GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells
GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells
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DOI：
10.3389/fcell.2020.00215
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发表时间：
2020-04-02
影响因子：
5.5
通讯作者：
Laird, Dale W.
中科院分区：
文献类型：
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作者：
Beach, Rianne;Abitbol, Julia M.;Laird, Dale W.
GJB2基因（编码Cx26）突变是听力损失的原因，强调了Corti器官中支持细胞中基于Cx26的通道信号的重要性。虽然大多数GJB2突变以常染色体隐性方式遗传，但其他突变以常染色体显性方式遗传，并导致综合征性听力损失和皮肤病。为了评估gjb2相关性听力损失的根源是共同的还是不同的机制，我们在来自Corti发育器官的耳蜗相关HEI-OC1细胞中表达了几个突变。由于Corti成熟哺乳动物器官的支持细胞中Cx43含量可忽略，而HEI-OC1细胞中Cx43含量丰富，因此我们首先利用CRISPR-Cas9将内源性Cx43烧除，从而建立一个连接蛋白缺失平台，以控制重新引入听力相关连接蛋白和Cx26突变体。当听力损失相关的Cx26突变体在耳蜗相关细胞中表达时，我们发现了三种不同的结果和细胞表型。显性综合征型Cx26突变体N54K具有运输缺陷，不能完全阻止野生型Cx26间隙连接斑块的形成，但当与Cx30共表达时，令人惊讶地形成了间隙连接。相比之下，优势综合征型S183F突变体形成了不能转移染料的间隙连接，并且如预期的那样，与野生型Cx26和Cx30共同定位在相同的间隙连接中，但也获得了在间隙连接中与Cx43混合的能力。两种隐性非综合征型Cx26突变体（R32H和R184P）均保留在细胞内囊泡（包括早期内体）中，且不与Cx30共定位。正如可以预测的那样，在富含Cx43的HEI-OC1细胞中，Cx26突变体都不能阻止Cx43间隙连接斑块的形成，而Cx43消融对听觉细胞分化相关内参基因的表达几乎没有影响。我们从耳蜗相关细胞的研究中得出结论，所选的Cx26突变体可能通过三种独立于Cx43状态的独特连接蛋白缺陷引起听力损失。
GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.