Lisek Malwina, Mackiewicz Joanna, Sobolczyk Marta, Ferenc Bozena, Guo Feng, Zylinska Ludmila, Boczek Tomasz
Department of Molecular Neurochemistry, Medical University of Lodz, Łódz, Poland.
Department of Pharmaceutical Toxicology, China Medical University, Shenyang, China.
Front Cell Neurosci. 2022 May 23;16:890827. doi: 10.3389/fncel.2022.890827. eCollection 2022.
PMCA2 is not expressed until the late embryonic state when the control of subtle Ca fluxes becomes important for neuronal specialization. During this period, immature neurons are especially vulnerable to degenerative insults induced by the N-methyl-D-aspartate (NMDA) receptor blocker, ketamine. As H19-7 hippocampal progenitor cells isolated from E17 do not express the PMCA2 isoform, they constitute a valuable model for studying its role in neuronal development. In this study, we demonstrated that heterologous expression of PMCA2b enhanced the differentiation of H19-7 cells and protected from ketamine-induced death. PMCA2b did not affect resting [Ca] in the presence or absence of ketamine and had no effect on the rate of Ca clearance following membrane depolarization in the presence of the drug. The upregulation of endogenous PMCA1 demonstrated in response to PMCA2b expression as well as ketamine-induced PMCA4 depletion were indifferent to the rate of Ca clearance in the presence of ketamine. Yet, co-expression of PMCA4b and PMCA2b was able to partially restore Ca extrusion diminished by ketamine. The profiling of NMDA receptor expression showed upregulation of the NMDAR1 subunit in PMCA2b-expressing cells and increased co-immunoprecipitation of both proteins following ketamine treatment. Further microarray screening demonstrated a significant influence of PMCA2b on GABA signaling in differentiating progenitor cells, manifested by the unique regulation of several genes key to the GABAergic transmission. The overall activity of glutamate decarboxylase remained unchanged, but Ca-induced GABA release was inhibited in the presence of ketamine. Interestingly, PMCA2b expression was able to reverse this effect. The mechanism of GABA secretion normalization in the presence of ketamine may involve PMCA2b-mediated inhibition of GABA transaminase, thus shifting GABA utilization from energetic purposes to neurosecretion. In this study, we show for the first time that developmentally controlled PMCA expression may dictate the pattern of differentiation of hippocampal progenitor cells. Moreover, the appearance of PMCA2 early in development has long-standing consequences for GABA metabolism with yet an unpredictable influence on GABAergic neurotransmission during later stages of brain maturation. In contrast, the presence of PMCA2b seems to be protective for differentiating progenitor cells from ketamine-induced apoptotic death.
PMCA2直到胚胎后期才表达，此时对细微钙通量的控制对神经元特化变得至关重要。在此期间，未成熟神经元特别容易受到N-甲基-D-天冬氨酸（NMDA）受体阻滞剂氯胺酮诱导的退行性损伤。由于从E17分离的H19-7海马祖细胞不表达PMCA2亚型，它们构成了研究其在神经元发育中作用的有价值模型。在本研究中，我们证明PMCA2b的异源表达增强了H19-7细胞的分化并保护其免受氯胺酮诱导的死亡。在存在或不存在氯胺酮的情况下，PMCA2b均不影响静息[Ca]，并且在存在该药物的情况下对膜去极化后的钙清除率没有影响。响应于PMCA2b表达而显示的内源性PMCA1的上调以及氯胺酮诱导的PMCA4耗竭对存在氯胺酮时的钙清除率无影响。然而，PMCA4b和PMCA2b的共表达能够部分恢复氯胺酮减少的钙外排。NMDA受体表达谱显示，在表达PMCA2b的细胞中NMDAR1亚基上调，并且在氯胺酮处理后两种蛋白的共免疫沉淀增加。进一步的微阵列筛选表明，PMCA2b对分化祖细胞中的GABA信号传导有显著影响，表现为对GABA能传递关键的几个基因的独特调控。谷氨酸脱羧酶的总体活性保持不变，但在存在氯胺酮的情况下，钙诱导的GABA释放受到抑制。有趣的是，PMCA2b的表达能够逆转这种效应。氯胺酮存在下GABA分泌正常化的机制可能涉及PMCA2b介导的对GABA转氨酶的抑制，从而将GABA的利用从能量目的转向神经分泌。在本研究中，我们首次表明发育控制的PMCA表达可能决定海马祖细胞的分化模式。此外，PMCA2在发育早期的出现对GABA代谢有长期影响，对脑成熟后期的GABA能神经传递有不可预测的影响。相比之下，PMCA2b的存在似乎对分化祖细胞免受氯胺酮诱导的凋亡死亡具有保护作用。
