Optic Nerve Regeneration Research Articles

Chemistry and Neurotrophic Activities of (-)-Talaumidin and Its Derivatives.

(–)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan isolated from Aristolochia arcuata Masters, exhibits significant neurite-outgrowth promotion and neuroprotection in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells. The first enantioselective total synthesis of 1 was achieved by a flexible and reliable synthetic pathway involving an Evans asymmetric aldol reaction, as well as a stereocontrolled hydroboration and Friedel–Crafts arylation, to construct the four contiguous chiral centers on the tetrahydrofuran (THF) ring of 1. In order to investigate the stereochemistry–activity relationship of 1, a systematic synthesis of all diastereomers of 1 was accomplished by applying the synthetic strategy used for natural product 1. The evaluation of neurite-outgrowth promotion by all of the synthesized diastereomers indicated that the (–)-(1S,2R,3S,4R)-isomer 1e was significantly more active than naturally occurring 1. Additionally, we established a synthetic methodology for talaumidin derivatives that could be used to prepare a variety of analogs in a few steps and on a large scale. The synthesized racemic analog rac-1e (56a) exhibited neurite-outgrowth promoting activity in NGF-differentiated PC12 cells to the same degree as the optically active (–)-1e, revealing that a relative configuration bearing all-cis- substituents is important for potent neurotrophic activity, whilst the absolute configuration does not affect activity. Fourteen analogs based on (±)-56a were prepared via the same synthetic methodology. Among them, 56b with a methylenedioxy group on both benzene rings was found to exhibit the most significant neurite outgrowth promotion. In addition, 56a and 56b induced regeneration of the mouse optic nerve in vivo, and their activity was higher than that of talaumidin, as well as their in vitro measured activity. Furthermore, the structure–activity relationship of 56b indicated that the two benzene rings were essential structures, and that the methyl groups on the THF ring could enhance the neurotrophic activity. This result suggests that the two benzene rings of the talaumidin derivatives are essential structures for neurotrophic activity, while the two methyl groups on the THF ring can enhance neurite-outgrowth activity. Finally, it was observed that 1 and derivatives 56a and 56b exhibited potent regenerative activity in the injured mouse optic nerve in vivo.

Optic Nerve Regeneration: How Will We Get There?

Restoration of vision in patients blinded by advanced optic neuropathies requires technologies that can either 1) salvage damaged and prevent further degeneration of retinal ganglion cells (RGCs), or 2) replace lost RGCs. Review of scientific literature. In this article, we discuss the different barriers to cell-replacement based strategies for optic nerve regeneration and provide an update regarding what progress that has been made to overcome them. We also provide an update on current stem cell-based therapies for optic nerve regeneration. As neuro-regenerative and cell-transplantation based strategies for optic nerve regeneration continue to be refined, researchers and clinicians will need to work together to determine who will be a good candidate for such therapies.

Knocking Out Non-muscle Myosin II in Retinal Ganglion Cells Promotes Long-Distance Optic Nerve Regeneration

SUMMARYIn addition to altered gene expression, pathological cytoskeletal dynamics in the axon are another key intrinsic barrier for axon regeneration in the central nervous system (CNS). Here, we show that knocking out myosin IIA and IIB (myosin IIA/B) in retinal ganglion cells alone, either before or after optic nerve crush, induces significant optic nerve regeneration. Combined Lin28a overexpression and myosin IIA/B knockout lead to an additive promoting effect and long-distance axon regeneration. Immunostaining, RNA sequencing, and western blot analyses reveal that myosin II deletion does not affect known axon regeneration signaling pathways or the expression of regeneration-associated genes. Instead, it abolishes the retraction bulb formation and significantly enhances the axon extension efficiency. The study provides clear evidence that directly targeting neuronal cytoskeleton is sufficient to induce significant CNS axon regeneration and that combining altered gene expression in the soma and modified cytoskeletal dynamics in the axon is a promising approach for long-distance CNS axon regeneration.

Activating Transcription Factor 3 (ATF3) Protects Retinal Ganglion Cells and Promotes Functional Preservation After Optic Nerve Crush.

