Cnicin promotes functional nerve regeneration.

BACKGROUND: The limited regenerative capacity of injured axons hinders functional recovery after nerve injury. Although no drugs are currently available in the clinic to accelerate axon regeneration, recent studies show the potential of vasohibin inhibition by parthenolide, produced in Tanacetum parthenium, to accelerate axon regeneration. However, due to its poor oral bioavailability, parthenolide is limited to parenteral administration. PURPOSE: This study investigates another sesquiterpene lactone, cnicin, produced in Cnicus benedictus for promoting axon regeneration. RESULTS: Cnicin is equally potent and effective in facilitating nerve regeneration as parthenolide. In culture, cnicin promotes axon growth of sensory and CNS neurons from various species, including humans. Neuronal overexpression of vasohibin increases the effective concentrations comparable to parthenolide, suggesting an interaction between cnicin and vasohibin. Remarkably, intravenous administration of cnicin significantly accelerates functional recovery after severe nerve injury in various species, including the anastomosis of severed nerves. Pharmacokinetic analysis of intravenously applied cnicin shows a blood half-life of 12.7 min and an oral bioavailability of 84.7 % in rats. Oral drug administration promotes axon regeneration and recovery after nerve injury in mice. CONCLUSION: These results highlight the potential of cnicin as a promising drug to treat axonal insults and improve recovery.

Inhibition of microtubule detyrosination by parthenolide facilitates functional CNS axon regeneration.

Injured axons in the central nervous system (CNS) usually fail to regenerate, causing permanent disabilities. However, the knockdown of Pten knockout or treatment of neurons with hyper-IL-6 (hIL-6) transforms neurons into a regenerative state, allowing them to regenerate axons in the injured optic nerve and spinal cord. Transneuronal delivery of hIL-6 to the injured brain stem neurons enables functional recovery after severe spinal cord injury. Here we demonstrate that the beneficial hIL-6 and Pten knockout effects on axon growth are limited by the induction of tubulin detyrosination in axonal growth cones. Hence, cotreatment with parthenolide, a compound blocking microtubule detyrosination, synergistically accelerates neurite growth of cultured murine CNS neurons and primary RGCs isolated from adult human eyes. Systemic application of the prodrug dimethylamino-parthenolide (DMAPT) facilitates axon regeneration in the injured optic nerve and spinal cord. Moreover, combinatorial treatment further improves hIL-6-induced axon regeneration and locomotor recovery after severe SCI. Thus, DMAPT facilitates functional CNS regeneration and reduces the limiting effects of pro-regenerative treatments, making it a promising drug candidate for treating CNS injuries.

Transneuronal Delivery of Cytokines to Stimulate Mammalian Spinal Cord Regeneration.

The spinal cord contains multiple fiber tracts necessary for locomotion. However, as a part of the central nervous system, they are extremely limited in regenerating after injury. Many of these key fiber tracts originate from deep brain stem nuclei that are difficult to access. Here we detail a new methodology that achieves functional regeneration in mice after a complete spinal cord crush, describing the crushing procedure itself, intracortical treatment application, and a set of appropriate validation steps. The regeneration is achieved by the one-time transduction of neurons in the motor cortex with a viral vector expressing the designer cytokine hIL-6. This potent stimulator of the JAK/STAT3 pathway and regeneration is transported in axons and then transneuronally delivered to critical deep brain stem nuclei via collateral axon terminals, resulting in previously paralyzed mice walking again after 3-6 weeks. With no previously known strategy accomplishing this degree of recovery, this model is well suited to studying the functional impact of compounds/treatments currently only known to promote anatomical regeneration.

[Regeneration of the optic nerve-Will it one day be reality?] / Regeneration des Sehnerven ­ Wird das einmal Realität?

BACKGROUND: Adult mammalian and human neurons of the central nervous system (CNS) lack the ability to spontaneously regenerate damaged axons. This dilemma of many CNS diseases is still an unsolved problem. OBJECTIVE: The purpose of this article is to examine the question which options have been investigated in more detail in recent years and offer approaches. METHODS: A web-based search of all articles published between 1958 to the present regarding regeneration of retinal ganglion cells was carried out. RESULTS: Over the last three decades it has been shown that axonal regeneration is possible under certain conditions when intrinsic and extrinsic factors are manipulated in retinal ganglion cells and in the optic nerve. Although there is still a long way to go, experimental regenerative approaches are already visible; however, it will take several years or decades before these can be approximately implemented in practice.

CXCR4/CXCL12-mediated entrapment of axons at the injury site compromises optic nerve regeneration.

