Stimulation of neuronal stress signaling in optic nerve crush

Dual Leucine-zipper Kinase (DLK)-dependent stress signaling is a critical determinant of neuronal survival and regenerative potential following axon damage, but it remains uncertain whether injury-activated DLK is adequate to initiate and maintain a pro-regenerative transcriptional response in the CNS. Using a drug-activatable DLK construct, we stimulated stress signaling for comparison of the retinal transcriptional response to, and in addition to, the response stimulated by mouse optic nerve injury in wildtype mice and in the context of partial axon regeneration enabled by disruption of the tumor suppressor PTEN. In a study to understand how augmenting stress signaling influences gene expression under conditions in which retinal ganglion cells (RGCs) undergo apoptosis in response to optic nerve. crush, mice were intravitreally injected with AAV2-hSyn1-mTagBFP2-ires-Cre and either a vector for the expression of drug-activatable DLK (AAV2-hSyn1-mTagBFP2-2A-ddDLK) or a control vector (AAV2-hSyn1-mTagBFP2-2A-ddYFP). Following optic nerve crush and repeated stimulation of ddDLK activity by systemic trimethoprim (TMP) injection, retinas were harvested 3 days post-crush for RNA-seq. In a parallel study to understand how augmenting stress signaling influences gene expression under conditions in which RGCs exhibit limited axon regeneration, PTEN conditional knockout mice were intravitreally injected with AAV2-hSyn1-mTagBFP2-ires-Cre and either a vector for the expression of drug-activatable DLK (AAV2-hSyn1-mTagBFP2-2A-ddDLK) or a control vector (AAV2-hSyn1-mTagBFP2-2A-ddYFP). Following optic nerve crush and repeated stimulation of ddDLK activity by systemic trimethoprim (TMP) injection, retinas were harvested at two time points (7 days post-crush and 10 days post-crush) for RNA-seq.