (LV-GFP-shGba #6+HsGBA). (I) Decrease in autophagic activity upon GBA knockdown in LV-GFP-shGba #6-infected neurons, as assessed by the ratio of LC3-II/ACTB. Activity was restored to a normal level upon overexpression of full-length GBA (LV-GFP-shGba #6+HsGBA) or rapamycin treatment (LV-GFP-shGba #6+Rapa). *P< 0.05, **P< 0.01 compared to LV-GFP-sh-con group, #P< 0.05, ###P< 0.001 compared to LV-GFP-shGba #6 group; n = 3.
Figure 3. Assessment of autophagic flux, following GBA knockdown. Autophagic flux was analyzed in GBA-deficient SK-N-SH neuroblastoma cells (A, B, C) and cortical neurons (D, E, F) treated with 100 nM BafA1 for 6 h or 10 mM NH4Cl for 1 h (G, H). The levels of LC3-II and SQSTM1 were normalized with ACTB. *P< 0.05, ***P< 0.001, ****P< 0.0001 vs. control; ###P< 0.001 vs. control+BafA1 group; *P< 0.05, **P< 0.01, ***P< 0.001 vs. LV-GFP-sh-con group; #P< 0.05 vs. LV-GFP-sh-con+BafA1 group; ###P< 0.001 vs. LV-GFP-sh-con+ NH4Cl group; ^P< 0.05, ^^^P< 0.001 vs. LV-GFP-shGba #6 group; ^^^P< 0.001 vs. shGBA #9848–1 group; n = 3. (I, J) LC3-II level was analyzed in response to starvation 2 h (Hank's balanced salt solution). ACTB was used as a loading control. **P< 0.01, ***P< 0.001 vs. LV-GFP-sh-con group; ###P< 0.001 vs. LV-GFP-sh-con+starvation group; ^^P< 0.01 vs. LV-GFP-shGba #6 group; n = 3.
Figure 4. GBA knockdown inhibits autophagosome formation. (A) SK-N-SH neuroblastoma cells were infected with LV-GFP-shGBA #9848–1. After 3 d, cells were fixed and stained with LAMP1 and LC3 primary antibodies. Immunolabeling was visualized with rabbit Alexa 647-conjugated and mouse Alexa Fluor 594-conjugated secondary antibodies, respectively. The nucleus was counterstained by DAPI. These detected cells were first verified to infect GFP. Then the channel was turned off. To better observe the colocalization of LAMP1 and LC3, LAMP1 was labeled as “green.” Scale bar =2 μm. (B) TEM images of autophagic vacuoles in primary rat cortical neurons. The cortical neurons were infected with LV-GFP-shGba #6 for 3 d The right panel is a high magnification image of the indicated portion. Arrows indicate autophagosomes. N, nuclei. Scale bar =500 nm. (C) Fluorescence images were analyzed using ImageJ “colocalization” plug-in software. A total of 30 cells were analyzed from 3 independent experiments (10 cells per experiment). The colocalization of LAMP1 and LC3 was decreased in GBA-deficient cells compared to control group, which was reversed by treatment with rapamycin (40 nM for 6 h). Also, the colocalization of LAMP1 and LC3 was decreased in GBA-deficient cells in response to starvation. *P< 0.05, ##P< 0.01, ^P< 0.05. (D) TEM revealed less autophagosomes in neurons infected with LV-GFP-shGba #6 than in LV-GFP-sh-con group. Twenty cells per experiment, n = 3. *P< 0.05.
