AhR inhibition promotes axon regeneration via a stress–growth switch | Nature

## Summary
Full size image a , STRING analysis shows no direct link between AhR and regeneration-associated TFs. b , Cytoplasm (cyt.) to nucleus (nuc.) AhR shuttling in cultured DRG neurons after 1.5 h exposure to ITE or CH (10 μM). n = 100 neurons per condition. d, days; Veh, vehicle. c , Ligand-mediated AhR activation induces target genes through the AHRE. d , RT–qPCR analysis of DRG neurons after 24 h agonist or antagonist treatment (25 μM). n = 4 cultures. Quantification of the presented immunoblot is shown; the experiment was repeated three times with similar results. f , IF analysis showing increased nuclear AhR in DRG neurons from ITE-treated mice (arrows). n = 10 neurons per DRG from 9 sciatic DRGs, 3 mice per group. g , RT–qPCR and IF analysis confirm Ahr knockdown in primary DRG neurons 48 h after siRNA. n = 4 cultures. To further corroborate these findings, we generated tamoxifen-inducible neuronal Ahr conditional knockout ( Ahr cKO ) mice by crossing Ahr fl/fl mice with Thy1-cre ERT2/eYFP (SLICK-H) mice (Fig. 1i ). eYFP expression confirmed widespread neuronal targeting in DRG neurons and sciatic axons (Extended Data Fig. 2a ), and efficient recombination was validated using the Rosa26-LSL-Sun1-GFP (INTACT) reporter line (Extended Data Fig. 2b ). Similarly, AhR protein was largely absent from DRG neurons of ITE-treated Ahr cKO mice, while glial AhR expression was unchanged (Extended Data Fig. 2g ).

## Article Content
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Subjects
Genetics of the nervous system
Neuroscience
Abstract
Axon regeneration is limited in the mammalian central nervous system
1
. Neurons must balance stress responses with regenerative demands after axonal injury
2
, but the mechanisms remain unclear. Here we identify aryl hydrocarbon receptor (AhR), a ligand-activated basic helix–loop–helix/PER-ARNT-SIM (bHLH-PAS) transcription factor, as a key regulator of this stress–growth switch. We show that ligand-mediated AhR signalling restrains axon growth, whereas neuronal deletion or pharmacological inhibition of AhR promotes axonal regeneration and functional recovery in both peripheral nerve and spinal cord injury models. Mechanistic studies reveal that axotomy-induced AhR activation in dorsal root ganglion neurons enforces proteostasis and stress-response programs to preserve tissue integrity. By contrast, AhR ablation redirects the neuronal response towards elevated de novo translation and pro-growth signalling, enabling axon regeneration. This growth-promoting effect requires HIF1α, with shared transcriptional targets enriched for metabolic and regenerative pathways. Single-cell and epigenomic analyses further revealed that the AhR regulon engages the integrated stress response and DNA hydroxymethylation to rewire neuronal injury-response programs. Together, our findings establish AhR as a neuronal brake on axon regeneration, integrating environmental sensing, protein homeostasis and metabolic signalling to control the balance between stress adaptation and axonal repair.
Main
Neural injury disrupts the extracellular milieu through tissue breakdown, neuroinflammation and altered oxygen tension. To cope with stress, injured neurons deploy safeguard mechanisms that adjust metabolism, synaptic activity and regenerative gene programs. However, how neurons coordinate the balance between stress adaptation and regenerative demands remains poorly understood.
Basic helix–loop–helix/PER-ARNT-SIM (bHLH-PAS) transcription factors (TFs) act as molecular sensors of environmental and physiological signals. Within this family, BMAL1 coordinates circadian rhythm, HIF1α mediates hypoxia responses and AhR detects xenobiotics and endogenous metabolites
3
,
4
. We recently showed that BMAL1 gates regenerative responses of dorsal root ganglion (DRG) neurons after peripheral axotomy
5
and others showed that intermittent hypoxia enhances axon regeneration through HIF1α activation
6
. AhR and HIF1α are structurally related α-subunits that share the dimerization partner ARNT (HIF1β), yet the roles of AhR or ARNT in axonal injury, and their interplay with HIF1α and BMAL1, remain largely unexplored.
AhR is unique among bHLH-PAS TFs as the only ligand-activated member
7
. Originally identified as a sensor of environmental toxins such as dioxin, AhR responds to diverse dietary, microbial and metabolic molecules
7
. After ligand binding, AhR translocates to the nucleus, dimerizes with ARNT and regulates transcription through AhR response elements (AHREs)
8
. Canonical AhR signalling induces xenobiotic-metabolizing enzymes of the P450 family and NRF2-dependent antioxidants. Multiple feedback mechanisms restrain AhR activity, including CYP1-mediated ligand metabolism
9
and induction of the AhR repressor (AhRR), underscoring the need for tight control to prevent toxicity from sustained AhR activation
8
.
Competition with HIF1α for ARNT provides an additional regulatory layer
10
. Depending on the temporal and cellular context, AhR and HIF1α can antagonize, cooperate or synergize
11
,
12
. Recent work suggests temporally gated access to ARNT, with HIF1α acting first and AhR subsequently taking over
12
.
Although AhR has been extensively studied in toxicology, barrier tissue biology and immunity, its neuronal functions, particularly in injury, remain poorly defined. DRG neurons offer an excellent model for axon regeneration: peripheral axotomy elicits robust regrowth, whereas central axotomy does not; however, a conditioning peripheral nerve lesion (PL) can prime central DRG axons for regeneration
13
,
14
.
Here we show that DRG neurons are responsive to ligand-mediated AhR signalling, which acts as a brake on axon regeneration. Neuronal deletion of AhR enhanced axonal regrowth in both peripheral nerve and spinal cord injury (SCI) models. Mechanistically, AhR activation induced an injury regulon that reinforces protein homeostasis (proteostasis), whereas AhR loss shifted neurons towards elevated protein translation, metabolism and pro-growth signalling. We further identify cross-talk between AhR, HIF1α and ARNT in balancing neuronal stress adaptation with regenerative growth. Together, these findings establish AhR as a central regulator of the stress–growth switch after axotomy and suggest that targeting AhR may provide therapeutic opportunities for neural repair after SCI.
DRG neurons respond to AhR signalling
To examine transcriptional regulatory networks after axonal inju

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## Expert Analysis

### Merits
- DRG neurons offer an excellent model for axon regeneration: peripheral axotomy elicits robust regrowth, whereas central axotomy does not; however, a conditioning peripheral nerve lesion (PL) can prime central DRG axons for regeneration 13 , 14 .

### Areas for Consideration
- Neurons must balance stress responses with regenerative demands after axonal injury 2 , but the mechanisms remain unclear.

### Implications
- Together, these findings establish AhR as a central regulator of the stress–growth switch after axotomy and suggest that targeting AhR may provide therapeutic opportunities for neural repair after SCI.
- FC, fold change. e , Immunoblot analysis of DRGs after in vivo ITE treatment (10 mg per kg, i.p.) showing CYP1B1 induction and AhR electrophoretic mobility shift.

### Expert Commentary
This article covers ahr, neurons, fig topics. Notable strengths include discussion of ahr. Areas of concern are also raised. Readability: Flesch-Kincaid grade 0.0. Word count: 2339.