Targeting Vasohibins to Promote Axon Regeneration.

Treatments accelerating axon regeneration in the nervous system are still clinically unavailable. However, parthenolide promotes adult sensory neurons' axon growth in culture by inhibiting microtubule detyrosination. Here, we show that overexpression of vasohibins increases microtubule detyrosination in growth cones and compromises growth in culture and in vivo. Moreover, overexpression of these proteins increases the required parthenolide concentrations to promote axon regeneration. At the same time, the partial knockdown of endogenous vasohibins or their enhancer SVBP in neurons facilitates axon growth, verifying them as pharmacological targets for promoting axon growth. In vivo, repeated intravenous application of parthenolide or its prodrug di-methyl-amino-parthenolide (DMAPT) markedly facilitates the regeneration of sensory, motor, and sympathetic axons in injured murine and rat nerves, leading to acceleration of functional recovery. Moreover, orally applied DMAPT was similarly effective in promoting nerve regeneration. Thus, pharmacological inhibition of vasohibins facilitates axon regeneration in different species and nerves, making parthenolide and DMAPT the first promising drugs for curing nerve injury.

In-depth analysis of Gαs protein activity by probing different fluorescently labeled guanine nucleotides.

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants.

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.

Synthesis and evaluation of radioiodinated estrogens for diagnosis and therapy of male urogenital tumours.

The preparation of 24 estrogens, their estrogen receptor (ER) affinity and studies of radioiodinated estrogen binding to ER-positive male bladder tumor cells (HTB9) are described. The estrogens with the highest affinity were selected using fluorescence anisotropy assays. A 2,2,2-trifluoroethyl group at the 11ß-position caused particularly promising affinity. (Radio)iodination was performed on the 17α-vinyl group. Binding studies on HTB9 cells revealed picomolar affinities of radioconjugates 19 and 31, indicating promising ability for targeting of urogenital tumors.

SLC22A11 Inserts the Uremic Toxins Indoxyl Sulfate and P-Cresol Sulfate into the Plasma Membrane.

Chronic kidney disease (CKD) is a global health concern affecting millions worldwide. One of the critical challenges in CKD is the accumulation of uremic toxins such as p-cresol sulfate (pCS) and indoxyl sulfate (IS), which contribute to systemic damage and CKD progression. Understanding the transport mechanisms of these prominent toxins is essential for developing effective treatments. Here, we investigated whether pCS and IS are routed to the plasma membrane or to the cytosol by two key transporters, SLC22A11 and OAT1. To distinguish between cytosolic transport and plasma membrane insertion, we used a hyperosmolarity assay in which the accumulation of substrates into HEK-293 cells in isotonic and hypertonic buffers was measured in parallel using LC-MS/MS. Judging from the efficiency of transport (TE), pCS is a relevant substrate of SLC22A11 at 7.8 ± 1.4 µL min-1 mg protein-1 but not as good as estrone-3-sulfate; OAT1 translocates pCS less efficiently. The TE of SLC22A11 for IS was similar to pCS. For OAT1, however, IS is an excellent substrate. With OAT1 and p-aminohippuric acid, our study revealed an influence of transporter abundance on the outcomes of the hyperosmolarity assay; very high transport activity confounded results. SLC22A11 was found to insert both pCS and IS into the plasma membrane, whereas OAT1 conveys these toxins to the cytosol. These disparate transport mechanisms bear profound ramifications for toxicity. Membrane insertion might promote membrane damage and microvesicle release. Our results underscore the imperative for detailed structural inquiries into the translocation of small molecules.

The Impact of Nε-Acryloyllysine Piperazides on the Conformational Dynamics of Transglutaminase 2.

