Department of Biological Sciences, Hunter College, Biology and Biochemistry Programs, Graduate Center, The City University of New York, New York, NY 10065, USA.
School of Biological Sciences, Queen's University Belfast, Belfast BT9 7BL, United Kingdom; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 119334, Russia.
Biochim Biophys Acta Mol Basis Dis. 2017 Jun;1863(6):1157-1170. doi: 10.1016/j.bbadis.2017.03.017. Epub 2017 Apr 1.
In Alzheimer's disease proteasome activity is reportedly downregulated, thus increasing it could be therapeutically beneficial. The proteasome-associated deubiquitinase USP14 disassembles polyubiquitin-chains, potentially delaying proteasome-dependent protein degradation. We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14) neuronal cultures treated with prostaglandin J2 (PGJ2). IU1 concentrations (IU1>25μM) reported by others to inhibit USP14 and be protective in non-neuronal cells, reduced PGJ2-induced Ub-protein accumulation in neurons. However, IU1 alone or with PGJ2 is neurotoxic, induces calpain-dependent Tau cleavage, and decreases E1Ub thioester levels and 26S proteasome assembly, which are energy-dependent processes. We attribute the two latter IU1 effects to ATP-deficits and mitochondrial Complex I inhibition, as shown herein. These IU1 effects mimic those of mitochondrial inhibitors in general, thus supporting that ATP-depletion is a major mediator of IU1-actions. In contrast, low IU1 concentrations (IU1≤25μM) or USP14 knockdown by siRNA in rat cortical cultures or loss of USP14 in cortical cultures from ataxia (Usp14) mice, failed to prevent PGJ2-induced Ub-protein accumulation. PGJ2 alone induces Ub-protein accumulation and decreases E1Ub thioester levels. This seemingly paradoxical result may be attributed to PGJ2 inhibiting some deubiquitinases (such as UCH-L1 but not USP14), thus triggering Ub-protein stabilization. Overall, IU1-concentrations that reduce PGJ2-induced accumulation of Ub-proteins are neurotoxic, trigger calpain-mediated Tau cleavage, lower ATP, E1~Ub thioester and E1 protein levels, and reduce proteasome activity. In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.
在阿尔茨海默病中，蛋白酶体的活性据报道被下调，因此增加其活性可能具有治疗益处。与蛋白酶体相关的去泛素化酶 USP14 可分解多泛素链，从而可能延迟蛋白酶体依赖性蛋白降解。我们评估了在前列腺素 J2 (PGJ2) 处理的大鼠和小鼠 (Usp14) 神经元培养物中抑制或下调 USP14 的保护效果。其他人报道的 IU1 浓度（IU1>25μM）抑制 USP14 并在非神经元细胞中具有保护作用，可减少 PGJ2 诱导的神经元中 Ub-蛋白积累。然而，IU1 单独或与 PGJ2 一起具有神经毒性，诱导钙蛋白酶依赖性 Tau 切割，并降低 E1Ub 硫酯水平和 26S 蛋白酶体组装，这些都是能量依赖的过程。我们将 IU1 的后两个作用归因于本文所示的 ATP 缺乏和线粒体复合物 I 抑制。这些 IU1 作用模拟了一般的线粒体抑制剂的作用，因此支持 ATP 耗竭是 IU1 作用的主要介导物。相比之下，在大鼠皮质培养物中用低浓度 IU1（IU1≤25μM）或 siRNA 敲低 USP14，或在共济失调（Usp14）小鼠的皮质培养物中缺失 USP14，均不能阻止 PGJ2 诱导的 Ub-蛋白积累。PGJ2 本身诱导 Ub-蛋白积累并降低 E1Ub 硫酯水平。这个看似矛盾的结果可能归因于 PGJ2 抑制了一些去泛素化酶（如 UCH-L1，但不是 USP14），从而触发 Ub-蛋白稳定。总的来说，降低 PGJ2 诱导的 Ub-蛋白积累的 IU1 浓度具有神经毒性，触发钙蛋白酶介导的 Tau 切割，降低 ATP、E1~Ub 硫酯和 E1 蛋白水平，并降低蛋白酶体活性。总之，药理学抑制（用低或高 IU1 浓度）或遗传下调 USP14 均不能增强神经元中 Ub-蛋白或 Tau 的蛋白酶体降解。