Figure 5. Reduction in ceramide production and downregulation of BECN1 protein expression upon GBA knockdown. (A) SK-N-SH neuroblastoma cells were infected with LV-GFP-shGBA #9848–1 for 3 d, and treated with an antibody against ceramide and stained with DAPI. Scale bar =50 μm. (B) Reduced ceramide production in cells infected with LV-GFP-shGBA #9848–1. Samples were imaged at the same exposure to allow direct comparisons; ceramide staining intensity was calculated using ImageJ software. Data were analyzed for 10 randomly selected fields of view from 3 independent experiments. **P< 0.01 vs. LV-GFP-sh-con group. (C) Endogenous C2 level was reduced in GBA-deficient SK-N-SH neuroblastoma cells infected with LV-GFP-shGBA #9848–1, which was reversed by exogenous C2 application (5 μM for 8 h), as determined by HPLC-MS/MS. **P< 0.01 vs. LV-GFP-sh-con group, ##P< 0.01 vs. LV-GFP-shGBA #9848–1 (n=3). (D, E) Downregulation of BECN1 protein expression in GBA knockdown neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. *P< 0.05, **P< 0.01 vs. normal; n = 3. (F, G) Decreased expression of BECN1 protein induced by GBA knockdown in cortical neurons is rescued by overexpression of full-length GBA (LV-GFP-shGba #6+HsGBA) or treatment with rapamycin (LV-GFP-shGba #6+Rapa; 40 nM for 6 h). **P< 0.01 vs. LV-GFP-sh-con group, #P< 0.05, ##P< 0.01 vs. LV-GFP-shGba #6 group; n = 3.
Figure 6. Inactivation of PPP2A via Tyr307 phosphorylation induced by loss of GBA function. (A) PPP2A activity was reduced in SK-N-SH neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. **P< 0.01 compared to normal; n = 3. (B) PPP2A activity was reduced in cortical neurons infected with LV-GFP-shGba #6, which was reversed by coinfection of cells with full-length GBA (LV-GFP-shGba #6+HsGBA) or C2 treatment (LV-GFP-shGba #6+C2; 5 μM for 8 h). **P< 0.01 compared to LV-GFP-sh-con group, ##P< 0.01 compared to LV-GFP-shGba #6 group; n = 3. ((C)to E) Increased phosphorylation of PPP2A at Tyr307 (p-PPP2A [Tyr307]) in neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. *P< 0.05 compared to normal; n = 3. ((F)to H) Increased level of p-PPP2A (Tyr307) in cortical neurons infected with LV-GFP-shGba #6; the effect was abolished by overexpression of full-length GBA (LV-GFP-shGba #6+HsGBA) or C2 treatment (LV-GFP-shGba #6+C2). **P< 0.01 compared to LV-GFP-sh-con group, ##P< 0.01 compared to LV-GFP-shGba #6 group; n = 3.
![Figure 6. Inactivation of PPP2A via Tyr307 phosphorylation induced by loss of GBA function. (A) PPP2A activity was reduced in SK-N-SH neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. **P< 0.01 compared to normal; n = 3. (B) PPP2A activity was reduced in cortical neurons infected with LV-GFP-shGba #6, which was reversed by coinfection of cells with full-length GBA (LV-GFP-shGba #6+HsGBA) or C2 treatment (LV-GFP-shGba #6+C2; 5 μM for 8 h). **P< 0.01 compared to LV-GFP-sh-con group, ##P< 0.01 compared to LV-GFP-shGba #6 group; n = 3. ((C)to E) Increased phosphorylation of PPP2A at Tyr307 (p-PPP2A [Tyr307]) in neuroblastoma cells transfected with shGBA #9847–1 and shGBA #9848–1. *P< 0.05 compared to normal; n = 3. ((F)to H) Increased level of p-PPP2A (Tyr307) in cortical neurons infected with LV-GFP-shGba #6; the effect was abolished by overexpression of full-length GBA (LV-GFP-shGba #6+HsGBA) or C2 treatment (LV-GFP-shGba #6+C2). **P< 0.01 compared to LV-GFP-sh-con group, ##P< 0.01 compared to LV-GFP-shGba #6 group; n = 3.](/cms/asset/6d2f280d-8e42-4574-b387-0ea516e76326/kaup_a_1086055_f0006_b.gif)
Figure 7. C2 treatment restores autophagic activity in SK-N-SH neuroblastoma cells with GBA knockdown. (A to C) Decreased LC3-II level, as well as increased PPP2A phosphorylation at Tyr307 (p-PPP2A [Tyr307]) in cells transfected with shGBA #9848–1 were reversed by C2 treatment (shGBA #9848–1+C2; 5 μM for 8 h). (D) Autophagic activity was restored in GBA-deficient cells in the presence of C2, as assessed by the ratio of LC3-II/ACTB. **P< 0.01, ***P< 0.001 compared to normal, ##P< 0.01 compared to shGBA #9848–1 group; n = 3.
