Abstract
Neuron-specific conventional protein kinase C (cPKC)γ mediates cerebral hypoxic preconditioning (HPC). In parallel, autophagy plays a prosurvival role in ischemic preconditioning (IPC) against ischemic stroke. However, the effect of cPKCγ on autophagy in IPC still remains to be addressed. In this study, adult and postnatal 1-day-old C57BL/6 J wild-type (cPKCγ+/+) and knockout (cPKCγ−/−) mice were used to establish in vivo and in vitro IPC models. The results showed that IPC pretreatment alleviated neuronal damage caused by lethal ischemia, which could be suppressed by autophagy inhibitor 3-MA or bafilomycin A1. Meanwhile, cPKCγ knockout blocked IPC-induced neuroprotection, accompanied by significant increase of LC3-I to LC3-II conversion and Beclin 1 protein level, and a significant decrease in p62 protein level. Immunofluorescent staining results showed a decrease of LC3 puncta numbers in IPC-treated cPKCγ+/+ neurons with fatal ischemia, which was reversed in cPKCγ−/− neurons. In addition, cPKCγ-modulated phosphorylation of mTOR at Ser 2448 and ULK1 at Ser 555, rather than p-Thr-172 AMPK, was detected in IPC-pretreated neurons upon lethal ischemic exposure. The present data demonstrated that cPKCγ-modulated autophagy via the mTOR-ULK1 pathway likely modulated IPC-induced neuroprotection.
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Data Availability
The data that support the findings of this study are available from the corresponding authors upon reasonable request.
Abbreviations
- cPKCγ:
-
Conventional protein kinase Cγ
- IPC:
-
Ischemic preconditioning
- MCAO:
-
Middle cerebral artery occlusion
- OGD:
-
Oxygen-glucose deprivation
- HBSS:
-
Hanks’ balanced salt solution
- DMEM:
-
Dulbecco’s modified Eagle’s medium
- 3-MA:
-
6-Amino-3-methylpurine
- Baf A1:
-
Bafilomycin A1
- MTT:
-
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- DAPI:
-
4′6-Diamidino-2-phenylindole
- DMSO:
-
Dimethyl sulfoxide
- SDS-PAGE:
-
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- LC3:
-
Microtubule-associated protein 1 light chain 3
- SQSTM1/p62:
-
Sequestosome 1
- mTOR:
-
Mechanistic target of rapamycin
- ULK1:
-
Unc-51 like autophagy activating kinase
- AMPK:
-
5’-Monophosphate-activated protein kinase
- HSP60:
-
Heat shock protein 60
- Tom20:
-
Translocase of the outer mitochondrial membrane 20
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Funding
This work was supported by the National Key R&D Program of China (No. 2017YFC1308401 to X. J.) and the National Natural Science Foundation of China (No. 82027802 to X. J., No. 31671205 and No. 31972911 to J. L.).
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Y. Z., J. L., and X. J. conceived and designed the experiments; Y. Z. and L. M. performed the experiments; Y. Z. and C. V. B. wrote and edited the manuscript; L. M., Y. Y., and L. Z. analyzed the data; D. W. and S. H. offered technical support.
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The experimental procedures were approved by the Experimental Animal Ethics Committee of Capital Medical University (AEEI-2020-144).
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This study was conducted according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as ARRIVE guidelines.
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Y. Z., L. M., Y. Y., L. Z., S. H., and J. L. belong to the Department of Neurobiology, Capital Medical University, who focus on the pathology of stroke and conditioning medicine. D. W. and X. J. work as an associate researcher and a clinical doctor respectively at Xuanwu Hospital, Capital Medical University. X. J. is interested in translational stroke research. C. V. B. is a professor at the Department of Neurosurgery and Brain Repair, University of South Florida. C. V. B. devotes himself to novel neuroprotective and neurorestorative therapies in brain injury.
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Zhang, Y., Ma, L., Yan, Y. et al. cPKCγ-Modulated Autophagy Contributes to Ischemic Preconditioning–Induced Neuroprotection in Mice with Ischemic Stroke via mTOR-ULK1 Pathway. Transl. Stroke Res. 14, 790–801 (2023). https://doi.org/10.1007/s12975-022-01094-5
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DOI: https://doi.org/10.1007/s12975-022-01094-5