Low-dose bafilomycin attenuates neuronal cell death associated with autophagy-lysosome pathway dysfunction

Abstract

We have shown previously that the plecomacrolide antibiotics bafilomycin A1 and B1 significantly attenuate cerebellar granule neuron death resulting from agents that disrupt lysosome function. To further characterize bafilomycin-mediated cytoprotection, we examined its ability to attenuate the death of naïve and differentiated neuronal SH-SY5Y human neuroblastoma cells from agents that induce lysosome dysfunction in vitro, and from in vivo dopaminergic neuron death in C. elegans. Low-dose bafilomycin significantly attenuated SH-SY5Y cell death resulting from treatment with chloroquine, hydroxychloroquine amodiaquine and staurosporine. Bafilomycin also attenuated the chloroquine-induced reduction in processing of cathepsin D, the principal lysosomal aspartic acid protease, to its mature 'active' form. Chloroquine induced autophagic vacuole accumulation and inhibited autophagic flux, effects that were attenuated upon treatment with bafilomycin and were associated with a significant decrease in chloroquine-induced accumulation of detergent-insoluble alpha-synuclein oligomers. In addition, bafilomycin significantly and dose-dependently attenuated dopaminergic neuron death in C. elegans resulting from in vivo over-expression of human wild-type alpha-synuclein. Together, our findings suggest that low-dose bafilomycin is cytoprotective in part through its maintenance of the autophagy-lysosome pathway, and underscores its therapeutic potential for treating Parkinson's disease and other neurodegenerative diseases that exhibit disruption of protein degradation pathways and accumulation of toxic protein species.

Fig. 1. Low-dose bafilomycin is not cytotoxic to SH-SY5Y cells

48h treatment with bafilomycin A1 (BafA1, A) or bafilomycin B1 (BafB1, B) decreases cell viability at concentrations ≥ 6 nM for BafA1 and ≥ 3 nM for BafB1. (C) BafA1 significantly increases caspase-3-like activity at concentrations ≥ 6 nM. Results represent mean ± SD from at least three independent experiments. *p<0.05 vs. 0 μM vehicle CTL; ^#p<0.05 vs.0-3 nM BafA1 (A) or 0-1 nM BafB1 (B).

Fig. 2. Low-dose bafilomycin attenuates chloroquine-induced cell death and apoptosis

48h treatment with bafilomycin A1 (BafA1, A) or bafilomycin B1 (BafB1, B) significantly attenuates chloroquine (CQ, 50 μM)-induced cell death from 0.1-6 nM for BafA1 (A) and from 0.1-1 nM for BafB1 (B). (C-D) Pretreatment with 1 nM BafA1 for either 12h (C) or 24h (D) significantly attenuates the reduction in viability following 48h post-treatment with 50 μM CQ. (E) 24h treatment with BafA1 (1 nM) significantly attenuates CQ-induced increase in caspase-3-like activity. (F) Co-treatment with the general caspase inhibitor BOC-Asp (OMe)-FMK (Boc-FMK, 30 μM) neither attenuates CQ-induced cell death nor enhances the cytoprotective effects of BafA1 against CQ-induced cell death. *p<0.05 vs. 0 μM vehicle CTL; ** p<0.05 vs. vehicle pre-treatment/CQ post-treatment (C-D) or CQ (F); ^#p<0.05 vs.0-3 nM & 3-6 nM BafA1 (A), 0-0.1 nM & 3-10 nM BafB1 (B), 0-0.1 nM & 6-10 nM BafA1 (C); ^##p<0.05 vs. CQ+Boc-FMK.

Fig. 3. Bafilomycin A1 attenuates chloroquine-induced inhibition of CD processing

(A) Whole cell lysates of SH-SY5Y cells were collected 24h following treatment with chloroquine (CQ, 50 μM) and/or bafilomycin A1 (BafA1, 1 nM), and subjected to western blot analysis for CD (pre-pro form, 50 kDa; pro form, 47 kDa; mature “active” form, 32 kDa). Blots were stripped and re-probed for GAPDH (37 kDa) loading control. Levels of (B) pre-pro CD, (C) pro CD and (D) mature CD were normalized to levels of β-tubulin. Results represent mean ± SD from at least three independent experiments. *p<0.05 vs. 0 μM vehicle CTL; **p<0.05 vs. CQ.

Fig. 4. Bafilomycin A1 attenuates chloroquine-induced increase in detergent-insoluble endogenous α-syn oligomers

(A) Representative western blot analysis of endogenous α-syn high molecular weight oligomers (34 & 51 kDa) in detergent–insoluble fractions, prepared from lysates of differentiated SH-SY5Y cells collected 48h after treatment with chloroquine (CQ, 50 μM) and/or bafilomycin A1 (BafA1, 1 nM). Blots were stripped and re-probed for actin (42 kDa) loading control. (B) Levels of insoluble α-syn high molecular weight oligomers were quantified and normalized to levels of actin. Results represent mean ± SD from five independent experiments. *p<0.05 vs. 0 μM vehicle CTL; **p<0.05 vs. CQ.

Fig. 5. Bafilomycin A1 attenuates chloroquine-induced AV accumulation

(A) Representative western blot analysis for LC3-I (cytosolic, 16 kDa) vs. LC3-II (AV membrane-specific, 14 kDa) in detergent-soluble (LEFT) vs. –insoluble (RIGHT) fractions prepared from differentiated SH-SY5Y cells 48h after treatment with chloroquine (CQ, 50 μM) and/or bafilomycin A1 (BafA1, 1 nM). Blots were stripped and re-probed for actin (42 kDa) loading control. Levels of LC3-II were quantified and normalized to actin for detergent-soluble (B) and detergent-insoluble (C) fractions. Note only detergent soluble fractions exhibited LC3-I immunoreactivity. Results represent mean ± SD from at least four independent experiments. *p<0.05 vs. 0 μM vehicle CTL; **p<0.05 vs. CQ.

Fig. 6. Bafilomycin A1 attenuates chloroquine-induced inhibition of autophagic flux

Differentiated SH-SY5Y were transiently transfected to over-express tfLC3 and following 24h recovery were treated for 8h with chloroquine (CQ, 50 μM) and/or bafilomycin A1 (BafA1, 1 μM) to observe effects of low-dose BafA1 on chloroquine-induced co-localization (merged image, right panel) of mRFP-LC3 (left panels) and eGFP-LC3 (center panels) fluorescent punctae, suggesting inhibition of autophagic flux. Cells were fixed and imaged via confocal microscopy as described in the Methods section. Panels a-c from CTL (A), BafA1 (B), CQ (C) and CQ+BafA1 are low magnification images; the box in each of these panels indicates higher magnification inset panels (d-f) for each. Images are representative of three independent experiments. Scale bar in Ac and Af = 10 μM.

Fig. 7. Bafilomycin attenuates the death of DA neurons in C. elegans following over-expression of wild-type human α-syn

(A, B) Worms over-expressing α-syn in DA neurons were acutely exposed to bafilomycin B1 (BafB1, 0-200 μg/ml) for 24h during larval development, and then subsequently scored for DA neuron loss at either seven days (A) or ten days (B) post-hatching (4 and 7 day adults, respectively). Results represent mean ± SD from at least three independent experiments, where 30 worms were analyzed for each experiment (n=90). *p<0.05 vs. 0 μg/ml vehicle CTL.