PALS1 is essential for retinal pigment epithelium structure and neural retina stratification

Abstract

The membrane-associated palmitoylated protein 5 (MPP5 or PALS1) is thought to organize intracellular PALS1-CRB-MUPP1 protein scaffolds in the retina that are involved in maintenance of photoreceptor-Müller glia cell adhesion. In humans, the Crumbs homolog 1 (CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. However, there is no clear genotype-phenotype correlation for CRB1 mutations, which suggests that other components of the CRB complex may influence the severity of retinal disease. Therefore, to understand the physiological role of the Crumbs complex proteins, especially PALS1, we generated and analyzed conditional knockdown mice for Pals1. Small irregularly shaped spots were detected throughout the PALS1 deficient retina by confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. The electroretinography a- and b-wave was severely attenuated in the aged mutant retinas, suggesting progressive degeneration of photoreceptors. The histological analysis showed abnormal retinal pigment epithelium structure, ectopic photoreceptor nuclei in the subretinal space, an irregular outer limiting membrane, half rosettes of photoreceptors in the outer plexiform layer, and a thinner photoreceptor synaptic layer suggesting improper photoreceptor cell layering during retinal development. The PALS1 deficient retinas showed reduced levels of Crumbs complex proteins adjacent to adherens junctions, upregulation of glial fibrillary acidic protein indicative of gliosis, and persisting programmed cell death after retinal maturation. The phenotype suggests important functions of PALS1 in the retinal pigment epithelium in addition to the neural retina.

Figure 1.

Decreased levels of PALS1 in the retina after conditional gene silencing. A, Schematic representation of shPals1-RxCre retinas after Cre-mediated recombination. After Cre recombination, the U6 promoter drives the expression of shRNA against the Pals1 gene as illustrated. The small arrow indicates the insertion of the short hairpin Pals1 sequence behind the second loxP. PGK, Phosphoglycerate kinase promoter; PURO, puromycin gene; U6, U6 promoter. B, Genotyping of the mice (RxCre, shPals1, and shPals1-RxCre) was done by long-distance-PCR on genomic DNA isolated from E15 or 3-month-old retinas. The upper PCR bands (1979 bp) are from non-recombined and the lower bands (466 bp) are from recombined DNA products. 3M, 3 Months of age; E15, embryonic day 15. C, PALS1 protein levels were decreased to 52 ± 3% in P6 shPals1-RxCre retinas. Total retina cell lysates were used for SDS-PAGE and Western blot analysis. PALS1 antibody was used to detect PALS1 protein in the retinas and actin antibody was used for loading control. Expression levels were normalized to actin.

Figure 2.

Decreased levels of PALS1 in shPals1-RxCre retinas caused early onset of loss of vision. A, cSLO and SD-OCT analysis from 1-, 3-, 6-, and 12-month-old shPals1-RxCre and control mice (RxCre). Spotty and patchy areas as well as vascular abnormalities (arrows in angiography) were found in shPals1-RxCre mouse retinas with cSLO fundus imaging. OCT analysis further revealed reduction of the retinal thickness in the outer retina as well as cellular mislocalization. Red lines indicate the thickness of the photoreceptor layer. Arrowheads indicate folded ONL regions and borders of photoreceptor degeneration. ICGA, Indocyanine green angiography; FLA, fluorescence angiography. B, Scotopic and photopic single-flash ERG responses from representative animals for a given genotype at the age of 3 months. The reduced scotopic ERG a-wave indicated photoreceptor degeneration (filled arrows in the right column). C, Single-flash ERG age series in shPals1-RxCre mutant (red), RxCre (green, control), or shPals1 (black, control) mice. Scotopic (SC, top) and photopic (PH, bottom) b-wave amplitudes were plotted as a function of the logarithm of the flash intensity. Data were obtained from 1-, 3-, 6-, and 12-month-old shPals1-RxCre, 1-, 3-, 6-, and 12-month-old RxCre, and 1- and 3-month-old control shPals1 animals. Boxes indicate the 25% and 75% quantile range, whiskers indicate the 5% and 95% quantiles, and solid lines connect the medians of the data. Note the decline of the b-wave amplitude in shPals1-RxCre mice with increasing age up to 12 months of age under both scotopic and photopic conditions, indicating degeneration of both rod and cone system components.

