Conditional loss of Spata7 in photoreceptors causes progressive retinal degeneration in mice

Abstract

The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7^Flox/-; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.

Keywords: Conditional knockout; Leber Congenital Amaurosis; Photoreceptors; RPE; Retinal degeneration; Retinal function; Retinitis Pigmentosa; SPATA7.

Figure 1. Generation of Spata7 conditional knockout mice

(A) Spata7 targeting strategy. Numbered rectangles represent Spata7 exons. (B) Genomic Southern blot of Spata7 targeted allele. Fragments of 5.8 kb and 11.4 kb are detected in a heterozygous mouse using 5’ (HindIII) and 3’ (EcoRV) probes, respectively. (C) Western blot of SPATA7 protein probed using anti-SPATA7 antibody using retinas isolated from wild-type and Spata7^Δ1–5 knockout mice. Anti-β-actin antibody was used as a loading control.

Figure 2. Loss of SPATA7 expression in the retina of Spata7^Flox/−; Crx-Cre mice

(A) Immunostaining for SPATA7 (green) in retinal sections obtained from Spata7^Flox/+; Crx-Cre control mice and Spata7^Flox/−; Crx-Cre mice at P28. (B) Immunostaining for Cre-recombinase in Spata7^Flox/− control mouse retina and Spata7^Flox/−; Crx-Cre adult cKO mouse retina at P28. Nuclei are counter-stained with DAPI (blue). Scale bar = 20 µm. CC: connecting cilium; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.

Figure 3. Progressive photoreceptor degeneration in Spata7^Flox/−; Crx-Cre conditional knockout mice

(A) Hematoxylin and eosin (H&E) staining of paraffin-embedded retinal sections obtained from Spata7^Flox/+ Control, Spata7^Flox/−; Crx-Cre, and Spata7^−/− mice at P28 (B) 4 months, and (C) 7 months of age. Progressive thinning of the ONL is observed in Spata7^Flox/−; Crx-Cre conditional KO mice and Spata7^−/− (germline null) mice starting at P28, while the retina appears normal in Spata7^Flox/+ control mice. (D) Retinal morphometry is presented by measuring the thickness of the ONL at 20 equally spaced positions in paraffin embedded sections along the vertical meridian of the retina for three retinas of the same genotype at P28. Three measurements were taken for each position, and a mean value was calculated. Each point represents the mean ±SEM obtained for each group (n ≥3 mouse retinas). Position 0 corresponds to the optic nerve head. Statistical analysis was performed using the Student’s t-test (NS= not significant, p>0.05; *p<0.05; **p<0.01). Scale bar = 40 µm. ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer.

Figure 4. Spata7^Flox/−; Crx-Cre mice exhibit reduced amplitudes of scotopic and photopic ERGs

Quantitative evaluation of scotopic (dark-adapted) a-wave and b-wave amplitudes was performed at (A, B) 1 month and (D, E) 4 months of age. Photopic (light-adapted cone response) amplitudes were also obtained at (C) 1 month and (F) 4 months of age from Spata7^Flox/+; Crx-Cre control mice and Spata7^Flox/−; Crx-Cre cKO mice. Spata7^Flox/+; Crx-Cre mice had normal scotopic and photopic ERG responses, while Spata7^Flox/−; Crx-Cre cKO mice exhibited a significantly reduced response to light, with a 50% reduction in the a-wave amplitude under scotopic conditions (A,B) and a 20–30% reduction under photopic conditions (C) at 1 month of age. At 4 months, Spata7^Flox/−; Crx-Cre cKO mice showed a further reduction in the scotopic response (D, E) and a consistent reduction in the photopic response (F). Statistical analysis was performed using the Student’s t-test (NS= not significant, p>0.05; *p<0.05; **p<0.01; ***p<0.001). n = 6 mice per genotype.

Figure 5. Spata7^Flox/−; Crx-Cre (cKO) mice exhibit Rhodopsin mislocalization

(A) SPATA7 was detected in retinal paraffin sections of Spata7^Flox/− mice, while little to no immunostaining was detectable in the retina of Spata7^Flox/−; Crx-Cre mice. (B, C) Localization of rhodopsin (Rho; red) is altered in Spata7^Flox/−; Crx-Cre cKO mice, where it is mislocalized to the inner segment and ONL of photoreceptor cells, compared to Rho localization in the OS of Spata7^Flox/+ control retinas at 7 month of age. Nuclei are counter-stained with DAPI (blue). Scale bar = 20 µm. OS: outer segment; CC: connecting cilium; IS: inner segment; ONL: outer nuclear layer.

Figure 6. RPGRIP1 mislocalization in Spata7^Flox/−; Crx-Cre retina

(A) Immunostaining for RPGRIP1 was performed in Spata7^Flox/+; Crx-Cre frozen retinal sections, where RPGRIP1 (green) co-localization was observed with acetylated-α tubulin (red) marker in the connecting cilium. (C) In Spata7^Flox/−; Crx-Cre retinas, mislocalization of RPGRIP1 is evident, where it is detectable in the IS and ONL regions at P40. (B, D) Surface plot quantification of RPGRIP1 and Acetylated- α tubulin intensity in the OS, CC, IS, and ONL compartments. OS: outer segment; CC: connecting cilium; IS: inner segment; ONL: outer nuclear layer. Scale bar = 10 µm.

Figure 7. Spata7^Flox/−; RPE-Cre conditional KO mice lack a retinal degeneration phenotype

(A) SPATA7 expression (green) in the connecting cilium (left) and overlay of expression with bright field imaging of frozen mouse retinal sections (right). DAPI was used for nuclear counterstaining (blue). (B) Enhanced re-focusing of the SPATA7 signal in the RPE and choroid region at high magnification. Expression of SPATA7 (green) is indicated in the RPE by arrows. Scale bar= 20 µm. (C) Cross sections of 2.5-month-old RPE-specific Spata7 cKO retinas show no obvious photoreceptor degeneration and appear normal compared to Spata7^Flox/+;Best1-Cre control retinal sections. (D) At 6.5 months, Spata7^Flox/−; RPE-Cre and Spata7^Flox/− ; RPE-Cre control mice consistently appear normal with no morphological defects present in retinas obtained from Spata7^Flox/− ; RPE-Cre mice. RPE= retinal pigment epithelium, CC= connecting cilium, ONL= outer nuclear layer, OS= outer segment, INL= inner nuclear layer. Scale bar = 20 µm.