Activating Injury-Responsive Genes with Hypoxia Enhances Axon Regeneration through Neuronal HIF-1α

Abstract

Injured peripheral neurons successfully activate a proregenerative transcriptional program to enable axon regeneration and functional recovery. How transcriptional regulators coordinate the expression of such program remains unclear. Here we show that hypoxia-inducible factor 1α (HIF-1α) controls multiple injury-induced genes in sensory neurons and contribute to the preconditioning lesion effect. Knockdown of HIF-1α in vitro or conditional knock out 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 re-innervation. This study demonstrates that HIF-1α represents a critical transcriptional regulator in regenerating neurons and suggests hypoxia as a tool to stimulate axon regeneration.

Figure 1. Statistical analysis of genes activated by axon injury

(A) Percentage of up-regulated genes after in vitro axon injury that are known HIF-1α target genes. The expression of 91 known HIF-1α target genes was compared to the genes up-regulated in injured cultured DRG neurons. 58 genes out of 91 (63.7%) were up-regulated over the 1.2 threshold at 3, 8, 12 or 40 h after in vitro axotomy. (B) As in A but comparison to predicted HIF-1α target genes. 61 genes out of 98 (62.2%) were up-regulated over the threshold at 3, 8, 12 or 40 h after in vitro axotomy. (C) Distribution of percentages of up-regulated genes over the threshold in 220,000 randomly selected groups of 189 genes from a total of 15310 genes detected in the microarray analysis (with detection p value below 0.01). The percentage of up-regulated genes over the threshold in each group was calculated and plotted (mean, 48.3%; SD, 3.4). The probability of obtaining 62.2% of up-regulated genes in a group of 189 genes is below 0.005%. (D) Quantitative PCR analysis of mRNA prepared from mouse DRG cultures at 0, 3, 8, 12 and 40 h after axotomy (n=3; mean±SEM). For each gene, the log₂-transformed expression fold change at the time after axotomy at which maximum fold change was detected is indicated.

Figure 2. Microarray analysis of genes regulated by HIF-1α in DRG neurons

(A) Microarray analysis of gene expression profiles from RNA samples of control DRG cultures, HIF-1α-knock down (KD) or HIF-1α-overexpression (OE) cultures (X-axis, arbitrary units of normalized expression levels of control; Y-axis, arbitrary units of normalized expression levels of HIF-1α-knockdown (left) or HIF-1α-overexpression (right); R², coefficient of determination). (B) Tree map of functional ontology from genes up-regulated by HIF-1α-overexpression (left) or down-regulated by HIF-1α-overexpression (right). (C) Schematic diagram of the screen identifying injury-responsive genes regulated by HIF-1α. (D) Functional gene ontology of HIF-1α-regulated injury-responsive genes.

Figure 3. HIF-1α is required for axon regeneration in vitro

(A) DRG spot-cultured neurons were infected with control shRNA (control), two different shRNA targeting HIF-1α (KD1, KD2) or HIF-1α–overexpressing lentivirus (OE), fixed and immunostained with SCG10 antibody 40 h after axotomy. Scale bar, 200µm. Dotted line indicates the axotomy site. (B) In vitro regeneration index was calculated from images in (A) (n=9, 14 and 16 for each condition; ***p<0.001 by one-way ANOVA with Tukey test; mean±SEM; ns, not significant). (C) In vitro regeneration assay was performed with the knockdown of selected candidate HIF-1α target genes (n=11, 12, 12, 17, 13, 15, 13 and 11 for Control, PDE1B, HMOX1, MAP3K1, ARHGEF3, ARHGAP29, NGFR and VEGFA; ***p<0.001, **p<0.01, *p<0.05 by one-way ANOVA with Tukey test; mean±SEM).

Figure 4. HIF-1α is required for axon regeneration in vivo

(A) Representative longitudinal sections of sciatic nerve from control or HIF1AcKO mice three days after crush injury stained with SCG10 and βIII tubulin (TUJ1) antibody. Scale bar, 500µm. Dotted lines indicate the crush site, identified as the maximal SCG10 intensity. (B and C) In vivo regeneration index was calculated from images in (A). SCG10 intensity was measured from the crush site towards the distal end and normalized to the intensity at the crush site. SCG10 intensity was plotted as a function of the distance from the crush site (n=7 and 10 for control and HIF1AcKO, respectively; ***p<0.001, *p<0.05 by one-way ANOVA with Tukey test; mean±SEM). (D) Representative longitudinal sections of mouse L4 DRGs dissected 24 h after sciatic nerve or dorsal root injury, immunostained with HIF-1α and βIII tubulin. Scale bar, 20µm. (E) Average of relative intensity of nuclear HIF-1α over total HIF-1α from images in (D) (n=20, 22 and 18 for no injury, sciatic nerve injury (SN) and dorsal root injury (DR), respectively; ***p<0.001 by one-way ANOVA with Tukey test; mean±SEM; ns, not significant). (F) Average percentage of DRG neurons displaying nuclear HIF-1α intensity over 30% (n=10, 12 and 12 for no injury, sciatic nerve injury (SN) and dorsal root injury (DR), respectively; ***p<0.001, *p<0.05 by one-way ANOVA with Tukey test; mean±SEM).

