Transcriptional Analysis of the Effects of Integrin-driven Axon Regeneration in the Spinal Cord

To elucidate the mechanisms and down-stream pathways affected by activated integrin expression, we performed transcriptomic analyses of dorsal root gangliona (DRG) sensory neurons transduced with a9 integrin and its activator kindlin-1(a9k1), which enable axons to interact with tenascin-C, or controls, with and without dorsal root/central axotomy. Overall design: Four experimental groups were created through injection of either AAV5-fGFP or AAV5-a9-V5 + AAV5-kindlin1-GFP into four cervical (left C5-C8) dorsal root ganglia (DRGs) with and without dorsal root crush. The groups were: i) a9-kindlin-1-GFP with dorsal root crush to enable axon regeneration in the spinal cord (a9k1-crush), ii) a9-kindlin-1-GFP with no axotomy in order to examine the effects of activated integrin expression without regeneration (a9k1-naïve), iii) GFP with axotomy to control for the effects of AAV injection and axotomy (GFP-crush), and iv) GFP with no axotomy to control for the effects of AAV injection (GFP-naïve). The DRGs were removed after 8 weeks, at which time many axons are actively regenerating in the spinal cord, and GFP-expressing neurons were dissociated and selected by fluorescence-activated cell sorting (FACS), then mRNA purified and profiled by sequencing.