Regenerative failure in the mammalian optic nerve is generally attributed to axotomy-induced retinal ganglion cell (RGC) death, an insufficient intrinsic regenerative capacity, and an extrinsic inhibitory environment. Here, we show that a chemoattractive CXCL12/CXCR4-dependent mechanism prevents the extension of growth-stimulated axons into the distal nerve. The chemokine CXCL12 is chemoattractive toward axonal growth cones in an inhibitory environment, and these effects are entirely abolished by the specific knockout of its receptor, CXCR4 (CXCR4-/-), in cultured regenerating RGCs. Notably, 8% of naïve RGCs express CXCL12 and transport the chemokine along their axons in the nerve. Thus, axotomy causes its release at the injury site. However, most osteopontin-positive α-RGCs, the main neuronal population that survives optic nerve injury, express CXCR4 instead. Thus, CXCL12-mediated attraction prevents growth-stimulated axons from regenerating distally in the nerve, indicated by axons returning to the lesion site. Accordingly, specific depletion of CXCR4 in RGC reduces aberrant axonal growth and enables long-distance regeneration. Likewise, CXCL12 knockout in RGCs fully mimics these CXCR4-/- effects. Thus, active CXCL12/CXCR4-mediated entrapment of regenerating axons to the injury site contributes to regenerative failure in the optic nerve.

Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice.

Spinal cord injury (SCI) often causes severe and permanent disabilities due to the regenerative failure of severed axons. Here we report significant locomotor recovery of both hindlimbs after a complete spinal cord crush. This is achieved by the unilateral transduction of cortical motoneurons with an AAV expressing hyper-IL-6 (hIL-6), a potent designer cytokine stimulating JAK/STAT3 signaling and axon regeneration. We find collaterals of these AAV-transduced motoneurons projecting to serotonergic neurons in both sides of the raphe nuclei. Hence, the transduction of cortical neurons facilitates the axonal transport and release of hIL-6 at innervated neurons in the brain stem. Therefore, this transneuronal delivery of hIL-6 promotes the regeneration of corticospinal and raphespinal fibers after injury, with the latter being essential for hIL-6-induced functional recovery. Thus, transneuronal delivery enables regenerative stimulation of neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI.

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.

Muscle LIM Protein Is Expressed in the Injured Adult CNS and Promotes Axon Regeneration.

Muscle LIM protein (MLP) has long been regarded as a muscle-specific protein. Here, we report that MLP expression is induced in adult rat retinal ganglion cells (RGCs) upon axotomy, and its expression is correlated with their ability to regenerate injured axons. Specific knockdown of MLP in RGCs compromises axon regeneration, while overexpression in vivo facilitates optic nerve regeneration and regrowth of sensory neurons without affecting neuronal survival. MLP accumulates in the cell body, the nucleus, and in axonal growth cones, which are significantly enlarged by its overexpression. Only the MLP fraction in growth cones is relevant for promoting axon extension. Additional data suggest that MLP acts as an actin cross-linker, thereby facilitating filopodia formation and increasing growth cone motility. Thus, MLP-mediated effects on actin could become a therapeutic strategy for promoting nerve repair.

Boosting CNS axon regeneration by harnessing antagonistic effects of GSK3 activity.

Implications of GSK3 activity for axon regeneration are often inconsistent, if not controversial. Sustained GSK3 activity in GSK3S/A knock-in mice reportedly accelerates peripheral nerve regeneration via increased MAP1B phosphorylation and concomitantly reduces microtubule detyrosination. In contrast, the current study shows that lens injury-stimulated optic nerve regeneration was significantly compromised in these knock-in mice. Phosphorylation of MAP1B and CRMP2 was expectedly increased in retinal ganglion cell (RGC) axons upon enhanced GSK3 activity, but, surprisingly, no GSK3-mediated CRMP2 inhibition was detected in sciatic nerves, thus revealing a fundamental difference between central and peripheral axons. Conversely, genetic or shRNA-mediated conditional KO/knockdown of GSK3ß reduced inhibitory phosphorylation of CRMP2 in RGCs and improved optic nerve regeneration. Accordingly, GSK3ß KO-mediated neurite growth promotion and myelin disinhibition were abrogated by CRMP2 inhibition and largely mimicked in WT neurons upon expression of constitutively active CRMP2 (CRMP2T/A). These results underscore the prevalent requirement of active CRMP2 for optic nerve regeneration. Strikingly, expression of CRMP2T/A in GSK3S/A RGCs further boosted optic nerve regeneration, with axons reaching the optic chiasm within 3 wk. Thus, active GSK3 can also markedly promote axonal growth in central nerves if CRMP2 concurrently remains active. Similar to peripheral nerves, GSK3-mediated MAP1B phosphorylation/activation and the reduction of microtubule detyrosination contributed to this effect. Overall, these findings reconcile conflicting data on GSK3-mediated axon regeneration. In addition, the concept of complementary modulation of normally antagonistically targeted GSK3 substrates offers a therapeutically applicable approach to potentiate the regenerative outcome in the injured CNS.