Figure 5. HIF-1α contributes to pre-conditioning lesion effects

(A) Representative longitudinal section images of sciatic nerve dissected 1 day after single injury (“single injury”). Sections from control or HIF1AcKO mice were immunostained with SCG10 and βIII tubulin. Scale bar, 500µm. Dotted lines indicate the crush site. (B) Representative longitudinal section images of mouse sciatic nerve 1 day after a second injury, given 3 days after a pre-conditioning injury (“double injury”). Sections from control or HIF1AcKO were immunostained with SCG10 and βIII tubulin. Scale bar, 500µm. Dotted lines indicate the crush site. (C) In vivo regeneration index was calculated from images in (B). SCG10 intensity was measured from the crush site towards the distal end and normalized to the intensity at the crush site. SCG10 intensity was plotted as a function of the distance from the crush site (n=6 for each condition; ***p<0.001 by one-way ANOVA with Tukey test; mean±SEM; ns, not significant). (D) The distance at which regenerating axons display 50% of the SCG10 intensity at the crush site was calculated (n=6 for each condition; ***p<0.001, **p<0.01 by one-way ANOVA with Tukey test; mean±SEM; ns, not significant). (E) Western blot of dissected L4 and L5 DRGs from control or HIF1AcKO mice that receveid (+Ax) or not (−Ax) a sciatic nerve axotomy 3 days earlier. (F) Average fold changes in intensity (+Ax/−Ax) (n=3 for each condition; ^#p>0.05, *p<0.05 by t- test; mean±SEM).

Figure 6. The HIF-1α target gene VEGFA enhances axon regeneration

(A) Representative longitudinal sections of sciatic nerves from wild type mice treated with vehicle or recombinant mouse VEGFA, dissected 3 days after injury and immunostained for SCG10 and βIII tubulin. Scale bar, 500nm. Dotted lines indicate the crush site. (B and C) In vivo regeneration index calculated from (A) (n=6 and 8 for vehicle and VEGFA; ***p<0.001, **p<0.01 by t-test; mean±SEM). (D) Representative longitudinal sections of sciatic nerves from HIF1AcKO mice treated with vehicle or recombinant mouse VEGFA, dissected 3 days after injury and immunostained for SCG10 and βIII tubulin. Scale bar, 500^m. Dotted lines indicate the crush site. (E and F) In vivo regeneration index calculated from (D) (n=12 for each condition; *p<0.05 by t-test; mean±SEM; ns, not significant).

Figure 7. Temporally controlled HIF-1α induction enhances axon regeneration in vitro

(A and B) Western blot analysis of tetracyclin-inducible tre-GFP-HIF-1α expression in cultured DRG neurons with different doxycycline concentrations (A) or different induction time (B). (C) Western blot analysis of tre-GFP-HIF-1α expression by doxycycline induction. The arrow indicates the exogenous GFP-HIF-1α and the arrowhead indicates the endogenous HIF-1α. (D) Representative images of in vitro regeneration of tre-GFP-HIF-1α-transduced DRG neurons without (−Dox) or with doxycycline induction (+Dox). Scale bar, 100^m. (E) In vitro regeneration index calculated from (H) (n=16 for each condition; ***p<0.001 by t-test; mean±SEM). (F) Western blot analysis of cultured DRG neurons treated with hypoxia for the indicated amount of time. (G) Quantification of (F) (n=4; *p<0.05 by one-way ANOVA with Tukey analysis; mean±SEM). (H and J) Experimental schemes for the in vitro regeneration assay with different hypoxia conditions. Numbers indicate hour at which hypoxia condition was started. In vitro axotomy was given at the 0 h, A refers to a 4 h hypoxia treatment before axotomy, B, refers to a 4 h of hypoxia treatment after axotomy. B, C and D refer to hypoxia starting 4, 8 or 12 h after axotomy, respectively. (I and K) In vitro regeneration index from the experimental schemes in C and E. N: normoxia. (n=15, 14 and 18 for N, A and B of (C); n=15, 18, 12, 12 and 16 for N, A, B, C and D of (E); **p<0.01 by one-way ANOVA with Tukey test; ns, not significant; mean±SEM).

Figure 8. Acute intermittent hypoxia (AIH) enhances axon regeneration in vivo

(A) Experimental scheme for in vivo AIH: 6 10min hypoxic episodes (8% oxygen) with equivalent 10min normoxic intervals. Green bar indicates 10 min hypoxic condition and black bar indicates 10 min normoxic condition (AIH total duration=120 min). Control mice followed the same 120 min regime under normoxia. SNC: sciatic nerve crush. (B) Western blot analysis of mouse L4 and L5 DRGs treated for 120 min of normoxia (No) or hypoxia (Hx) using the AIH protocol. (C) Quantifications of protein level of HIF-1α and p-JNKs (n=3 for each condition; ***p<0.001, **p<0.01 by t-test; mean±SEM). (D) Representative longitudinal sections of sciatic nerves from control or HIF1AcKO mice, dissected 3 days after injury with or without daily AIH and immunostained for SCG10 and βIII tubulin. Scale bar, 500µm. (E and F) In vivo regeneration index calculated from (D) (n=7 and 10 for normoxia and hypoxia from control mice, n=10 and 8 for normoxia and hypoxia from HIF1AcKO mice; *p<0.05 by one-way ANOVA with Tukey test; mean±SEM; ns, not significant). (G) Motor axons reinnervation assays. EHL muscles of thy1-YFP-16 mice dissected 12 days after sciatic nerve injury with or without daily AIH were stained with Alexa Fluor 647-conjugated α-Bungarotoxin (αBTX). Scale bar, 100µm. (H) Quantification of percentage of axon-non-occupied (N) and axon-re-occupied NMJ end plates (O) (n=15 for each condition; box, 25 to 75%; whisker, standard deviation; closed circle, mean; ***p<0.001 by one-way ANOVA with Tukey test; ns, not significant).