Ablation of Htra1 leads to sub-RPE deposits and photoreceptor abnormalities

Abstract

The high-temperature requirement A1 (HTRA1), a serine protease, has been demonstrated to play a pivotal role in the extracellular matrix (ECM) and has been reported to be associated with the pathogenesis of age-related macular degeneration (AMD). To delineate its role in the retina, the phenotype of homozygous Htra1-KO (Htra1-/-) mice was characterized to examine the effect of Htra1 loss on the retina and retinal pigment epithelium (RPE) with age. The ablation of Htra1 led to a significant reduction in rod and cone photoreceptor function, primary cone abnormalities followed by rods, and atrophy in the RPE compared with WT mice. Ultrastructural analysis of Htra1-/- mice revealed RPE and Bruch's membrane (BM) abnormalities, including the presence of sub-RPE deposits at 5 months (m) that progressed with age accompanied by increased severity of pathology. Htra1-/- mice also displayed alterations in key markers for inflammation, autophagy, and lipid metabolism in the retina. These results highlight the crucial role of HTRA1 in the retina and RPE. Furthermore, this study allows for the Htra1-/- mouse model to be utilized for deciphering mechanisms that lead to sub-RPE deposit phenotypes including AMD.

Keywords: Genetics; Mouse models; Neurodegeneration; Ophthalmology; Retinopathy.

Figure 1. Photoreceptor function in Htra1^–/– mice.

(A–E) Mean photopic and scotopic responses of 1.5, 3, 5, 15, and 21m Htra1^–/– mice compared and normalized to age-matched WT mice at 1.09 log cd·s/m², 2.00 log cd·s/m², and –3.5 log cd·s/m² stimulation intensities. n = 5 mice per age point and genotype. Two-way ANOVA followed by the Tukey-Kramer for pairwise comparisons, with P values adjusted using the Bonferroni Correction. Age-matched comparisons are displayed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Assessment of cones and cell death in Htra1^–/– mice.

(A) Representative images of M-opsin–expressing (red) and S-opsin–expressing (green) cones (white arrows) in 1.5m, 3m, 5m, and 21m WT (n = 4 mice per age) and Htra1^–/– mice (n = 4 mice per age). Scale bars: 10 μm. (B–D) Cone outer segment lengths in 21m WT versus Htra1^–/– mice in the dorsal, central, and ventral regions of the retina. Cone outer segment measurements were analyzed on n = 3 sections per genotype and averaged. Two-tailed t tests were performed. ****P < 0.0001. (E and F) Representative images of TUNEL assay of 21m WT and Htra1^–/– mice and quantification of TUNEL⁺ cells (n = 3 mice per genotype). A 2-tailed t test was performed. ***P < 0.001. TUNEL-labeled cells are indicated by white arrow. Scale bars: 10 μm.

Figure 3. Expression of photoreceptor and RPE-specific genes in Htra1^–/– mice.

(A–F) Opn1mw, Opn1sw, Rho, Best1, Mitf, and Rpe65 transcripts in 1.5m, 3m, 5m, 15m, and 21m Htra1^–/– mice compared with age-matched WT mice (n = 3 mice per genotype and age point). Actb was used as the housekeeping control. Two-way ANOVA followed by the Tukey-Kramer for pairwise comparisons, with P values adjusted using the Bonferroni Correction. Age-matched comparisons are displayed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. RPE abnormalities in Htra1^–/– mice with age.

(A) WT mice (5m) with choriocapillaris (CC), BM, and RPE labeled. (B–D) Htra1^–/– mice (5m). Pink arrows indicate areas of vacuole-like structures. Blue arrows indicate phagosomes bearing undigested material from the photoreceptor outer segments. (E) WT mice (21m) with labeled CC, BM, and RPE. (F–H) Htra1^–/– mice (21m). Yellow arrowhead indicates thickening of the BM. Cyan arrowhead shows a large phagosome. n = 3 mice per age and genotype.

Figure 5. Sub-RPE deposition in Htra1^–/– mice.

(A) WT mice (21m) show intact basal infoldings (indicated by yellow arrow) and lack of RPE-BM abnormalities. (B) At 5m, sub-RPE deposits were present in the RPE-BM of Htra1^–/– mice. (C) Htra1^–/– mice (21m) show more sub-RPE deposits and abnormal basal infoldings. Representative images are displayed. n = 3 mice per age and genotype. Black arrows indicate sub-RPE deposit.

Figure 6. Abnormal age-related changed in Htra1^–/– mice.

(A–T) Representative retinal sections from 5m and 21m WT and Htra1^–/– mice were stained with IBA1 (A–D), LGALS3 (E–H), p62 (I–L), APOE (M–P), and VTN (Q–T) antibodies. n = 3 mice per age and genotype were evaluated. DAPI was used as a counterstain. White arrows indicate signal detected. Scale bars: 50 μm.