Grant R J, Sellings L H L, Crocker S J, Melloni E, Park D S, Clarke P B S
Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir-William-Osler Room 1325, Montreal, Quebec, Canada H3G 1Y6.
Neuroscience. 2009 Jan 23;158(2):558-69. doi: 10.1016/j.neuroscience.2008.10.023. Epub 2008 Nov 1.
The neurotoxin 6-hydroxydopamine has been widely used to model aspects of Parkinson's disease in rodents, but the mechanisms underlying toxin-induced dopaminergic degeneration and functional impairment have not been fully elucidated. The main aim of the present study was to assess a possible role for calpains in neurochemical and behavioral deficits following unilateral infusion of intrastriatal 6-hydroxydopamine in adult rats. Toxin administration produced a profound dopaminergic denervation, as indicated by a 90-95% reduction in dopamine transporter radiolabeling measured in the caudate-putamen at 2 weeks post-lesion. Treatment with 6-hydroxydopamine also resulted in calpain activation in both caudate-putamen and substantia nigra, as measured by the appearance of calpain-specific spectrin breakdown products. Calpain activation peaked at 24 h after 6-hydroxydopamine infusion and remained elevated at later time points. In contrast, caspase-3-mediated spectrin cleavage subsided within 48 h in both brain areas. In a subsequent experiment, calpain inhibition was achieved by intrastriatal infusion of an adenovirus expressing the endogenous calpain inhibitor, calpastatin. Calpastatin delivery abolished the lesion-induced calpain-mediated spectrin cleavage and alleviated forelimb asymmetries resulting from unilateral intrastriatal 6-hydroxydopamine. Unexpectedly, dopamine transporter and tyrosine hydroxylase labeling revealed significant neuroprotection, not in the nigrostriatal pathway but rather in the ventral tegmental area. These findings support a role for calpain activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons. However, after near-total dopaminergic depletion, the primary benefit of calpain inhibition may not occur within the nigrostriatal dopaminergic pathway itself.
神经毒素6-羟基多巴胺已被广泛用于建立啮齿动物帕金森病某些方面的模型，但毒素诱导的多巴胺能神经元变性和功能损害的潜在机制尚未完全阐明。本研究的主要目的是评估钙蛋白酶在成年大鼠单侧纹状体内注射6-羟基多巴胺后神经化学和行为缺陷中可能发挥的作用。毒素给药导致了严重的多巴胺能去神经支配，损伤后2周在尾状核-壳核中测量的多巴胺转运体放射性标记减少了90%-95%，这表明了这一点。用6-羟基多巴胺处理还导致尾状核-壳核和黑质中钙蛋白酶的激活，这通过钙蛋白酶特异性血影蛋白降解产物的出现来测量。钙蛋白酶激活在6-羟基多巴胺注射后24小时达到峰值，并在随后的时间点保持升高。相比之下，caspase-3介导的血影蛋白裂解在两个脑区均在48小时内消退。在随后的实验中，通过纹状体内注射表达内源性钙蛋白酶抑制剂钙蛋白酶抑制蛋白的腺病毒来实现钙蛋白酶抑制。钙蛋白酶抑制蛋白的递送消除了损伤诱导的钙蛋白酶介导的血影蛋白裂解，并减轻了单侧纹状体内注射6-羟基多巴胺导致的前肢不对称。出乎意料的是，多巴胺转运体和酪氨酸羟化酶标记显示出显著的神经保护作用，不是在黑质纹状体通路，而是在腹侧被盖区。这些发现支持钙蛋白酶激活在6-羟基多巴胺诱导的多巴胺能神经元变性中发挥作用。然而，在多巴胺能几乎完全耗竭后，钙蛋白酶抑制的主要益处可能不会出现在黑质纹状体多巴胺能通路本身。
