NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development

Abstract

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

Figure 1.

The kidney morphology in Nphp4^{nmf192/nmf192} is normal despite the nonsense Leu104Ter mutation. (A) By direct sequence analysis, a single base pair substitution (T to A) was found in exon 4 of the Nphp4 gene of nmf192/nmf192 mice. The mutation is predicted to cause amino acid Leu104 to become a termination codon. (B) To examine the effects of the mutation on the Nphp4 gene product, an antibody was raised against 188 amino acids from the N-terminus of NPHP4. Western analysis of retinal lysates from 2-month-old control and mutant mice stained with anti-NPHP4 was conducted. Besides a major full size product at ∼150 kDa, multiple smaller bands were observed in lysates from control retinas. The largest NPHP4 band was not detected in Nphp4^{nmf192/nmf192} mutant retinas, however, smaller bands were found in both mutant and control retinas suggesting alternative splice forms or non-specific interaction with the polyclonal NPHP4 antibody. Staining with anti-γ-tubulin served as a loading control. (C) Nphp4^{nmf192/nmf192} mice have normal kidney morphology. H&E staining of kidneys from 6-month-old male control (left) and Nphp4^{nmf192/nmf192} (right) mice. Bar = 50 μm.

Figure 2.

Longitudinal studies examining photoreceptor degeneration. (A) Fundus photographs of Nphp4^{nmf192/nmf192} mice at 2 and 10m with littermate control at 2m. Retinas of Nphp4^{nmf192/nmf192} mutants appear grainy with large patches of depigmentation. (B) Nphp4^{nmf192/nmf192} mutant retinas show thin OS at 2w without a notable ONL reduction, whereas at 4w only one layer of cell bodies remained in the ONL. (C) Pan-retinal ONLT index (±SEM) was obtained from three retinas each of control (open symbols) and of mutant (closed symbols) mice. In the central retina, ONLT index of mutants were significantly reduced. (D) Average retinal thickness (±SEM) of the ONL and the INL of control and mutant mice at P19 and P21. n= 5. GC, ganglion cell layer; INL, inner nuclear layer; OPL, outer plexiform layer; IS, inner segment; ONL, outer nuclear layer; RPE, retinal pigmented epithelium. Bar = 50 μm.

Figure 3.

Mislocalization of OS proteins. Rhodopsin (RHO; A and B) and ROM1 (C and D) mislocalized to the IS and ONL in retinas from 2-week-old mice. By whole-mount staining, the majority of green opsin was found to mislocalize to the IS and ONL in Nphp4^{nmf192/nmf192} retina (E and F). However, staining was also found in the OS-like structures. OS, outer segment. Bars = 50 μm.

Figure 4.

NPHP4 localization and CC marker analysis. NPHP4 localization was examined by co-staining with RP1 (A and B) and γ-tubulin (γ-TUB; C and D). Insets represent ×6 magnified images. NPHP4 staining was not detected in Nphp4^{nmf192/nmf192} CC, whereas RP1 and γ-tubulin were similar to that observed in controls (B and D). Photo-transduction of rod-transducin (GNAT-1; E–H) and arrestin (I–L) in dark-adapted (E, G, I, K) and light-stimulated (F, H, J, L) conditions. Arrows indicate OS in Nphp4^{nmf192/nmf192} retina. The localization of acetylated tubulin (Ac-TUB; M and N) and RPGRIP1 (O and P) were examined. All retinal sections were obtained at P14. Bars = 20 μm (A–L).

Figure 5.

Ultrastructural analysis of the developing photoreceptor in mutant and WT mice. Rudimentary OS that failed to elongate were observed by transmission electron microscopy in mutant retinas at P14 (arrows, A and B). Basal bodies (arrow heads) were similar in structure in control and mutant retinas. A cross-section of mutant CC at P10 in insert of (B) shows the normal nine sets of doublets. Scanning electron microscopy revealed comparable CC number and length at P7 (C and D). But by P12, short and rudimentary OS were observed in mutant while well-organized and elongated paddle shape OS were found in littermate controls (E and F). NC, nascent cilia. Bars indicated 2 μm (A and B), 200 nm (insert of B) and 10 μm (C–F).

