CNS Regeneration Research Articles

Progress and Setbacks in the Study of CNS Axonal Plasticity and Regeneration

Understanding of basic mechanisms underlying the refractory state of axonal regeneration in the CNS has advanced remarkably in the last 50 years. Mechanisms that underlie the failure of central regeneration include: 1) an absence of permissive extracellular matrices in lesion sites, 2) insufficient secretion of trophic molecules, 3) the presence of growth‐inhibitory molecules in the CNS, and 4) an intrinsic failure of adult CNS neurons to upregulate extensive sets of growth‐related genes. Addressing several of these mechanisms simultaneously, it is possible to elicit growth of select spinal cord axonal populations into and beyond lesion sites, and to reinnervate normal targets. However, several critical challenges remain in SCI research, including: 1) elicitation of regeneration or corticospinal axonal systems; 2) remyelination of regenerating axons; 3) elicitation of substantial growth at chronic stages after SCI, and 4) translation of these findings to primate systems, given fundamental differences in axonal organization and function between rodent and primate models. This talk will summarize these topics and highlight future directions of research.

Postnatal Deamidation of 4E-BP2 in Brain Enhances Its Association with Raptor and Alters Kinetics of Excitatory Synaptic Transmission

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Research Highlights

Research Highlights

NgR expression in macrophages promotes nerve regeneration after spinal cord injury in rats

This study aimed to investigate the expression of Nogo-66 receptor (NgR) in macrophages after SCI and clarify its role in neuron regeneration. Macrophages harvested from injured spine cord of rats were stained by double immunofluorescence labeling technique to observe the expression of NgR at histological and cellular levels. Macrophages which expressed NgR were constructed in vitro, and then the effects of NgR on macrophage phagocytosis and neuraxon regeneration in three groups (NgR-macrophages group, mock group and normal macrophages group) were studied using Western blot, micro-MTT colorimetry, and LDH assay separately. The results showed that CD68-positive macrophages in injured tissue of spine cord expressed NgR after double immunofluorescence staining on day 7 after SCI, and so did macrophages isolated and cultured from the injured spine cord. The results of Western blot showed that phagocytosis of macrophages in NgR-macrophages group was much better than that in mock group and normal macrophage group (p < 0.05). And the results of Micro-MTT colorimetry and LDH assay indicated that the capacity of neuraxon regeneration in NgR-macrophages group was significantly higher than that in the other two groups (p < 0.05). The results suggested that there was NgR expressing in the infiltrated macrophages following SCI, which increased phagocytosis of the macrophages, and promoted post-SCI CNS regeneration in vitro.

Tuba1a gene expression is regulated by KLF6/7 and is necessary for CNS development and regeneration in zebrafish

We report that knockdown of the α1 tubulin isoform Tuba1a, but not the highly related Tuba1b, dramatically impedes nervous system formation during development and RGC axon regeneration following optic nerve injury in adults. Within the tuba1a promoter, a G/C-rich element was identified that is necessary for tuba1a induction during RGC differentiation and optic axon regeneration. KLF6a and 7a, which we previously reported are essential for optic axon regeneration (Veldman et al., 2007), bind this G/C-rich element and transactivate the tuba1a promoter. In vivo knockdown of KLF6a and 7a attenuate regeneration-dependent activation of the endogenous tuba1a and p27 genes. These results suggest tuba1a expression is necessary for CNS development and regeneration and that KLF6a and 7a mediate their effects, at least in part, via transcriptional control of tuba1a promoter activity.

Effect of biodegradable fibrin scaffold on survival, migration, and differentiation of transplanted bone marrow stromal cells after cortical injury in rats