PurposeTo investigate the possible role of activating transcription factor 3 (ATF3) in retinal ganglion cell (RGC) neuroprotection and optic nerve regeneration after optic nerve crush (ONC).MethodsOverexpression of proteins of interest (ATF3, phosphatase and tensin homolog [PTEN], placental alkaline phosphatase, green fluorescent protein) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs) expressing corresponding proteins. The number of RGCs and αRGCs was evaluated by immunostaining retinal sections and whole-mount retinas with antibodies against RNA binding protein with multiple splicing (RBPMS) and osteopontin, respectively. Axonal regeneration was assessed via fluorophore-coupled cholera toxin subunit B labeling. RGC function was evaluated by recording positive scotopic threshold response.ResultsThe level of ATF3 is preferentially elevated in osteopontin+/RBPMS+ αRGCs following ONC. Overexpression of ATF3 by intravitreal injection of rAAV 2 weeks before ONC promoted RBPMS+ RGC survival and preserved RGC function as assessed by positive scotopic threshold response recordings 2 weeks after ONC. However, overexpression of ATF3 and simultaneous downregulation of PTEN, a negative regulator of the mTOR pathway, combined with ONC, only moderately promoted short distance RGC axon regeneration (200 μm from the lesion site) but did not provide additional RGC neuroprotection compared with PTEN downregulation alone.ConclusionsThese results reveal a neuroprotective effect of ATF3 in the retina following injury and identify ATF3 as a promising agent for potential treatments of optic neuropathies.

Longitudinal evaluation of immediate inflammatory responses after intravitreal AAV2 injection in rats by optical coherence tomography

Gene therapy has been proposed as a feasible strategy for RGC survival and optic nerve regeneration. Some preclinical and clinical studies revealed intraocular inflammation after intravitreal injection of adeno-associated virus (AAV) by slit-lamp or indirect ophthalmoscope. Here we evaluate the longitudinal profile of immediate inflammatory responses after AAV2 injection in rat retina and vitreous body by optical coherence tomography (OCT). Adult Fischer F344 rats were intravitreally injected once with saline, AAV2 or zymosan. Retinal thickness and cell infiltration were recorded by OCT longitudinally for 2 months and verified by histological analysis. The transduction rate of single intravitreal AAV2 injection was 21.3 ± 4.9% of whole retina, and the transduction efficiency on RGCs was 91.5 ± 2.5% in the transduced area. Significant increase in cell infiltration was observed from Day 1–3 after AAV2 injection, compared to very few infiltrating cells observed in the saline-injected group. The infiltrating cells ceased at Day 5 after intravitreal injection and remained absent at 2 months. The thicknesses of total and inner retina were increased along Day 1–3 after AAV2 injection, but reverted to normal afterwards. The surviving RGCs in the AAV2-injected groups at Day 14 showed no significant difference compared to saline-injected group. In summary, this study revealed the immediate inflammatory responses and retinal edema after intravitreal AAV2 injection in normal rats, without influencing long-term retinal thickness and RGC survival. OCT can be implemented for the time-lapse in vivo evaluation of inflammatory response after AAV-mediated gene therapy through intravitreal injection.

Development of New Neurotrophic Compounds Based on Talaumidin

(-)-2S,3S,4S,5S-Talaumidin (1) exhibits potent neurotrophic activities such as neurite-outgrowth promotion and neuroprotection in primary cultured rat cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells. To examine the stereochemistry-activity relationship of 1, systematic syntheses of seven stereoisomers of 1 were accomplished via Evans aldol reaction, diastereoselective hydroboration, and Mitsunobu reaction. All stereoisomers showed moderate-to-potent neurite-outgrowth promotion in NGF-differentiated PC12 cells. In particular, (-)-2S,3R,4S,5R-1e having all-cis-substituted configuration exhibited the most significant neurite-outgrowth promotion. Subsequently, we developed a short-step synthetic route for talaumidin derivatives so as to explore new neurotrophic compounds that could be obtained on a large scale. First, we synthesized derivative 2a, which is a racemic compound of (-)-1e, and compared its neurotrophic activity with (-)-1e. It was found that the neurotrophic activity of racemic 2a was similar to that of (-)-1e. Using the same synthetic route, several talaumidin derivatives were synthesized to optimize the oxy-functionality on aromatic rings. Thereby, bis(methylenedioxybenzene) derivative 2b was found to exhibit the highest neurotrophic activity. In addition, examination of the structure-activity relationship of 2b indicated that the 2,5-diphenyl structure was crucial moiety, and the two methyl groups on the tetrahydrofuran (THF) ring could enhance the neurotrophic activity. Furthermore, 2a and 2b were found to induce mouse optic nerve regeneration in vivo.

