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.

噪声引起的听力损失（NIHL）使全球数百万人残疾。 NIHL的个人风险 在相似的暴露条件下，人之间因人而异，这表明遗传因素有助于 敏感性。我们发现，缺乏称为Foxo3的转录因子的小鼠变得严重变化，并且 噪音暴露后，永久性聋，只会短暂影响其野生型窝窝。 FOXO3有 其他细胞类型的多个功能，包括减少氧化应激，自噬和直接诱导 凋亡。最近的一项研究将FOXO3中的人类遗传变异与对职业的敏感性更大 NIHL。但是，与NIHL驱动相关的FOXO3等位基因增加了FOXO3的表达。因此，那里 有证据表明FOXO3对于听力保存很重要，但也有证据表明 FOXO3驱动NIHL。在这笔赠款中，我们试图通过研究的机制来解决这一知识差距 FOXO3功能，使用翻译组测序，特异性FOXO3条件敲除（CKO）和CRISPR 修改以生成可用于研究人NIHL连接FOXO3等位基因的小鼠线。 在FOXO3-KNOCKOUT（KO）小鼠中，噪声消除了高频外毛细胞（OHC）。我们表明 这是通过称为Parthanatos的快速细胞死亡程序发生的，parthanatos是与caspase无关的凋亡。 Parthanatos表明，在没有任何噪声损害的情况下，FoxO3-KO OHC被置于死亡。大部分 来自控制FOXO3-KO和野生型同窝仔的RNA-seq数据没有显示出氧化变化的证据 应力减少器已知由FOXO3调节。相反，我们看到表达的肌动蛋白结合基因的变化 在OHCS。在AIM 1中，我们建议验证此屏幕并确定FOXO3-KO中OHC遇险的标记 通过翻译组测序。在AIM 2中，我们建议使细胞特异性FOXO3-CKO识别细胞 FOXO3的作用。野生型小鼠在OHC和周围支撑架上表达FOXO3蛋白 细胞（SC）。通过使用特定于OHC或SC的诱导型DNA重组酶系线，我们可以消灭 FOXO3在任何一个单元格中的功能。我们将使这样的foxo3-cko小鼠暴露于噪声中，并确定其NIHL 敏感性。最后，在AIM 3中，我们使用了CRISPR遗传修饰技术来创建两只鼠标 线，一个具有控制序列（FOXO3-T-Allele小鼠）的线，以及与人Foxo3同源的线条 赋予NIHL敏感性的等位基因（FOXO3-G-Allele小鼠）。我们将验证FOXO3-G-Allele鼠标 噪声暴露后，线在耳蜗细胞中的FOXO3水平增加。我们假设这是 修改促进FOXO3激活中的细胞凋亡，我们将通过暴露FOXO3-- G-Allele小鼠到噪声，测量其听力并分析潜在的细胞损失。总而言之 功能丧失和功能收益实验，我们将分析FOXO3在噪声中听力丧失中的作用。

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.