Role of Neuropilin-1/Semaphorin-3A signaling in the functional and morphological integrity of the cochlea

Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

神经纤毛蛋白-1（Neuropilin-1，简称Nrp1）编码跨膜细胞受体神经纤毛蛋白-1，该蛋白与心血管及神经发育密切相关，且在我们此前针对小鼠年龄相关性听力损失（age-related hearing loss, ARHL）的全基因组关联研究（Genome-Wide Association Study, GWAS）中，其位点位于8号染色体的峰值SNP区间内。
本研究构建并鉴定了内耳特异性Nrp1条件性敲除（conditional knockout, CKO）小鼠品系，原因在于组成型敲除Nrp1会引发胚胎致死。
原位杂交实验结果显示，胚胎耳蜗发育后期的Nrp1 mRNA表达水平较弱，但在出生后早期阶段（此时耳蜗毛细胞的神经支配模式已逐步成熟），其表达量显著升高。
出生后第5天的Nrp1 CKO小鼠表现出外螺旋束紊乱、血管纹（stria vascularis, SV）微血管扩张，但螺旋神经节细胞（spiral ganglion cell, SGN）密度及突触前带数量均保持正常；然而，在4月龄Nrp1 CKO小鼠中，我们观察到其血管纹微血管进一步扩张、螺旋神经节细胞密度降低，且外毛细胞区域的突触前带数量出现减少。
此外，与2月龄野生型小鼠相比，2月龄和4月龄Nrp1 CKO小鼠在4至32kHz频段的听阈均显著升高。上述数据表明，内耳中Nrp1的条件性缺失会导致小鼠出现进行性听力损失。
我们还证实，携带Nrp1截短变异体的小鼠会出现耳蜗轴突导向缺陷；且作为已知的神经纤毛蛋白-1受体激动剂，外源性信号素-3A（semaphorin-3A）可在体外排斥螺旋神经节细胞轴突。上述数据提示，神经纤毛蛋白-1/信号素-3A信号通路可能在发育中的耳蜗神经元寻路过程中发挥关键作用。
综上，本研究结果支持如下模型：神经纤毛蛋白-1/信号素-3A信号通路对耳蜗的功能与形态完整性至关重要，且Nrp1可能在年龄相关性听力损失的发生发展中扮演重要角色。