271. Strategies Based on NexGen AAV2 to Improve AKT3-Induced CNS Axon Regeneration In Vivo
Authors: Yang Hu, Linqing Miao, Liu Yang, Haoliang Huang, Feisi Liang, Chen Ling
Journal: Molecular Therapy (2016)

Injuries of mature central nervous system (CNS) axons result in loss of vital functions due to the failure of CNS axons to regenerate. AKT, a family of serine/threonine-specific protein kinases, induces CNS axon regeneration through activation of the mTOR signaling. However, mTOR participates in two distinct complexes, mTOR complex 1 (mTORC1) and complex 2 (mTORC2). How these two complexes interact with regard to axon regeneration is unknown. To exploit the significant advantages of specific tropism of adeno-associated virus serotype 2 (AAV2) for retinal ganglion cells (RGC) after intravitreal injection, we generated a series of triple tyrosine-optimized AAV2 (NexGen AAV2) vectors carrying different AKT variants and mutants. We first confirmed that the NexGen viral vectors showed significant more infection efficiency compared to vectors based on wild-type AAV2 capsids. More than 90% RGC cells in whole-mount retina was transduced, based on triple immunofluorescent staining of transgene, RGC marker, and active form of AKT. Then, the optic nerve was crushed as an in vivo axon regeneration model in mice 2 weeks post-intravitreal injection of AAV vectors. It was observed that the predominant AKT isoform in brain and retina, AKT3, induces much better axon regeneration than AKT1 and AKT2. Next, we evaluated the roles of mTORC1 and mTORC2 in AKT3-induced CNS axon regeneration. NexGen AAV2 vectors carrying AKT3 and Cre were injection into eyes of either mTOR floxed mice, RAPTOR floxed mice, or RICTOR floxed mice. RAPTOR is unique to mTORC1, whose deletion totally blocks mTORC1 activity; whilst RICTOR is a rapamycin-insensitive component essential for mTORC2 activity. As expected, deletion of mTOR or RAPTOR significantly inhibited AKT3-induced axon regeneration. Interestingly, AKT3 and RICTOR KO produced even more extensive and fine axon regeneration than AKT3 alone, suggesting that mTORC2 is inhibitory for AKT3-induced CNS axon regeneration. Furthermore, NexGen AAV2-mediated delivery of dominant negative mutant of ribosomal protein S6 kinase (S6K1) or constitutively active mutant of factor 4E-binding protein (4E-BP1), two best-characterized substrates of mTORC1, also inhibited AKT3-induced axon regeneration, further corroborating our conclusions. In addition, since S472 of AKT3 is phosphorylated by mTORC2, we generated NexGen AAV2 vectors carrying an AKT3-S472A mutant gene. Other mutant genes, such as AKT3 kinase dead mutant (K177M) and T305A mutant, were used as appropriate controls. Our results indicated that neither of the control mutants induced axon regeneration. However, the AKT3-S472A mutant produced a significant increase in axon regeneration compared to wild-type AKT3. Taken together, our study revealed an AKT-based neuron-intrinsic balancing mechanism involving mTORC1 and mTORC2, which coordinates positive and negative cues to regulate adult CNS axon regeneration, respectively. Our results should provide promising therapeutic targets for CNS injuries. Taken together, our study revealed an AKT-based neuron-intrinsic balancing mechanism involving mTORC1 and mTORC2, which coordinates positive and negative cues to regulate adult CNS axon regeneration, respectively. Our results should provide promising therapeutic targets for CNS injuries.