The role of TMTC4, endoplasmic reticulum Ca2+ flux, and the unfolded protein response in noise-induced hearing loss

TMTC4、内质网 Ca2 通量和未折叠蛋白反应在噪声性听力损失中的作用

• 批准号: 10599869
• 负责人: Dylan Chan
• 金额: $ 65.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599869
• 关键词: Affect; American; Animal Model; Apoptosis; Apoptotic; Attenuated; Biochemical; Biological Models; Ca(2+)-Transporting ATPase; Calcium; Caspase; Cell Death; Cell Line; Cell Survival; Cell model; Cells; Cellular Stress; Chemicals; Clinical; Cochlea; Critical Pathways; Cytoplasm; Cytosol; Data; Development; Ear; Endoplasmic Reticulum; Equilibrium; Event; Exhibits; FDA approved; Family; Foundations; Functional disorder; Gene Expression; Genes; Genetic; Genetic Models; Genetic Techniques; Genetic Transcription; Gerbils; Grant; Hair Cells; Hearing; Heterozygote; Homeostasis; Hour; Human; Image; Impairment; Induction of Apoptosis; Knock-out; Knockout Mice; Learning; Link; Loudness; Macromolecular Complexes; Measures; Mediating; Mediator; Messenger RNA; Modeling; Mus; Mutation; Neonatal; Noise; Noise-Induced Hearing Loss; Pathway interactions; Pattern; Pharmaceutical Preparations; Phase; Physiological; Predisposition; Process; Proteins; Public Health; Publishing; Research Proposals; Role; Series; Signal Transduction; Specificity; Stimulus; Supporting Cell; Testing; Time; Translational Research; Trauma; Up-Regulation; Variant; Wild Type Mouse; arm; cell type; clinically relevant; conditional knockout; deaf; deafness; endoplasmic reticulum stress; experimental study; genetic deafness; hearing impairment; in vivo; in vivo Model; insight; multidisciplinary; noise exposure; normal hearing; novel; ototoxicity; pharmacologic; positive allosteric modulator; prevent; prevent hearing loss; progressive hearing loss; protein folding; release of sequestered calcium ion into cytoplasm; response; reuptake; segregation; sound; targeted treatment; therapeutic development; therapeutic target

Noise-induced hearing loss (NIHL) is a significant public health problem, affecting nearly 40 million Americans. We have made the exciting discovery that NIHL may be linked to the unfolded protein response (UPR), a critical early response mechanism to cellular stress that has downstream effectors that can promote both cell survival and apoptosis. In support of this, we have additionally identified and characterized a novel deafness gene in mice, Tmtc4, which has also been recently identified as a potential deafness gene in a human family. Mice in which Tmtc4 is genetically absent (Tmtc4 knockout (KO) mice) hear normally at the onset of hearing but rapidly become deaf within 2 weeks and have markedly increased susceptibility to NIHL. We have found that Tmtc4 is broadly expressed in cochlear hair cells and supporting cells, both of which degenerate over time in Tmtc4 KO mice. We have shown that Tmtc4 is part of a macromolecular complex involved in clearing calcium (Ca2+) from the cytoplasm into the endoplasmic reticulum (ER), and that cochlear cells from Tmtc4 KO mice have impairments in intracellular Ca2+ homeostasis and dynamics. This impairment in Ca2+ management leads to upregulation of the UPR and cell death in the Tmtc4 KO cochlea. In parallel with this genetic deafness model of UPR dysregulation, we have found that NIHL in wild-type (WT) mice results in UPR upregulation within 2 hours of noise exposure; this hearing loss could be prevented in part by treatment with one drug, ISRIB, that specifically targets the UPR, or a second drug, CDN1163, that facilitates Ca2+ reuptake into the ER. These preliminary findings strongly implicate the UPR as an early mediator of cellular stress in the cochlea, upstream of other previously studied apoptotic mechanisms, and thus is a potential therapeutic target for a wide range of acquired and genetic forms of hearing loss. In this proposal, our specific aims are to investigate 1) how, in cell lines, TMTC4 dysfunction, including human variants associated with hearing loss, affect ER Ca2+ flux and, subsequently, UPR activation; 2) how, in the cochlea, noise-induced trauma in the form of hair-cell tip-link disruption and ER Ca2+ depletion activate the UPR to induce hair-cell loss; and 3) how, in in vivo models of hearing loss, the UPR is modulated to give rise to different patterns of hearing loss and hair-cell death. These Aims will be achieved using a multidisciplinary set of physiologic, biochemical, pharmacologic, and genetic techniques including ER Ca2+ imaging, mRNA transcriptional analysis, and genetic TMTC4 conditional knockout mice. Through these experiments, we will gain valuable insight into the mechanisms by which ER Ca2+ flux and the UPR are involved in genetic and noise-induced hearing loss, laying the foundation for development of targeted therapies for NIHL, a critical unmet clinical need.

噪声引起的听力损失（NIHL）是一个重大的公共卫生问题，影响了近4000万美国人。 我们已经提出了令人兴奋的发现，即NIHL可能与未展开的蛋白质反应（UPR）有关 对具有下游效应子的细胞应激的关键早期反应机制，可以促进两个细胞 生存和凋亡。为此，我们还确定并表征了一种新颖的耳聋 小鼠中的基因TMTC4，该基因最近也被确定为人类家庭中潜在的耳聋基因。 TMTC4在遗传上不存在的小鼠（TMTC4敲除（KO）小鼠）正常听到听力开始时听到 但是在2周内迅速变得聋，并显着提高了对NIHL的敏感性。我们找到了 TMTC4在人工耳蜗细胞和支持细胞中广泛表达，这两个都随着时间而退化 在TMTC4 KO小鼠中。我们已经表明，TMTC4是与清除有关的大分子复合物的一部分 钙（Ca2+）从细胞质进入内质网（ER），并从TMTC4 KO中的耳蜗细胞 小鼠在细胞内Ca2+稳态和动力学方面有损害。 CA2+管理中的这种损害 导致TMTC4 KO耳蜗中UPR和细胞死亡的上调。与这种遗传耳聋并行 UPR失调的模型，我们发现野生型（WT）小鼠的NIHL导致UPR上调 在噪音暴露的2小时内；可以通过一种药物治疗来阻止这种听力损失， 伊斯里布（Isrib）专门针对UPR或第二种药物CDN1163，可促进Ca2+再摄取到ER中。 这些初步发现强烈暗示了UPR是耳蜗中细胞应激的早期介体， 其他先前研究的凋亡机制的上游，因此是A 听力损失的广泛获得和遗传形式。 在此提案中，我们的具体目的是研究1）在细胞系中，如何使用TMTC4功能障碍，包括 与听力丧失相关的人类变异，影响ER Ca2+通量，然后会影响UPR激活； 2）如何，在 耳蜗诱导的手电孔尖端连接的形式的耳蜗诱导的创伤和ER Ca2+耗竭激活 UPR诱导发丝损失； 3）在体内听力损失模型中，如何调制UPR以产生 听力损失和发胶死亡的不同模式。这些目标将使用多学科集合来实现 生理，生化，药理学和遗传技术，包括ER CA2+成像，mRNA 转录分析和遗传TMTC4条件基因敲除小鼠。通过这些实验，我们将 对ER Ca2+通量和UPR参与遗传和UPR的机制获得宝贵的见解 噪声引起的听力损失，为NIHL的靶向疗法开发奠定了基础，这是一个关键 未满足的临床需求。