Axon Regeneration In Adult Central Nervous System Research Articles

Adult Mouse Retina Explants: From ex vivo to in vivo Model of Central Nervous System Injuries.

In mammals, adult neurons fail to regenerate following any insult to adult central nervous system (CNS), which leads to a permanent and irreversible loss of motor and cognitive functions. For a long time, much effort has been deployed to uncover mechanisms of axon regeneration in the CNS. Even if some cases of functional recovery have been reported, there is still a discrepancy regarding the functionality of a neuronal circuit upon lesion. Today, there is a need not only to identify new molecules implicated in adult CNS axon regeneration, but also to decipher the fine molecular mechanisms associated with regeneration failure. Here, we propose to use cultures of adult retina explants to study all molecular and cellular mechanisms that occur during CNS regeneration. We show that adult retinal explant cultures have the advantages to (i) recapitulate all the features observed in vivo, including axon regeneration induced by intrinsic factors, and (ii) be an ex vivo set-up with high accessibility and many downstream applications. Thanks to several examples, we demonstrate that adult explants can be used to address many questions, such as axon guidance, growth cone formation and cytoskeleton dynamics. Using laser guided ablation of a single axon, axonal injury can be performed at a single axon level, which allows to record early and late molecular events that occur after the lesion. Our model is the ideal tool to study all molecular and cellular events that occur during CNS regeneration at a single-axon level, which is currently not doable in vivo. It is extremely valuable to address unanswered questions of neuroprotection and neuroregeneration in the context of CNS lesion and neurodegenerative diseases.

Lab review: Molecular dissection of the signal transduction pathways associated with PTEN deletion-induced optic nerve regeneration.

Permanent loss of vital functions after central nervous system (CNS) injury occurs in part because axons in the adult mammalian CNS do not regenerate after injury. PTEN was identified as a prominent intrinsic inhibitor of CNS axon regeneration about 10 years ago. The PTEN negatively regulated PI3K-AKT-mTOR pathway, which has been intensively explored in diverse models of axon injury and diseases and its mechanism for axon regeneration is becoming clearer. It is timely to summarize current knowledge about the PTEN/AKT/mTOR pathway and discuss future directions of translational regenerative research for neural injury and neurodegenerative diseases. Using mouse optic nerve crush as an in vivo retinal ganglion cell axon injury model, we have conducted an extensive molecular dissection of the PI3K-AKT-mTORC1/mTORC2 pathway to illuminate the cross-regulating mechanisms in axon regeneration. AKT is the nodal point that coordinates both positive (PI3K-PDK1-pAKT-T308) and negative (PI3K-mTORC2-pAKT-S473) signals to regulate adult CNS axon regeneration through two parallel pathways, activating mTORC1 and inhibiting GSK3β. However, mTORC1/S6K1-mediated feedback inhibition after PTEN deletion prevents potent AKT activation. A key permissive signal from an unidentified AKT-independent pathway is required for stimulating the neuron-intrinsic growth machinery. Future studies into this complex neuron-intrinsic balancing mechanism involving necessary and permissive signals for axon regeneration is likely to lead to safe and effective regenerative strategies for CNS repair.

271. Strategies Based on NexGen AAV2 to Improve AKT3-Induced CNS Axon Regeneration In Vivo

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.

Sustained axon regeneration induced by co-deletion of PTEN and SOCS3

A formidable challenge in neural repair in the adult central nervous system (CNS) is the long distances that regenerating axons often need to travel in order to reconnect with their targets. Thus, a sustained capacity for axon regeneration is critical for achieving functional restoration. Although deletion of either Phosphatase and tensin homolog (PTEN), a negative regulator of mammalian target of rapamycin (mTOR), or suppressor of cytokine signaling 3 (SOCS3), a negative regulator of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, in adult retinal ganglion cells (RGCs) individually promoted significant optic nerve regeneration, such regrowth tapered off around two weeks after the crush injury1,2. Remarkably, we now find that simultaneous deletion of both PTEN and SOCS3 enables robust and sustained axon regeneration. We further show that PTEN and SOCS3 regulate two independent pathways that act synergistically to promote enhanced axon regeneration. Gene expression analyses suggest that double deletion not only results in the induction of many growth-related genes, but also allows RGCs to maintain the expression of a repertoire of genes at the physiological level after injury. Our results reveal concurrent activation of mTOR and STAT3 pathways as a key for sustaining long-distance axon regeneration in adult CNS, a crucial step toward functional recovery.

Generation of an OMgp allelic series in mice.

The very limited ability to regenerate axons after injury in the mature mammalian central nervous system (CNS) has been partly attributed to the growth restrictive nature of CNS myelin. Oligodendrocyte myelin glycoprotein (OMgp) was identified as a major myelin-derived inhibitor of axon growth. However, its role in axon regeneration in vivo is poorly understood. Here we describe the generation and molecular characterization of an OMgp allelic series. With a single gene targeting event and Cre/FLP mediated recombination, we generated an OMgp null allele with a LacZ reporter, one without a reporter gene, and an OMgp conditional allele. This allelic series will aid in the study of OMgp in adult CNS axon regeneration using mouse models of spinal cord injury. The conditional allele will overcome developmental compensation when employed with an inducible Cre, and allows for the study of temporal and tissue/cell type-specific roles of OMgp in CNS injury-induced axonal plasticity.