Draf Clara, Wyrick Taylor, Chavez Eduardo, Pak Kwang, Kurabi Arwa, Leichtle Anke, Dazert Stefan, Ryan Allen F
Department of Surgery/Otolaryngology, University of California, San Diego, San Diego, CA, United States.
Department of Otolaryngology, St. Elisabeth-Hospital, Ruhr University Bochum, Bochum, Germany.
Front Cell Dev Biol. 2021 Oct 26;9:762751. doi: 10.3389/fcell.2021.762751. eCollection 2021.
Autophagy is a degradative pathway to safely break down and recycle dysfunctional cellular components. There is prior evidence of autophagy participation during hair cell (HC) damage. Our goal was to screen compounds targeting different aspects of autophagy for their effects on HC loss due to an ototoxic aminoglycoside, gentamicin (GM). The SELLECKChem autophagy compound library, consisting of 154 compounds with defined autophagy inducing or inhibitory activity, was used for targeted screening model of ototoxicity. Organ of Corti from postnatal days 3-5 pou4f3/GFP transgenic mice (HCs express green fluorescent protein) were utilized. The organs were micro-dissected, and basal and middle turns divided into micro-explants individually placed into the single wells of a 96-well plate. Samples were treated with 200 μM of GM plus three dosages of tested compound and cultured for 72 h. Negative controls were treated with media only; positive ototoxicity controls were treated with GM only. The majority of the library compounds had no effect on GM-induced HC loss. However, 18 compounds exhibited a significant, protective effect, two compounds were protective at low dosage but showed enhanced GM toxicity at higher doses and one compound was toxic to HCs in the absence of GM. This study evaluated many autophagy compounds that have not been tested previously on HCs. The disparate results obtained underscore the complexity of autophagy events that can influence HC responses to aminoglycosides, but also implicate the proteosome as an important damage mechanism. The screening results can serve as basis for further studies with protective compounds as potential drug targets.
自噬是一种降解途径，可安全地分解和回收功能失调的细胞成分。先前有证据表明自噬参与毛细胞（HC）损伤过程。我们的目标是筛选针对自噬不同方面的化合物，以研究其对由耳毒性氨基糖苷类药物庆大霉素（GM）导致的HC损失的影响。使用了SELLECKChem自噬化合物库，该库由154种具有明确自噬诱导或抑制活性的化合物组成，用于耳毒性的靶向筛选模型。利用出生后3 - 5天的pou4f3/GFP转基因小鼠（HC表达绿色荧光蛋白）的柯蒂氏器。将这些器官进行显微解剖，将基底转和中转分别分成微切片，单独放入96孔板的单个孔中。样品用200μM的GM加三种剂量的受试化合物处理，并培养72小时。阴性对照仅用培养基处理；阳性耳毒性对照仅用GM处理。该库中的大多数化合物对GM诱导的HC损失没有影响。然而，有18种化合物表现出显著的保护作用，两种化合物在低剂量时具有保护作用，但在高剂量时显示出增强的GM毒性，还有一种化合物在没有GM的情况下对HC有毒性。本研究评估了许多以前未在HC上测试过的自噬化合物。所获得的不同结果强调了自噬事件的复杂性，这些事件可影响HC对氨基糖苷类药物的反应，但也表明蛋白酶体是一种重要的损伤机制。筛选结果可为进一步研究将保护性化合物作为潜在药物靶点提供依据。
