CDK5 inhibition protects against OGDR induced mitochondrial fragmentation and apoptosis through regulation of Drp1S616 phosphorylation

Chunli Chen; Xiaoxia Peng; Jiayu Tang; Zhiping Hu; Jieqiong Tan; Liuwang Zeng

doi:10.1016/j.lfs.2021.119062

CDK5 inhibition protects against OGDR induced mitochondrial fragmentation and apoptosis through regulation of Drp1S616 phosphorylation

Life Sci. 2021 Mar 15:269:119062. doi: 10.1016/j.lfs.2021.119062. Epub 2021 Jan 18.

Authors

Chunli Chen¹, Xiaoxia Peng², Jiayu Tang³, Zhiping Hu¹, Jieqiong Tan², Liuwang Zeng⁴

Affiliations

¹ Department of Neurology, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China.
² Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan 410078, China; Hunan Key Laboratory of Medical Genetics, Central South University, Changsha, Hunan 410078, China; Hunan Key Laboratory of Animal Model for Human Diseases, Central South University, Changsha, Hunan 410078, China.
³ Department of Neurology, The Second people's Hospital of Hunan Province, Changsha, Hunan 410007, China.
⁴ Department of Neurology, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China. Electronic address: zengliuwang@csu.edu.cn.

PMID: 33476635
DOI: 10.1016/j.lfs.2021.119062

Abstract

Aims: Cyclin-dependent kinase 5 (CDK5) is a potential target for the treatment of cerebral ischemia. CDK5 is one of the upstream regulators for Dynamin-related protein 1 (Drp1) phosphorylation. This study intends to discuss whether CDK5 inhibition conferring neuroprotection in cerebral ischemia through regulating Drp1 phosphorylation.

Materials and methods: Mouse neuroblastoma N2a cells and N1E-115 cells were cultured and subjected to oxygen-glucose deprivation/reperfusion (OGDR). N2a cells and N1E-115 cells were treated with Roscovitine, a pharmacological inhibitor of CDK5, or transfected with CDK5 siRNA to knock down CDK5 expression. N2a cells were transfected with different plasmids (Drp1-Myc, the dephosphorylation-mimic mutant Drp1S616A-Myc and the phosphorylation-mimic mutant Drp1S616D-Myc). The expression of CDK5 and its activator p35, Drp1 and phosphorylated Drp1 on S616 was determined by western blot. The morphology of mitochondria was detected by immunofluorescence staining and the proportion of N2a cells with apoptosis was detected by flow cytometry analysis.

Key findings: Expression of CDK5, p35 and phosphorylated Drp1 on S616 was strongly upregulated after 4 h and 12 h reperfusion following 4 h oxygen-glucose deprivation (OGD) at protein level. CDK5 inhibition by pre-treated with Roscovitine or transfection with CDK5 siRNA significantly ameliorated OGDR induced mitochondrial fragmentation and apoptosis. Overexpression of the phosphorylation-mimic mutant Drp1S616D abrogated the protective effect of CDK5 inhibition against OGDR induced mitochondrial fragmentation and apoptosis.

Significance: Our data indicate that the neuroprotective effect of CDK5 inhibition against OGDR induced neuronal damage is Drp1S616 phosphorylation dependent. A better understanding of the neuroprotective mechanisms of CDK5 inhibition in cerebral ischemia will help to develop safe and efficacious drugs targeting CDK5 signaling for clinical use.

Keywords: Cyclin-dependent kinase 5 (CDK5); Dynamin-related protein 1 (Drp1); Mitochondria; Oxygen-glucose deprivation/reperfusion (OGDR); Phosphorylation.

MeSH terms

Animals
Apoptosis*
Cyclin-Dependent Kinase 5 / antagonists & inhibitors*
Cyclin-Dependent Kinase 5 / genetics
Cyclin-Dependent Kinase 5 / metabolism
Dynamins / genetics
Dynamins / metabolism*
Glucose / deficiency*
Mice
Mitochondria / metabolism
Mitochondria / pathology*
Neuroblastoma / metabolism
Neuroblastoma / pathology
Neuroblastoma / prevention & control*
Neuroprotective Agents
Oxygen / metabolism*
Phosphorylation
Reperfusion Injury / complications
Signal Transduction
Tumor Cells, Cultured

Substances

Neuroprotective Agents
Cyclin-Dependent Kinase 5
Cdk5 protein, mouse
Dnm1l protein, mouse
Dynamins
Glucose
Oxygen