Activating injury-responsive genes with hypoxia enhances axon regeneration through neuronal HIF-1α

Injured peripheral neurons successfully activate a pro-regenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators coordinate the expression of such programs remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the pre-conditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knockout in vivo impairs sensory axon regeneration. The HIF-1α target gene Vascular Endothelial Growth Factor A (VEGFA) is expressed in injured neurons and contributes to stimulate axon regeneration. Induction of HIF-1α using hypoxia enhances axon regeneration in vitro and in vivo in sensory neurons. Hypoxia also stimulates motor neuron regeneration and accelerates neuromuscular junction reinnervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration. To study the genes regulated by HIF-1α in DRG neurons, we examined changes in gene expression in cultured DRG by microarray analysis, comparing DRG control condition to DRG overexpressing HIF-1α or to DRG in which HIF-1α was knocked down, in uninjured conditions as well as 3 and 12 h after in vitro axotomy. To express HIF-1α in cultured DRG neurons, human HIF-1α cDNA (Addgene, 18949) was sub-cloned into FUGW lentiviral vector for constitutively overexpression. HA-HIF1alpha-pcDNA3 was a gift from Dr. William Kaelin (Addgene plasmid #18949 (Kondo et al., 2002). To knock down HIF-1α MISSION shRNAs from Sigma were used and lentiviruses were generated as previously described (Cho and Cavalli, 2012). MISSION shRNAs from Sigma were used: HIF-1α, TRCN0000232222, TRCN0000232223. Total RNA was extracted 0, 3 and 12 h after axotomy and purified using PureLink RNA mini kit (Life Technologies) according to the manufacturer’s protocol. The RNA quality was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies; Palo Alto, CA, USA) using the Eukaryotic Total RNA Nano Assay. Samples were then run using the Mouse Ref-8 v2.0 Expression BeadChip (Illumina, Inc.; San Diego, CA, USA) by the Genome Technology Access Center at Washington University.