Roy Tapasi, Banerjee Rachana, Chatterjee Abhishek, Swarnakar Snehasikta
Infectious Diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Jadavpur, Kolkata, West Bengal, 700032, India.
JIS Institute of Advanced Studies and Research, JIS University, JIS School of Medical Science and Research Campus, 51, South Nayabaz, GIP Colony, Santragachi, Howrah, West Bengal, 711112, India.
Neurochem Res. 2025 Mar 4;50(2):111. doi: 10.1007/s11064-025-04362-6.
The death of dopaminergic neurons, a hallmark event during Parkinson's disease (PD), leads to increased dopamine concentration in the neuronal micro-environment. Keeping this in mind, we intend to understand the impact of elevated dopamine concentration on molecular interactions among proteins and the stability of the neuronal cytoskeleton. We used differentiated N2A cells and exposed them to 100 µM DA for 24 h. Evaluations of cell death, measurement of the concentration of DA oxidation products and reactive oxygen species (ROS), conventional RT-PCR, western blotting, zymography, reverse zymography, co-immunoprecipitation, mitochondrial transmembrane potential, confocal imaging, and in-silico studies were performed thereon. We observed that a significant number of viable N2A cells underwent ROS-dependent apoptotic cell death under elevated media DA concentrations. An altered transcriptional pattern of alpha-synuclein, UCHL1, and cofilin genes and their respective gene products were also observed. The activity and expression of matrix metalloproteinases9 (MMP9), involved in neuro-inflammation, was enhanced upon DA-exposure. Further, DA exposure also led to degradation of actin cytoskeleton. In silico studies revealed that interactions of Cofilin with UCHL1 and MMP9 were altered in dopamine-rich microenvironment. This result was further validated by co-immunoprecipitation experiments. Collectively our observations with murine neuroblastoma cells suggest that DA toxicity alters interaction patterns among intracellular proteins and degrades neuronal cytoskeleton that finally leads to cell death. Our study unveils a new frontier in PD treatment by paving the way for the development of specific drugs targeting the DA altered protein interactions.
多巴胺能神经元的死亡是帕金森病（PD）的标志性事件，会导致神经元微环境中多巴胺浓度升高。考虑到这一点，我们旨在了解多巴胺浓度升高对蛋白质间分子相互作用以及神经元细胞骨架稳定性的影响。我们使用分化的N2A细胞，并将它们暴露于100 µM多巴胺中24小时。对其进行了细胞死亡评估、多巴胺氧化产物和活性氧（ROS）浓度测量、常规逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹法、酶谱分析、反向酶谱分析、免疫共沉淀、线粒体跨膜电位、共聚焦成像和计算机模拟研究。我们观察到，在培养基多巴胺浓度升高的情况下，大量存活的N2A细胞经历了依赖ROS的凋亡性细胞死亡。还观察到α-突触核蛋白、泛素羧基末端水解酶L1（UCHL1）和丝切蛋白基因及其各自基因产物的转录模式发生了改变。暴露于多巴胺后，参与神经炎症的基质金属蛋白酶9（MMP9）的活性和表达增强。此外，多巴胺暴露还导致肌动蛋白细胞骨架的降解。计算机模拟研究表明，在富含多巴胺的微环境中，丝切蛋白与UCHL1和MMP9的相互作用发生了改变。免疫共沉淀实验进一步验证了这一结果。总体而言，我们对小鼠神经母细胞瘤细胞的观察表明，多巴胺毒性会改变细胞内蛋白质之间的相互作用模式，并降解神经元细胞骨架，最终导致细胞死亡。我们的研究通过为开发针对多巴胺改变的蛋白质相互作用的特异性药物铺平道路，为帕金森病治疗开辟了一个新领域。
