Supplementary Material for: Gαi Proteins are Indispensable for Hearing

Background/Aims: From invertebrates to mammals, Gαi proteins act together with their common binding partner Gpsm2 to govern cell polarization and planar organization in virtually any polarized cell. Recently, we demonstrated that Gαi3-deficiency in pre-hearing murine cochleae pointed to a role of Gαi3 for asymmetric migration of the kinocilium as well as the orientation and shape of the stereociliary (“hair”) bundle, a requirement for the progression of mature hearing. We found that the lack of Gαi3 impairs stereociliary elongation and hair bundle shape in high-frequency cochlear regions, linked to elevated hearing thresholds for high-frequency sound. How these morphological defects translate into hearing phenotypes is not clear. Methods: Here, we studied global and conditional Gnai3 and Gnai2 mouse mutants deficient for either one or both Gαi proteins. Comparative analyses of global versus Foxg1-driven conditional mutants that mainly delete in the inner ear and telencephalon in combination with functional tests were applied to dissect essential and redundant functions of different Gαi isoforms and to assign specific defects to outer or inner hair cells, the auditory nerve, satellite cells or central auditory neurons. Results: Here we report that lack of Gαi3 but not of the ubiquitously expressed Gαi2 elevates hearing threshold, accompanied by impaired hair bundle elongation and shape in high-frequency cochlear regions. During the crucial reprogramming of the immature inner hair cell (IHC) synapse into a functional sensory synapse of the mature IHC deficiency for Gαi2 or Gαi3 had no impact. In contrast, double-deficiency for Gαi2 and Gαi3 isoforms results in abnormalities along the entire tonotopic axis including profound deafness associated with stereocilia defects. In these mice, postnatal IHC synapse maturation is also impaired. In addition, the analysis of conditional versus global Gαi3-deficient mice revealed that the amplitude of ABR wave IV was disproportionally elevated in comparison to ABR wave I indicating that Gαi3 is selectively involved in generation of neural gain during auditory processing. Conclusion: We propose a so far unrecognized complexity of isoform-specific and overlapping Gαi protein functions particular during final differentiation processes.

背景与目的：从无脊椎动物到哺乳动物，Gαi蛋白（Gαi proteins）可与其通用结合伴侣Gpsm2协同作用，在几乎所有极化细胞中调控细胞极化与平面组织构建。本团队前期研究证实，听阈前小鼠耳蜗中Gαi3缺失，会影响动纤毛的不对称迁移，以及静纤毛（毛）束的取向与形态——而这是成熟听觉形成的必要条件。我们发现，Gαi3缺失会损伤高频耳蜗区域的静纤毛伸长与毛束形态，进而导致高频听觉阈值升高，但目前尚不明确此类形态学缺陷如何转化为听觉表型。方法：本研究构建了全局敲除及条件性敲除Gnai3与Gnai2的小鼠突变体，分别缺失单一或两种Gαi蛋白亚型。通过对全局敲除小鼠与主要在内耳及端脑内进行Foxg1驱动的条件性敲除小鼠开展对比分析，并结合功能实验，旨在解析不同Gαi亚型的必需功能与冗余功能，并明确特定缺陷累及外毛细胞、内毛细胞（IHC）、听神经、卫星细胞还是中枢听觉神经元。结果：本研究发现，仅Gαi3缺失（而非普遍表达的Gαi2缺失）会升高听觉阈值，同时伴随高频耳蜗区域的静纤毛伸长与毛束形态异常。在未成熟内毛细胞（IHC）突触向成熟功能性感觉突触重编程的关键时期，Gαi2或Gαi3缺失均未产生影响。与之相反，Gαi2与Gαi3双敲除会导致整个听拓扑轴出现异常，包括伴随静纤毛缺陷的重度耳聋；此类小鼠的出生后内毛细胞突触成熟过程同样受损。此外，通过对比条件性与全局Gαi3敲除小鼠，我们发现听觉脑干反应（Auditory Brainstem Response, ABR）波IV的振幅相较于ABR波I出现不成比例升高，提示Gαi3在听觉处理过程中选择性参与神经增益的生成。结论：本研究揭示了此前未被认知的Gαi蛋白亚型特异性与重叠性功能的复杂性，尤其在终末分化过程中。