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

噪声性听力损失(NIHL)是一个重大的公共健康问题，影响着近4000万美国人。 我们已经有了一个令人兴奋的发现，NIHL可能与未折叠蛋白反应(UPR)有关，一个 对细胞应激的关键早期反应机制，其下游效应物可以促进两种细胞 生存和凋亡。为了支持这一点，我们还鉴定并表征了一种新的耳聋 小鼠的Tmtc4基因，最近也被发现是人类家族中的一个潜在的耳聋基因。 Tmtc4基因缺失的小鼠(Tmtc4基因敲除(KO)小鼠)在听力开始时正常听力 但在2周内迅速失聪，并明显增加了对NIHL的易感性。我们发现了 Tmtc4在耳蜗毛细胞和支持细胞中广泛表达，两者都随着时间的推移而退化 在Tmtc4KO小鼠中。我们已经证明Tmtc4是参与清除的大分子复合体的一部分。 钙(Ca~(2+))从细胞质进入内质网(ER)，耳蜗细胞从Tmtc4KO 小鼠细胞内钙动态平衡和动态平衡受损。钙离子管理中的这种损害 导致Tmtc4 KO耳蜗内UPR表达上调和细胞死亡。伴随着这种遗传性耳聋 通过建立UPR异常调节模型，我们发现野生型(WT)小鼠的NIHL导致UPR表达上调 在噪音暴露后2小时内；这种听力损失可以通过一种药物治疗而部分预防， ISRIB，它专门针对UPR，或者第二种药物CDN1163，它促进钙重新摄取到内质网。 这些初步发现强烈暗示UPR是耳蜗细胞应激的早期调节因子， 在其他先前研究的凋亡机制的上游，因此是一种潜在的治疗靶点 各种后天性和遗传性听力损失。 在这项提案中，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，影响ER钙流量，并随后影响UPR激活；2)如何，在 以毛细胞尖端连接断裂和内质网钙离子耗竭为形式的噪声所致的耳蜗损伤激活了 以及3)在听力损失的活体模型中，UPR是如何被调节以引起 不同的听力损失和毛细胞死亡模式。这些目标将使用多学科集合来实现 生理、生化、药理和遗传技术，包括ER、Ca~(2+)成像、 转录分析和遗传TMTC4条件性基因敲除小鼠。通过这些实验，我们将 深入了解ER、Ca~(2+)通量和UPR参与基因和蛋白质合成的机制。 噪声性听力损失，为开发针对NIHL的靶向治疗奠定了基础，NIHL是一种关键的 未得到满足的临床需求。

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.