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cPKCγ-Modulated Autophagy Contributes to Ischemic Preconditioning–Induced Neuroprotection in Mice with Ischemic Stroke via mTOR-ULK1 Pathway

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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.).

Author information

Authors and Affiliations

  1. Department of Neurobiology, Capital Medical University, #10 You An Men Wai Xi Tou Tiao, Fengtai District, Beijing, 100069, People’s Republic of China

    Ying Zhang, Longhui Ma, Yi Yan, Li Zhao, Song Han & Junfa Li

  2. Beijing Key Laboratory of Hypoxia Conditioning Translational Medicine, Beijing, 100053, China

    Ying Zhang, Di Wu & Xunming Ji

  3. Beijing Institute for Brain Disorders, Capital Medical University, Beijing, 100069, China

    Ying Zhang, Longhui Ma, Yi Yan, Li Zhao, Song Han, Di Wu, Junfa Li & Xunming Ji

  4. Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, 33612, USA

    Cesar V. Borlongan

  5. Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, 100053, China

    Xunming Ji

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  1. Ying Zhang
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Contributions

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.

Corresponding authors

Correspondence to Junfa Li or Xunming Ji.

Ethics declarations

Ethical Approval and Consent to Participate

The experimental procedures were approved by the Experimental Animal Ethics Committee of Capital Medical University (AEEI-2020-144).

Human and Animal Ethics

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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Not applicable.

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The authors declare no competing interests.

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Authors’ Information

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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