Abstract
Methamphetamine (METH), a psychoactive-stimulant facilitates massive accumulation of autophagosomes and causes autophagy-associated neuronal death. However, the underlying mechanisms involving METH-induced auto-phagosome accumulation remain poorly understood. In the current study, autophagic flux was tracked by mRFP-GFP-LC3 adenovirus, 900 μM METH treatment was found to significantly disrupt autophagic flux, which was further validated by remarkable increase of co-localized of LC3 and SQSTM1/p62, enhancement of LC3-II and SQSTM1/p62 protein levels, and massive autophagosome puncta aggregation. With the cycloheximide (CHX) treatment, METH treatment was displayed a significant inhibition of SQSTM1/p62 degradation. Therefore, the mRNAs associated with vesicle degradation were screened, and syntaxin 17 (Stx17) and dynein-dynactin mRNA levels significantly decreased, an effect was proved in protein level as well. Intriguingly, METH induced autophagosome accumulation and autophagic flux disturbance was incredibly retarded by overexpression of Stx17, which was validated by the restoration of the fusion autophagosome–late endosome/lysosome fusion. Moreover, Stx17 overexpression obviously impeded the METH-induced decrease of co-localization of the retrograded motor protein dynein/dynactin and autophagosome-late endosome, though the dynein/dynactin proteins were not involved in autophagosome–late endosome/lysosome fusion. Collectively, our findings unravel the mechanism of METH-induced autophagosome accumulation involving autophagosome–late endosome/lysosome fusion deficiency and that autophagy-enhancing mechanisms such as the overexpression of Stx17 may be therapeutic strategies for the treatment of METH-induced neuronal damage.
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Abbreviations
- 3-MA:
-
3-Methyladenine
- BAF:
-
Bafilomycin
- BCA:
-
Bicinchoninic acid
- c-caspase 3:
-
Cleaved caspase 3
- CTSB:
-
Cathepsin B
- CQ:
-
Chloroquine
- CHX:
-
Cycloheximide
- DCTN1:
-
Dynactin subunit 1
- GFP:
-
Green fluorescence
- HOPS:
-
Homotypic fusion and protein sorting complex
- LAMP1:
-
Lysosomal-associated membrane protein 1
- METH:
-
Methamphetamine
- PARP:
-
Poly (ADP -ribose) polymerase
- RFP:
-
Red fluorescence
- Rapa:
-
Rapamycin
- SNAP29:
-
Synaptosomal-associated protein 29
- SNARE:
-
Soluble NSF attachment protein receptor
- Stx17:
-
Syntaxin 17
- TUNEL:
-
Terminal deoxynucleotidyl transferase dUTP nick end labeling
- VAMP8:
-
Vesicle-associated membrane protein 8
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This work received financial support from the National Natural Science Foundation of China (82073584, 82072159) and the Natural Science Foundation of Jiangsu Province (BK20191349).
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JW designed the experiments and revised the manuscript. HSX collected and analyzed the data and wrote the manuscript. YHZ and XFC collected and analyzed the data. TYY, XW, XS and X.X.X collected the data. MYH and LJ provided technical assistance. JC and RG revised the manuscript.
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Xu, H., Zhu, Y., Chen, X. et al. Mystery of methamphetamine-induced autophagosome accumulation in hippocampal neurons: loss of syntaxin 17 in defects of dynein–dynactin driving and autophagosome–late endosome/lysosome fusion. Arch Toxicol 95, 3263–3284 (2021). https://doi.org/10.1007/s00204-021-03131-y
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DOI: https://doi.org/10.1007/s00204-021-03131-y