In this study the authors' aim was to assess whether fibrin matrix could act as an injectable, valuable scaffold in bone marrow stromal cell (BMSC) transplantation for injured CNS tissue. Both clotting time and 3D structure of fibrin matrix were analyzed with various concentrations of fibrinogen and CaCl(2). The BMSCs were harvested from green fluorescent protein-transgenic mice and cultured. A cortical lesion was produced in rats by application of a very cold rod to the right cerebral hemisphere. The BMSCs, fibrin matrix, or BMSC-fibrin matrix complex was transplanted into the lesion though a small bur hole 7 days after the insult. Using immunohistochemical analysis, the authors evaluated the survival, migration, and differentiation of the transplanted cells 4 weeks after transplantation. Based on in vitro observations, the concentrations of fibrinogen and CaCl(2) were fixed at 2.5 mg/ml and 2 microM in animal experiments, respectively. Fibrin matrix almost completely disappeared 4 weeks after transplantation. However, immunohistochemical analysis revealed that fibrin matrix exclusively enhanced the retention of the transplanted cells within the lesion, migration toward the lesion boundary zone, and differentiation into the neurons and perivascular cells. Injectable fibrin matrix enhanced the survival, migration, and differentiation of the BMSCs transplanted into the cortical lesion in rats. The authors believe that it is one of the promising candidates for a potential, minimally invasive scaffold for CNS disorders. The present findings strongly suggest that such a strategy of tissue engineering could be a therapeutic option for CNS regeneration in patients with CNS injuries.

Functional electrical stimulation helps replenish progenitor cells in the injured spinal cord of adult rats

Functional electrical stimulation (FES) can restore control and offset atrophy to muscles after neurological injury. However, FES has not been considered as a method for enhancing CNS regeneration. This paper demonstrates that FES dramatically enhanced progenitor cell birth in the spinal cord of rats with a chronic spinal cord injury (SCI). A complete SCI at thoracic level 8/9 was performed on 12 rats. Three weeks later, a FES device to stimulate hindlimb movement was implanted into these rats. Twelve identically-injured rats received inactive FES implants. An additional control group of uninjured rats were also examined. Ten days after FES implantation, dividing cells were marked with bromodeoxyuridine (BrdU). The “cell birth” subgroup (half the animals in each group) was sacrificed immediately after completion of BrdU administration, and the “cell survival” subgroup was sacrificed 7 days later. In the injured “cell birth” subgroup, FES induced an 82–86% increase in cell birth in the lumbar spinal cord. In the injured “cell survival” subgroup, the increased lumbar newborn cell counts persisted. FES doubled the proportion of the newly-born cells which expressed nestin and other markers suggestive of tripotential progenitors. In uninjured rats, FES had no effect on cell birth/survival. This report suggests that controlled electrical activation of the CNS may enhance spontaneous regeneration after neurological injuries.

Olfactory Ensheathing Cell : a peculiar glial cell

Olfactory Ensheathing Cells (OECs) are glial cells that ensheathe the small nonmyelinated axons of the olfactory receptor neurons. Several studies reveal that OECs produce various growth factors and adhesion molecules that are involved in glia-axon adhesion. Immunocytochemical techniques have demonstrated that OECs express an high number of markers, such as GFAP, p75NTR, S100, vimentin, nestin, neuropeptide Y and calponin. During the last years, OECs have drawn considerable interest for their exceptional ability to promote regeneration in the injured CNS and for their functional plasticity. These peculiarities suggest that OECs could be used as potential restoration agents and provide trophic support to CNS injury. In this study we have evaluated: 1) the effect of rat OECs on the CNS neurons in culture; 2) the expression of different markers in OECs grown in different conditions. 1) We have focused our researches on hypothalamic and hippocampal neuronal survival and neurite outgrowth in the presence of OECs, compared to the action of some trophic factors, such as bFGF, NGF and GDNF in a co-culture model. Each individual population of cells was identified by immunocytochemical procedures. Our results have demonstrated that both in hypothalamic neurons/OECs co-cultures and in hippocampal neurons/OECs co-cultures, the number of neurons was significantly increased compared to controls exhibiting a dense axonal outgrowth. Moreover, we show that in both co-culture models, bFGF, NGF and GDNF support them differently. In conclusion, our data report that OECs exert a positive effect on survival and growth of neurons in vitro suggesting that they might be considered a better approach for functional restoration and for neural plasticity. 2) In our studies, we examined the expression of different markers in rat OECs grown both in serum containing medium and in serum-free medium added with different growth factors (bFGF and GDNF). OECs cultures were immunostained for calponin, nestin, Protein Gene Product (PGP 9.5) and Microtubule Associate Protein-2 (MAP-2). Our results have shown a different expression of nestin and calponin, depending on the presence of bFGF or GDNF. Moreover, the expression of neuronal markers (PGP 9.5 and MAP-2) increased in OECs grown in serum-free medium both treated with growth factors and without them. This result suggests that the highest expression of neuronal markers in serum-free medium might be explained by serum containing molecules that inhibit the effect of growth factors on the PGP 9.5 and MAP-2 expression.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Transcriptional regulatory regions of gap43 needed in developing and regenerating retinal ganglion cells