In addition to the classic functions of proteins, such as acting as a biocatalyst or binding partner, the conformational states of proteins and their remodeling upon stimulation need to be considered. A prominent example of a protein that undergoes comprehensive conformational remodeling is transglutaminase 2 (TGase 2), the distinct conformational states of which are closely related to particular functions. Its involvement in various pathophysiological processes, including fibrosis and cancer, motivates the development of theranostic agents, particularly based on inhibitors that are directed toward the transamidase activity. In this context, the ability of such inhibitors to control the conformational dynamics of TGase 2 emerges as an important parameter, and methods to assess this property are in great demand. Herein, we describe the application of the switchSENSE® principle to detect conformational changes caused by three irreversibly binding Nε-acryloyllysine piperazides, which are suitable radiotracer candidates of TGase 2. The switchSENSE® technique is based on DNA levers actuated by alternating electric fields. These levers are immobilized on gold electrodes with one end, and at the other end of the lever, the TGase 2 is covalently bound. A novel computational method is introduced for describing the resulting lever motion to quantify the extent of stimulated conformational TGase 2 changes. Moreover, as a complementary biophysical method, native polyacrylamide gel electrophoresis was performed under similar conditions to validate the results. Both methods prove the occurrence of an irreversible shift in the conformational equilibrium of TGase 2, caused by the binding of the three studied Nε-acryloyllysine piperazides.

Human retinal ganglion cell axon regeneration by recapitulating developmental mechanisms: effects of recruitment of the mTOR pathway.

The poor axon regeneration in the central nervous system (CNS) often leads to permanent functional deficit following disease or injury. For example, degeneration of retinal ganglion cell (RGC) axons in glaucoma leads to irreversible loss of vision. Here, we have tested the hypothesis that the mTOR pathway regulates the development of human RGCs and that its recruitment after injury facilitates axon regeneration. We observed that the mTOR pathway is active during RGC differentiation, and using the induced pluripotent stem cell model of neurogenesis show that it facilitates the differentiation, function and neuritogenesis of human RGCs. Using a microfluidic model, we demonstrate that recruitment of the mTOR pathway facilitates human RGC axon regeneration after axotomy, providing evidence that the recapitulation of developmental mechanism(s) might be a viable approach for facilitating axon regeneration in the diseased or injured human CNS, thus helping to reduce and/or recover loss of function.

Protective effects of 4-aminopyridine in experimental optic neuritis and multiple sclerosis.

Chronic disability in multiple sclerosis is linked to neuroaxonal degeneration. 4-aminopyridine (4-AP) is used and licensed as a symptomatic treatment to ameliorate ambulatory disability in multiple sclerosis. The presumed mode of action is via blockade of axonal voltage gated potassium channels, thereby enhancing conduction in demyelinated axons. In this study, we provide evidence that in addition to those symptomatic effects, 4-AP can prevent neuroaxonal loss in the CNS. Using in vivo optical coherence tomography imaging, visual function testing and histologic assessment, we observed a reduction in retinal neurodegeneration with 4-AP in models of experimental optic neuritis and optic nerve crush. These effects were not related to an anti-inflammatory mode of action or a direct impact on retinal ganglion cells. Rather, histology and in vitro experiments indicated 4-AP stabilization of myelin and oligodendrocyte precursor cells associated with increased nuclear translocation of the nuclear factor of activated T cells. In experimental optic neuritis, 4-AP potentiated the effects of immunomodulatory treatment with fingolimod. As extended release 4-AP is already licensed for symptomatic multiple sclerosis treatment, we performed a retrospective, multicentre optical coherence tomography study to longitudinally compare retinal neurodegeneration between 52 patients on continuous 4-AP therapy and 51 matched controls. In line with the experimental data, during concurrent 4-AP therapy, degeneration of the macular retinal nerve fibre layer was reduced over 2 years. These results indicate disease-modifying effects of 4-AP beyond symptomatic therapy and provide support for the design of a prospective clinical study using visual function and retinal structure as outcome parameters.

Advances in chemistry and bioactivity of parthenolide.

Review covering up to 07/2019(-)-Parthenolide is a germacrane sesquiterpene lactone, available in ample amounts from the traditional medical plant feverfew (Tanacetum parthenium). Acting as a covalently reactive compound, it displays anti-inflammatory, redox-modulating, and epigenetic activities, as well as selective cytotoxicity towards cancer stem and progenitor cells. Furthermore, parthenolide was found to modulate microtubule dynamics by interfering with the detyrosination of α-tubulin, a specific posttranslational modification of the cytoskeleton. This review interfaces recently achieved parthenolide syntheses with strategies for bioactivity-based derivatization. Furthermore, chemical probe development from parthenolide is discussed, leading to a qualified summary of reported biological activities and implicated or identified targets. Special emphasis is given to parthenolide-induced microtubule modulation and the recently characterized tubulin carboxypeptidase enzymes involved in nerve (re)growth, cardiac muscle cell function, and metastasis development.

