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RNA-seq of rd1 retinal microglia after treatment with AAV8-GFP vs. AAV8-GFP + AAV8-TGFB1

ABSTRACT: Transcriptional profiling of microglia (seven biological replicates per experimental condition) or non-microglia (four biological replicates total). One thousand microglia (CD11b+ Ly6G/Ly6C-) or non-microglia cells (CD11b-) from P30 rd1 (FVB) retinas infected with either AAV8-GFP or AAV8-GFP + AAV8-TGFB1 were sorted into 10 µL of Buffer TCL (Qiagen) containing 1% beta-mercaptoethanol and immediately frozen on dry ice. Samples were subsequently sent to the Broad Institute Genomics Platform for cDNA library synthesis and sequencing using a modified Smart-Seq2 protocol with an expected coverage of ~6 million reads per sample. Prior to analysis, reads were subjected to quality control measures and mapped to the GRCm38.p6 reference genome. Reads assigned to each gene were then quantified using featureCounts and normalized and analyzed for differential expression using DESeq2.

ORGANISM(S): Mus musculus

PROVIDER: GSE145601 | GEO | 2020/02/21

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.

Dataset Information

RNA-seq of rd1 retinal microglia after treatment with AAV8-GFP vs. AAV8-GFP + AAV8-TGFB1

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