Mammals and fish differ in their ability to express axon growth-associated genes in response to CNS injury, which contributes to the differences in their ability for CNS regeneration. Previously we demonstrated that for the axon growth-associated gene, gap43, regions of the rat promoter that are sufficient to promote reporter gene expression in the developing zebrafish nervous system are not sufficient to promote expression in regenerating retinal ganglion cells in zebrafish. Recently, we identified a 3.6-kb gap43 promoter fragment from the pufferfish, Takifugu rubripes (fugu), that can promote reporter gene expression during both development and regeneration. Using promoter deletion analysis, we have found regions of the 3.6-kb fugu gap43 promoter that are necessary for expression in regenerating, but not developing, retinal ganglion cells. Within the 3.6-kb promoter, we have identified elements that are highly conserved among fish, as well as elements conserved among fish, mammals, and birds.

Mst3b, an Ste20-like kinase, regulates axon regeneration in mature CNS and PNS pathways

Mammalian sterile 20-like kinase-3b (Mst3b/Stk24) regulates axon outgrowth in embryonic cortical neurons in culture, but its role in vivo and in neural repair is unknown. Here we show that Mst3b mediates the axon-promoting effects of trophic factors in mature rat retinal ganglion cells (RGCs) and dorsal root ganglion (DRG) neurons, and is essential for axon regeneration in vivo. Reducing Mst3b levels using shRNA prevented RGCs and DRG neurons from regenerating axons in response to growth factors in culture, as did expression of a kinase-dead Mst3b mutant. Conversely, expression of constitutively active Mst3b enabled both types of neurons to extend axons without growth factors. In vivo, RGCs lacking Mst3b failed to regenerate injured axons when stimulated by intraocular inflammation. DRG neurons regenerating axons in vivo showed elevated Mst3b activity, and reducing Mst3b expression attenuated regeneration and p42/44 MAPK activation. Thus, Mst3b regulates axon regeneration in both CNS and PNS neurons.

Considering the evolution of regeneration in the central nervous system

For many years the mammalian CNS has been seen as an organ that is unable to regenerate. However, it was also long known that lower vertebrate species are capable of impressive regeneration of CNS structures. How did this situation arise through evolution? Increasing cellular and molecular understanding of regeneration in different animal species coupled with studies of adult neurogenesis in mammals is providing a basis for addressing this question. Here we compare CNS regeneration among vertebrates and speculate on how this ability may have emerged or been restricted.

Combined inhibition of Cdk5 and ROCK additively increase cell survival, but not the regenerative response in regenerating retinal ganglion cells

CNS regeneration is limited by lesion-induced neuronal apoptosis and an environment inhibiting axonal elongation. Inhibition of ROCK has been previously shown to promote regeneration in retinal ganglion cells (RGC) whereas Cdk5 inhibition mainly promoted survival. Therefore, we have evaluated the effects of combined treatment with inhibitors of ROCK and Cdk5. We show that in vitro, the co-application of the Cdk5 inhibitor, Indolinone A, and the ROCK inhibitor, Y-27632, potentiated the survival-promoting effect of either substance alone. However, neurite outgrowth in vitro was promoted only by the presence of Y-27632, not by Indolinone A alone. In the ex vivo explant and the in vivo optic nerve crush model the combination of both inhibitors significantly increased neurite outgrowth at small distances, but this effect leveled off for longer neurites. In summary, the combined treatment with the Cdk5 inhibitor Indolinone A and the ROCK inhibitor Y-27632 results in a strong additive effect on neuronal survival, but is not able to increase the regenerative response beyond the effect of the ROCK inhibitor.