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.

Monitoring retinal changes with optical coherence tomography predicts neuronal loss in experimental autoimmune encephalomyelitis.

BACKGROUND: Retinal optical coherence tomography (OCT) is a clinical and research tool in multiple sclerosis, where it has shown significant retinal nerve fiber (RNFL) and ganglion cell (RGC) layer thinning, while postmortem studies have reported RGC loss. Although retinal pathology in experimental autoimmune encephalomyelitis (EAE) has been described, comparative OCT studies among EAE models are scarce. Furthermore, the best practices for the implementation of OCT in the EAE lab, especially with afoveate animals like rodents, remain undefined. We aimed to describe the dynamics of retinal injury in different mouse EAE models and outline the optimal experimental conditions, scan protocols, and analysis methods, comparing these to histology to confirm the pathological underpinnings. METHODS: Using spectral-domain OCT, we analyzed the test-retest and the inter-rater reliability of volume, peripapillary, and combined horizontal and vertical line scans. We then monitored the thickness of the retinal layers in different EAE models: in wild-type (WT) C57Bl/6J mice immunized with myelin oligodendrocyte glycoprotein peptide (MOG35-55) or with bovine myelin basic protein (MBP), in TCR2D2 mice immunized with MOG35-55, and in SJL/J mice immunized with myelin proteolipid lipoprotein (PLP139-151). Strain-matched control mice were sham-immunized. RGC density was counted on retinal flatmounts at the end of each experiment. RESULTS: Volume scans centered on the optic disc showed the best reliability. Retinal changes during EAE were localized in the inner retinal layers (IRLs, the combination of the RNFL and the ganglion cell plus the inner plexiform layers). In WT, MOG35-55 EAE, progressive thinning of IRL started rapidly after EAE onset, with 1/3 of total loss occurring during the initial 2 months. IRL thinning was associated with the degree of RGC loss and the severity of EAE. Sham-immunized SJL/J mice showed progressive IRL atrophy, which was accentuated in PLP-immunized mice. MOG35-55-immunized TCR2D2 mice showed severe EAE and retinal thinning. MBP immunization led to very mild disease without significant retinopathy. CONCLUSIONS: Retinal neuroaxonal damage develops quickly during EAE. Changes in retinal thickness mirror neuronal loss and clinical severity. Monitoring of the IRL thickness after immunization against MOG35-55 in C57Bl/6J mice seems the most convenient model to study retinal neurodegeneration in EAE.

Synthesis and biological profiling of parthenolide ether analogs.

Parthenolide (PTL) strongly inhibits the detyrosination of microtubules and accelerates neuronal growth. In order to access cyclic ether derivatives of PTL, ring-closing metathesis (RCM) was investigated in comparison to intramolecular sulfone alkylation. Incompatibility of RCM with epoxides was found in this setting. Biological evaluation for tubulin carboxypeptidase inhibition indicated that the epoxide is crucial for parthenolide's activity.

Microglia Are Irrelevant for Neuronal Degeneration and Axon Regeneration after Acute Injury.

The role of microglia in degenerative and regenerative processes after damage of the nervous system remains ambiguous, partially due to the paucity of appropriate investigative methods. Here, we show that treatment with the pharmacological colony stimulating factor 1 receptor inhibitor PLX5622 specifically eliminated microglia in murine retinae and optic nerves with high efficiency. Interestingly, time course and extent of retinal ganglion cell (RGC) degeneration after optic nerve crush remained unaffected upon microglia depletion, although remnants of prelabeled apoptotic RGCs were not cleared from the retina in these animals. In addition, microglia depletion neither affected the induction of regeneration associated genes upon optic nerve injury nor the increased regenerative potential of RGCs upon lens injury (LI). However, although the repopulation of the optic nerve lesion site by astrocytes was significantly delayed upon microglia depletion, spontaneous and LI-induced axon regeneration were unaffected by PLX5622 treatment or peripheral macrophage depletion by clodronate liposome treatment. Only concurrent double depletion of microglia and infiltrated macrophages slightly, but significantly, compromised optic nerve regeneration. Therefore, microglia are not essentially involved in RGC degeneration or axonal regeneration after acute CNS injury.SIGNIFICANCE STATEMENT The roles of microglia, the phagocytosing cells of the CNS, and invading macrophages in degenerative and regenerative processes after injury are still controversial and insufficiently characterized. Here, we show that application of a CSF1R inhibitor eliminated virtually all microglia from the visual system, whereas macrophages were spared. Specific microglia depletion impaired the removal of dead labeled retinal ganglion cells after optic nerve crush, but remarkable had no influence on their degeneration. Similarly, optic nerve regeneration was completely unaffected, although repopulation of the lesion site by astrocytes was delayed significantly. Therefore, contrary to previous reports, this experimental approach revealed that microglia seemingly neither promote nor inhibit neuronal degeneration or axonal regrowth within the injured visual system.

