Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors

Abstract

Purpose: Loss of retinoschisin (RS1) function underlies X-linked retinoschisis (XLRS) pathology. In the retina, both photoreceptor inner segments and bipolar cells express RS1. However, the loss of RS1 function causes schisis primarily in the inner retina. To understand these cell type-specific phenotypes, we decoupled RS1 effects in bipolar cells from that in photoreceptors.

Methods: Bipolar cell transgene RS1 expression was achieved using two inner retina-specific promoters: (1) a minimal promoter engineered from glutamate receptor, metabotropic glutamate receptor 6 gene (mini-mGluR6/ Grm6) and (2) MiniPromoter (Ple155). Adeno-associated virus vectors encoding RS1 gene under either the mini-mGluR6 or Ple-155 promoter were delivered to the XLRS mouse retina through intravitreal or subretinal injection on postnatal day 14. Retinal structure and function were assessed 5 weeks later: immunohistochemistry for morphological characterization, optical coherence tomography and electroretinography (ERG) for structural and functional evaluation.

Results: Immunohistochemical analysis of RS1expression showed that expression with the MiniPromoter (Ple155) was heavily enriched in bipolar cells. Despite variations in vector penetrance and gene transfer efficiency across the injected retinas, those retinal areas with robust bipolar cell RS1 expression showed tightly packed bipolar cells with fewer cavities and marked improvement in inner retinal structure and synaptic function as judged by optical coherence tomography and electroretinography, respectively.

Conclusions: These results demonstrate that RS1 gene expression primarily in bipolar cells of the XLRS mouse retina, independent of photoreceptor expression, can ameliorate retinoschisis structural pathology and provide further evidence of RS1 role in cell adhesion.

Trial registration: ClinicalTrials.gov NCT02317887.

Conflict of interest statement

Disclosure: C. Vijayasarathy, None; Y. Zeng, None; D. Marangoni, None; L. Dong, None; Z.-H. Pan, co-inventor of the improved mGluR6 promoter; E.M. Simpson, None; R.N. Fariss, None; P.A. Sieving, co-inventor of the AAV8-RS1 construct and participates in the NIH XLRS gene therapy trial (ClinicalTrials.Gov NCT02317887); Co-Founder of VegaVect Therapeutics, Inc. with intent to conduct work in the XLRS clinical space

Figures

Figure 1.

Human and mouse XLRS. (A) Retina photograph of a 17-year-old XLRS subject with 20/63 acuity showing classic macular retinoschisis with a subtle spoke wheel pattern radiating from the fovea. (B) Representative dark-adapted ERG combined responses (arising from photoreceptors and bipolar cells) of the XLRS subject show characteristic b-wave reduction disproportionate to a-wave reduction. (C) RS1 expression in mouse retina and XLRS phenotype. Retinal cryosections were immunolabeled with antibodies against RS1 (red, 1:1000); Na/K ATPase a3 subunit (green, 1:1000). The nuclei were counterstained with DAPI (blue). In WT retina RS1 is profusely expressed in photoreceptor IS and in inner retinal layers. In XLRS mouse retina, loss of Rs1 expression results in splitting of the inner retina cell layers. (D) RS1 expression on mouse retina bipolar cells and its colocalization with Na/K-ATPase, a plasma membrane marker. (E) Discoidin domain has been shown to be involved in cell adhesion during the streaming and aggregation of D.discoideum. During the growth phase of development, D.discoideum amoeboid cells feed on bacteria and replicate by binary fission. The development cycle is initiated upon starvation (resource depletion), and aggregation occurs when starving cells secrete cyclic AMP to recruit additional cells. Discoidin I is synthesized profusely as cells stream together into aggregate to form slug and fruiting body. OPL, outer plexiform layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Fig 1E is reproduced from Dunn JD, Bosmani C, Barisch C, et al. Eat prey, live: Dictyostelium discoideum as a model for cell-autonomous defenses. Front Immunol. 2018;8:1906. Copyright © 2018 Dunn, Bosmani, Barisch, Raykov, Lefrançois, Cardenal-Muñoz, López-Jiménez and Soldati; open-access, distributed under the terms of the Creative Commons Attribution License (CC BY).

Figure 2.

