Unlike in the peripheral nervous system (PNS), axons in the adult mammalian central nervous system (CNS) usually do not spontaneously regenerate after injury. The failure of axon regeneration is one of the major obstacles for functional recovery after neurotrauma such as spinal cord or optic nerve injury. This phenomenon has been attributed to two properties of the adult CNS, the inhibitory extrinsic environment and a diminished intrinsic regenerative capacity of mature neurons. During my postdoc period, by collaborating with other people, we have found that down-regulation of neuronal mechanistic target of rapamycin (mTOR) activity represents a roadblock in preventing the axonal regeneration after injury. Deletion of Pten(phosphatase and tensin homolog), a negative regulator of the pathway, in retinal ganglion cells (RGCs), not only promotes robust axon regeneration butalso cell survival after optic nerve crush. With Pten deletion, even corticospinal tract (CST) axons, especially refractory to regeneration, can regrow through the lesion site after a severe spinal cord injury. These exciting findings provide an opportunity to study the function of regenerating axonal tracts, rebuild disrupted circuitries, and develop therapeutic strategies after CNS injury.
During past years, my lab has been focusing on the intrinsic mechanisms regulating axon regeneration especially on the mTOR signaling, and worked on several interrelated questions that I believe are important in the axon regeneration field.
1. Can corticospinal tract axons regenerate after chronic spinal cord injury?
2. What could be an alternative strategy to activate mTOR and promote axon regeneration other than Pten deletion?
3. Is differential mTOR activity in CNS and PNS neurons linked to their differential regenerative capacity?
4. Can regenerated axons make functional reconnection or even result infunctional recovery?
1. Pten deletion promotes CST regeneration after chronic spinal cord injury.
Spinal cord injury (SCI) often causes permanent functional deficits due to the failure of axon regeneration. Many attempts have been made to promote axon regeneration in the spinal cord, mostly before or immediately after injury. Whether the injury-induced down-regulation of the growth capacity can be reversed in adult CSMNs and whether chronically injured CST axons can regenerate remained elusive. Through Cre-expressing, self-complementary AAV serotype 2/1, a large number of corticospinal motor neurons (CSMNs)can be targeted in adult mice, and biotin dextran amine (BDA) tracing can be effectively used to assess the axon sprouting or regeneration.We tested the effect of this strategy in three injury paradigms. Firstly, we applied a unilateral pyramidotomy in adult Ptenf/f mice, where CST axons from one side of the sensorimotor cortex were transected at the level of the medulla above the pyramidal decussation. The uninjured side of the cortex was injected with AAV-Cre or AAV-GFP 1 week after lesion. In 10 weeks, we found that Pten deletion elicited extensive trans-midline sprouting of adult CST axons from the intact side of the spinal cord into the denervated side.Secondly, we assessed whether Pten deletion after spinal cord injury would re-initiate CST growth. A severe T8 crush injury transecting all CST axons was performed, and 4 weeks later we injected AAV-Cre or AAV-GFP into the right side of the sensorimotor cortex. CST was traced 4 months later with Biotin dextran amine (BDA). No CST axons extended beyond the lesion site in control mice. When Pten was deleted in CSMNs, 4 months after the AAV-Creinjection, many axons grew into the lesion site and beyond, some up to 2.5mm. No labeled axons were detected beyond 3 mm distal to the lesion sites,which indicates that regeneration is limited and provides further evidence that the lesions were complete.Then, we moved to a chronic lesion model. A T8 crush was carried out, and12 months later we injected AAV-Cre or AAV-GFP into one side of the hemisphere. CST was then traced 4 months later by BDA. In the controls rarely could a few fibers be seen approaching the injury site. In Pten-deleted mice, we did not see the obvious extension of the main CST bundle. However,a few axons grew into and even beyond the lesion, up to 1.5 mm. 7 months after the AAV-Cre injection, the main CST bundle extended up to the boundary of the lesion, and evident axons regrew into the injury site and even across the lesion for up to 3 mm. Some synapses reformed by regenerating axons could be detected by immunostaining of BDA, VGlut1, and Homer1.As one of the long descending tracts controlling voluntary movement, the CSTplays an important role for functional recovery after spinal cord injury. The regeneration of CST has been a major challenge in the field, especially after chronic injuries. Here we developed a strategy to modulate Pten/mTOR signaling in adult corticospinal motor neurons in the post-injury paradigm. Itnot only promoted the sprouting of uninjured CST axons, but also enabled the regeneration of injured axons past the lesion in a mouse model of severe spinal cord injury, even when treatment was delayed up to 1 year after the original injury. The results considerably extend the window of opportunity for regenerating CST axons severed in spinal cord injuries. The work was published in Journal of Neuroscience in July 2015. We are currently trying to understand one immediate question after the chronic study and that is why it takes such a long time for CST to regenerate comparing with acute injury.
