Neuroprotective Mitochondrial Remodeling by AKAP121/PKA Protects HT22 Cell from Glutamate-Induced Oxidative Stress
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Protein kinase A (PKA) is a ser/thr kinase that is critical for maintaining essential neuronal functions including mitochondrial homeostasis, bioenergetics, neuronal development, and neurotransmission. The endogenous pool of PKA is targeted to the mitochondrion by forming a complex with the mitochondrial scaffold A-kinase anchoring protein 121 (AKAP121). Enhanced PKA signaling via AKAP121 leads to PKA-mediated phosphorylation of the fission modulator Drp1, leading to enhanced mitochondrial networks and thereby blocking apoptosis against different toxic insults. In this study, we show for the first time that AKAP121/PKA confers neuroprotection in an in vitro model of oxidative stress induced by exposure to excess glutamate. Unexpectedly, treating mouse hippocampal progenitor neuronal HT22 cells with an acute dose or chronic exposure of glutamate robustly elevates PKA signaling, a beneficial compensatory response that is phenocopied in HT22 cells conditioned to thrive in the presence of excess glutamate but not in parental HT22 cells. Secondly, redirecting the endogenous pool of PKA by transiently transfecting AKAP121 or transfecting a constitutively active mutant of PKA targeted to the mitochondrion (OMM-PKA) or of an isoform of AKAP121 that lacks the KH and Tudor domains (S-AKAP84) are sufficient to significantly block cell death induced by glutamate toxicity but not in an oxygen deprivation/reperfusion model. Conversely, transient transfection of HT22 neuronal cells with a PKA-binding-deficient mutant of AKAP121 is unable to protect against oxidative stress induced by glutamate toxicity suggesting that the catalytic activity of PKA is required for AKAP121’s protective effects. Mechanistically, AKAP121 promotes neuroprotection by enhancing PKA-mediated phosphorylation of Drp1 to increase mitochondrial fusion, elevates ATP levels, and elicits an increase in the levels of antioxidants GSH and superoxide dismutase 2 leading to a reduction in the level of mitochondrial superoxide. Overall, our data supports AKAP121/PKA as a new molecular target that confers neuroprotection against glutamate toxicity by phosphorylating Drp1, to stabilize mitochondrial networks and mitochondrial function and to elicit antioxidant responses.
KeywordsProtein kinase A A-kinase anchoring protein 121 Dynamin-related protein 1 (Drp1) Oxidative stress HT22 Mitochondrion
Superoxide dismutase 2
A-kinase anchor protein 121
Cyclic adenosine monophosphate
cAMP response element-binding protein
Dynamin-related protein 1
Dual specificity A-kinase anchoring protein 1
We thank Dr. Antonio Feliciello (Department of Molecular Medicine & Medical Biotechnology, University of Naples, Naples, Italy), Xingshun Xu (Institute of Neuroscience, Soochow University, China), Steven Green (Biology Department, University of Iowa, IA, USA), and Stefan Strack (Department of Pharmacology, University of Iowa Carver College of Medicine, IA, USA) for providing vectors and the HT22 cell line. We apologize to all colleagues whose important contributions to this field could not be cited due to space limitations.
Availability of Data and Materials
The raw data files used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Conceptualization and design of the study: Jingdian Zhang, Ming Zhang, Dagda RK, and Ying Zhang; methodology and investigation: Jingdian Zhang, Dagda RK, Feng Wang, Yumeng Wang, Xu Wang, Di Ma, Xiang Yin, Chunxiao Li; providing important research resources, materials, and scientific input: Jiachun Feng, Dagda RK, Ming Zhang, and Ying Zhang; writing the original manuscript draft: Jingdian Zhang and Dagda RK; review and editing: all authors; supervision: Ming Zhang, Dagda RK, Jiachun Feng, and Ying Zhang. All authors read and approved the final manuscript.
This work was supported by the Natural Science Foundation of Jilin Province Science and Technology Development Plan (No. 20180101154JC) (to Ying Zhang), by NIH grants 1R01NS105783-01 and GM103554 (to Ruben K. Dagda), National Natural Science Foundation of China (No. 81771257) (to Jiachun Feng), and National Natural Science Foundation for Young Scientists of China (No. 81701158) (to Di Ma).
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The authors declare that they have no competing interests.
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