Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling
Hairong Shi, Hongshun Wang, Cheng Zhang, Yajie Lu, Jun Yao, Zhibin Chen, Guangqian Xing, Qinjun Wei, Xin Cao
Hairong Shi, Hongshun Wang, Cheng Zhang, Yajie Lu, Jun Yao, Zhibin Chen, Guangqian Xing, Qinjun Wei, Xin Cao
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Research Article Genetics Otology

Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling

Abstract

Defective primary cilia cause a range of diseases called ciliopathies, which include hearing loss (HL). Variants in the human oxysterol-binding protein like 2 (OSBPL2/ORP2) are responsible for autosomal dominant nonsyndromic HL (DFNA67). However, the pathogenesis of OSBPL2 deficiency has not been fully elucidated. In this study, we show that the Osbpl2-KO mice exhibited progressive HL and abnormal cochlear development with defective cilia. Further research revealed that OSBPL2 was located at the base of the kinocilia in hair cells (HCs) and primary cilia in supporting cells (SCs) and functioned in the maintenance of ciliogenesis by regulating the homeostasis of PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) on the cilia membrane. OSBPL2 deficiency led to a significant increase of PI(4,5)P2 on the cilia membrane, which could be partially rescued by the overexpression of INPP5E. In addition, smoothened and GL13, the key molecules in the Sonic Hedgehog (Shh) signaling pathway, were detected to be downregulated in Osbpl2-KO HEI-OC1 cells. Our findings revealed that OSBPL2 deficiency resulted in ciliary defects and abnormal Shh signaling transduction in auditory cells, which helped to elucidate the underlying mechanism of OSBPL2 deficiency in HL.

Authors

Hairong Shi, Hongshun Wang, Cheng Zhang, Yajie Lu, Jun Yao, Zhibin Chen, Guangqian Xing, Qinjun Wei, Xin Cao

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Figure 4

OSBPL2 regulated ciliogenesis by maintaining PI homeostasis on the ciliary membrane.

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OSBPL2 regulated ciliogenesis by maintaining PI homeostasis on the cilia...
(A) Immunofluorescence staining of cilia in Osbpl2–/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2. Cells were stained with anti-acetylated tubulin (red), anti–gamma-tubulin (green), and DAPI (blue). Scale bar: 5 μm. (B) The ciliary length of Osbpl2–/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2 (50 cells per genotype, each dot represents a cell in a microscope field. *P < 0.05; **P < 0.01; ns: not significant; tested by 1-way ANOVA). (C) The proportion of ciliated cells in Osbpl2–/– HEI-OC1 cells transiently expressing Flag-ΔFFAT, Flag-ΔORD, and Flag-OSBPL2 (each dot represents the proportion of ciliated cells in a microscope field; ns: not significant; tested by 1-way ANOVA). (D) Immunofluorescence staining of HEI-OC1 cells with anti-PI(4,5)P2 (green), anti-acetylated tubulin (red), and DAPI (blue). Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. (E) Quantification of ciliary PI(4,5)P2 intensity in HEI-OC1 cells (50 cells per genotype, each dot represents a cell. ***P < 0.05 by 2-tailed Student’s t test). (F) The proportion of PI(4,5)P2 positive cilia in Osbpl2–/– HEI-OC1 cells (each dot represents the proportion of PI(4,5)P2 positive cilia in a microscope field. ns: not significant by 2-tailed Student’s t test). (G) Immunofluorescence staining of HEI-OC1 cells with anti-PI4P (green), anti-acetylated tubulin (red), and DAPI (blue). PI4P showed no significant difference in Osbpl2–/– and WT HEI-OC1 cells. Dashed frames denote the locally zoomed regions in solid frames (bottom right). Scale bar: 5 μm. (H) Quantification of ciliary PIP4 intensity in HEI-OC1 cells (50 cells per genotype, each dot represents a cell. ns: not significant by 2-tailed Student’s t test). (I) The proportion of PI4P positive cilia in Osbpl2–/– HEI-OC1 cells (each dot represents the proportion of PI4P positive cilia in a microscope field. ns: not significant by 2-tailed Student’s t test).