Figure 3.

Decreased levels of PALS1 in shPals1-RxCre retinas caused retinal degeneration and abnormal RPE structure. A, Technovit retinal sections of control RxCre mice at postnatal day 6 (P6), P10, P18, and P30 and 12 months of age (12M), and shPals1-RxCre mice at P6, P10, P18, and P30 and 3, 6, and 12 months of age (3M, 6M, 12M). The phenotype in shPals1-RxCre retinas is detectable initially at the peripheral part of the retina at P6, and it spreads to the entire retina following retinal maturation. Even though the retinal layers (ONL, INL, GCL) are separated, retinal folds and half rosettes of the ONL are detected, and photoreceptor nuclei protruded into the subretinal space and ingressed into the OPL from P10 onwards. In aged mutant retinas (3–12M), the ONL and OPL become thinner due to gradual retinal degeneration. The ONL is also severely damaged and abnormal large vacuoles are detected in the RPE layer. Scale bar, 50 μm. B, Representative PALS1 staining images from immuno-EM and IHC analysis are shown from wild-type at P10. a, IHC flat mount of RPE cells stained with anti-PALS1. At immuno-EM level, the RPE monolayer has apical microvilli toward the photoreceptor outer segments and the cells are interconnected by tight junctions (b, c). Arrowheads indicate the basolateral membrane. b, c, Arrows indicate PALS1 at the tight junction (b) and in apical microvilli surrounding a photoreceptor outer segment (c). Scale bars: 20 μm (a), 0.5 μm (b, c). C, EM analysis of RxCre and shPals1-RxCre mice at P10. RPE cells appeared to be disorganized with short microvilli (double-headed arrows in d), reduced/disorganized basal infoldings (arrow in c), and in addition, numerous vacuoles were present within the cells (c, asterisk). N, RPE cell nucleus; OS, photoreceptor outer segments; Mv, microvilli; BM, Bruch's membrane; BI, basal infoldings. Scale bars: 5 μm (a, c), 1 μm (b, d).

Figure 4.

Decreased levels of PALS1 in shPals1-Chx10Cre retinas caused late onset of loss of vision. A, Genotyping of the mice (Chx10Cre, shPals1, and shPals1-Chx10Cre) was done on genomic DNA isolated from 3-month-old retinas. The lower bands (466 bp) are from shPals1-Chx10Cre recombined DNA products. B, PALS1 protein levels were decreased to 66 ± 4% in P6 shPals1-Chx10Cre retinas. PALS1 antibody was used to detect PALS1 protein in the retinas and actin antibody was used for loading control. Expression levels were normalized to actin. C, cSLO and OCT analysis of 12-month-old shPals1-Chx10Cre retinas. shPals1-Chx10Cre retinas showed almost normal fundus appearance and regular vascular pattern in cSLO fundus imaging but some mislocalized structures in the OPL are found with OCT imaging. Red lines indicate the thickness of the photoreceptor layer and arrowhead indicates folded ONL regions. D, Electroretinographic data from 12-month-old shPals1-Chx10Cre mutant mice and control Chx10Cre mice showing scotopic (SC, left) and photopic (PH, right) b-wave amplitudes from controls (green) and shPals1-Chx10Cre mice (red) as a function of the logarithm of the flash intensity. Boxes indicate the 25% and 75% quantile range, whiskers indicate the 5% and 95% quantiles, and solid lines connect the medians of the data. Insets, Scotopic (left) and photopic (right) single-flash ERGs with 1.5 log cd · s/m² intensity of a Chx10Cre control mouse (green traces) and a shPals1-Chx10Cre mouse (red traces). The 12-month-old shPals1-Chx10Cre mice showed only slightly reduced scotopic and photopic responses compared to the Chx10Cre littermate controls. E, Morphological analysis of control Chx10Cre and shPals1-Chx10Cre retinas. At 3 months of age (3M), the shPals1-Chx10Cre retinas showed mild and sporadic phenotype. At 12 months of age (12M), the mutant retinas showed disturbed ONL and INL layers and also reduced width of the OPL. Scale bar, 50 μm. F, EM analysis of shPals1-Chx10Cre at P10. The RPE of shPals1-Chx10Cre showed normal cell structure as Chx10Cre controls. N, RPE cell nucleus; Mv, microvilli; BM, Bruch's membrane; BI, basal infoldings. Scale bar, 5 μm.