Anti-angiogenesis therapy of diabetic macular edema in patients with primary open-angle glaucoma

To study the effect of anti-VEGF therapy on the structural and functional state of the retina and optic nerve in patients with DME and POAG. The study included 72 patients (132 eyes): the 1st group - 22 patients (40 eyes) with stage I POAG and DME, the 2nd group - 25 patients (46 eyes) with DME receiving anti-VEGF therapy. The 3rd group (control) consisted of 25 patients (46 eyes) with stage I POAG. The observation period lasted 24 months. The average number of injections was 8.48±3.65. The indicators for evaluation were: visual acuity, tonometry, perimetry, optical coherence tomography (OCT) of the optic nerve and macular region. By the end of the observation period, the increase in IOP in the groups was +0.82 (4.4%), 0.41 (2.4%), 0.65 (3.6%) mm Hg. In the group of comorbid patients, a small-scale increase trend of BCVA was noted: +0.05 (6.6%), a decrease in MD by -2.48 Db (92.1%), an increase in excavation volume by 0.16 (43.2%) mm3, decrease in the area of RA by 0.3 mm2 (12.7%). A decrease in retinal nerve fibers layer (RNFL) thickness of 6.55 μm (7.8%), mainly the superior (9.2%), inferior (7.3%) and nasal sectors (7.9%). Loss of GCL+IPL 8.68 μm (12.7%) in the superior (19%), superonasal (20.2%) and inferonasal (20.7%) sectors. The combined course of POAG and DME is accompanied by a decrease in the functional and structural parameters of the retina and optic nerve, and a higher rate of progression of glaucomatous optic neuropathy. Long-term results did not reveal a significant deterioration in the structural parameters of the optic disc and retina against the background of anti-VEGF therapy when comparing the study groups.

Development of a New Large-Animal Model for Composite Face and Whole-Eye Transplantation: A Novel Application for Anatomical Mapping Using Indocyanine Green and Liquid Latex.

Recent advances in the field of vascularized composite allotransplantation have revolutionized reconstructive surgery and demonstrated opportunity for restoration of orbital content and perhaps vision. The development of an animal model that would facilitate study of surgical technique, nerve regeneration, vision restoration, and immunosuppression is needed. The aim of this study is to describe three different large-animal cadaveric models suitable for composite face and whole-eye transplantation. In addition, the authors introduce a novel colored liquid latex and indocyanine green mixture for vasculature identification and visualization. The authors studied three different flap designs using cadaveric Yorkshire swine and modified them for face and whole-eye transplant harvest. Flaps harvested included a chimeric flap, a monobloc flap, and a bipedicled free flap. The authors injected selected vasculature with colored latex to better delineate vascular anatomy supplying the orbit and face. Indocyanine green was added to the latex solution to allow for visualization of the vascular supply using near-infrared imaging. Colored latex and indocyanine green injections were successfully visualized in all cadaveric dissections. All three modified flap designs showed a well-defined and consistent vascular network within each face and whole-eye transplantation flap using laser-assisted near-infrared imaging. The authors present the feasibility of composite face and whole-eye transplantation models using a novel mixture of liquid latex and indocyanine green in a porcine cadaver. Further study in large animals is needed to appraise the surgical feasibility of this procedure and potential for clinically relevant outcomes, including vision restoration. The authors believe this study establishes a foundation for translation into live animal models for optic nerve regeneration.

Optic nerve regeneration: A long view.

The optic nerve conveys information about the outside world from the retina to multiple subcortical relay centers. Until recently, the optic nerve was widely believed to be incapable of re-growing if injured, with dire consequences for victims of traumatic, ischemic, or neurodegenerative diseases of this pathway. Over the past 10-20 years, research from our lab and others has made considerable progress in defining factors that normally suppress axon regeneration and the ability of retinal ganglion cells, the projection neurons of the retina, to survive after nerve injury. Here we describe research from our lab on the role of inflammation-derived growth factors, suppression of inter-cellular signals among diverse retinal cell types, and combinatorial therapies, along with related studies from other labs, that enable animals with optic nerve injury to regenerate damaged retinal axons back to the brain. These studies raise the possibility that vision might one day be restored to people with optic nerve damage.

