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Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

ABSTRACT: The goals of this study are to analyze the effects of RS1 mutation on retinal transcriptome profiling during retina differentiation. We used next generation sequencing to analyze the retinal mRNA profiles in a panel of time-course differentiated 3D retinal cups (RCs) from control donor- and XLRS patient-derived iPSCs, as well as 150-day differentiated RCs from CRISPR/Cas9-reparied patient-derived iPSCs. The transcriptomes of control-RCs and patient-RCs considerably diverged along the time course of differentiation, with the difference becoming more pronounced at the later time points. In contrast, analysis of repaired RCs revealed transcriptomes that were highly similar to those of control-RCs and significantly different from those of patient-RCs. Because RS1 is only expressed in later stage of differentiation, we focued on days 90, 120, and 150 post-induction to identify the genes whose expression was reduced at least 2-fold in patient-RCs. Among all of these genes, 326 genes were overexpressed in normal RCs at all of these stages, emphasizing their importance. According to GSEA annotation, nine out of these 326 genes were implicated in eye development. Among these nine genes, the expression of RS1, IQCB1, and OPA1 was rescued in CRISPR repaired RCs. The relative loss of expression became even more prominent in later stages of retinal differentiation. Our transcriptome analysis revealed significant changes in gene expression between patient-derived and control-RCs that become more pronounced at the later stages of development. This implies that RS1 is essential for normal retinal development at the systemic level. These results reflect the progressive nature of the disease phenotypes observed in RS-1-mutant RCs, mirror the observed ciliary phenotype, and reveal potential links of XLRS to other hereditary retinopathies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE137910 | GEO | 2022/09/03

REPOSITORIES: GEO

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Project description:To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death.

Project description:G protein coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3 kb-Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone which were accompanied by an increase in OB lineage cells, especially immature OBs, indicated by an expansion of cells expressing Osterix and Runx2 in the whole femur. In this study, we further evaluated how Gs signaling in OBs affects intramembranous bone formation by examining calvariae of one-and nine-week-old Col1(2.3)/Rs1 mice. Rs1 calvariae displayed a dramatic increase in total volume and trabecular bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space in Rs1 expressing mice while Osteocalcin was expressed predominantly in cells along bone surfaces. These findings resembled that previously seen in Rs1 femoral bones, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb-Col I promoter could influence early OB commitment, differentiation, and/or proliferation. However, it is still unclear how G protein signaling in mature OBs leads to the observed alterations in bone mass. In this study, we investigated the cellular basis of the skeletal changes by assessing the effect of Rs1 expression in vivo on the transcriptome of mature OBs. We identified the complete set of Gs-GPCRs and other GPCRs that are expressed on OBs which may contribute to the observed skeletal phenotype. Candidate paracrine mediators of the effect of Gs signaling in OBs were determined. Genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. Our results identify novel candidate mediators of the anabolic response to the skeleton to Gs signaling in mature OBs. We utilized a microarray approach to examine Rs1-induced alterations in the OB transcriptome. The approach used to identify an approximate snapshot of the OB transcriptome at the time of sacrifice by isolating the OB population that expresses Rs1 by GFP labeling, without the use of cell culture. We compared gene expression between control OBs and OBs-expressing Rs1 in triplicates.

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Morphological and Molecular Defects in Human Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

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