Reversal of injury-associated retinal ganglion cell gene expression by a phosphodiesterase anchoring disruptor peptide

Abstract

Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased RGC cAMP signaling is neuroprotective. We have shown that displacement of the cAMP-specific phosphodiesterase PDE4D3 from an RGC perinuclear compartment by expression of the modified PDE4D3 N-terminal peptide 4D3(E) increases perinuclear cAMP and protein kinase A activity in cultured neurons and in vivo RGC survival after optic nerve crush (ONC) injury. To explore mechanisms by which PDE4D3 displacement promotes neuroprotection, in this study mice intravitreally injected with an adeno-associated virus to express an mCherry-tagged 4D3(E) peptide were subjected to ONC injury and analyzed by single cell RNA-sequencing (scRNA-seq). 4D3(E)-mCherry expression was associated with an attenuation of injury-induced changes in gene expression, thereby supporting the hypothesis that enhanced perinuclear PKA signaling promotes neuroprotective RGC gene expression.

Keywords: Gene expression; Neuroprotection; Retinal ganglion cell; cAMP.

Figure.. scRNA-seq analysis of RGC gene expression in mice treated with AAV2.4D3(E)-mcherry and subjected to optic nerve crush injury.

A. Experimental paradigm. C57BL/6J mice were injected with 10¹⁰ viral genomes AAV2.4D3(E)-mcherry (4D3) or control AAV2.mCherry (Con) two weeks prior to optic nerve crush (ONC) or sham injury. RGCs with detectable mCherry and RBPMS expression were analyzed by scRNA-seq. B. The numbers of RGCs identified for each of two duplicate cohorts (#) are indicated. C. The effect on RGC gene expression of 4D3(E)-mCherry during injury (Con-ONC vs. 4D3-ONC) is compared by linear regression to the effect of ONC injury (Con-Sham vs. Con-ONC). Log2 fold-change is graphed for the 1181 genes for which the unadjusted p-value for Con-Sham vs. Con-ONC was < 0.05. D. Heat map of log2 fold change for the 1181 genes studied in B for the indicated cohort comparisons. The genes are sorted by log2 fold change for the Con-Sham vs. Con-ONC comparison. E. Pearson correlation analysis for the log2 fold change for the 1181 genes studied in B for the indicated cohort comparisons. r-values are shown in bold, p-values below. F. Gene Ontology analysis (https://geneontology.org/) was performed for the 981 of the 1181 injury-induced genes that were improved in expression towards the non-ONC state by 4D3(E)-mCherry expression. Analysis was performed separately for the genes that were up- and down-regulated by injury. Selected gene ontologies are shown based upon a maximum p-value of 10⁻⁴ and deletion of ontologies that were highly redundant.