Stimulation of Axon Regeneration in the Mature Optic Nerve by Intravitreal Application of the Toll-like Receptor 2 Agonist Pam3Cys

After injury of the optic nerve, mature retinal ganglion cells (RGCs) are normally unable to regenerate axons and undergo apoptosis. However, inflammatory stimulation in the eye induced by the release of beta/gamma-crystallins from the injured lens or intravitreal zymosan injection transforms RGCs into an active regenerative state, protecting these neurons from cell death and allowing them to regenerate axons back into the optic nerve. The authors tested whether intravitreal application of the selective, water-soluble, toll-like receptor 2 agonist Pam(3)Cys can delay axotomized RGC cell death and stimulate the regeneration of axons using an in vitro and in vivo paradigm. Intravitreal injection of Pam(3)Cys, as lens injury (LI), induced the upregulation of ciliary neurotrophic factor and glial fibrillary acidic protein expression in retinal glia accompanied by the activation of the JAK/STAT3 pathway in RGCs. As a consequence, RGCs switched to a regenerative state, indicated by a significant upregulation of GAP43 expression and increased neurite outgrowth of RGCs in culture. Repeated intravitreal Pam(3)Cys application in vivo induced neuroprotective effects and caused stronger axon regeneration in the injured optic nerve than observed after LI. Pam(3)Cys may be a suitable agent for stimulating CNS regeneration.

NG2 and phosphacan are present in the astroglial scar after human traumatic spinal cord injury

BackgroundA major class of axon growth-repulsive molecules associated with CNS scar tissue is the family of chondroitin sulphate proteoglycans (CSPGs). Experimental spinal cord injury (SCI) has demonstrated rapid re-expression of CSPGs at and around the lesion site. The pharmacological digestion of CSPGs in such lesion models results in substantially enhanced axonal regeneration and a significant functional recovery. The potential therapeutic relevance of interfering with CSPG expression or function following experimental injuries seems clear, however, the spatio-temporal pattern of expression of individual members of the CSPG family following human spinal cord injury is only poorly defined. In the present correlative investigation, the expression pattern of CSPG family members NG2, neurocan, versican and phosphacan was studied in the human spinal cord.MethodsAn immunohistochemical investigation in post mortem samples of control and lesioned human spinal cords was performed. All patients with traumatic SCI had been clinically diagnosed as having "complete" injuries and presented lesions of the maceration type.ResultsIn sections from control spinal cord, NG2 immunoreactivity was restricted to stellate-shaped cells corresponding to oligodendrocyte precursor cells. The distribution patterns of phosphacan, neurocan and versican in control human spinal cord parenchyma were similar, with a fine reticular pattern being observed in white matter (but also located in gray matter for phosphacan). Neurocan staining was also associated with blood vessel walls. Furthermore, phosphacan, neurocan and versican were present in the myelin sheaths of ventral and dorsal nerve roots axons. After human SCI, NG2 and phosphacan were both detected in the evolving astroglial scar. Neurocan and versican were detected exclusively in the lesion epicentre, being associated with infiltrating Schwann cells in the myelin sheaths of invading peripheral nerve fibres from lesioned dorsal roots.ConclusionNG2 and phosphacan were both present in the evolving astroglial scar and, therefore, might play an important role in the blockade of successful CNS regeneration. Neurocan and versican, however, were located at the lesion epicentre, associated with Schwann cell myelin on regenerating peripheral nerve fibres, a distribution that was unlikely to contribute to failed CNS axon regeneration. The present data points to the importance of such correlative investigations for demonstrating the clinical relevance of experimental data.

Central nervous system regeneration: from leech to opossum

A major problem of neurobiology concerns the failure of injured mammalian spinal cord to repair itself. This review summarizes work done on two preparations in which regeneration can occur: the central nervous system of an invertebrate, the leech, and the spinal cord of an immature mammal, the opossum. The aim is to understand cellular and molecular mechanisms that promote and prevent regeneration. In the leech, an individual axon regrows successfully to re-establish connections with its synaptic target, while avoiding other neurons. Functions that were lost are thereby restored. Moreover, pairs of identified neurons become re-connected with appropriate synapses in culture. It has been shown that microglial cells and nitric oxide play key roles in leech CNS regeneration. In the opossum, the neonatal brain and spinal cord are so tiny that they survive well in culture. Fibres grow across spinal cord lesions in neonatal animals and in vitro, but axon regeneration stops abruptly between postnatal days 9 and 12. A comprehensive search has been made in spinal cords that can and cannot regenerate to identify genes and establish their locations. At 9 days, growth-promoting genes, their receptors and key transcription molecules are up-regulated. By contrast at 12 days, growth-inhibitory molecules associated with myelin are prominent. The complete sequence of the opossum genome and new methods for transfecting genes offer ways to determine which molecules promote and which inhibit spinal cord regeneration. These results lead to questions about how basic research on mechanisms of regeneration could be 'translated' into effective therapies for patients with spinal cord injuries.

