Dysregulated Arginine Metabolism Is Linked to Retinal Degeneration in Cep250 Knockout Mice

Abstract

Purpose: Degeneration of retinal photoreceptors is frequently observed in diverse ciliopathy disorders, and photoreceptor cilium gates the molecular trafficking between the inner and the outer segment (OS). This study aims to generate a homozygous global Cep250 knockout (KO) mouse and study the resulting phenotype.

Methods: We used Cep250 KO mice and untargeted metabolomics to uncover potential mechanisms underlying retinal degeneration. Long-term follow-up studies using optical coherence tomography (OCT) and electroretinography (ERG) were performed.

Results: OCT and ERG results demonstrated gradual thinning of the outer nuclear layer (ONL) and progressive attenuation of the scotopic ERG responses in Cep250-/- mice. More TUNEL signal was observed in the ONL of these mice. Immunostaining of selected OS proteins revealed mislocalization of these proteins in the ONL of Cep250-/- mice. Interestingly, untargeted metabolomics analysis revealed arginine-related metabolic pathways were altered and enriched in Cep250-/- mice. Mis-localization of a key protein in the arginine metabolism pathway, arginase 1 (ARG1), in the ONL of KO mice further supports this model. Moreover, adeno-associated virus (AAV)-based retinal knockdown of Arg1 led to similar architectural and functional alterations in wild-type retinas.

Conclusions: Altogether, these results suggest that dysregulated arginine metabolism contributes to retinal degeneration in Cep250-/- mice. Our findings provide novel insights that increase understanding of retinal degeneration in ciliopathy disorders.

Figure 1.

Segmentation of the retinal layers in WT and Cep250 KO mice. (A) Representative images of WT and Cep250 KO mice along with nasal and temporal orientation. Scale bars: 100 µm. WT, Wild-types; HO, Homozygotes. (B–D) Measurements of the thicknesses of the ONL (B), OS/IS (C), and the whole retina (D) from OCT images of mice at P30, P90 and P180. ONH, optic nerve head. Normalized values represent mean ± SEM.*P < 0.05, **P < 0.01, and ***P < 0.001, Student's t test. n = 6.

Figure 2.

Evaluation of retinal function in Cep250 KO mice. (A) Representative ERG waves at dark 3.0, dark 10.0 and light 3.0. (B–E) Statistical analysis of b-wave amplitudes at dark 3.0, dark 10.0 and light 3.0. Experimental time points were P30 (B), P90 (C), P180 (D) and P270 (E). n = 8. Error bars represent SEM. *P < 0.05, **P < 0.01, and ***P < 0.001. HO, Homozygotes.

Figure 3.

TUNEL staining and immunostaining of Rhodopsin, M-opsin and S-opsin in WT and Cep250 KO mouse retinas. (A–C) Representative images of Rhodopsin (A), M-opsin (B), and S-opsin (C) immunostaining results in WT (left) and HO (right) mouse retinas. White arrows indicate the dislocation of photosensitive proteins in the ONL. Scale bar: 20 µm. (D) Representative images of TUNEL staining results in WT (left) and HO (right) mouse retinas. n = 3. Scale bar: 50 µm. HO, Homozygotes.

Figure 4.

Metabolic pathway and enrichment analysis of differentially expressed metabolites between WT and Cep250 KO mouse retinas at P30. (A, B) Pathway analysis and enrichment of differentially expressed metabolites detected in positive ion mode. (C, D) Pathway analysis and enrichment of differentially expressed metabolites detected in negative ion mode. Enrichment analysis module conduct metabolite set enrichment analysis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) library. Pathway analysis integrate enrichment analysis and pathway topology analysis. The ratio represents KO/WT. n = 3.

Figure 5.

Immunostaining of ARG1 and ARG2 at P30. (A, B) Representative images of ARG1 (A) and ARG2 (B) in Cep250 KO mouse and WT control retinas. Scale bar: 20 µm and 50 µm. Red arrows indicate the mislocation of ARG1 in the ONL. HO, Homozygotes. n = 3.

Figure 6.

AAV-based retinal Arg1 knockdown experiments in WT mice. (A) Schematic representation of knockdown experiments. (B) Representative fundus photographs of AAV-injected retinas at P60. Strong GFP signals were indicative of successful subretinal injection and AAV expression. (C) Retinal arginine quantification of the AAV injected mouse retinas. (D) The short red lines on the right sides of the OCT images indicate that ONL layer thickness was decreased in the retinas of Arg1 knockdown mice. Scale bar: 100 µm. (E) Measurements of the thicknesses of the ONL from OCT images of mice at P90. ONH, optic nerve head. Normalized values represent mean ± SEM.*P < 0.05, and **P < 0.01, Student's t test. n = 5. (F–I) Statistical analysis of a-wave and b-wave amplitudes at dark 3.0 (F, G) and dark 10.0 (H, I) in WT mice injected with AAV-Control and AAV-Arg1-RNAi.