PTEN regulates microtubule polymerization via mTORC2, but not mTORC1, in peripheral sensory neurons

Peripheral neuropathy affects over 18 million adults in the U.S. (Hicks et al 2021), but therapeutic outcomes are poor due to a lack of regenerative treatments. Development of novel, disease-modifying therapies is hindered by poor understanding of the mechanisms promoting axonal regeneration in peripheral neurons. Pten is a strong negative regulator of cell growth, and Pten-KO drives axonal regeneration in various neuronal subtypes potentially via downstream regulation of stability of the microtubule (MT) cytoskeleton, a vital component of axonal growth. While Pten-KO accelerates MT polymerization rates in the axonal growth cone, it remains unknown whether this action is dependent on mTORC1 or mTORC2 signaling, and whether regeneration under Pten-KO is dependent on MT activation. Here, we perform in vitro codeletions of either Raptor (mTORC1) or Rictor (mTORC2) alongside Pten-KO in mouse peripheral sensory neuron cultures of either sex to isolate the effects of each pathway on the MT cytoskeleton. We use Pten-KO to increase MT polymerization and neuronal outgrowth, and then show that suppression of mTORC2, but not mTORC1, is sufficient to reduce the accelerated MT polymerization and neuronal hypertrophy to wild-type levels. These results are specific to the axonal growth cone, and MT dynamics in the proximal axon shaft are not impacted by Pten-KO, mTORC1 suppression, or mTORC2 suppression. Our results help elucidate the mechanism by which Pten regulates the MT cytoskeleton and axonal outgrowth in peripheral sensory neurons, localize where this occurs in the axon, and highlight the MT cytoskeleton as a potential molecular target for regenerative therapies.Significance statement Pten-KO promotes neuron growth, but Pten has several downstream effectors, and it is unclear which effectors drive axonal regeneration. One of these downstream targets is the microtubule cytoskeleton, which is vital for normal axonal growth and development. We demonstrate that Pten-KO increases MT polymerization rates in the growth cone, but not proximal shaft, in axons of peripheral sensory neurons. This effect is reversed by co-deletion of mTORC2, but not mTORC1, and the loss of mTORC2 primarily inhibits elongation of the distal axon, suggesting that the regulation of MT polymerization is specific to the growth cone. These results highlight MT dynamics in the axonal growth cone as a vital component of peripheral neuron regeneration and a potential therapeutic target.