Department of Biochemistry and Physiology, University of Oklahoma Health Sciences Center, OK, USA; Center for Geroscience and Healthy Brain Aging, University of Oklahoma Health Sciences Center, OK, USA; Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA.
Department of Biochemistry and Physiology, University of Oklahoma Health Sciences Center, OK, USA.
Cell Calcium. 2024 May;119:102854. doi: 10.1016/j.ceca.2024.102854. Epub 2024 Feb 6.
The necroptotic effector molecule MLKL accumulates in neurons over the lifespan of mice, and its downregulation has the potential to improve cognition through neuroinflammation, and changes in the abundance of synaptic proteins and enzymes in the central nervous system. Notwithstanding, direct evidence of cell-autonomous effects of MLKL expression on neuronal physiology and metabolism are lacking. Here, we tested whether the overexpression of MLKL in the absence of cell death in the neuronal cell line Neuro-2a recapitulates some of the hallmarks of aging at the cellular level. Using genetically-encoded fluorescent biosensors, we monitored the cytosolic and mitochondrial Ca levels, along with the cytosolic concentrations of several metabolites involved in energy metabolism (lactate, glucose, ATP) and oxidative stress (oxidized/reduced glutathione). We found that MLKL overexpression marginally decreased cell viability, however, it led to reduced cytosolic and mitochondrial Ca elevations in response to Ca influx from the extracellular space. On the contrary, Ca signals were elevated after mobilizing Ca from the endoplasmic reticulum. Transient elevations in cytosolic Ca, mimicking neuronal stimulation, lead to higher lactate levels and lower glucose concentrations in Neuro-2a cells when overexpressing MLKL, which suggest enhanced neuronal glycolysis. Despite these alterations, energy levels and glutathione redox state in the cell bodies remained largely preserved after inducing MLKL overexpression for 24-48 h. Taken together, our proof-of-concept experiments are consistent with the hypothesis that MLKL overexpression in the absence of cell death contributes to both Ca and metabolic dyshomeostasis, which are cellular hallmarks of brain aging.
细胞程序性坏死效应分子 MLKL 在小鼠的整个生命周期中在神经元中积累，其下调有可能通过神经炎症、中枢神经系统中突触蛋白和酶丰度的变化来改善认知。然而，缺乏 MLKL 表达对神经元生理学和代谢的直接细胞自主效应的证据。在这里，我们测试了在神经元细胞系 Neuro-2a 中不发生细胞死亡的情况下过表达 MLKL 是否能再现细胞水平衰老的一些特征。使用遗传编码的荧光生物传感器，我们监测了胞质和线粒体 Ca 水平，以及涉及能量代谢（乳酸、葡萄糖、ATP）和氧化应激（氧化/还原型谷胱甘肽）的几种代谢物的胞质浓度。我们发现 MLKL 的过表达略微降低了细胞活力，但导致胞质和线粒体 Ca 对细胞外空间 Ca 内流的升高反应降低。相反，动员内质网中的 Ca 后，Ca 信号升高。在过表达 MLKL 时，胞质 Ca 的短暂升高，模拟神经元刺激，导致乳酸水平升高和葡萄糖浓度降低，这表明神经元糖酵解增强。尽管存在这些变化，但在诱导 MLKL 过表达 24-48 小时后，细胞体中的能量水平和谷胱甘肽氧化还原状态仍基本保持不变。综上所述，我们的概念验证实验与假设一致，即在没有细胞死亡的情况下过表达 MLKL 会导致 Ca 和代谢失调，这是大脑衰老的细胞特征。