Figure 6.

Synaptic ribbon defects in the OPL of Nphp4^{nmf192/nmf192} mice. (A) The shortening of OPL was not obvious by light microscopy at P10 (i), whereas the shortening of OS was observable by P11 (ii). (B) Synaptophysin staining at P8 (i) and P12 (ii). Synaptophysin staining was discontinuous (arrows) and ectopic staining in the ONL (arrowhead) was observed at P12 (ii). (C) PSD95 was similar between mutant and controls at P12 (i) but reduced in staining intensity at P14 (ii). v indicates blood vessels stained with anti-mouse secondary antibodies. (D) Reduced PNA cone pedicle staining (arrows) and ectopic staining in ONL (arrowhead) were observed at P14 (i). Normally PSD95 and PNA do not colocalize at P14 (ii, left) but in the Nphp4^{nmf192/nmf192} OPL some co-localization of these biomarkers were observed (arrows, ii), indicating the shortening of the OPL. m indicates the mislocalized PNA staining of the cone nucleus. (E) NPHP4 antibody stains rod and cone ribbons as indicated by double-staining with PNA (i), Bassoon (ii) and RIBEYE (iii). Arrows indicate cone pedicles stained with PNA. (F) Synaptic ribbons were stained with RIBEYE at P8 (i) and P12 (ii). OPL shortening was observed by staining with pre-synaptic marker kinesin-II (KIN) at P14 (iii). The typical horse-shoe shape of the ribbon synaptic terminal revealed by anti-kinesin-II staining was not well preserved in Nphp4^{nmf192/nmf192} mutant retinas. Bars = 50 μm (A–D) and 20 μm (E and F).

Figure 7.

Ultrastructure of ribbon synapses is affected in Nphp4^{nmf192/nmf192} mice. Developing ribbon synapses were examined by TEM at P10 (A) and P14 (B) in control and Nphp4^{nmf192/nmf192} retinas. Ribbons are indicated by arrows, and N indicates the photoreceptor nucleus. Bar = 500 nm.

Figure 8.

Ectopic horizontal cell processes are observed in Nphp4^{nmf192/nmf192} mice. (A) The ectopic horizontal cell processes were observed as early as P11 in Nphp4^{nmf192/nmf192} retina. Arrows indicate horizontal cell processes in the ONL. (B) Calbindin staining was observed in retinal sections from various photoreceptor degeneration models, which are not associated with cilia structure or function per se. Two Pde6a models, which show a very rapid degeneration, and Mfrp^rd6 mutants, which show a relatively slow degeneration, were examined for ectopic horizontal cell processes. The tissues were collected at an early stage of degeneration when most of the cell bodies of the photoreceptor were retained in each animal model. None of the other models developed ectopic horizontal cell processes at the time points sampled. INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. Bar = 50 μm.

Figure 9.

Male infertility was observed in Nphp4^{nmf192/nmf192} mice. Testes size was normal in Nphp4^{nmf192/nmf192} compared with control (A), however spermatozoa counts (B) and amount of motile sperm (D) were extremely low, with little forward movement (E, *P < 0.01), and SMI necessary for fertilization was nonexistent (C, ****P < 0.0001). TEM revealed that Nphp4^{nmf192/nmf192} spermatozoa had intact axoneme in both longitudinal (F, left panel) and cross-sections, with 9+2 flagella structure in sperm tails (F, right panel). Normal early spermatogenesis, albeit few fully mature spermatozoa (insets), was observed in Nphp4^{nmf192/nmf192} compared with control testis by toluene blue staining (G). (H) NPHP4 did not colocalize with γ-tubulin, the centriole marker in testicular sperm. Insert is a ×2 magnified image and sperm head is visualized with a dashed line. Bars = 50 μm (G), 25 μm (insets in G), 500 nm (F, left panel), 100 nm (F right panel) and 10 μm (H).