Activation of multiple pathways during photoreceptor apoptosis in the rd mouse
Activation of multiple pathways during photoreceptor apoptosis in the rd mouse
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DOI：
10.1167/iovs.05-0248
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发表时间：
2005-10-01
影响因子：
4.4
通讯作者：
Cotter, TG
中科院分区：
文献类型：
--
作者：
Doonan, F;Donovan, M;Cotter, TG
目的。本研究的主要目的是研究RD小鼠光感受器细胞凋亡的特征。鉴于细胞凋亡是许多视网膜变性的最终共同途径，延缓甚至阻止这一过程的能力可能会改善视网膜疾病，如视网膜色素变性(RP)。没有任何公认的治疗方法强调了这样一个事实，即对所涉及的分子事件的详细了解对于确定合理的治疗干预靶点是必要的。方法：使用流式细胞术来测量光感受器群体的物理和化学特征。单个细胞悬浮流过一个或多个激光，散射光并发出荧光。Western印迹技术显示了钙蛋白酶特异性底物的裂解。视网膜外植体培养用于抑制物研究。结果培养后第10天(P-10)视网膜无视网膜色素上皮(RPE)附着至P-17。结果胞浆及线粒体钙超载。随后，在细胞死亡高峰期检测到线粒体膜去极化和活性氧(ROS)。对下游事件的分析表明，钙激活的钙调蛋白酶在早期被激活。用钙蛋白酶抑制剂N-乙酰-亮氨酸-亮氨酸-NLE-CHO(ALLN)处理RD视网膜外植体后，成功地抑制了Calain诱导的α-fodrin裂解，但不能防止光感受器退化。结论天门冬氨酸蛋白酶前体和加工型组织蛋白表达水平均升高，钙内流过多是启动钙激活蛋白水解酶的早期事件。然而，这些蛋白水解酶并不是导致死亡的特殊原因，因为它们的抑制并不能阻止细胞凋亡。事实上，这里提出的结果表明，涉及多个途径，并且可能必须解决这些成分中的每一个才能成功地抑制细胞死亡。
PURPOSE. The primary purpose of this study was to characterize photoreceptor apoptosis in the rd mouse. Given that apoptosis is the final common pathway in many cases of retinal degeneration, the ability to retard or even arrest this process may ameliorate retinal disorders such as retinitis pigmentosa ( RP). The absence of any recognized therapy emphasizes the fact that a detailed knowledge of the molecular events involved is necessary to identify rational targets for therapeutic intervention.METHODS. Flow cytometry was used to measure physical and chemical characteristics in the photoreceptor population. Individual cells flow in suspension past one or more lasers, scattering light and emitting fluorescence. Western blot techniques demonstrated cleavage of calpain-specific substrates. Retinal explant cultures were used for inhibitor studies. Postnatal day 10 (P-10) rd retinas were cultured without retinal pigment epithelium (RPE) attached up to P-17.RESULTS. This study demonstrated calcium overload in the cytosol and subsequently in mitochondria. Mitochondrial membrane depolarization and reactive oxygen species ( ROS) were detected later, during the peak of cell death. Analysis of downstream events indicated early activation of calcium-activated calpains. Treatment of rd retinal explants with the calpain inhibitor N-acetyl- Leu- Leu- Nle-CHO (ALLN) successfully inhibited calpain-induced alpha-fodrin cleavage, yet it did not protect against photoreceptor degeneration. Finally, the results demonstrate an increase in the levels of both precursor and processed forms of the aspartate protease cathepsin D.CONCLUSIONS. Excessive calcium influx is an early event that initiates the activation of calcium-activated proteases. However, these proteases are not singularly the cause of death, because their inhibition does not prevent apoptosis. Indeed, the results presented herein suggest that multiple pathways are involved and that each of these components may have to be addressed for cell death to be successfully inhibited.