Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, PL 31-343 Krakow, Poland.
Neurotoxicology. 2011 Dec;32(6):845-56. doi: 10.1016/j.neuro.2011.05.013. Epub 2011 Jun 1.
The dysfunction of the proteasome system is implicated in the pathomechanism of several chronic neurodegenerative diseases. Lactacystin (LC), an irreversible proteasome inhibitor, induces cell death in primary cortical neurons, however, the molecular mechanisms of its neurotoxic action has been only partially unraveled. In this study we aimed to elucidate an involvement of the key enzymatic pathways responsible for LC-induced neuronal cell death. Incubation of primary cortical neurons with LC (0.25-50 μg/ml) evoked neuronal cell death in concentration- and time-dependent manner. Lactacystin (2.5 μg/ml; 6.6μM) enhanced caspase-3 activity, but caspase-3 inhibitor, Ac-DEVD-CHO did not attenuate the LC-evoked cell damage. Western blot analysis showed a time-dependent, prolonged activation of MAPK/ERK1/2 pathway after LC exposure. Moreover, inhibitors of MAPK/ERK1/2 signaling, U0126 and PD98052 attenuated the LC-evoked cell death. We also found that LC-treatment resulted in the induction of calpains and calpain inhibitors (MDL28170 and calpeptin) protected neurons against the LC-induced cell damage. Neuroprotective action of MAPK/ERK1/2 and calpain inhibitors were connected with attenuation of LC-induced DNA fragmentation measured by Hoechst 33342 staining and TUNEL assay. However, only MAPK/ERK1/2 but not calpain inhibitors, attenuated the LC-induced AIF (apoptosis inducing factor) release. Further studies showed no synergy between neuroprotective effects of MAPK/ERK1/2 and calpain inhibitors given in combination when compared to their effects alone. The obtained data provided evidence for neuroprotective potency of MAPK/ERK1/2 and calpain, but not caspase-3 inhibition against the neurotoxic effects of LC in primary cortical neurons and give rationale for using these inhibitors in the treatment of neurodegenerative diseases connected with proteasome dysfunction.
蛋白酶体系统功能障碍与几种慢性神经退行性疾病的发病机制有关。乳酰基肽（LC）是一种不可逆的蛋白酶体抑制剂，可诱导原代皮质神经元细胞死亡，但它的神经毒性作用的分子机制尚未完全阐明。在这项研究中，我们旨在阐明参与 LC 诱导的神经元细胞死亡的关键酶途径。用 LC（0.25-50μg/ml）孵育原代皮质神经元以浓度和时间依赖的方式引起神经元细胞死亡。乳酰基肽（2.5μg/ml；6.6μM）增强了 caspase-3 的活性，但 caspase-3 抑制剂 Ac-DEVD-CHO 并不能减轻 LC 诱导的细胞损伤。Western blot 分析显示，LC 暴露后 MAPK/ERK1/2 途径呈时间依赖性、持续激活。此外，MAPK/ERK1/2 信号通路抑制剂 U0126 和 PD98052 减弱了 LC 诱导的细胞死亡。我们还发现，LC 处理导致钙蛋白酶及其抑制剂（MDL28170 和 calpeptin）的诱导，这些抑制剂保护神经元免受 LC 诱导的细胞损伤。MAPK/ERK1/2 和钙蛋白酶抑制剂的神经保护作用与 Hoechst 33342 染色和 TUNEL 测定法测量的 LC 诱导的 DNA 片段化的减弱有关。然而，只有 MAPK/ERK1/2 而不是钙蛋白酶抑制剂，减轻了 LC 诱导的 AIF（凋亡诱导因子）释放。进一步的研究表明，与单独使用相比，MAPK/ERK1/2 和钙蛋白酶抑制剂联合使用时，其神经保护作用没有协同作用。这些数据为 MAPK/ERK1/2 和钙蛋白酶的神经保护效力提供了证据，但 caspase-3 抑制对原代皮质神经元中 LC 的神经毒性作用无效，并为使用这些抑制剂治疗与蛋白酶体功能障碍有关的神经退行性疾病提供了依据。
