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Ying Zhu, Bogdan Tanasa, Michael Nahmou, Jeffrey L Goldberg, Michael Kapiloff; Single cell RNA sequencing of retinal ganglion cells with enhanced perinuclear cAMP signaling after optic nerve crush injury. Invest. Ophthalmol. Vis. Sci. 2021;62(8):3006.
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Enhanced cyclic-AMP (cAMP) signaling promotes neuronal survival after injury. cAMP signaling is highly compartmentalized in cells, and distinct compartments regulate different cellular processes. We recently showed that elevation of cAMP in a perinuclear compartment organized by the scaffold protein muscle A-kinase anchoring protein α (mAKAPα) is sufficient to protect retinal ganglion cells (RGCs) in the mouse optic nerve crush model. However, the underlying mechanisms remain unclear. In this study, we aim to identify neuroprotective gene expression regulated by perinuclear cAMP in RGCs.
We intravitreally injected 4-week-old C57BL/6J mice with the adeno-associated virus gene therapy vector AAV2.4D3(E)-mCherry to express in RGCs the type 4D3 phosphodiesterase anchoring disruptor peptide 4D3(E)-mCherry, which displaces PDE4D3 from mAKAPα signalosomes in neurons and elevates cAMP in that compartment. Control mice were injected with AAV2.mCherry. Two weeks later the mice were subjected to right-sided optic nerve crush. RGCs pooled from 8 eyes for each cohort were isolated by immunopanning with Thy-1 antibody one day after crush. Cell suspensions were submitted to the Stanford Genome Sequencing Service Center (GSSC) for 10X Chromium Single Cell 3’ library preparation and HiSeq 4000 sequencing. scRNA-seq analysis was carried out with Seurat using R.
3800-6000 cells were sequenced for each condition, among which 15%-25% expressed RBPMS and were identified as RGCs. About half of the RGCs in each group expressed the mCherry reporter gene. Analysis of differentially expressed genes showed that expression of the 4D3-mCherry anchoring disruptor reversed many of the changes in gene expression induced by crush injury (p=1e-16). In particular, Npy, Mmp12, Ucn and Timp2 were differentially regulated by elevation of cAMP in the perinuclear compartment.
Our study shows that elevation of perinuclear cAMP can reverse changes in gene expression following acute optic nerve crush injury, providing an explanation for the pro-survival effects of PDE4D3 anchoring disruption. In addition, our results provide candidate therapeutic targets for subsequent testing. In the future, we will apply this approach to derive further insight into the diversity of gene expression programs relevant to RGC neuroprotection and axon regeneration therapies.
This is a 2021 ARVO Annual Meeting abstract.
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