Signaling the pathway to regeneration

Robust axon regeneration occurs after peripheral nerve injury through coordinated activation of a genetic program and local intracellular signaling cascades. Although regeneration-associated genes are being identified with increasing frequency, most aspects of regeneration-associated intracellular signaling remain poorly understood. Two independent studies now report that upregulation of cAMP is a component of the PNS regeneration program that can be exploited to enhance axon regeneration through the normally inhibitory CNS environment.     

Signaling mechanisms of the myelin inhibitors of axon regeneration

One of the major obstacles to successful axon regeneration in the adult CNS is the presence of inhibitory molecules that are associated with myelin. Recent studies have identified several major myelin-associated inhibitors along with the relevant signaling molecules. Such advances have not only enhanced our understanding of the signaling mechanisms that are involved in the inhibition of axon regeneration in the adult CNS but also allowed us to assess the therapeutic potential of blocking these inhibitory influences to promote axon regeneration.     

Myelin suppresses axon regeneration by PIR-B/SHP-mediated inhibition of Trk activity

Paired immunoglobulin-like receptor B (PIR-B) partially mediates the regeneration-inhibiting effects of the myelin-derived protein Nogo, myelin-associated glycoprotein (MAG), and oligodendrocyte-myelin glycoprotein (OMgp). In this study, we report that inhibition of the PIR-B signaling cascades in neurons enhances axon regeneration in the central nervous system (CNS). Binding of MAG to PIR-B led to the association of PIR-B with tropomyosin receptor kinase (Trk) neurotrophin receptors. Src homology 2-containing protein tyrosine phosphatase (SHP)-1 and SHP-2, which were recruited to PIR-B upon MAG binding, functioned as Trk tyrosine phosphatases. Further, SHP-1 and SHP-2 inhibition reduced MAG-induced dephosphorylation of Trk receptors and abolished the inhibitory effect of MAG on neurite growth. Thus, PIR-B associated with Trk to downregulate basal and neurotrophin-regulated Trk activity through SHP-1/2 in neurons. Moreover, in vivo transfection of small interfering RNA (siRNA) for SHP-1 or SHP-2 induced axonal regeneration after optic nerve injury in mice. Our results thus identify a new molecular target to enhance regeneration of the injured CNS.     

Counteraction of axonal growth inhibitory properties of Semaphorin 3A and myelin-associated proteins by a synthetic neurotrophic compound

One of the reasons for the lack of nerve regeneration in the CNS is the formation of a glial scar over-expressing multiple inhibitory factors including myelin-associated proteins and members of the Semaphorin family. Innovative therapeutic strategies must stimulate axon extension across the lesion site despite this inhibitory molecular barrier. We recently developed a synthetic neurotrophic compound combining an omega-alkanol with a retinol-like cycle (3-(15-hydroxy-pentadecyl)-2,4,4,-trimethyl-cyclohexen-2-one (tCFA15)). Here, we demonstrate that tCFA15 is able to promote cortical axon outgrowth in vitro even in the presence of the inhibitory Semaphorin 3A and myelin extracts. This growth-promoting effect is selectively observed in axons and requires multiple growth-associated intracellular pathways. Our results illustrate the potential use of synthetic neurotrophic compounds to promote nerve regeneration by counteracting the axonal growth inhibition triggered by glial scar-associated inhibitory factors.     

中枢神经再生抑制因子及免疫治疗研究

成体哺乳动物中枢神经损伤后早期轴突再生失败的一个主要原因是由于髓磷脂抑制分子的存在。Nogo、髓磷脂相关糖蛋白以及少突胶质细胞髓磷脂糖蛋白等神经再生抑制因子的发现,大大促进了中枢神经再生分子机制的研究。它们均能独立通过Nogo-66受体产生对轴突再生的抑制效应,髓磷脂抑制分子及其信号转导机制的研究日益成为中枢神经再生的研究热点,髓磷脂及其信号转导分子特别是Nogo-66受体、p75神经营养素受体成为损伤后促进轴突再生、抑制生长锥塌陷的主要治疗靶点。抑制上述抑制因子及相关受体NgR或p75NTR可能有助于中枢神经损伤的修复,围绕这些抑制因子及其相关受体介导的信号转导途径,人们提出了多种治疗中枢神经损伤的新思路,其中免疫学方法尤其受到关注。     

New roles for old proteins in adult CNS axonal regeneration

The past year has yielded many insights and a few surprises in the field of axonal regeneration. The identification of oligodendrocyte-myelin glycoprotein as an inhibitor of axonal growth, and the discovery that the three major myelin-associated inhibitors of CNS regeneration share the same functional receptor, has launched a new wave of studies that aim to identify the signaling components of these inhibitory pathways. These findings also offer new avenues of research directed toward blocking possible therapeutic targets that inhibit regeneration and toward encouraging axonal regeneration in the CNS after injury.     

