Sánchez C, Arellano J I, Rodríguez-Sánchez P, Avila J, DeFelipe J, Díez-Guerra F J
Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
Neuroscience. 2001;107(1):25-33. doi: 10.1016/s0306-4522(01)00338-4.
Microtubule-associated protein 2 (MAP2) is an abundant component of the neuronal cytoskeleton whose function is related to the outgrowth and stability of neuronal processes, to synaptic plasticity and neuronal cell death. We have sought to study whether abnormal patterns of neuronal activity which are characteristic of epileptic patients are associated to alterations of MAP2 phosphorylation. An antibody (305) that selectively recognizes a phosphorylated epitope in a proline-rich region of the MAP2 molecule has been used to analyze neocortical biopsy samples from temporal lobe epileptic patients, whose electrocorticogram activity had been previously monitored. Immunoblot analysis showed that samples with greater spiking activity displayed significantly diminished MAP2 phosphorylation. Immunocytochemical analysis revealed the occurrence of discrete areas in the neocortex with highly decreased or no immunostaining for antibody 305, which showed a clear, although non-significant, tendency to appear more frequently in areas with greater spiking activity. To further support an association between epileptiform activity and MAP2 dephosphorylation an experimental model of epileptiform activity in cultures of rat hippocampal neurons was used. Neurons were cultured during 15 days in the presence of kynurenic acid, an antagonist of glutamate receptors. At this time, kynurenic acid was removed from the culture medium and neurons developed seizure-like activity. Using antibody 305, we found a decrease of MAP2 phosphorylation that was already visible after 15 min of kynurenic acid withdrawal. We therefore propose that MAP2 phosphorylation is decreased in the neocortex of epileptic patients and that this decrease is a likely consequence of seizure activity. Also, MAP2 dephosphorylation may lead to alterations of the neuronal cytoskeleton and eventually to neuronal damage and loss, which is typical of epileptic patients.
微管相关蛋白2（MAP2）是神经元细胞骨架的一种丰富成分，其功能与神经元突起的生长和稳定性、突触可塑性以及神经元细胞死亡有关。我们试图研究癫痫患者所特有的异常神经元活动模式是否与MAP2磷酸化的改变有关。一种能选择性识别MAP2分子富含脯氨酸区域中磷酸化表位的抗体（305）已被用于分析颞叶癫痫患者的新皮质活检样本，这些患者的皮质电图活动此前已被监测。免疫印迹分析表明，具有更高棘波活动的样本显示MAP2磷酸化显著减少。免疫细胞化学分析显示，新皮质中存在离散区域，对抗体305的免疫染色高度降低或无免疫染色，这些区域在棘波活动更强的区域出现的频率虽无明显差异，但有明显的增加趋势。为了进一步支持癫痫样活动与MAP2去磷酸化之间的关联，我们使用了大鼠海马神经元培养物中的癫痫样活动实验模型。神经元在犬尿氨酸（一种谷氨酸受体拮抗剂）存在的情况下培养15天。此时，将犬尿氨酸从培养基中去除，神经元出现癫痫样活动。使用抗体305，我们发现犬尿氨酸去除15分钟后，MAP2磷酸化就已明显降低。因此，我们提出癫痫患者新皮质中的MAP2磷酸化降低，这种降低可能是癫痫活动的结果。此外，MAP2去磷酸化可能导致神经元细胞骨架改变，并最终导致神经元损伤和丢失，这是癫痫患者的典型特征。