Boosting Central Nervous System Axon Regeneration by Circumventing Limitations of Natural Cytokine Signaling.

Retinal ganglion cells (RGCs) do not normally regenerate injured axons, but die upon axotomy. Although IL-6-like cytokines are reportedly neuroprotective and promote optic nerve regeneration, their overall regenerative effects remain rather moderate. Here, we hypothesized that direct activation of the gp130 receptor by the designer cytokine hyper-IL-6 (hIL-6) might induce stronger RGC regeneration than natural cytokines. Indeed, hIL-6 stimulated neurite growth of adult cultured RGCs with significantly higher efficacy than CNTF or IL-6. This neurite growth promoting effect could be attributed to stronger activation of the JAK/STAT3 and PI3K/AKT/mTOR signaling pathways and was also observed in peripheral dorsal root ganglion neurons. Moreover, hIL-6 abrogated axon growth inhibition by central nervous system (CNS) myelin. Remarkably, continuous hIL-6 expression upon RGC-specific AAV transduction after optic nerve crush exerted stronger axon regeneration than other known regeneration promoting treatments such as lens injury and PTEN knockout, with some axons growing through the optic chiasm 6 weeks after optic nerve injury. Combination of hIL-6 with RGC-specific PTEN knockout further enhanced optic nerve regeneration. Therefore, direct activation of gp130 signaling might be a novel, clinically applicable approach for robust CNS repair.

Promotion of Functional Nerve Regeneration by Inhibition of Microtubule Detyrosination.

Functional recovery of injured peripheral neurons often remains incomplete, but the clinical outcome can be improved by increasing the axonal growth rate. Adult transgenic GSK3α(S/A)/ß(S/A) knock-in mice with sustained GSK3 activity show markedly accelerated sciatic nerve regeneration. Here, we unraveled the molecular mechanism underlying this phenomenon, which led to a novel pharmacological approach for the promotion of functional recovery after nerve injury.In vitroandin vivoanalysis of GSK3 single knock-in mice revealed the unexpected contribution of GSK3α in addition to GSK3ß, as both GSK3(S/A) knock-ins improved axon regeneration. Moreover, growth stimulation depended on overall GSK3 activity, correlating with increased phosphorylation of microtubule-associated protein 1B and reduced microtubule detyrosination in axonal tips. Pharmacological inhibition of detyrosination by parthenolide or cnicin mimicked this axon growth promotion in wild-type animals, although it had no effect in GSK3α(S/A)/ß(S/A) mice. These results support the conclusion that sustained GSK3 activity primarily targets microtubules in growing axons, maintaining them in a more dynamic state to facilitate growth. Accordingly, further manipulation of microtubule stability using either paclitaxel or nocodazole compromised the effects of parthenolide. Strikingly, either local or systemic application of parthenolide in wild-type mice dose-dependently acceleratedin vivoaxon regeneration and functional recovery similar to GSK3α(S/A)/ß(S/A) mice. Thus, reducing microtubule detyrosination in axonal tips may be a novel, clinically suitable strategy to treat nerve damage. SIGNIFICANCE STATEMENT: Peripheral nerve regeneration often remains incomplete, due to an insufficient growth rate of injured axons. Transgenic mice with sustained GSK3 activity showed markedly accelerated nerve regeneration upon injury. Here, we identified the molecular mechanism underlying this phenomenon and provide a novel therapeutic principle for promoting nerve repair. Analysis of transgenic mice revealed a dependence on overall GSK3 activity and reduction of microtubule detyrosination in axonal tips. Pharmacological inhibition of detyrosination by parthenolide fully mimicked this axon growth promotion in wild-type mice. Strikingly, local or systemic treatment with parthenolidein vivomarkedly accelerated axon regeneration and functional recovery. Thus, pharmacological inhibition of microtubule detyrosination may be a novel, clinically suitable strategy for nerve repair with potential relevance for human patients.

Sustained GSK3 activity markedly facilitates nerve regeneration.