Recombinant DNA vaccine encoding multiple domains related to inhibition of neurite outgrowth: a potential strategy for axonal regeneration

Myelin-derived proteins, such as tenascin-R (TN-R), myelin associate glycoprotein (MAG), and Nogo-A, inhibit the CNS regeneration. By targeting specifically the inhibitory epitopes, we have investigated whether vaccination with a recombinant DNA molecule encoding multiple domains of myelin inhibitors may be useful in CNS repair. We show here that the recombinant DNA vaccine is able to activate the immune system but does not induce experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Importantly, it promotes axonal regeneration in a spinal cord injury model. Thus, the application of DNA vaccine, encoding multiple specific domains of major inhibitory proteins and/or their receptors, provides another promising approach to overcome the inhibitory barriers during CNS regeneration.     

Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

Myelin associated glycoprotein cross-linking triggers its partitioning into lipid rafts, specific signaling events and cytoskeletal rearrangements in oligodendrocytes

Myelin-associated glycoprotein (MAG) has been implicated in inhibition of nerve regeneration in the CNS. This results from interactions between MAG and the Nogo receptor and gangliosides on the apposing axon, which generates intracellular inhibitory signals in the neuron. However, because myelin-axon signaling is bidirectional, we undertook an analysis of potential MAG-activated signaling in oligodendrocytes (OLs). In this study, we show that antibody cross-linking of MAG on the surface of OLs (to mimic axonal binding) leads to the redistribution of MAG into detergent (TX-100)-insoluble complexes, hyperphosphorylation of Fyn, dephosphorylation of serine and threonine residues in specific proteins, including lactate dehydrogenase and the beta subunit of the trimeric G-protein-complex, and cleavage of alpha-fodrin followed by a transient depolymerization of actin. We propose that these changes are part of a signaling cascade in OLs associated with MAG function as a mediator of axon-glial communication which might have implications for the mutual regulation of the formation and stability of axons and myelin.     

Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration

Gaining a basic understanding of the inhibitory molecules and the intracellular signaling involved in axon development and repulsion after neural lesions is of clear biomedical interest. In recent years, numerous studies have described new molecules and intracellular mechanisms that impair axonal outgrowth after injury. In this scenario, the role of glycogen synthase kinase 3 beta (GSK3β) in the axonal responses that occur after central nervous system (CNS) lesions began to be elucidated. GSK3β function in the nervous tissue is associated with neural development, neuron polarization, and, more recently, neurodegeneration. In fact, GSK3β has been considered as a putative therapeutic target for promoting functional recovery in injured or degenerative CNS. In this review, we summarize current understanding of the role of GSK3β during neuronal development and regeneration. In particular, we discuss GSK3β activity levels and their possible impact on cytoskeleton dynamics during both processes.     

Signal transduction by the chemokine receptor CXCR5: structural requirements for G protein activation analyzed by chimeric CXCR1/CXCR5 molecules.

The human chemokine receptors CXCR5 and CXCR1 activate signaling pathways via pertussis toxin-sensitive as well as insensitive G proteins. CXCR5 induces Ca2+ signaling and chemotaxis independently of inhibitory G proteins, whereas the same signaling pathways are entirely dependent on inhibitory G proteins for CXCR1. In contrast, activation of the MAP kinase cascade via ERK1/2 is a pertussis toxin-sensitive signaling event for both receptors. Using chimeric CXCR1/CXCR5 receptors we investigated structural requirements for the activation of signal transduction pathways by CXCR5. Individual or multiple intracellular domains of CXCR1 were exchanged for the corresponding sequences of CXCR5, leading to receptors resembling CXCR5 at the cytoplasmic surface to a varying extent. Replacing the second intracellular domain of CXCR1 had a major influence on signaling mediated by inhibitory G proteins, whereas the exchange of the third or carboxy-terminal intracellular domain had only minor effects on signal transduction. Activation of the MAP kinase cascade via ERK1/2 and chemotaxis are largely reduced in chimeras comprising the second intracellular domain of CXCR5, although coupling to inhibitory G proteins is retained in all chimeric receptors. In summary, these data characterize the contribution of the intracellular domains of CXCR5 to receptor signaling, thereby disclosing unique structural requirements that modulate G protein coupling by the receptor.     