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.

Immunological considerations and concerns as pertinent to whole eye transplantation.

The advent of clinical vascularized composite allotransplantation (VCA), offers hope for whole eye transplantation (WET) in patients with devastating vison loss that fails or defies current treatment options. Optic nerve regeneration and reintegration remain the overarching hurdles to WET. However, the realization of WET may indeed be limited by our lack of understanding of the singular immunological features of the eye as pertinent to graft survival and functional vision restoration in the setting of transplantation. Like other VCA, such as the hand or face, the eye includes multiple tissues with distinct embryonic lineage and differential antigenicity. The ultimate goal of vision restoration through WET requires optimal immune modulation of the graft for successful optic nerve regeneration. Our team is exploring barriers to our understanding of the immunology of the eye in the context of WET including the role of immune privilege and lymphatic drainage on rejection, as well as the effects ischemia, reperfusion injury and rejection on optic nerve regeneration. Elucidation of the unique immunological responses in the eye and adnexa after WET will provide foundational clues that will help inform therapies that prevent immune rejection without hindering optic nerve regeneration or reintegration.

Axon transport as a target for promoting RGC survival and optic nerve regeneration

AbstractAdult CNS neurons have a weak intrinsic capacity for regeneration after injury, but the underlying cellular mechanisms preventing regeneration are not completely understood. Increasing intrinsic growth ability is a critical part of enabling injured optic nerve axons to regenerate. The work presented here is part of a large body of research into understanding the role of membrane protein trafficking and transport in the control of regenerative ability. We have found that as CNS neurons mature, there is a developmental decline in the axonal transport of integrins (cell surface adhesion and guidance molecules) and their endosomal transporters, Rab11 positive recycling endosomes. This is controlled by a molecular switch called ARF6. Switching off ARF6 in mature CNS axons leads to an increase in the anterograde axonal transport of integrins and Rab11 endosomes, leading to a restoration of the regenerative capacity that is lost as axons mature. We have been targeting these mechanisms to stimulate axon regeneration in the optic nerve, and neuroprotection of retinal ganglion neurons during raised IOP. Here we present novel data from two new lines of investigation into facilitating CNS axonal transport, neuroprotection and regeneration, focusing on signaling pathways and organelle motility. Firstly, by investigating signaling upstream of ARF6, we have identified an isoform of PI3 Kinase which we have used to stimulate axon transport, neuroprotection and regeneration; both after laser axotomy in vitro and after optic nerve crush. Secondly, we present exciting new data from studies into a trafficking molecule which resides in the endoplasmic reticulum (ER) and demonstrate that this can be targeted to enable robust optic nerve regeneration and neuroprotection in the retina. These findings confirm integrins and Rab11 as critical facilitators of optic nerve regeneration whilst establishing an important new role for the axonal ER in this process.

GSK3/CRMP2 signaling confers PTEN knockout mediated axonal regeneration

AbstractKnockout of phosphatase and tensin homolog (PTEN‐/‐) is neuroprotective and promotes axon regeneration in mature neurons. Elevation and maintenance of mTOR activity in injured neurons have been proposed as the main underlying mechanism. Here we demonstrate that PTEN‐/‐ also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in GSK3S/A knockin mice significantly compromised PTEN‐/‐‐mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. GSK3S/A knockin or CRMP2 inhibition also decreased PTEN‐/‐ mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN‐/‐ mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration

Expression of Developmentally Important Axon Guidance Cues in the Adult Optic Chiasm.

