Axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons

Abstract Sensory dorsal root ganglion (DRG) neurons have a unique pseudo-unipolar morphology in which a stem axon bifurcates into a peripheral and a central axon, with different regenerative abilities. Whereas peripheral DRG axons regenerate, central axons are unable to regrow. Central axon regeneration can however be elicited by a prior conditioning lesion to the peripheral axon. How DRG axon asymmetry is established, remains unknown. Here we developed a rodent in vitro system replicating DRG pseudo-unipolarization and asymmetric axon regeneration. Using this model, we observed that from early development, central DRG axons have a higher density of growing microtubules. This asymmetry was also present in vivo and was abolished by a conditioning lesion that decreased microtubule polymerization of central DRG axons. An axon-specific microtubule-associated protein (MAP) signature, including the severases spastin and katanin and the microtubule regulators CRMP5 and tau, was found and shown to adapt upon conditioning lesion. Supporting its significance, interfering with the DRG MAP signature either in vitro or in vivo, readily abolished central-peripheral asymmetries in microtubule dynamics and regenerative ability. In summary, our data unveil that axon-specific microtubule regulation drives asymmetric regeneration of sensory neuron axons. Impact statement Sensory neurons have a stem axon that bifurcates originating two axons with different properties. This work shows that DRG axons have a specific protein signature underlying microtubule and regeneration asymmetries. It also provides an in vitro system replicating DRG biology. Competing Interest Statement The authors have declared no competing interest. Footnotes The manuscript was revised to include two additional figures: Figure 1B and Figures 3H and 3K. The initial section of the discussion was substantially restructured to address reviewers concerns regarding the in vitro model, including its significance, applicability to major DRG neuron subtypes, the role of intrinsic and extrinsic factors in pseudounipolarization, and comparisons to in vivo and compartmentalized DRG models. We emphasized the model's value in studying DRG axon biology and diseases, particularly for visualizing structures like the stem axon and T-junction, which are challenging to observe in vivo with current microscopy techniques. Additional points were addressed, including the potential use of other knockout models for katanin and CRMP5 to strengthen claims regarding MAP asymmetry and its role in regenerative capacity differences. The methods section was expanded to provide clarity on how pseudo-unipolar neurons were selected in vitro and to detail the approach used to define the spinal cord lesion border.