Foxo3 Mechanisms in Noise Damage

Foxo3 噪声损害机制

• 批准号: 10640125
• 负责人: Patricia M. White
• 金额: $ 31.48万
• 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/10640125
• 关键词: Ablation; Actin-Binding Protein; Actins; Address; Adult; Affect; Alleles; American; Animal Model; Apoptosis; Autophagocytosis; Binding; Biological Assay; Biological Markers; Breeding; 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; Induction of Apoptosis; Inner Hair Cells; 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; RiboTag; Ribosomes; Risk; Role; Single Nucleotide Polymorphism; Stimulus; Supporting Cell; Technology; Testing; Validation; Variant; Wild Type Mouse; Work; Workplace; biological adaptation to stress; biomarker identification; cell type; conditional knockout; deaf; disability; experimental study; gain of function; genetic variant; hearing impairment; hearing preservation; loss of function; mouse model; mutant; noise exposure; novel; overexpression; 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) 已导致全球数百万人残疾。 NIHL 的个体风险 在相似的暴露条件下，人与人之间存在差异，这表明遗传因素有助于 易感性。我们发现，缺乏一种名为 FOXO3 的转录因子的小鼠会变得严重且 在噪音暴露后，它们会永久失聪，而这种噪音只会短暂地影响它们的野生型同窝动物。 FOXO3 有 其他细胞类型中的多种功能，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 FOXO3 基因变异与对职业的更易感性联系起来 NIHL。然而，与 NIHL 相关的 FOXO3 等位基因驱动 FOXO3 表达增加。因此，有 有证据表明 FOXO3 对听力保护很重要，但也有证据表明，过量 FOXO3 驱动 NIHL。在这笔赠款中，我们寻求通过研究机制来解决这一知识差距 FOXO3 功能，使用翻译组测序、细胞特异性 Foxo3 条件敲除 (cKO) 和 CRISPR 修改以生成可用于研究人类 NIHL 连接的 FOXO3 等位基因的小鼠品系。 在 Foxo3 基因敲除 (KO) 小鼠中，噪音消除了高频外毛细胞 (OHC)。我们表明 这是通过一种称为“parthanatos”的快速细胞死亡程序发生的，该程序是不依赖半胱天冬酶的细胞凋亡。 Parthanatos 指出，在没有任何噪音损害的情况下，Foxo3-KO OHC 已准备好死亡。大部分 来自对照 Foxo3-KO 和野生型同窝小鼠的 RNA-Seq 数据没有显示氧化变化的证据 已知受 FOXO3 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 中。在目标 1 中，我们建议验证该筛选并识别 Foxo3-KO 中 OHC 应激的标记物 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 FOXO3 发挥作用。野生型小鼠在 OHC 和周围支持区域中表达 FOXO3 蛋白 细胞（SC）。通过使用 OHC 或 SC 特异性的诱导型 DNA 重组酶系，我们可以消除 FOXO3 在任一细胞类型中均发挥作用。我们将这些 Foxo3-cKO 小鼠暴露在噪音中并确定它们的 NIHL 易感性。最后，在目标3中，我们利用CRISPR基因改造技术创造了两只小鼠 品系，一种具有控制序列（Foxo3-T 等位基因小鼠），以及一种与人类 FOXO3 同源 赋予 NIHL 易感性的等位基因（Foxo3-G 等位基因小鼠）。我们将验证 Foxo3-G 等位基因小鼠 噪音暴露后，耳蜗细胞中的 FOXO3 水平增加。我们假设这 修饰促进 FOXO3 激活引起的细胞凋亡，我们将通过暴露 Foxo3- 来检验该假设 G 等位基因小鼠接受噪音，测量它们的听力并分析潜在的细胞损失。总而言之，通过两者 通过功能丧失和功能获得实验，我们将分析 FOXO3 在噪声引起的听力损失中的作用。

项目成果

Semi-Automated Analysis of Peak Amplitude and Latency for Auditory Brainstem Response Waveforms Using R.

• DOI: 10.3791/64737
• 发表时间: 2022-12-09
• 期刊: Journal of visualized experiments : JoVE
• 作者: Na D;White PM
• 通讯作者: White PM

Single cell RNA sequencing analysis of mouse cochlear supporting cell transcriptomes with activated ERBB2 receptor indicates a cell-specific response that promotes CD44 activation.

• DOI: 10.3389/fncel.2022.1096872
• 发表时间: 2022
• 期刊: FRONTIERS IN CELLULAR NEUROSCIENCE
• 影响因子: 5.3
• 作者: Piekna-Przybylska, Dorota;Na, Daxiang;Zhang, Jingyuan;Baker, Cameron;Ashton, John M.;White, Patricia M.
• 通讯作者: White, Patricia M.