PurposeRegeneration of optic nerve axons after injury can be facilitated by several approaches, but misguidance at the optic chiasm is often observed. We characterized guidance cues in the embryonic visual system and adult optic chiasm before and after optic nerve crush (ONC) injury to better understand barriers to optic nerve regeneration in adults.MethodsRadial glial (RC2/BLBP/Slit1), developmental (Pax2) and extracellular markers (CSPG: H2B/CS-56) were assessed in C57BL/6J mice by immunohistochemistry. RC2, BLBP, Slit1, and CSPG are known inhibitory guidance cues while Pax2 is a permissive guidance cue.ResultsAt embryonic day 15.5 (E.15.5), RC2 and BLBP were identified superior to, and extending through, the optic chiasm. The optic chiasm was BLBP−ve in adult uninjured mice but BLBP+ve in adult mice 10 days after ONC injury. The reverse was true for RC2. Both BLBP and RC2 were absent in adult mice 6 weeks post-ONC. Slit1 was present in the optic chiasm midline and optic tracts in embryonic samples but was absent in uninjured adult tissue. Slit1 was observed superior to and at the midline of the optic chiasm 10 days post-ONC but absent 6 weeks after injury. Pax2 was expressed at the junction between the optic nerve and optic chiasm in embryonic brain tissue. In embryonic sections, CS-56 was observed at the junction between the optic chiasm and optic tract, and immediately superior to the optic chiasm. Both 2H6 and CS-56 staining was absent in uninjured and ONC-injured adult brains.ConclusionDifferences in guidance cue expression during development, in adulthood and after injury may contribute to misguidance of regenerating RGC axons in the adult optic chiasm.

Sema3A inhibits axonal regeneration of retinal ganglion cells via ROCK2

Successful regeneration of injured axons in the adult mammalian central nervous system (CNS) is mainly limited by lesion-induced neuronal apoptosis and the inhibitory environment consisting of numerous extrinsic and intrinsic factors. Semaphorin 3A (Sema3A), a classic axonal guidance cue, contributes to the failure of axonal regeneration and can be neutralized to enhance axonal regeneration. Previous studies have suggested that blockage of rho-associated protein kinase 2 (ROCK2) also exerts a protective effect on the survival and axonal regeneration of retinal ganglion cells (RGC, RGCs) after injury. Yet unresolved question is the interaction between the two factors. We thus evaluated the role of Sema3A and ROCK2 in RGC axonal regeneration. In this study, we first examined the expression of Sema3A and ROCK2 against optic nerve crush in vivo and oxygen-glucose deprivation insult to RGCs in vitro at different time points. Then Sema3A, ROCK2 inhibitor Y-27632, combination of both and phosphate-buffered saline (PBS) only were injected into the vitreous cavity after optic nerve crush at various times in different experiments. In order to assess axonal regeneration, we detected the mRNA levels of small proline-rich protein 1A (Sprr1A) and growth-associated protein 43 (GAP43) by quantitative real time-polymerase chain reaction (RT-qPCR), evaluated visual function by Flash Visual Evoked Potentials (F-VEPs), and checked the protein level of GAP43 by immunofluorescent staining. Our results demonstrated that Sema3A significantly suppressed optic nerve regeneration and this effect can be attenuated via blocking ROCK2. Moreover, Sema3A promoted the phosphorylation of myosin light chain 2 (MLC2) (specific downstream effector of ROCK2 concerning neurite growth). Collectively, Sema3A may negatively regulate axonal regeneration through ROCK2 in RGCs.

Telomerase Reverse Transcriptase and p53 Regulate Mammalian Peripheral Nervous System and CNS Axon Regeneration Downstream of c-Myc.

Although several genes have been identified to promote axon regeneration in the CNS, our understanding of the molecular mechanisms by which mammalian axon regeneration is regulated is still limited and fragmented. Here by using female mouse sensory axon and optic nerve regeneration as model systems, we reveal an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. We also provide evidence that TERT and p53 act downstream of c-Myc to control sensory axon regeneration. More importantly, overexpression of p53 in sensory neurons and retinal ganglion cells is sufficient to promote sensory axon and optic never regeneration, respectively. The study reveals a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches promoting CNS axon regeneration.SIGNIFICANCE STATEMENT Despite significant progress during the past decade, our understanding of the molecular mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented. By using sensory axon and optic nerve regeneration as model systems, the study revealed an unexpected role of telomerase reverse transcriptase (TERT) in regulation of axon regeneration. The results also delineated a c-Myc-TERT-p53 pathway in controlling axon growth. Last, our results demonstrated that p53 alone was sufficient to promote sensory axon and optic nerve regeneration in vivo Collectively, the study not only revealed a new mechanisms underlying mammalian axon regeneration, but also expanded the pool of potential targets that can be manipulated to enhance CNS axon regeneration.

