Foxo3 Mechanisms in Noise Damage

噪声性听力损失(NIHL)已使全世界数百万人致残。NIHL的个体风险 在相似的暴露条件下，不同的人不同，这表明遗传因素对 敏感度。我们发现，缺乏一种名为FOX03的转录因子的小鼠会变得严重和 在噪声暴露后永久耳聋，只会短暂地影响它们的野生子。FOXO_3有 其他类型细胞的多种功能，包括减少氧化应激、自噬和直接诱导 细胞凋亡。最近的一项研究将人类FOXO_3基因变异与职业病易感性联系起来 NIHL。然而，与NIHL相关的FOXO 3等位基因增加了FOXO 3的表达。因此，在那里 有证据表明FOXO_3对听力保护很重要，但也有证据表明过量 FOXO_3驱动NIHL。在这笔赠款中，我们试图通过研究 FOX03功能，使用翻译组测序、细胞特异性Foxo3条件基因敲除(CKO)和CRISPR 修改以产生可用于研究人类NIHL连锁FOX03等位基因的小鼠品系。 在Foxo3基因敲除(KO)小鼠中，噪声消除了高频外毛细胞(OHC)。我们证明了 这是通过一种称为甲状旁腺激素的快速细胞死亡程序来实现的，这是一种不依赖于半胱氨酸酶的细胞凋亡。 ParThanatos表明，在没有任何噪音伤害的情况下，Foxo3-KO毛囊已经准备好死亡。散装 来自对照Foxo3-KO和野生型窝种的RNA-Seq数据显示，没有证据表明氧化作用发生了变化 已知的受FOXO_3调控的减压剂。相反，我们看到肌动蛋白结合基因表达的变化 在急诊室里。在目标1中，我们建议验证这一筛查，并在Foxo3-KO中识别OHC痛苦的标志 通过翻译组测序。在目标2中，我们建议制作细胞特异性Foxo3-CKO来识别 FOXO_3的作用。野生型小鼠在毛细胞和周围支持细胞中都表达FOXO_3蛋白 细胞(SC)。通过使用针对OHC或SCS的可诱导DNA重组酶系，我们可以消融 FOX03在任何一种细胞类型中都起作用。我们将把这些Foxo3-CKO小鼠暴露在噪声中，并测定它们的NIHL 敏感度。最后，在目标3中，我们使用CRISPR基因改造技术创造了两只小鼠 一株具有控制序列(Foxo3-T等位基因小鼠)，以及一株与人类FOX03同源 使NIHL易感的等位基因(Foxo3-G等位基因小鼠)。我们将验证Foxo3-G等位基因小鼠 LINE在噪声暴露后耳蜗细胞中FOXO_3水平升高。我们假设这是 修饰促进了FOXO_3激活的细胞凋亡，我们将通过暴露FOXO_3- G等位基因小鼠对噪音的反应，测量它们的听力，并分析潜在的细胞损失。总而言之，通过这两个 在功能丧失和功能获得实验中，我们将分析FOXO_3在噪声性听力损失中的作用。

Noise induced hearing loss (NIHL) has disabled millions of people world-wide. Individual risk for NIHL varies from person to person under similar exposure conditions, suggesting that genetic factors contribute to susceptibility. We have found that mice lacking a transcription factor called FOXO3 become severely and permanently deafened after a noise exposure that only briefly affects their wild-type littermates. FOXO3 has multiple functions in other cell types, including oxidative stress reduction, autophagy, and directly inducing apoptosis. A recent study linked human genetic variations in FOXO3 to a greater susceptibility to occupational NIHL. However, the FOXO3 alleles associated with NIHL drive increased expression of FOXO3. Thus, there is evidence to indicate that FOXO3 is important for hearing preservation, but there is also evidence that excess FOXO3 drives NIHL. In this grant, we seek to address this knowledge gap by researching the mechanisms of FOXO3 function, using translatome sequencing, cell-specific Foxo3 conditional knockouts (cKO), and CRISPR modifications to generate mouse lines that can be used to investigate the human NIHL-linked FOXO3 allele. In Foxo3-knockout (KO) mice, noise eliminates high-frequency outer hair cells (OHCs). We show that this occurs through a rapid cell death program called parthanatos, which is caspase-independent apoptosis. Parthanatos indicates that in the absence of any noise damage, Foxo3-KO OHCs are primed for death. Bulk RNA-Seq data from control Foxo3-KO and wild-type littermates show no evidence for changes in oxidative stress reducers known to be regulated by FOXO3. Instead, we see changes in actin binding genes expressed in OHCs. In Aim 1, we propose to validate this screen and identify markers of OHC distress in the Foxo3-KO through translatome sequencing. In Aim 2, we propose to make cell-specific Foxo3-cKO to identify the cells in which FOXO3 acts. Wild-type mice express FOXO3 protein in both OHCs and in the surrounding supporting cells (SCs). By using inducible DNA recombinases lines specific to either OHCs or SCs, we can ablate FOXO3 function in either cell type. We will expose such Foxo3-cKO mice to noise and determine their NIHL susceptibility. Finally, in Aim 3, we have used CRISPR genetic modification technology to create two mouse lines, one with control sequences (Foxo3-T-allele mice), as well as one homologous to the human FOXO3 allele that confers NIHL susceptibility (Foxo3-G-allele mice). We will validate that the Foxo3-G-allele mouse line has increased levels of FOXO3 in cochlear cells after noise exposure. We hypothesize that this modification promotes apoptosis from FOXO3 activation, and we will test that hypothesis by exposing Foxo3- G-allele mice to noise, measuring their hearing and analyzing potential cellular losses. In sum, through both loss-of-function and gain-of-function experiments, we will analyze FOXO3's role in hearing loss from noise.