2. Enhancing neuronal activity by modulating melanopsin/GPCRsignaling promotes axon regeneration.
Pten inhibition activates mTOR and promotes corticospinal tract regeneration after acute or chronic spinal cord injury, indicating the translational potential of this strategy. However, Pten inhibition also activates additional pathways that likely contribute to its oncogenic activity, which may not be an ideal therapeutic target. We demonstrated that axon regenerative capacity could be boosted with the right stimulants on neuronal activity through either an optogenetic or a chemogenetic approach. Several hints lead us to test the effect of melanopsin and Designer Receptor Exclusively Activated byDesigner Drugs (DREADD) in axon regeneration. It has been shown that neurotrophic factors coupled with electric stimulation boost axonal elongation in postnatal RGCs in vitro. Neuronal activity has also been shown to regulate the phosphorylation of S6 ribosomal protein (pS6) in animals, which often serves as an mTOR activity indicator. In addition, we found that RGCsexpressing high-level melanopsin often co-localize with pS6. Melanopsin and DREADD-Gq have previously been used to modulate neuronal activity and animal behaviors.We found that overexpression of melanopsin in retinas of mice could enhance neuronal firing of RGCs and promote axonal regeneration after optic nerve crush. Either blocking daily light or silencing neurons by Kir2.1 can dramatically suppress the growth effect. Melanopsin overexpression sustains neuronal activity-dependent mTOR signaling in injured RGCs, and mTOR is also functionally required for the regeneration. Knocking down both Gq andG11 inhibits the growth effect. We then took a chemogenetic approach to activate Gq signaling by overexpressing DREADD-Gq, widely adopted as a tool to enhance neuronal activity. A significant increase in axonal growth was detected as well after daily administration of clozapine-n-oxide, a synthetic ligand to activate DREADD. The results showed that melanopsin activatesGq/11 signaling that subsequently increases neuronal activity and calcium influx to a degree that may be necessary to sustain long-term mTOR activation in RGCs. Our work identifies a mechanistic link between axon regeneration and neuronal activity in vivo and provides an intrinsic factor that can be further exploited to promote neural repair after injury. The result was published in PNAS in February 2016.