Promotion of axonal growth of injured DRG neurons improves the functional recovery associated with peripheral nerve regeneration. Both isoforms of glycogen synthase kinase 3 (GSK3; α and ß) are phosphorylated and inactivated via phosphatidylinositide 3-kinase (PI3K)/AKT signalling upon sciatic nerve crush (SNC). However, the role of GSK3 phosphorylation in this context is highly controversial. Here we use knock-in mice expressing GSK3 isoforms resistant to inhibitory PI3K/AKT phosphorylation, and unexpectedly find markedly accelerated axon growth of DRG neurons in culture and in vivo after SNC compared with controls. Moreover, this enhanced regeneration strikingly accelerates functional recovery after SNC. These effects are GSK3 activity dependent and associated with elevated MAP1B phosphorylation. Altogether, our data suggest that PI3K/AKT-mediated inhibitory phosphorylation of GSK3 limits the regenerative outcome after peripheral nerve injury. Therefore, suppression of this internal 'regenerative break' may potentially provide a new perspective for the clinical treatment of nerve injuries.

Neuronal expression of muscle LIM protein in postnatal retinae of rodents.

Muscle LIM protein (MLP) is a member of the cysteine rich protein family and has so far been regarded as a muscle-specific protein that is mainly involved in myogenesis and the organization of cytoskeletal structure in myocytes, respectively. The current study demonstrates for the first time that MLP expression is not restricted to muscle tissue, but is also found in the rat naive central nervous system. Using quantitative PCR, Western blot and immunohistochemical analyses we detected MLP in the postnatal rat retina, specifically in the somas and dendritic arbors of cholinergic amacrine cells (AC) of the inner nuclear layer and the ganglion cell layer (displaced AC). Induction of MLP expression started at embryonic day 20 and peaked between postnatal days 7 and 14. It subsequently decreased again to non-detectable protein levels after postnatal day 28. MLP was identified in the cytoplasm and dendrites but not in the nucleus of AC. Thus, retinal MLP expression correlates with the morphologic and functional development of cholinergic AC, suggesting a potential role of this protein in postnatal maturation and making MLP a suitable marker for these neurons.

TGF-ß signaling protects retinal neurons from programmed cell death during the development of the mammalian eye.

We investigated the influence of transforming growth factor-ß (TGF-ß) signaling on developmental programmed cell death in the mouse retina by direct and specific molecular targeting of TGF-ß type II receptor (TßRII) and Smad7 in retinal progenitor cells. Mice were generated carrying a conditional deletion of the TßRII in cells that originate from the inner layer of the optic cup. The animals showed a significant decrease of phosphorylated Smad3 in both the central and peripheral retina, which indicates the diminished activity of TGF-ß signaling. TßRII deficiency significantly increased the apoptotic death of retinal neurons during embryonic and postnatal development without affecting their proliferation. In contrast, treatment with TGF-ß2 inhibited cell death of retinal ganglion cells in dissociated retinal cell cultures, an effect that was blocked by inhibiting the phosphorylation of Smad3. The increase in apoptosis during development resulted in a significant reduction in the number of neurons in adult TßRII-deficient mice. The effect was most pronounced in the inner retina neurons and resulted in functional deficits as determined by electroretinography. In contrast, a conditional deletion of TGF-ß-inhibiting Smad7 in retinal neurons significantly enhanced Smad3 phosphorylation and significantly decreased apoptosis of retinal neurons in embryos and pups. Moreover, the number of retinal ganglion cells was significantly higher in Smad7-deficient mice compared with control littermates. TßRII-deficient pups showed a lower level of nerve growth factor (NGF) in its mRNA; however, higher levels were observed in Smad7-deficient pups, which strongly suggests that the protective effects of TGF-ß signaling on developmental cell death are mediated through NGF.

Do growth-stimulated retinal ganglion cell axons find their central targets after optic nerve injury? New insights by three-dimensional imaging of the visual pathway.

Retinal ganglion cells (RGCs) do not normally regenerate injured axons. However, several strategies to transform RGCs into a potent regenerative state have been developed in recent years. Intravitreal CNTF application combined with conditional PTEN and SOCS3 deletion or zymosan-induced inflammatory stimulation together with cAMP analogue injection and PTEN-deletion in RGCs induce long-distance regeneration into the optic nerve of adult mice. A recent paper by the Benowitz group (de Lima et al.) claimed that the latter treatment enables full-length regeneration, with axons correctly navigating to their central target zones and partial recovery of visual behaviors. To gain a more detailed view of the extent and the trajectories of regenerating axons, Luo et al. applied a tissue clearing method and fluorescent microscopy to allow the tracing of naïve and regenerating RGC axons in whole ON and all the way to their brain targets. Using this approach, the authors found comparable axon regeneration in the optic nerve after both above-mentioned experimental treatments. Regeneration was accompanied by prevalent aberrant axon growth in the optic nerve and significant axonal misguidance at the optic chiasm. Less than 120 axons per animal reached the optic chiasm and only few entered the correct optic tract. Importantly, no axons reached visual targets in the olivary pretectal nucleus, the lateral geniculate nucleus or the superior colliculus, thereby contradicting and challenging previous claims by the Benowitz group. The data provided by Luo et al. rather suggest that potent stimulation of axonal growth per se is insufficient to achieve functional recovery and underscore the need to investigate regeneration-relevant axon guidance mechanisms in the mature visual system.

