Displacement of PDE4D3 with an anchoring disruptor peptide is RGC protective after injury

Loss of retinal ganglion cells (RGCs) is central to the pathogenesis of optic neuropathies such as glaucoma. Increased cAMP signaling in RGCs is neuroprotective, as has been previously demonstrated in multiple animal models, including optic nerve crush (ONC) injury. 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 protein kinase A activity in cultured neurons and RGC survival in vivo after ONC injury. To explore potential mechanisms by which 4D3(E) expression promotes neuroprotection, 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) for changes in RGC gene expression. 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. scRNA-seq was performed using RGCs isolated from C57BL/6J mice 1 day after crush injury. 4-5 week old mice (Jackson Laboratories Strain 000664) of both sexes were injected intravitreally with AAV2.4D3(E)-mCherry or AAV2.mCherry control. 2 weeks after AAV administration, paired cohorts of 8 mice were subjected to optic nerve crush or sham injury. The following day RGCs were isolated by a modified two-step immunopanning protocol and analyzed by scRNA-seq. The entire experiment was performed twice, resulting in biological duplicates for each of the four cohorts.