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Abstract：
Leukocyte common antigen-related protein tyrosine phosphatase (LAR) is a member of the protein tyrosine phosphatase family that serves as a key regulator of cellular survival. It is also involved in neurodevelopment and brain disorders. This study was designed to investigate the role of LAR in a cell-based model of Parkinson\'s disease (PD) in which U251 and SH-SY5Y cells were used as models of astrocytes and dopaminergic neurons, respectively. Cell viability, cell death, cell morphology, protein phosphorylation and expression, ATP levels, reactive oxygen species (ROS) generation, and mitochondrial membrane potential were analyzed in the wild-type (WT) and heterozygous LAR-knockout astrocytoma U251 cells to assess the cell state, signal transduction, and mitochondrial function. LAR downregulation showed a protective effect in rotenone-exposed U251 cells by increasing cell viability, reducing cell mortality, and restoring appropriate cellular morphology. LAR downregulation enhanced IGF-1R phosphorylation and downstream signal transduction as evidenced by increases in the Akt and GSK-3β phosphorylation, as well as the upregulation of NRF2 and HO-1. The downregulation of LAR also augmented DJ-1 levels in these cells. The enhanced Akt and GSK-3β phosphorylation contributed to a reduced Bax/Bcl2 ratio and suppressed apoptosis after rotenone exposure. Heterozygous LAR-knockout U251 cells exhibited higher mitochondrial function evidenced by increased mitochondrial membrane potential, ATP contents, and reduced ROS production compared to the WT cells following rotenone exposure. Further studies showed that the astrocytic protection mediated by the heterozygous knockout of LAR was associated with the activation of Akt. A specific Akt inhibitor, MK2206, reduced the cell viability, Akt and GSK3β phosphorylation, and HO-1 and NRF2 expression in U251 cells exposed to rotenone. Astrocytes provide structural and metabolic support to maintain neuronal health. Astrocytic glial cell-derived neurotrophic factor (GDNF) production is vital for dopaminergic neuron survival. Heterozygous LAR-knockout U251 cells produced higher amounts of GDNF than the WT cells. The SH-SY5Y cells cocultured with heterozygous LAR-knockout U251 cells exhibited greater viability than that of cells cocultured with WT U251 cells in response to rotenone. Together, these findings demonstrate that the heterozygous knockout of LAR in astrocytes can play a key role in protecting both astrocytic cells and cocultured neurons in a rotenone-induced cell-based model of PD. This neuroprotective effect is attributable to the augmentation of IGF1R-Akt-GDNF signaling and the maintenance of astrocytic mitochondrial function.
摘要：
白细胞共同抗原相关蛋白酪氨酸磷酸酶（LAR）是蛋白酪氨酸磷酸酶家族的成员，是细胞存活的关键调节剂。它还参与神经发育和脑部疾病。本研究旨在研究LAR在帕金森病（PD）细胞模型中的作用，其中U251和SH-SY5Y细胞被用作星形胶质细胞和多巴胺能神经元的模型。分别。细胞活力，细胞死亡,细胞形态学,蛋白质磷酸化和表达，ATP水平,活性氧（ROS）的产生，在野生型（WT）和杂合子LAR敲除星形细胞瘤U251细胞中分析线粒体膜电位，以评估细胞状态，信号转导，和线粒体功能。LAR下调通过增加细胞活力在鱼藤酮暴露的U251细胞中显示出保护作用，降低细胞死亡率，并恢复适当的细胞形态。LAR下调增强IGF-1R磷酸化和下游信号转导，如Akt和GSK-3β磷酸化增加所证明，以及NRF2和HO-1的上调。LAR的下调也增加了这些细胞中的DJ-1水平。增强的Akt和GSK-3β磷酸化有助于降低Bax/Bcl2比率并抑制鱼藤酮暴露后的细胞凋亡。杂合LAR敲除U251细胞表现出更高的线粒体功能，线粒体膜电位增加证明，ATP含量,与接触鱼藤酮后的WT细胞相比，ROS产生减少。进一步的研究表明，LAR杂合敲除介导的星形胶质细胞保护与Akt的激活有关。一种特定的Akt抑制剂，MK2206，降低了细胞活力，Akt和GSK3β磷酸化，以及暴露于鱼藤酮的U251细胞中HO-1和NRF2的表达。星形胶质细胞提供结构和代谢支持以维持神经元健康。星形胶质细胞源性神经营养因子（GDNF）的产生对于多巴胺能神经元的存活至关重要。杂合子LAR敲除U251细胞比WT细胞产生更大量的GDNF。与杂合LAR敲除U251细胞共培养的SH-SY5Y细胞比与WTU251细胞共培养的细胞在响应鱼藤酮时表现出更高的活力。一起,这些发现表明,在鱼藤酮诱导的PD细胞模型中,星形胶质细胞中LAR的杂合子敲除在保护星形胶质细胞和共培养神经元方面发挥关键作用.这种神经保护作用归因于IGF1R-Akt-GDNF信号传导的增强和星形细胞线粒体功能的维持。
