Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin-1 (CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT-PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single-cell RNA-sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock-in mouse with a hemagglutinin (HA) epitope-tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1-specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

中文翻译：

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Clarin-1 在成年小鼠和人类视网膜中的表达突出了 Müller 胶质细胞在 Usher 综合征中的作用。

Usher 综合征 3 型 (USH3) 是一种常染色体隐性遗传病，由 clarin-1 (CLRN1) 基因突变引起，可导致进行性听力损失和视网膜退化。CLRN1 在视网膜中的细胞分布仍然不确定，要么是因为它的表达水平低，要么是因为它的表位被掩盖了。事实上，在成年小鼠视网膜中，Clrn1 mRNA 在发育过程中被下调，只能通过 RT-PCR 检测到。在这项研究中，我们使用高灵敏度的 RNAscope 原位杂交测定和单细胞 RNA 测序技术来研究小鼠和人类视网膜中 Clrn1 和 CLRN1 的分布。我们发现小鼠组织中的 Clrn1 转录本在出生后发育和成年阶段定位于视网膜内层。Clrn1 mRNA 细胞表达模式在小鼠和成人视网膜中相似，CLRN1 转录本位于 Müller 神经胶质细胞中，而不是光感受器中。我们生成了一种带有血凝素 (HA) 表位标记的 CLRN1 的新型敲入小鼠，并表明 CLRN1 在视网膜的蛋白质水平上持续表达。在酶促去糖基化和免疫印迹分析之后，我们在小鼠和人类视网膜的匀浆中检测到单个 CLRN1 特异性蛋白条带，其大小与主要 CLRN1 亚型一致。综上所述，我们的结果表明 Müller 胶质细胞与 USH3 病理学有关，将这种细胞类型置于未来机制和治疗研究的中心，以防止这种疾病的视力丧失。© 2019 作者。John Wiley 出版的 The Journal of Pathology &