Department of Drug Discovery and Biomedical Sciences, Medical University of South Carolina, Charleston, SC.
Department of Ophthalmology, Medical University of South Carolina, Charleston, SC.
Mol Vis. 2021 Apr 2;27:151-160. eCollection 2021.
Recent reports linking HDAC6 to mitochondrial turnover and neurodegeneration led us to hypothesize that an inhibitor such as Vorinostat (suberoylanilide hydroxamic acid, SAHA) may reduce mitochondrial damage found in retinitis pigmentosa (RP), a progressive neurodegenerative disease of the eye. Here we tested the efficacy of SAHA for its ability to protect photoreceptors in in-vitro and in-situ models of RP. As the stressor, we focused on calcium overload. Calcium is one of the main drivers of cell death, and is associated with rod loss in the rd1 mouse retina, which harbors a mutation in the Pde6b gene similar to that found in human patients suffering from autosomal recessive RP.
Murine photoreceptor cell line (661W) were exposed to agents that led to calcium stress. Cell survival and redox capacity were measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, real-time changes in cellular metabolism were assessed using the Seahorse Biosciences XF24 analyzer, and mitochondrial fission-fusion using imaging. In-situ, neuroprotection was assessed in RPE/retina organ cultures of the rd1 mouse. SAHA effects on cell survival were compared in 661W cells with those of the specific HDAC6 inhibitor tubastatin A, and those on protein acetylation by Western blotting.
In stressed 661W cells, SAHA was found to increase cell survival that was associated with improved mitochondrial respiration and reduced mitochondrial fission. The protective effects of SAHA were also observed on photoreceptor cell survival in whole retinal organ explants of the rd1 mouse. Even though tubastatin A was ineffective in increasing cell survival in 661W cells, HDAC6 activity was confirmed in 661W cells after SAHA treatment with protein acetylation specific for HDAC6, defined by an increase in tubulin, but not histone acetylation.
SAHA was found to protect mitochondria from damage, and concomitantly reduced photoreceptor cell death in cell and organ cultures. The lack of activity of tubastatin A suggests that there must be an additional mechanism of action involved in the protective mechanism of SAHA that is responsible for its neuroprotection. Overall, SAHA may be a useful treatment for the prevention of photoreceptor degeneration associated with human RP. The results are discussed in the context of the effects of inhibitors that target different classes and members of the HDAC family and their effects on rod versus cone survival.
最近有研究报告将 HDAC6 与线粒体转化和神经退行性变联系起来，这促使我们假设，一种抑制剂，如伏立诺他（丁酸钠，SAHA），可能会减少视网膜色素变性（RP）中发现的线粒体损伤，RP 是一种进行性眼部神经退行性疾病。在这里，我们测试了 SAHA 保护体外和原位 RP 模型中光感受器的功效。作为应激源，我们专注于钙超载。钙是细胞死亡的主要驱动因素之一，与 rd1 小鼠视网膜中的杆状细胞丢失有关，rd1 小鼠的 Pde6b 基因中存在突变，类似于人类常染色体隐性 RP 患者的突变。
将鼠光感受器细胞系（661W）暴露于导致钙应激的试剂中。使用 3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴化物（MTT）测定法测量细胞存活率和氧化还原能力，使用 Seahorse Biosciences XF24 分析仪实时评估细胞代谢变化，并使用成像评估线粒体分裂融合。在 rd1 小鼠的 RPE/视网膜器官培养物中进行原位神经保护评估。在 661W 细胞中比较了 SAHA 对细胞存活率的影响与特定的 HDAC6 抑制剂 tubastatin A 的影响，并通过 Western blot 比较了它们对蛋白质乙酰化的影响。
在应激的 661W 细胞中，发现 SAHA 可增加细胞存活率，这与改善线粒体呼吸和减少线粒体分裂有关。SAHA 的保护作用也在 rd1 小鼠的整个视网膜器官外植体中的光感受器细胞存活中得到了观察。尽管 tubastatin A 不能增加 661W 细胞中的细胞存活率，但在 SAHA 处理后，661W 细胞中的 HDAC6 活性得到了确认，这是通过增加微管蛋白（而非组蛋白）乙酰化来定义的 HDAC6 特异性蛋白乙酰化。
发现 SAHA 可保护线粒体免受损伤，并同时减少细胞和器官培养物中的光感受器细胞死亡。tubastatin A 无活性表明，SAHA 的保护机制中必须涉及其他作用机制，这是其神经保护作用的原因。总体而言，SAHA 可能是预防与人类 RP 相关的光感受器变性的有用治疗方法。在讨论抑制剂的效果时，将考虑针对不同类别和成员的 HDAC 家族的抑制剂及其对杆状细胞与锥状细胞存活的影响。
