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）是一个严重的公共卫生问题，影响着近4000万美国人。 我们已经取得了令人兴奋的发现，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，具有下游效应物，可以促进细胞 存活和凋亡。为了支持这一点，我们还发现了一种新的耳聋， 小鼠中的Tmtc 4基因最近也被确定为人类家族中的潜在耳聋基因。 Tmtc 4基因缺失的小鼠（Tmtc 4敲除（KO）小鼠）在听力开始时听力正常 但在2周内迅速变聋，并且对NIHL的易感性显著增加。我们发现 Tmtc 4在耳蜗毛细胞和支持细胞中广泛表达，这两种细胞都随着时间的推移而退化 在Tmtc 4 KO小鼠中。我们已经表明Tmtc 4是参与清除的大分子复合物的一部分， 钙离子（Ca 2+）从细胞质进入内质网（ER），并且来自Tmtc 4 KO的耳蜗细胞 小鼠在细胞内Ca 2+稳态和动力学方面有损伤。这种钙离子管理的损害 导致Tmtc 4 KO耳蜗中UPR的上调和细胞死亡。与这种遗传性耳聋并行的是 在UPR失调模型中，我们发现野生型（WT）小鼠的NIHL导致UPR上调 噪声暴露2小时内;这种听力损失可以通过一种药物治疗部分预防， ISRIB，特异性靶向UPR，或第二种药物，CDN 1163，促进Ca 2+再摄取到ER中。 这些初步发现强烈暗示UPR是耳蜗细胞应激的早期介质， 上游的其他先前研究的凋亡机制，因此是一个潜在的治疗目标， 广泛的后天性和遗传性听力损失。 在这项提案中，我们的具体目标是调查1）如何在细胞系中，TMTC 4功能障碍，包括 与听力损失相关的人类变异，影响ER Ca 2+通量，随后影响UPR激活; 2）如何， 耳蜗，以毛细胞尖端连接破坏和ER Ca 2+耗竭形式的噪声诱导的创伤激活了耳蜗内的 UPR诱导毛细胞损失;以及3）在听力损失的体内模型中，UPR如何被调节以引起 听力损失和毛细胞死亡的不同模式。这些目标将通过一套多学科的 生理学、生物化学、药理学和遗传学技术，包括ER Ca 2+成像、mRNA 转录分析和遗传TMTC 4条件性敲除小鼠。通过这些实验，我们将 获得有价值的洞察ER Ca 2+通量和UPR参与遗传和 噪声引起的听力损失，为NIHL的靶向治疗奠定了基础，这是一个关键的 未满足的临床需求。

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.