Inhibitors of neuronal regeneration: mediators and signaling mechanisms

Neuritogenesis and its inhibition are opposite and balancing processes during development as well as pathological states of adult neuron. In particular, the inability of adult central nervous system (CNS) neurons to regenerate upon injury has been attributed to both a lack of neuritogenic ability and the presence of neuronal growth inhibitors in the CNS environment. I review here recent progress in our understanding of neuritogenic inhibitors, with particular emphasis on those with a role in the inhibition of neuronal regeneration in the CNS, their signaling cascades and signal mediators. Neurotrophines acting through the tropomyosin-related kinase (Trk) family and p75 receptors promote neuritogenesis, which appears to require sustained activation of the mitogen activated protein (MAP) kinase pathway, and/or the activation of phosphotidylinositol 3-kinase (PI3 kinase). During development, a plethora of guidance factors and their receptors navigate the growing axon. However, much remained to be learned about the signaling receptors and pathways that mediate the activity of inhibitors of CNS regeneration. There is growing evidence that neuronal guidance molecules, particularly semaphorins, may also have a role as inhibitors of CNS regeneration. Although direct links have not yet been established in many cases, signals from these agents may ultimately converge upon the modulators and effectors of the Rho-family GTPases. Rho-family GTPases and their effectors modulate the activities of actin modifying molecules such as cofilin and profilin, resulting in cytoskeletal changes associated with growth cone extension or retraction.     

Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain

Chondroitin sulphate proteoglycans (CSPGs) are axon growth inhibitory molecules present in the glial scar that play a part in regeneration failure after damage to the CNS and which restrict CNS plasticity. Removal of chondroitin sulphate glycosaminoglycan (GAG) chains with chondroitinase-ABC (chABC) in models of CNS injury promotes both axon regeneration and plasticity. We have analysed the immediate and long-term effects of a single injection of chABC on CSPGs, GAGs and axon regeneration. We made unilateral nigrostriatal lesions in adult rats accompanied by an adjacent infusion of either chABC or a bacterial-derived control enzyme (penicillinase). Within 24 h of chABC treatment there was digestion of GAGs, including hyaluronan, and a reduction in neurocan in an area extending 1.5 mm around the injection site. Around 50% of GAG is inaccessible to chABC digestion, even in tissue digested in vitro, which probably represents intracellular stores. In control penicillinase treated animals, total GAGs recovered from the lesioned brains were up-regulated by 4-fold 7 days after injury and gradually decreased to normal at 28 days post-lesion. In chondroitinase-treated animals, the total GAG remained at low level throughout the 28-day experimental period. This suggests the persistence of active chABC for at least 10 days after injection which is able to digest CSPGs released from cells during this time. This was confirmed by immunological detection of enzyme for 10 days and by retrieval of active enzyme from the brain at 10 days after injection. Our results suggest that a single injection of chABC can produce an environment conducive to CNS repair for over 10 days.     

Role of taurine in the central nervous system

Taurine demonstrates multiple cellular functions including a central role as a neurotransmitter, as a trophic factor in CNS development, in maintaining the structural integrity of the membrane, in regulating calcium transport and homeostasis, as an osmolyte, as a neuromodulator and as a neuroprotectant. The neurotransmitter properties of taurine are illustrated by its ability to elicit neuronal hyperpolarization, the presence of specific taurine synthesizing enzyme and receptors in the CNS and the presence of a taurine transporter system. Taurine exerts its neuroprotective functions against the glutamate induced excitotoxicity by reducing the glutamate-induced increase of intracellular calcium level, by shifting the ratio of Bcl-2 and Bad ratio in favor of cell survival and by reducing the ER stress. The presence of metabotropic taurine receptors which are negatively coupled to phospholipase C (PLC) signaling pathway through inhibitory G proteins is proposed, and the evidence supporting this notion is also presented.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.     