In XLRS mouse, AAV2/2-Y444F–mediated mCherry expression is restricted to cells in the inner retina. Retinal expression of fluorescent transgenes mCherry was examined 5 weeks after intravitreal injection of In4s-In3-200En-mGluR500P-mCherry (mini-mGluR6-mCherry) into XLRS mouse retina. mCherry expression was restricted to cells in the inner retina, most predominately in RBCs. There is no labeling of mCherry in the photoreceptor layer. Vertical sections of XLRS mouse retinas were colabelled for mCherry (red), protein kinase Cα (green), RBC marker; calretinin (green), amacrine and ganglion cell marker. DAPI was used as a nuclear counterstain (blue).

Figure 3.

(A) RS1 expression variability and schisis cavity closure in XLRS mouse retina under mini-mGluR6 promoter. RS1 expression in WT and XLRS mouse retinas 5 weeks after intravitreal injection of AAV2/2-Y444F vector encoding human RS1 gene under bipolar cell specific mini-mGluR6 (In4s-In3-200En-mGluR500P) promoter. Retinal sections from injected XLRS mice along with age matched C57BL/6 WT mice were immunolabeled with antibodies raised against RS1 (red), Na/K ATPase a3 subunit (green), and DAPI counterstain for nuclei (blue). In WT retina, Rs1 is profusely present in the photoreceptor ISs, outer plexiform layer (OPL), bipolar cell layer, and IPL. In XLRS mouse retinas, intravitreal injection of viral vector leads to RS1expression specifically in inner retina with Rs1 immunoreactivity in cell processes within the IPL with moderate to modest cavity closure. RS1 localization is also seen in IS of photoreceptors. Retina 3 has the least cavity closure even though it has the most RS1 labeling. (B) RS1 expression in transgenic mice under mini mGluR6 promoter. In transgenic mice the mini-mGluR6 promoter drives RS1 gene expression in the inner retina but the transgenic mice tended to have significantly more variability in gene expression levels with no effect on cavity closure. RS1 labeling is also seen in photoreceptor IS. Creation of transgenic mice is described in Materials and Methods section. (C) RS1 localization on photoreceptor IS. Mini-mGluR6-RS1 injected XLRS mice retinas showed immunolabeling of Na/K ATPase (green) in photoreceptor IS, OPL and IPL, the labeling being intense in IPL. Merged images confirmed a high degree of colocalization of the Na/K ATPase (green) and RS1 (red) in all layers of the retina.

Figure 4.

MiniPromoter Ple155 drives bipolar cell specific RS1 gene expression in XLRS mice retinas. Retinal sections from representative vector injected and uninjected XLRS mice 5 weeks after subretinal injection of AAV8-Ple155-RS1 vector were immunolabeled with anti-RS1 antibody (red), RBC marker protein kinase Cα (green), Müller glia marker glutamine synthetase (GS), (green) and DAPI counterstain for nuclei (blue). Subretinal injection of AAV8-Ple155-RS1 at postnatal day 14, led to robust RS1 expression in bipolar cells (a, e) as confirmed by costaining with the bipolar cell marker PKCα (b). Compared with uninjected mice retina (c, d), which showed no RS1 labeling and displayed large schisis cavities, injected retinas showed schisis closure and marked improvement in inner retina structure. In AAV 8-Ple155-RS1 injected retinas, RS1 staining juxtaposed in close physical association with parallel Müller cell processes as revealed with anti-GS staining (f, h). Representative images for 4 mice in each group. The images in the lower panel show RS1 expression variability and photoreceptor localization in Ple155 injected retinas (n = 4).

Figure 5.

Functional and structural changes in XLRS mice retinas after injection of AAV8 vector encoding RS1 under Ple155 promoter. (A) Representative ERG waveforms recorded in scotopic and photopic conditions in 3 XLRS mice between 5 and 6 weeks after unilateral subretinal injection of AAV8-PLe155-RS1. The flash luminances at which the dark- and light-adapted ERGs shown in the figure were elicited were −0.82 log sc cd-s/m2 and 1.0 log sc cd-s/m2, respectively. Both scotopic and photopic ERG responses in the injected eye (red waveforms) were increased in amplitude when compared with the uninjected eye (black waveforms). All three study animals showed larger scotopic and photopic b-wave amplitudes and a shorter scotopic a-wave implicit time in the treated eyes, indicating a selective improvement of the postphotoreceptorial element function, including the photoreceptor–bipolar cell synapse. The a-wave amplitude is measured from baseline (0 ms) to the negative trough; b-wave amplitude is measured from the a-wave trough to the positive peak. Stimulus flash occurs at 0 ms. (B) Analysis of retinal structure by OCT in the same 3 animals showed a reduction in intraretinal cystic cavities in the eye injected with the vector and almost complete restoration of the normal retinal architecture.