Gelsolin dysfunction causes photoreceptor loss in induced pluripotent cell and animal retinitis pigmentosa models

Abstract

Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause X-linked RP (XLRP), an untreatable, inherited retinal dystrophy that leads to premature blindness. RPGR localises to the photoreceptor connecting cilium where its function remains unknown. Here we show, using murine and human induced pluripotent stem cell models, that RPGR interacts with and activates the actin-severing protein gelsolin, and that gelsolin regulates actin disassembly in the connecting cilium, thus facilitating rhodopsin transport to photoreceptor outer segments. Disease-causing RPGR mutations perturb this RPGR-gelsolin interaction, compromising gelsolin activation. Both RPGR and Gelsolin knockout mice show abnormalities of actin polymerisation and mislocalisation of rhodopsin in photoreceptors. These findings reveal a clinically-significant role for RPGR in the activation of gelsolin, without which abnormalities in actin polymerisation in the photoreceptor connecting cilia cause rhodopsin mislocalisation and eventual retinal degeneration in XLRP.Mutations in the Retinitis Pigmentosa GTPase Regulator (RPGR) cause retinal dystrophy, but how this arises at a molecular level is unclear. Here, the authors show in induced pluripotent stem cells and mouse knockouts that RPGR mediates actin dynamics in photoreceptors via the actin-severing protein, gelsolin.

Fig. 1

Three-dimensional patterning of induced pluripotent stem cells produces mature photoreceptors. Free floating aggregates grown using a retinal differentiation protocol^, self-organise into budding spheroids with a single bud shown in a (arrow) and multiple buds from one spheroid in b. Buds invaginate (c) to form optic cups which mature over 100 days (d). Pigmented retinal pigment epithelium (RPE) emerges externally (arrow; d) while a radial arrangement of Recoverin (e) and Rhodopsin (f) expressing photoreceptors organise internally, forming an outer nuclear-like layer. Electron microscopy studies reveal a classic “9 + 0” microtubule doublet formation in these cilia (g, inset h shows cross-section). RPGR is present in photoreceptor cilia (arrow; i) whilst electron microcopy shows the production of membranous material, as required for outer segment formation (arrow, j). Scale bars: 400 μm (b); 200 μm (a); 50 μm (c, d); 10 μm (e, f); 100 nm (g); 200 nm (h); 5 μm (i); 1 μm (j)

Fig. 2

RPGR-mutant, iPSC-derived, 3-dimensional photoreceptor cultures display perturbed actin regulation. a–d RPGR-mutant photoreceptors display increased actin polymerisation, as evidenced by increased phalloidin binding in the Recoverin-positive photoreceptors of patient-derived cultures (panels c, d) as compared to photoreceptors from the control patient (panels a, b). See text for details of quantification of pixel intensities. Nuclei stained with Hoechst (blue). e The mean phosphorylation level of cytoskeletal regulatory proteins was higher in RPGR/XLRP photoreceptors compared to control photoreceptors (red dots; 0.092±0.0033, n = 56 proteins). This was significantly more than the mean ratio of phosphorylation levels of non-cytoskeletal proteins when RPGR/XLRP photoreceptors were compared to control (blue dots; −0.008±0.01, n = 583 proteins, unpaired, two-tailed t-test, p = 0.004 (** = p < 0.01))

Fig. 3

The Rpgr KO mouse retina demonstrates actin dysregulation and rhodopsin mislocalisation in the photoreceptor layer prior to degeneration. a–c Outer nuclear layer (ONL—photoreceptor) degeneration develops in the Rpgr KO mouse (bracket represents the region measured). This change is significant by 4 months of age (compared to 4-month old wild-type) as indicated by the number of rows of photoreceptor nuclei (b; Figures denote mean±SEM, Kruskal–Wallis test [H = 30.8, 4 d.f., p < 0.0001], Dunn’s multiple comparisons test, p < 0.05, * = p < 0.05) and ONL thickness (c; Figures denote mean±SEM, one-way ANOVA [F(4,76) = 10.7, p < 0.0001], Tukey’s multiple comparisons test, p < 0.01 ** = p < 0.01). The WT control shown is 4 months of age. d, e Reactive gliosis as reflected by increased GFAP immunolabeling throughout the radial length of Müller cells in the ONL is apparent as early as 3 weeks of age (arrow; d) while rhodopsin, normally only present in the outer segments outside (above in the figure) the ONL, is now mislocalised into other regions of the photoreceptor in the outer plexiform layer (OPL—arrowhead; e) and peri-nuclear area (arrow; e) at this timepoint, prior to the visible onset of photoreceptor degeneration. f Outer segment length is reduced by 3 weeks of age (compared to 4-month old wild-type; Figures denote mean±SEM, *** = p < 0.001) after which they become markedly disorganised. g Increased F-actin is seen in the connecting cilium (arrowhead) of the Rpgr KO mouse photoreceptor compared to wild type (Images representative of n = 1–10 analysed)