Bcl-2 enhances Ca(2+) signaling to support the intrinsic regenerative capacity of CNS axons

At a certain point in development, axons in the mammalian CNS undergo a profound loss of intrinsic growth capacity, which leads to poor regeneration after injury. Overexpression of Bcl-2 prevents this loss, but the molecular basis of this effect remains unclear. Here, we report that Bcl-2 supports axonal growth by enhancing intracellular Ca(2+) signaling and activating cAMP response element binding protein (CREB) and extracellular-regulated kinase (Erk), which stimulate the regenerative response and neuritogenesis. Expression of Bcl-2 decreases endoplasmic reticulum (ER) Ca(2+) uptake and storage, and thereby leads to a larger intracellular Ca(2+) response induced by Ca(2+) influx or axotomy in Bcl-2-expressing neurons than in control neurons. Bcl-x(L), an antiapoptotic member of the Bcl-2 family that does not affect ER Ca(2+) uptake, supports neuronal survival but cannot activate CREB and Erk or promote axon regeneration. These results suggest a novel role for ER Ca(2+) in the regulation of neuronal response to injury and define a dedicated signaling event through which Bcl-2 supports CNS regeneration.     

Multiple signals regulate axon regeneration through the nogo receptor complex

Several myelin-derived proteins have been identified as components of central nervous system (CNS) myelin, which prevents axonal regeneration in the adult vertebrate CNS. The discovery of the receptor for these proteins was a major step toward understanding the failure of axon regeneration. The receptor complex consists of at least three elements: the p75 receptor (p75^NTR), the Nogo receptor and LINGO-1. Downstream from the receptor complex, RhoA activation has been shown to be a key element of the signaling mechanism of these proteins. Rho activation arrests axon growth, and blocking Rho activation promotes axon regeneration in vivo. Recent studies have identified conventional protein kinase C as an additional necessary component for axon growth inhibition. Possible crosstalk downstream of these signals should be explored to clarify all the inhibitory signals and may provide an efficient molecular target against injuries to the CNS.     

Nogo-A, a potent inhibitor of neurite outgrowth and regeneration

The lack of regrowth of injured neurons in the adult central nervous system (CNS) of higher vertebrates was accepted as a fact for many decades. In the last few years a very different view emerged; regeneration of lesioned fibre tracts in vivo could be induced experimentally, and molecules that are responsible for inhibition and repulsion of growing neurites have been defined. Mechanisms that link cellular phenomena like growth cone turning or growth cone collapse to intracellular changes in second messenger systems and cytoskeletal dynamics became unveiled. This article reviews recent developments in this field, focusing especially on one of the best characterised neurite out-growth inhibitory molecules found in CNS myelin that was recently cloned: Nogo-A. Nogo-A is a high molecular weight transmembrane protein and an antigen of the monoclonal antibody mAb IN-1 that was shown to promote long-distance regeneration and functional recovery in vivo when applied to spinal cord-injured adult rats. Nogo-A is expressed by oligodendrocytes in white matter of the CNS. With the molecular characterisation of this factor new possibilities open up to achieve structural and functional repair of the injured CNS.     

Neurotrophin signaling among neurons and glia during formation of tripartite synapses

Synapse formation in the CNS is a complex process that involves the dynamic interplay of numerous signals exchanged between pre- and postsynaptic neurons as well as perisynaptic glia. Members of the neurotrophin family, which are widely expressed in the developing and mature CNS and are well-known for their roles in promoting neuronal survival and differentiation, have emerged as key synaptic modulators. However, the mechanisms by which neurotrophins modulate synapse formation and function are poorly understood. Here, we summarize our work on the role of neurotrophins in synaptogenesis in the CNS, in particular the role of these signaling molecules and their receptors, the Trks, in the development of excitatory and inhibitory hippocampal synapses. We discuss our results that demonstrate that postsynaptic TrkB signaling plays an important role in modulating the formation and maintenance of NMDA and GABAA receptor clusters at central synapses, and suggest that neurotrophin signaling coordinately modulates these receptors as part of mechanism that promotes the balance between excitation and inhibition in developing circuits. We also discuss our results that demonstrate that astrocytes promote the formation of GABAergic synapses in vitro by differentially regulating the development of inhibitory presynaptic terminals and postsynaptic GABAA receptor clusters, and suggest that glial modulation of inhibitory synaptogenesis is mediated by neurotrophin-dependent and -independent signaling. Together, these findings extend our understanding of how neuron-glia communication modulates synapse formation, maintenance and function, and set the stage for defining the cellular and molecular mechanisms by which neurotrophins and other cell-cell signals direct synaptogenesis in the developing brain.