![Figure 7. C2 treatment restores autophagic activity in SK-N-SH neuroblastoma cells with GBA knockdown. (A to C) Decreased LC3-II level, as well as increased PPP2A phosphorylation at Tyr307 (p-PPP2A [Tyr307]) in cells transfected with shGBA #9848–1 were reversed by C2 treatment (shGBA #9848–1+C2; 5 μM for 8 h). (D) Autophagic activity was restored in GBA-deficient cells in the presence of C2, as assessed by the ratio of LC3-II/ACTB. **P< 0.01, ***P< 0.001 compared to normal, ##P< 0.01 compared to shGBA #9848–1 group; n = 3.](/cms/asset/709f4de9-1323-43ee-8214-729d5195c64a/kaup_a_1086055_f0007_b.gif)
Figure 8. Loss of GBA function leads to inhibition of autophagy via downregulation of PPP2A activity, resulting in SNCA accumulation in cortical neurons. (A, B) Increased SNCA protein expression, and decreased autophagic activity (LC3-II/ACTB), in neurons infected with LV-GFP-shGba #6 are reversed by overexpression of full-length GBA (LV-GFP-shGba #6+HsGBA), or by treatment with C2 (LV-GFP-shGba #6+C2; 5 μM for 8 h) or rapamycin (LV-GFP-shGba #6+Rapa; 40 nM for 6 h). (C) Expression of SNCA protein in GBA-deficient neurons was restored to normal levels by overexpression of full-length GBA, or treatment with PPP2A or autophagy agonists. **P< 0.01 compared to LV-GFP-sh-con group, #P< 0.05, ##P< 0.01, ###P< 0.001 compared to LV-GFP-shGba #6 group; n = 3.
Figure 9. Loss of GBA function leads to SNCA accumulation through inhibition of autophagy involving PPP2A inactivation in rats. (A) Confocal immunofluorescence microscopy reveals an enhancement of GFP signal following LV-GFP-shGba #6 injection into the left striatum of rats, with no fluorescence detected on the contralateral (uninjected) side. Scale bar = 500 μm. (B, C) LV gene transfer vectors encoding GFP-shGba #6 (LV-GFP-shGba #6) and scrambled negative control (LV-GFP-sh-con) were injected into the left striatum of rats; 1 wk later, RNA was extracted from whole striatal tissue homogenates for analysis of Gba and Snca mRNA expression by quantitative RT-PCR. (D, E) GBA and PPP2A activities were reduced in the striatum of rats infected with LV-GFP-shGba #6. (F) GBA expression, autophagy level, phosphorylation level of PPP2A at Tyr307, and SNCA expression were assessed by western blotting. (G) GBA protein expression was reduced in the striatum of rats infected with LV-GFP-shGba #6. ((H)to K) Downregulation of BECN1 protein expression, increased phosphorylation of PPP2A at Tyr307, decreased LC3-II level, and increased SNCA protein expression were observed in the striatum infected with LV-GFP-shGba #6. *P< 0.05, **P< 0.01 vs. contralateral group; n = 3.
Figure 10. SNCA oligomerization and aggregation by loss of GBA function. (A, B) The accumulation of high-molecular-weight SNCA species were significantly increased in the TritonX-100 insoluble fraction in LV-GFP-shGba #6-infected neurons, and this effect was abolished by treatment with C2 (5 μM for 8 h). (C) Neurons were infected with LV-GFP-shGba #6 or LV-GFP-sh-con for 3 d, and SNCA aggregation was assessed by thioflavin-S. Nuclei were stained with DAPI. Scale bar = 10 μm. (D) The number of cells containing SNCA aggregates was quantified in 6 randomly chosen microscopic fields. SNCA aggregation was observed in cortical neurons upon GBA knockdown, which was alleviated upon treatment with rapamycin (40 nM for 6 h). *P< 0.05, **P< 0.01 vs. LV-GFP-sh-con group, #P< 0.05, vs. LV-GFP-shGba #6 group; n = 3.
Table 1 ShRNA-plasmids and shRNA-lentivirus used in this study