Differential Gamma-Synuclein Expression in Acute and Chronic Retinal Ganglion Cell Death in the Retina and Optic Nerve.

We used genetic naturally occurring glaucoma (DBA/2J) and experimentally induced optic nerve crush (ONC) as models to study gamma-synuclein expression change in retinal ganglion cells and optic nerves. Gene chip microarray analysis demonstrated downregulated expression of the gamma-synuclein gene in DBA/2J mice as they developed age-associated glaucoma with concomitant with retinal ganglion cell loss. Real-time PCR, Western blot, and immunostaining results confirmed that the expression of gamma-synuclein at the mRNA and protein level was significantly reduced in the retinas and optic nerves of aged DBA/2J mice. We also observed similar reduced expression of gamma-synuclein in the retinas from mice after optic nerve crush. Surprisingly, the expression of gamma-synuclein was increased in optic nerves after crush. This is the first study demonstrating gamma-synuclein-expressing cells accumulate in the optic nerve crush site. Gamma-synuclein was found in axons colocalizing largely with neurofilaments in control mice without injury but was found inside cells within the scar in the crush site. Gamma-synuclein expression is predominantly expressed at the optic nerve crush site associated with CD68+ macrophage-like cells, not GFAP-expressing astroglial cells, suggesting gamma-synuclein expression is associated with glial scar formation inhibitory to optic nerve regeneration. We propose gamma-synuclein labels macrophage-like cells recruited to the site of acute optic nerve injury.

GSK3-CRMP2 signaling mediates axonal regeneration induced by Pten knockout

Knockout of phosphatase and tensin homolog (PTEN−/−) is neuroprotective and promotes axon regeneration in mature neurons. Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN−/− also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3S/A knockin mice significantly compromised PTEN−/−-mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. Gsk3S/A knockin or CRMP2 inhibition also decreased PTEN−/− mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN−/− mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration.

Optic nerve regeneration in larval zebrafish exhibits spontaneous capacity for retinotopic but not tectum specific axon targeting.

In contrast to mammals, retinal ganglion cells (RGC) axons of the optic nerve even in mature zebrafish exhibit a remarkable capacity for spontaneous regeneration. One constraint of using adult zebrafish is the limited ability to visualize the regeneration process in live animals. To dynamically visualize and trace the degree of target specific optic nerve regeneration, we took advantage of the optical transparency still preserved in post developmental larval zebrafish. We developed a rapid and robust assay to physically transect the larval optic nerve and find that by 96 hours post injury RGC axons have robustly regrown onto the optic tectum. We observe functional regeneration by 8 days post injury, and demonstrate that similar to adult zebrafish, optic nerve transection in larval zebrafish does not prominently induce cell death or proliferation of RGC neurons. Furthermore, we find that partial optic nerve transection results in axonal growth predominantly to the original, contralateral tectum, while complete transection results in innervation of both the correct contralateral and ‘incorrect’ ipsilateral tectum. Axonal tracing reveals that although regenerating axons innervate the ‘incorrect’ ipsilateral tectum, they successfully target their topographically appropriate synaptic areas. Combined, our results validate post developmental larval zebrafish as a powerful model for optic nerve regeneration, and reveal intricate mechanistic differences between axonal growth, midline guidance and synaptic targeting during zebrafish optic nerve regeneration.

Regulation of adult mammalian intrinsic axonal regeneration by NF-κB/STAT3 signaling cascade.

The inflammatory response is a critical regulator for the regeneration of axon following nervous system injury. Nuclear factor-kappa B (NF-κB) is characteristically known for its ubiquitous role in the inflammatory response. However, its functional role in adult mammalian axon growth remains elusive. Here, we found that the NF-κB signaling pathway is activated in adult sensory neurons through peripheral axotomy. Furthermore, inhibition of NF-κB in peripheral sensory neurons attenuated their axon growth in vitro and in vivo. Our results also showed that NF-κB modulated axon growth by repressing the phosphorylation of STAT3. Furthermore, activation of STAT3 significantly promoted adult optic nerve regeneration. Taken together, the findings of our study indicated that NF-κB/STAT3 cascade is a critical regulator of intrinsic axon growth capability in the adult nervous system.