Department of Neurology and Neuroscience Center, First Hospital of Jilin University, Xinmin Street No. 71, Changchun, 130000, China.
National-Local Joint Engineering Laboratory of Animal Models for Human Diseases, Changchun, Jilin, China.
Mol Neurobiol. 2019 Aug;56(8):5586-5607. doi: 10.1007/s12035-018-1464-3. Epub 2019 Jan 16.
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.
蛋白激酶 A（PKA）是一种丝氨酸/苏氨酸激酶，对于维持包括线粒体稳态、生物能量学、神经元发育和神经递质传递在内的基本神经元功能至关重要。PKA 的内源性池通过与线粒体支架 A-激酶锚定蛋白 121（AKAP121）形成复合物而靶向线粒体。通过 AKAP121 增强 PKA 信号导致 PKA 介导的分裂调节剂 Drp1 的磷酸化，导致增强的线粒体网络，从而阻止不同毒性损伤的细胞凋亡。在这项研究中，我们首次表明 AKAP121/PKA 在体外过量谷氨酸诱导的氧化应激模型中提供神经保护。出乎意料的是，用急性剂量或慢性暴露于谷氨酸处理小鼠海马祖细胞 HT22 细胞会强烈增加 PKA 信号，这是一种有益的补偿反应，在适应在过量谷氨酸存在下茁壮成长的 HT22 细胞中被模拟，但在亲本 HT22 细胞中则不然。其次，通过瞬时转染 AKAP121 或转染靶向线粒体的 PKA 的组成型活性突变体（OMM-PKA）或缺乏 KH 和 Tudor 结构域的 AKAP121 的同工型（S-AKAP84），将内源性 PKA 池重定向足以显著阻止谷氨酸毒性诱导的细胞死亡，但不能阻止氧剥夺/再灌注模型中的细胞死亡。相反，瞬时转染 HT22 神经元细胞的 AKAP121 的 PKA 结合缺陷突变体不能防止谷氨酸毒性诱导的细胞死亡，表明 PKA 的催化活性是 AKAP121 保护作用所必需的。从机制上讲，AKAP121 通过增强 PKA 介导的 Drp1 磷酸化来促进神经保护作用，以增加线粒体融合，提高 ATP 水平，并引起抗氧化剂 GSH 和超氧化物歧化酶 2 的水平增加，从而降低线粒体超氧化物的水平。总的来说，我们的数据支持 AKAP121/PKA 作为一种新的分子靶点，通过磷酸化 Drp1 赋予神经保护作用，以稳定线粒体网络和线粒体功能，并引发抗氧化反应，从而对抗谷氨酸毒性。