Early alpha-lipoic acid therapy protects from degeneration of the inner retinal layers and vision loss in an experimental autoimmune encephalomyelitis-optic neuritis model.

BACKGROUND: In multiple sclerosis (MS), neurodegeneration is the main reason for chronic disability. Alpha-lipoic acid (LA) is a naturally occurring antioxidant which has recently been demonstrated to reduce the rate of brain atrophy in progressive MS. However, it remains uncertain if it is also beneficial in the early, more inflammatory-driven phases. As clinical studies are costly and time consuming, optic neuritis (ON) is often used for investigating neuroprotective or regenerative therapeutics. We aimed to investigate the prospect for success of a clinical ON trial using an experimental autoimmune encephalomyelitis-optic neuritis (EAE-ON) model with visual system readouts adaptable to a clinical ON trial. METHODS: Using an in vitro cell culture model for endogenous oxidative stress, we compared the neuroprotective capacity of racemic LA with the R/S-enantiomers and its reduced form. In vivo, we analyzed retinal neurodegeneration using optical coherence tomography (OCT) and the visual function by optokinetic response (OKR) in MOG35-55-induced EAE-ON in C57BL/6J mice. Ganglion cell counts, inflammation, and demyelination were assessed by immunohistological staining of retinae and optic nerves. RESULTS: All forms of LA provided equal neuroprotective capacities in vitro. In EAE-ON, prophylactic LA therapy attenuated the clinical EAE score and prevented the thinning of the inner retinal layer while therapeutic treatment was not protective on visual outcomes. CONCLUSIONS: A prophylactic LA treatment is necessary to protect from visual loss and retinal thinning in EAE-ON, suggesting that a clinical ON trial starting therapy after the onset of symptoms may not be successful.

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.

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.

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.

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.

Small fibre integrity and axonal pathology in the rat model of experimental autoimmune neuritis.

Experimental autoimmune neuritis is a common animal model for acute human immune-mediated polyneuropathies. Although already established in 1955, a number of pathophysiological mechanisms remain unknown. In this study, we extensively characterize experimental autoimmune neuritis progression in Lewis rats, including new insights into the integrity of small nerve fibres, neuropathic pain and macrophage activation. Acute experimental autoimmune neuritis was induced with P253-78 peptide and consequently investigated using the gait analysis system CatWalk XT, electrophysiological and histopathological analyses, quantitative polymerase chain reaction (PCR), dorsal root ganglia outgrowth studies, as well as the von Frey hair and Hargreaves tests. For the longitudinal setup, rats were sacrificed at Day (d) 10 (onset), d15 (peak), d26 (recovery) and d29 (late recovery). We confirmed the classical T-cell and macrophage-driven inflammation and the primarily demyelinating nature of the experimental autoimmune neuritis. The dual role of macrophages in experimental autoimmune neuritis is implicated by the high number of remaining macrophages throughout disease progression. Furthermore, different subpopulations of macrophages based on Cx3-motif chemokine receptor 1 (Cx3cr1), platelet factor 4 (Pf4) and macrophage galactose-type lectin-1 (Mgl1) expressions were identified. In addition, modulation of the sensory system in experimental autoimmune neuritis was detected. An outgrowth of small fibres in the plantar skin at the onset and peak of the experimental autoimmune neuritis was evident parallel to the development of acute hyperalgesia mediated through transient receptor potential vanilloid 1 modulation. Our data depict experimental autoimmune neuritis as a primary demyelinating disease with implicated axonal damage, a small unmyelinated fibre impairment throughout the disease progression course, and underline the pivotal role of macrophages in the effector and during the recovery stage.

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.