CNS Regeneration: Only on One Condition

The mammalian CNS is usually not capable of regeneration. However, conditioning dorsal root ganglion neurons by first lesioning their peripheral axons allows for regeneration of their central axons later on within the spinal cord. New work shows that, even if the sequence of lesioning is reversed, regeneration through the CNS lesion can rapidly occur under certain conditions.

Blockade of Nogo Receptor Ligands Promotes Functional Regeneration of Sensory Axons after Dorsal Root Crush

A major impediment for regeneration of axons within the CNS is the presence of multiple inhibitory factors associated with myelin. Three of these factors bind to the Nogo receptor, NgR, which is expressed on axons. Administration of exogenous blockers of NgR or NgR ligands promotes the regeneration of descending axonal projections after spinal cord hemisection. A more detailed analysis of CNS regeneration can be made by examining the growth of specific classes of sensory axons into the spinal cord after dorsal root crush injury. In this study, we assessed whether administration of a soluble peptide fragment of the NgR (sNgR) that binds to and blocks all three NgR ligands can promote regeneration after brachial dorsal root crush in adult rats. Intraventricular infusion of sNgR for 1 month results in extensive regrowth of myelinated sensory axons into the white and gray matter of the dorsal spinal cord, but unmyelinated sensory afferents do not regenerate. In concert with the anatomical growth of sensory axons into the cord, there is a gradual restoration of synaptic function in the denervated region, as revealed by extracellular microelectrode recordings from the spinal gray matter in response to stimulation of peripheral nerves. These positive synaptic responses are correlated with substantial improvements in use of the forelimb, as assessed by paw preference, paw withdrawal to tactile stimuli and the ability to grasp. These results suggest that sNgR may be a potential therapy for restoring sensory function after injuries to sensory roots.

TACE‐induced cleavage of Neogenin disinhibits CNS axon/neurite growth by RGM in rat primary cortical neuron

Neogenin is multifunctional receptor implicated in axon navigation, neuronal differentiation, morphogenesis, and cell death. Recently, neogenin appears to be a high‐affinity receptor for the repulsive guidance molecule (RGM), and RGMa‐neogenin interaction inhibits axon regeneration. While performing a two‐hybrid screen using the intracellular domain of neogenin, we observed that neogenin bound to ADAM17, also called TACE (TNF‐alpha converting enzyme). TACE is a 70‐kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases, and plays a role in proteolytic processing of some kinds of receptors, ligands, and enzymes. This process is of physiological importance. We show here that the cleavage of neogenin by TACE attenuates RGMa‐dependent neurite outgrowth inhibition in rat primary cortical neuron. Thus, TACE can be used as a molecular target for clinical conditions characterized by a failure of CNS regeneration

Focal laser-lesions activate an endogenous population of neural stem/progenitor cells in the adult visual cortex

CNS lesions stimulate adult neurogenic niches. Endogenous neural stem/progenitor cells represent a potential resource for CNS regeneration. Here, we investigate the response to unilateral focal laser-lesions applied to the visual cortex of juvenile rats. Within 3 days post-lesion, an ipsilateral increase of actively cycling cells was observed in cortical layer one and in the callosal white matter within the lesion penumbra. The cells expressed the neural stem/progenitor cell marker Nestin and the 473HD-epitope. Tissue prepared from the lesion area by micro-dissection generated self-renewing, multipotent neurospheres, while cells from the contralateral visual cortex did not. The newly formed neural stem/progenitor cells in the lesion zone might support neurogenesis, as suggested by the expression of Pax6 and Doublecortin, a marker of newborn neurons. We propose that focal laser-lesions may induce the emergence of stem/progenitor cells with neurogenic potential. This could underlie the beneficial effects of laser application in neurosurgery.