LoPachin Richard M, He Deke, Reid Maria L
Department of Anesthesiology, Albert Einstein College of Medicine, Montefiore Medical Center, Moses Research Tower-7, 111 E. 210th St., Bronx, NY 10467, USA.
Neurotoxicology. 2005 Mar;26(2):229-40. doi: 10.1016/j.neuro.2004.09.007.
Axon atrophy is the principle morphological feature of the peripheral neuropathy induced by 2,5-hexanedione (HD). Axon caliber is determined by a stationary neurofilamentous cytoskeleton that is maintained through dynamic interactions with mobile neurofilament (NF) subunits. To determine the effects of HD on the stationary and mobile NF pools, groups of rats were exposed to HD at dosing schedules (175 mg/kg x 101 days or 400 mg/kg x 26 days) that produced moderate levels of neurological deficits and, as assessed by previous studies, prevalent axon atrophy in peripheral nerve. Sciatic and tibial nerves from HD-intoxicated rats and their age-matched controls were triton-extracted and separated by differential centrifugation into a high-speed pellet (P1) of NF polymer and a corresponding supernatant fraction (S1), which presumably contained mobile monomer. Cytoskeletal proteins (NF-L, NF-M, NF-H and beta-tubulin) in each fraction were determined by immunoblot analysis. Results show that regardless of HD dose-rate, triton-soluble NF subunits in the supernatant fractions were significantly reduced, whereas triton-insoluble proteins in the corresponding pellets were inconsistently affected. Beta-tubulin also exhibited inconsistent fractional changes, while abnormal higher molecular weight NF proteins were detected primarily in the triton-insoluble fraction. Studies with antibodies directed against phosphorylated (RT97) and non-phosphorylated (SMI32) epitopes on NF-H did not reveal major changes in subunit phosphorylation. These results suggest that HD intoxication is primarily associated with depletion of soluble NF proteins, which could produce axon atrophy through disruption of cytoskeletal turnover and maintenance.
轴突萎缩是2,5 -己二酮（HD）诱发的周围神经病变的主要形态学特征。轴突管径由固定的神经丝细胞骨架决定，该骨架通过与可移动的神经丝（NF）亚基的动态相互作用得以维持。为了确定HD对固定和可移动NF库的影响，将几组大鼠按照能产生中度神经功能缺损水平的给药方案（175 mg/kg×101天或400 mg/kg×26天）暴露于HD，并且如先前研究所评估的，这些方案会导致周围神经普遍出现轴突萎缩。对HD中毒大鼠及其年龄匹配的对照大鼠的坐骨神经和胫神经进行曲拉通抽提，并通过差速离心分离成NF聚合物的高速沉淀（P1）和相应的上清液部分（S1），上清液部分可能含有可移动的单体。通过免疫印迹分析测定每个部分中的细胞骨架蛋白（NF-L、NF-M、NF-H和β-微管蛋白）。结果表明，无论HD剂量率如何，上清液部分中曲拉通可溶的NF亚基显著减少，而相应沉淀中曲拉通不溶的蛋白受到的影响不一致。β-微管蛋白也表现出不一致的部分变化，而异常的高分子量NF蛋白主要在曲拉通不溶部分中检测到。针对NF-H上磷酸化（RT97）和非磷酸化（SMI32）表位的抗体研究未发现亚基磷酸化有重大变化。这些结果表明，HD中毒主要与可溶性NF蛋白的耗竭有关，这可能通过破坏细胞骨架周转和维持而导致轴突萎缩。
