Evaluating anti-oxidant and autophagy compounds as potential therapies for mammalian aminoglycoside-induced hair cell loss

人口发展概述了老年人疾病的日益重要性，例如感音神经性听力损失（SNHL）（>65岁高达50%）。目前尚无法预防由大多数原因（如药物、遗传或衰老）引起的SNHL。噪声性听力损失可以通过耳塞或保护器来预防，但在必要时通常不会佩戴。SNHL很难治疗，因为它会造成灵敏度的丧失和感知声音的失真。这部分是由于与外毛细胞（HC）损失相关的频率特异性损失，但也由于内毛细胞上的传入突触损失，这可能不会反映在听力损失中（Kujawa和Liberman，2015）。助听器可以增加灵敏度，但通常不能克服声音失真（Silverman，1998）。因此，迫切需要新的SNHL预防和治疗方法。先前使用体外干预的研究已经表征了导致HC损伤和丢失（Battaglia，2003; Brand，2015）或影响HC再生（增田，2006）的许多途径。该知识库为保护HC的药物干预提供了机会。提交的项目涉及抗氧化剂和自噬化合物的评估，这些化合物可以预防和保护HC免受氨基糖苷（AG）诱导的毛细胞损失。目的：短期目标是确定I）抗氧化剂在体内保护HC和听力免受损伤的相对潜力。II）自噬化合物在体外影响HC损伤的相对潜力。该项目的长期目标是增加对不同类型的SNHL（如AG诱导的HC损伤和噪声诱导的SNHL）的预防和改善治疗选择。将每只小鼠暴露于105 dB SPL（声压级）的倍频程噪声（12-24 kHz）中30分钟。暴露产生约65分贝的阈值移动测量后立即噪音，和40分贝的永久性阈值移动测量两周后，和损失主要是外部HC。实验方案II：对于体外自噬筛选，将使用Pou 4f 3/GFP转基因小鼠，其中HC选择性表达绿色荧光蛋白（GFP）。将对Corti器官进行显微解剖，并丢弃对AG相对不敏感的顶圈。基部和中部将被分成短片段或微外植体。通过靶向筛选，我们将使用与先前体外氧化还原筛选相同的测定来评估筛选孔自噬文库，以鉴定具有影响AGS诱导的HC损伤的能力的化合物。

Demographic development outlines an increasing importance for diseases of the elderly, such as sensorineural hearing loss (SNHL) (>65 years up to 50%). Prevention is not currently possible for SNHL induced by most causes such as drugs, genetics or aging. Noise-induced hearing loss can be prevented with ear plugs or protectors, but they are frequently not worn when necessary. SNHL is difficult to treat, since it creates both loss of sensitivity and distortion of perceived sounds. This is partly due to loss of frequency specifity associated with outer hair cell (HC) loss, but also to loss of afferent synapses on the inner HCs, that may not be reflected in loss of hearing sensitivity (Kujawa and Liberman, 2015). Hearing aids increase sensitivity, but typically do not overcome sound distortion (Silverman, 1998). New methods for SNHL prevention and treatment are urgently needed. Prior research using in vitro intervention has characterized a number of pathways that contribute to HC damage and loss (Battaglia, 2003; Brand, 2015) or influence HC regeneration (Masuda, 2006). This knowledge base provides opportunities for pharmacological intervention to protect HCs. The submitted project deals with the evaluation of anti-oxidant and autophagy compounds, which may prevent and protect HCs against aminoglycoside (AG)-induced hair cell loss. Aim:The short-term objective is to determineI) relative potential of antioxidants to protect HCs and hearing from damage in vivo.II) relative potential of autophagy compounds to influence HC damage in vitro.The long term goal of this project is to increase prevention and improve treatment options for different types of SNHL such as AG-induced HC damage and noise- induced SNHL.Methods:Experimental Plan I:For the in vivo test of anti-oxidants, FVB mice will be employed, which do not display age-related hearing loss or increased noise sensitivity. Each mouse will be exposed to octave-band noise (12-24 kHz) at 105 dB SPL (sound pressure level) for 30 minutes. The exposure produces approximately 65 dB of threshold shift measured immediately after noise, and 40 dB of permanent threshold shift measured two weeks later, and loss primarily of outer HCs. The effects of perilymph delivery of three dosages of the top four antioxidant molecules, identified as most effective in a previous in vitro screen, will be compared.Experimental Plan II:For the in vitro autophagy screen, Pou4f3/GFP transgenic mice will be used, in which HCs selectively express green fluorescent protein (GFP). The organs of Corti will be micro-dissected and the apical turns, relatively insensitive to AGs, will be discarded. The basal and middle turns will be divided into short segments, or micro-explants. By means of targeted screening we will assess the Screen-Well Autophagy Library, using an identical assay to that used for the prior in vitro redox screen, to identify compounds with the ability to influence AGS-induced HC damage.