Fig. 4

Studies of iPSC-derived photoreceptor cultures reveal perturbation of gelsolin and cofilin activity in RPGR-mutant lines. a A phosphoarray comparing phosphorylation levels of cytoskeletal proteins in patient and control photoreceptor cultures identified cofilin as hyperphosphorylated on Serine 3 in RPGR-mutant photoreceptors (results presented as a heat map, with a patient:control ratio of phosphorylation for cofilin of 3.16). Note that only 3 other proteins (cortactin, FAK and PLCbeta3) show similar levels of hyperphosphorylation. b This increase in cofilin phosphorylation was confirmed by western blotting of repeat cultures using a phospho-serine 3 specific antibody. c F-actin-bound (active) gelsolin is reduced in RPGR-mutant photoreceptors

Fig. 5

Knockout mouse studies confirm a role for Gelsolin in photoreceptor maintenance. a–c Outer nuclear layer (ONL) degeneration develops in the gelsolin knockout mouse at 5 months of age (a; bracket represents ONL length measured); significant for ONL thickness (b; n = 4, Figures denote mean±SEM, unpaired two-tailed t-test, ** = p < 0.01) and rows of nuclei (c; n = 4, Figures denote mean±SEM, unpaired two-tailed t-test, ** = p < 0.01). As in the Rpgr-KO mouse shown in Fig. 3, rhodopsin is mislocalised to the outer plexiform layer (arrowhead; a) and peri-nuclear area (arrow; a). d Increased GFAP immunolabeling throughout the radial length of Müller cells in the ONL is apparent (arrow) at 5 months of age. e Increased F-actin is seen in the connecting cilium (arrowhead) of Gelsolin KO mouse photoreceptor compared to wild-type. (Images representative of n = 3 analysed). Scale bars: 20 μm a, e

Fig. 6

Loss-of-function and gain-of-function experiments support a role for gelsolin in RPGR mediated cilia maintenance. a Immunoprecipitation (IP) of RPGR (upper panel) and gelsolin (lower panel) demonstrates an endogenous RPGR-gelsolin interaction in bovine retina, as evidenced by immunoblotting (IB) showing the presence of gelsolin (86 kD) and the 127 kD retina-specific form of RPGR respectively in the immunoprecipitates. b Immunopreciptation (IP) of gelsolin followed by immunoblotting (IB) for RPGR reveals that the RPGR-gelsolin interaction is present in control iPSC-derived photoreceptors but is perturbed in both XLRP patient-derived RPGR-mutant cultures—note in the righthand set of three lanes that RPGR is present only in the IPs from the control patient. The lower panel shows a longer exposure of the immunoblot to show that both the constitutively-expressed (90 kD, encoded by exons 1–19, arrowhead) and retina-specific (127 kD—encoded by exons 1–14 and specific open reading frame ORF 15, arrow) forms of RPGR are present in the control and mutant cell lysates shown in the three left hand lanes. Far right panel shows equal amounts of gelsolin are pulled down from each sample by gelsolin IP. c siRNA-mediated RPGR knockdown. Note that expression of both the constitutively-expressed (90 kD—Ex 1–19) and retina-specific (127 kD—ORF 15) forms of RPGR are reduced by the siRNA while the non-specific band at 100 kD is unaffected in the hTERT-RPE cell line. d, e Immmunolabeling of the hTERT-RPE cell line after siRNA-mediated RPGR knockdown reveals a loss of ciliogenesis (Mean percentage of ciliated cells 37.62%±3.113 vs 16.48%±3.762 (SEM), unpaired two-tailed t-test, p = 0.0124; identified by acetylated α-tubulin immunoreactivity in green), as shown by a comparison of the control (left) and RPGR knockdown (centre) panels. The defect is rescued by overexpression of constitutively active gelsolin, as shown in the right panel (Mean percentage of ciliated cells 16.48%±3.762 vs 38.33%±5.349 (SEM), unpaired two-tailed t-test, p = 0.0288.) The tracks below and to the right of the micrographs in d show z-planes of the image to confirm the presence of elongated cilia. Scale bars: 10 μm (d)

Fig. 7

Proposed model for RPGR mediated actin turnover in the photoreceptor connecting cilium. We propose two possible mechanisms of action for RPGR. First, RPGR’s regulation of actin turnover could mediate rhodopsin trafficking into the photoreceptor connecting cilium (left panels). An actin bundle connects the periciliary membrane complex to the basal body, along which Myosin VIIa actively transports visual pigments. Whirlin regulates this actin network at the connecting cilium base and interacts with both gelsolin and RPGR, suggesting a model whereby mutant RPGR disrupts the complex, perturbing actin turnover and compromising Myosin VIIa-mediated rhodopsin transport (left panels). The resulting rhodopsin mislocalisation to the inner segment results in cell stress and degeneration. Second, RPGR’s regulation of actin turnover could facilitate outer segment disc formation (right panels). Actin exerts an influence on both ciliogenesis and maintenance and is localised to the site of photoreceptor disc budding. The disorganised discs and hyperstabilised actin seen in the Rpgr knockout mouse supports a model whereby RPGR facilitates outer segment disc budding or the completion of disc formation by gelsolin-mediated actin turnover (right panels). The compromised disc morphogenesis seen with mutant RPGR would mislocalise rhodopsin to the inner segment, resulting in cell stress and degeneration