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.

噪声性听力损失 (NIHL) 是一个重大的公共卫生问题，影响着近 4000 万美国人。 我们有了令人兴奋的发现，即 NIHL 可能与未折叠蛋白反应 (UPR) 有关，这是一种 对细胞应激的关键早期反应机制，其下游效应器可以促进细胞 存活和凋亡。为了支持这一点，我们还发现并描述了一种新型耳聋 小鼠基因 Tmtc4，最近也被鉴定为人类家族中潜在的耳聋基因。 Tmtc4 基因缺失的小鼠（Tmtc4 敲除 (KO) 小鼠）在开始听力时听力正常 但在 2 周内迅速失聪，并且对 NIHL 的易感性显着增加。我们发现 Tmtc4 在耳蜗毛细胞和支持细胞中广泛表达，这两种细胞都会随着时间的推移而退化 在 Tmtc4 KO 小鼠中。我们已经证明 Tmtc4 是参与清除的大分子复合物的一部分 钙（Ca2+）从细胞质进入内质网（ER），并且来自Tmtc4 KO的耳蜗细胞 小鼠细胞内 Ca2+ 稳态和动力学受损。 Ca2+ 管理受损 导致 Tmtc4 KO 耳蜗中 UPR 上调和细胞死亡。与这种遗传性耳聋并行 UPR 失调模型中，我们发现野生型 (WT) 小鼠中的 NIHL 会导致 UPR 上调 噪声暴露后 2 小时内；这种听力损失可以通过一种药物治疗来部分预防， ISRIB 专门针对 UPR，或另一种药物 CDN1163，可促进 Ca2+ 再摄取到 ER 中。 这些初步发现强烈暗示 UPR 作为耳蜗细胞应激的早期调节剂， 其他先前研究的细胞凋亡机制的上游，因此是细胞凋亡的潜在治疗靶点 各种获得性和遗传性听力损失。 在本提案中，我们的具体目标是研究 1) 在细胞系中，TMTC4 功能障碍是如何发生的，包括 与听力损失相关的人类变异会影响 ER Ca2+ 通量，进而影响 UPR 激活； 2）如何，在 耳蜗、噪音引起的毛细胞尖端连接破坏和 ER Ca2+ 耗竭形式的创伤会激活 UPR 诱导毛细胞损失； 3）在听力损失的体内模型中，UPR如何被调节以产生 不同模式的听力损失和毛细胞死亡。这些目标将通过多学科组合来实现 生理、生化、药理学和遗传技术，包括 ER Ca2+ 成像、mRNA 转录分析和基因 TMTC4 条件敲除小鼠。通过这些实验，我们将 获得对 ER Ca2+ 通量和 UPR 参与遗传和发育的机制的宝贵见解 噪音引起的听力损失，为开发 NIHL 的靶向治疗奠定了基础，NIHL 是一个关键的疾病 未满足的临床需求。