3. Genetic evidence that mTOR signaling regulates axon regeneration in adult peripheral neurons.
While ample evidence from others and ours suggests that neuronal mTOR activity is a critical determinant of the intrinsic regenerative ability of mature neurons in the adult CNS, it is unknown whether its action may also apply toPNS neurons after injury. It has become an interesting hypothesis that high mTOR activity may mediate high growth capacity in PNS neurons. However, recent pharmacological studies by using rapamycin, a specific mTOR inhibitor, in peripheral sensory neurons have yielded conflicting results.To address this issue, we took genetic approaches to determine the role of the mTOR signaling in the axon regeneration of mouse dorsal root ganglion(DRG) neurons. We show that knocking out mTOR in DRG neurons suppressed the axon regeneration, induced by a conditioning lesion, from both the peripheral and the central branches. The mTOR kinase exists in two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2, and these complexes functionally depend on their Raptor (mTORC1) or Rictor(mTORC2) subunits. To establish whether the impact of mTOR on axon regeneration results from functions of either complex, we conditionally deleted either Raptor or Rictor in DRG neurons. We found that Raptor deletion dramatically decreased the conditioning lesion effect in vivo, whereas Rictordeletion had a modest effect. Ablating Pten or TSC1, negative regulators of mTOR, enhanced the ability of axons to regrow into the lesion site after dorsal column crush.Unexpectedly, examination of several conditioning lesion induced proregenerative pathways revealed that mTOR or Raptor deletion but not rapamycin, a widely used specific mTOR inhibitor, suppressed STAT3 activity in neurons. These results demonstrate cross-talk between mTOR and STAT3signaling in mediating the conditioning lesion effect in DRGs, different from retinal ganglion cells whereas the two signaling pathways independently regulate axon regeneration. Thus, our results provide genetic evidence that the rapamycin-resistant mTOR activity contributes to the intrinsic axon growth capacity in adult peripheral neurons after injury. The work has been submitted for publication.
4. Regenerated axons reform active synapses with target neurons.
One of the major goals of studying axon regeneration is to achieve functional recovery after CNS injury. However, it is very challenging to comprehensively demonstrate that axons across the lesion site can reconnect with target neurons with functional synapses, at the levels of ultrastructure, circuitry, electrophysiology especially with the post-synaptic response, and behavior, by using an appropriate injury model.Combining several strategies to boost the intrinsic growth capacity of RGCsleads to robust and long-distance regeneration within the optic nerve after intraorbital crush, a type of lesion close to the eyeball. However, only a few axons innervate the brain, which makes it difficult to assess the potential functional outcome. The limited innervation could be due to the long distance for axons to travel, misguidance along the path, crossing the optic chiasm, and continuous neuronal loss months after lesion. To test the hypothesis that regenerated axons can make functional connections, one strategy to bypass the long path is to move the lesion site closer to the target neurons. Such effort has been carried out for sensory axons to reach dorsal nuclei, and also recently optic tract to reach superior colliculi.Our aim was to develop an injury model to examine whether a large number of regenerating retinal axons can reinnervate appropriate target area, and form active connections with appropriate neurons at the synaptic and functional level. We managed to crush the optic nerve at the point right before it enters the chiasm. This model gives us an opportunity to examine the potential recovery of both the visual and non-visual functions after injury. We started the initial experiment by deleting both Pten and Socs3 in retinal ganglion cells, and found that, after prechiasm lesion, retinal axons robustly reinnervate the hypothalamus. Many axons projected ectopically around the core region of the suprachiasmatic nucleus (SCN), suggesting an abnormal innervation. More axons in the core region were found 4 months after lesion.Some of the regenerated axons reform synapses with neurons in SCN. Both the pre- and post-synaptic markers are co-localized with terminals of regenerated axons by immunofluorescence labeling. To convincingly demonstrate the formation of the new synapses, we overexpressed GFP in regenerated axons using AAV, and performed immunogold electromicroscopy analysis. We found some structures with key features of asymmetric synapses. Through trans-neuronal tracing, we also showed that axons make connections with SCN neurons with the existing circuitry. Furthermore, electric stimulation of the regenerated optic nerve induces excitatory postsynaptic currents (EPSCs) in neurons at SCN. Our results support that boosting the intrinsic growth capacity through inhibiting both Pten and Socs3 in injured neurons promotes axonal reinnervation and rewiring. However both the innervation pattern and evoked responses are not completely restored by there generating axons, suggesting combining with other strategies may be necessary. The initial result was published in Neurobiology of Disease in October 2014. The prechiasm lesion model provides us a base to carry out functional studies after injury. We are currently investigating whether there generated axons can promote visual and non-visual functional recovery.My goal is to establish a research program to study axon regeneration at the molecular, cellular, and system levels, and contribute new knowledge to the field.