Foxo3 Mechanisms in Noise Damage

Foxo3 噪声损害机制

• 批准号: 10301834
• 负责人: Patricia M. White
• 金额: $ 35.1万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10301834
• 关键词: Actin-Binding Protein; Actins; Address; Adult; Affect; Alleles; American; Animal Model; Apoptosis; Autophagocytosis; Binding; Biological Assay; Biological Markers; CRISPR/Cas technology; Caspase; Cell Death; Cell Survival; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Cochlea; DNA; Data; Disabled Persons; Distress; Enterobacteria phage P1 Cre recombinase; FOXO3A gene; Frequencies; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Goals; Grant; Hearing; Hearing Protection; Hearing Tests; Heat-Shock Proteins 70; Homeostasis; Hour; Human; Human Genetics; Individual; Knock-out; Knockout Mice; Knowledge; Link; LoxP-flanked allele; Measures; Methods; Modeling; Modification; Mus; Neurons; Noise; Noise-Induced Hearing Loss; Nuclear; Nucleic Acid Regulatory Sequences; Occupational; Occupational Noise; Ontology; Outer Hair Cells; Oxidative Stress; Persons; Population; Predisposition; Proteins; RNA; RNA analysis; Recovery; Research; RiboTag; Ribosomes; Risk; Role; Single Nucleotide Polymorphism; Stimulus; Sum; Supporting Cell; Technology; Testing; Validation; Variant; Wild Type Mouse; Work; Workplace; biological adaptation to stress; cell type; conditional knockout; deaf; disability; experimental study; gain of function; genetic variant; hearing impairment; hearing preservation; loss of function; mouse model; mutant; novel; preservation; programs; protein expression; rat Pres protein; recombinase; response; sound; spiral ganglion; stress reduction; transcription factor; transcriptome sequencing; translatome

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.

噪声性听力损失（NIHL）已使全球数百万人致残。NIHL的个体风险 在类似的暴露条件下，每个人的情况都不同，这表明遗传因素有助于 易感性我们发现，缺乏一种称为FOXO 3的转录因子的小鼠会变得严重， 在噪声暴露后永久性地退化，仅短暂地影响它们的野生型同窝仔。FOXO 3具有 在其他细胞类型中的多种功能，包括氧化应激减少、自噬和直接诱导 凋亡最近的一项研究将人类FOXO 3的遗传变异与职业性疾病的易感性联系起来。 NIHL。然而，与NIHL相关的FOXO 3等位基因驱动FOXO 3表达增加。因此 有证据表明FOXO 3对听力保护很重要，但也有证据表明， FOXO 3驱动NIHL。在这项资助中，我们试图通过研究 FOXO 3功能，使用翻译组测序，细胞特异性Foxo 3条件性敲除（cKO）和CRISPR 修饰以产生可用于研究人NIHL连锁的FOXO 3等位基因的小鼠系。 在Foxo 3基因敲除（KO）小鼠中，噪音消除了高频外毛细胞（OHC）。我们证明了 这是通过称为parthanatos的快速细胞死亡程序发生的，这是半胱天冬酶非依赖性细胞凋亡。 Parthanatos指出，在没有任何噪音损害的情况下，Foxo 3-KO OHC是死亡的准备。散装 来自对照Foxo 3-KO和野生型同窝仔的RNA-Seq数据显示没有证据表明氧化修饰的改变。 已知由FOXO 3调节的减压剂。相反，我们看到肌动蛋白结合基因表达的变化， 在OHC。在目标1中，我们建议验证该筛选并识别Foxo 3-KO中OHC困扰的标记物 通过translatome测序。在目的2中，我们提出了制备细胞特异性Foxo 3-cKO以鉴定细胞中的细胞。 FOXO 3的作用。野生型小鼠在OHC和周围支持细胞中表达FOXO 3蛋白。 细胞（SC）。通过使用对OHC或SC特异的诱导型DNA重组酶系，我们可以消融 FOXO 3在任一细胞类型中发挥作用。我们将这些Foxo 3-cKO小鼠暴露于噪声中，并测定其NIHL。 易感性最后，在目标3中，我们使用CRISPR基因修饰技术创造了两种小鼠， 一个具有对照序列（Foxo 3-T-等位基因小鼠），以及一个与人FOXO 3同源 赋予NIHL易感性的等位基因（Foxo 3-G-等位基因小鼠）。我们将验证Foxo 3-G等位基因小鼠 噪声暴露后耳蜗细胞FOXO 3水平升高。我们假设这 修饰促进FOXO 3激活引起的凋亡，我们将通过暴露FOXO 3- G等位基因小鼠暴露于噪音，测量它们的听力并分析潜在的细胞损失。总之，通过这两个 通过功能丧失和功能获得实验，我们将分析FOXO 3在噪声性听力损失中的作用。