Genetic Rescue of X-Linked Retinoschisis Mouse (Rs1^−/y) Retina Induces Quiescence of the Retinal Microglial Inflammatory State Following AAV8-RS1 Gene Transfer and Identifies Gene Networks Underlying Retinal Recovery

Abstract

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. Clinical Trials. gov Identifier NTC 02317887.

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

“…RT-PCR analysis revealed no changes in mRNA gene expression as the cause for altered protein levels of Kv2.1 and Kv8.2. This is in line with a previous study by Vijayasarathy and colleagues, who used microarray-based genome-wide expression profiling and observed no mRNA expression differences for Kcnb1 and Kcnv2 as well as for Atp1a 3 and Atp1b2 in wildtype and retinoschisin-deficient retinae of P12 and P21 old mice [ 59 ]. Accordingly, the effect of retinoschisin-deficiency on Kv2.1 and Kv8.2 subunits should be attributed to a posttranslational process.…”

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors

“…Interestingly, RT-PCR analysis revealed no changes in mRNA gene expression as the cause for altered protein levels. This is in line with a previous study by Vijayasarathy and colleagues, who used microarray-based genome-wide expression pro ling and observed no mRNA expression differences for Kv2.1 and Kv8.2 as well as for Atp1a3 and Atp1b2 in wildtype and retinoschisin-de cient retinae of P12 and P21 old mice [37]. Accordingly, the effect of retinoschisinde ciency on Kv2.1 and Kv8.2 subunits should be attributed to a posttranslational process.…”

Retinal Proteomic Alterations and Combined Transcriptomic-Proteomic Analysis in the Early Stages of Progression of a Mouse Model of X-Linked Retinoschisis

“…To reveal the connections between the transcriptome and the proteome, and to discover important pathways and functional proteins, we performed a combined analysis of proteomic and published transcriptomic data [ 6 ]. The correlation analysis revealed that the proteome of RS1-KO retinas at P15 exhibited a modest correlation with the transcriptome at P12 but not with the transcriptome at P21 ( Figure 7 a,b).…”

“…Even though visual acuity may remain relatively stable in XLRS boys for years, an acceleration of vision loss due to central retinal atrophy in the fourth to fifth decade of life occurs [ 4 , 5 ]. Previous studies suggest that early interventions may alleviate the progress of XLRS in humans and animals [ 6 , 7 , 8 , 9 ]. Nevertheless, the manner in which the malfunction of the RS1 protein induces the degeneration of the inner and outer retinal neurons remains poorly understood.…”

“…By using RNA sequencing, Sieving et al investigated the transcriptomic changes in RS1-KO mice at P12 and P21. Only a few alterations were observed at P12, whereas 358 genes were differentially expressed at P21 [ 6 ]. In contrast to the transcriptomics research, deep proteomic analysis of RS1-KO retinas has not yet been conducted.…”