CXCL12/SDF-1 facilitates optic nerve regeneration.

Mature retinal ganglion cells (RGCs) do not normally regenerate injured axons, but undergo apoptosis soon after axotomy. Besides the insufficient intrinsic capability of mature neurons to regrow axons inhibitory molecules located in myelin of the central nervous system as well as the glial scar forming at the site of injury strongly limit axon regeneration. Nevertheless, RGCs can be transformed into a regenerative state upon inflammatory stimulation (IS), enabling these neurons to grow axons into the injured optic nerve. The outcome of IS stimulated regeneration is, however, still limited by the inhibitory extracellular environment. Here, we report that the chemokine CXCL12/SDF-1 moderately stimulates neurite growth of mature RGCs on laminin in culture and, in contrast to CNTF, exerts potent disinhibitory effects towards myelin. Consistently, co-treatment of RGCs with CXCL12 facilitated CNTF stimulated neurite growth of RGCs on myelin. Mature RGCs express CXCR4, the cognate CXCL12 receptor. Furthermore, the neurite growth promoting and disinhibitory effects of CXCL12 were abrogated by a specific CXCR4 antagonist and by inhibition of the PI3K/AKT/mTOR-, but not the JAK/STAT3-pathway. In vivo, intravitreal application of CXCL12 sustained mTOR activity in RGCs upon optic nerve injury and moderately stimulated axon regeneration in the optic nerve without affecting the survival of RGCs. Importantly, intravitreal application of CXCL12 also significantly increased IS triggered axon regeneration in vivo. These data suggest that the disinhibitory effect of CXCL12 towards myelin may be a useful feature to facilitate optic nerve regeneration, particularly in combination with other axon growth stimulatory treatments.

Promoting optic nerve regeneration.

Vision is the most important sense for humans and it is irreversibly impaired by axonal damage of retinal ganglion cells (RGCs) in the optic nerve due to the lack of axonal regeneration. The failure of regeneration is partially attributable to factors located in the inhibitory environment of the forming glial scar and myelin as well as an insufficient intrinsic ability for axonal regrowth. Moreover, RGCs undergo apoptotic cell death after optic nerve injury, eliminating any chance for regeneration. In this review, we discuss the different aspects that cause regenerative failure in the optic nerve. Moreover, we describe discoveries of the last two decades demonstrating that under certain circumstances mature RGCs can be transformed into an active regenerative state allowing these neurons to survive axotomy and to regenerate axons in the injured optic nerve. In this context we focus on the role of the cytokines ciliary neutrophic factor (CNTF) and leukemia inhibitory factor (LIF), their receptors and the downstream signaling pathways. Furthermore, we discuss strategies to overcome inhibitory signaling induced by molecules associated with optic nerve myelin and the glial scar as well as the regenerative outcome after combinatorial treatments. These findings are encouraging and may open the possibility that clinically meaningful regeneration may become achievable one day in the future.

Role of mTOR in neuroprotection and axon regeneration after inflammatory stimulation.

Mature retinal ganglion cells (RGCs) do not normally regenerate injured axons, but degenerate after axotomy. However, inflammatory stimulation (IS) enables RGCs to survive axotomy and regenerate axons in the injured optic nerve. Similar effects are achieved by the genetic deletion of phosphatase and tensin homolog (PTEN) and subsequent mammalian target of rapamycin (mTOR) activation. Here, we report that IS prevents the axotomy-induced decrease of mTOR activity in RGCs in a CNTF/LIF-dependent manner. Inactivation of mTOR significantly reduced the number of long axons regenerating in the optic nerve, but surprisingly, did not affect the initial switch of RGCs into the regenerative state, or the neuroprotective effects associated with IS. In vitro, inhibition of mTOR activity reduced regeneration on myelin or chondroitin sulfate proteoglycans (CSPGs), but not on a growth-permissive substrate. Thus, mTOR activity is not generally required for neuroprotection or switching mature neurons into an active regenerative state, but it is important for the maintenance of the axonal growth state and overcoming of inhibitory effects caused by myelin and CSPGs.