Figure 5.

PALS1 localizes with CRB1 and MUPP1 to the subapical region adjacent to adherens junctions at the outer limiting membrane. Immunohistochemistry on 3-month-old retinas showing confocal images of staining for proteins associated with the Crumbs complex, PALS1, CRB1, and MUPP1, and for GFAP, a Müller glial cell marker. Staining for the Crumbs complex shows fragmented staining (arrows) of PALS1, CRB1, and MUPP1 in the shPals1-RxCre retinas, which indicate the dislocalization of the Crumbs complex proteins at the SAR adjacent to adherens junctions at the OLM. GFAP staining shows increased reactive gliosis in the mutant retinas. Scale bar, 50 μm.

Figure 6.

Retinal cell specification is normal, but cell patterning is aberrant in shPals1-RxCre retinas. At P10, in RxCre control retina, the staining of PKCα (green), a bipolar cell marker, and recoverin (red), a photoreceptor cell and cone bipolar cell marker, show well separated, properly layered cells. At P10, in shPals1-RxCre retinas, even though the retina layers are folded and some photoreceptor cells are dislocated into the subretinal space, most retina cells are separated and properly located in the retina. TO-PRO-3 was used for nuclear DNA staining (blue). Scale bar, 50 μm.

Figure 7.

Reduced levels of PALS1 do not affect the size of cone photoreceptor outer segments. A, PNA staining (red) for cone outer segment on representative sections of RxCre control retina and two different regions (less disorganized, left panel, and more disorganized, right panel) from the same shPals1-RxCre mutant retina at P18. The subapical region adjacent to the OLM is stained by anti-PALS1 (green). Scale bar, 50 μm. B, Histogram shows the length of cone outer segments in the mutant retinas and control retinas at P10, P18, and P30. The PNA stained cone outer segments at less disturbed regions were measured to verify the involvement of PALS1 in the growth of cone outer segment during development, but there was no significant difference in P10 and P18 except for in P30 (B). Asterisk (*) indicates significant difference compared to the control (Student's t test, p < 0.0007). Error bars indicate +SEM.

Figure 8.

Retinas lacking PALS1 undergo persistent programmed cell death at maturation of the retina. A, Immunohistochemistry for c-CAS3 on representative sections of shPals1-RxCre and RxCre retinas at P6 and P18. Apoptotic cells were stained with c-CAS3 antibodies (red). Nuclear DNA was counterstained with TO-PRO-3 (blue). Scale bar, 50 μm. B, Histogram depicting the number of c-CAS3-positive cells in shPals1-RxCre and control retinas at P6 and P18. Asterisk (*) indicates significant difference compared to the control (Student's t test, p < 0.0017 for P18 OLM, p < 0.0029 for P18 INL). Error bars indicate +SEM. C, Immunohistochemistry for apoptotic cells and bipolar cells on representative sections of shPals1-RxCre and control RxCre retinas at 12 months of age. Apoptotic cells were stained with c-CAS3 antibodies (red, arrowheads), and bipolar cells with anti-PKCα (green). Nuclear DNA was counterstained with TO-PRO-3 (blue). Dashed quadrants in the upper panels indicate examples of a c-CAS3-positive cell, and are shown enlarged in the panel down on the left (scale bar, 10 μm). The number of photoreceptor and bipolar cells decreased over time. Scale bar, 50 μm. D, Quantification of the outer nuclear layer thickness. ONL thickness measured every 500 μm from the optic nerve to an area near the peripheral edge of retinal sections in control (12M) and shPals1-RxCre (3, 6, 12M). Error bars indicate ±SEM.