Eupatilin rescues ciliary transition zone defects to ameliorate ciliopathy-related phenotypes
Yong Joon Kim, … , Ho Jeong Kwon, Joon Kim
Yong Joon Kim, … , Ho Jeong Kwon, Joon Kim
Published July 23, 2018
Citation Information: J Clin Invest. 2018;128(8):3642-3648. https://doi.org/10.1172/JCI99232.
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Concise Communication Development Ophthalmology

Eupatilin rescues ciliary transition zone defects to ameliorate ciliopathy-related phenotypes

Abstract

Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.

Authors

Yong Joon Kim, Sungsoo Kim, Yooju Jung, Eunji Jung, Ho Jeong Kwon, Joon Kim

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Figure 4

Eupatilin restores centrosomal NPHP5 levels in CEP290null RPE1 cells by inhibiting NPHP5-calmodulin interaction.

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Eupatilin restores centrosomal NPHP5 levels in CEP290null RPE1 cells by ...
(A) Fluorescence micrographs visualizing centrosomal localization of NPHP5 in CEP290WT and CEP290null RPE1 cells treated with DMSO or eupatilin (20 μM) for 24 hours in the absence of serum. (B) Quantification (arbitrary unit) of the experiment presented in A. (C) DARTS–Western blot analysis that shows eupatilin-mediated protection of calmodulin (CAM) against pronase (62.5 ng [+], 250 ng [++], or 1 μg [+++] per 100 μg total cell protein). (D) Immunoprecipitation of NPHP5 after 48 hours DMSO or eupatilin treatment and coprecipitated calmodulin detected by Western blotting. (E) Immunoblot analysis of NPHP5 after 48 hours DMSO or eupatilin treatment. (F and G) Effect of simultaneous knock down of 3 calmodulin genes on ciliogenesis and centrosomal NPHP5 recruitment in CEP290null RPE1 cells. Reduction of total calmodulin levels was confirmed by Western blotting. (H–J) Effect of simultaneous knock down of 3 calmodulin genes on ciliogenesis and cilium elongation in CEP290null RPE1 cells treated with 20 μM eupatilin. Scale bars indicate 5 μm. Error bars represent SEM (B, G, J: >50 cells of each condition from 3 independent experiments were analyzed; F, H: n = 3 independent experiments; **P < 0.01, t test).