Guidance Molecules in Axon Regeneration

The regenerative capacity of injured adult mammalian central nervous system (CNS) tissue is very limited. Disease or injury that causes destruction or damage to neuronal networks typically results in permanent neurological deficits. Injury to the spinal cord, for example, interrupts vital ascending and descending fiber tracts of spinally projecting neurons. Because neuronal structures located proximal or distal to the injury site remain largely intact, a major goal of spinal cord injury research is to develop strategies to reestablish innervation lost as a consequence of injury. The growth inhibitory nature of injured adult CNS tissue is a major barrier to regenerative axonal growth and sprouting. An increasing complexity of molecular players is being recognized. CNS inhibitors fall into three general classes: members of canonical axon guidance molecules (e.g., semaphorins, ephrins, netrins), prototypic myelin inhibitors (Nogo, MAG, and OMgp) and chondroitin sulfate proteoglycans (lecticans, NG2). On the other end of the spectrum are molecules that promote neuronal growth and sprouting. These include growth promoting extracellular matrix molecules, cell adhesion molecules, and neurotrophic factors. In addition to environmental (extrinsic) growth regulatory cues, cell intrinsic regulatory mechanisms exist that greatly influence injury-induced neuronal growth. Various degrees of growth and sprouting of injured CNS neurons have been achieved by lowering extrinsic inhibitory cues, increasing extrinsic growth promoting cues, or by activation of cell intrinsic growth programs. More recently, combination therapies that activate growth promoting programs and at the same time attenuate growth inhibitory pathways have met with some success. In experimental animal models of spinal cord injury (SCI), mono and combination therapies have been shown to promote neuronal growth and sprouting. Anatomical growth often correlates with improved behavioral outcomes. Challenges ahead include testing whether some of the most promising treatment strategies in animal models are also beneficial for human patients suffering from SCI.     

Human Genetic Disorders of Axon Guidance

This article reviews symptoms and signs of aberrant axon connectivity in humans, and summarizes major human genetic disorders that result, or have been proposed to result, from defective axon guidance. These include corpus callosum agenesis, L1 syndrome, Joubert syndrome and related disorders, horizontal gaze palsy with progressive scoliosis, Kallmann syndrome, albinism, congenital fibrosis of the extraocular muscles type 1, Duane retraction syndrome, and pontine tegmental cap dysplasia. Genes mutated in these disorders can encode axon growth cone ligands and receptors, downstream signaling molecules, and axon transport motors, as well as proteins without currently recognized roles in axon guidance. Advances in neuroimaging and genetic techniques have the potential to rapidly expand this field, and it is feasible that axon guidance disorders will soon be recognized as a new and significant category of human neurodevelopmental disorders.The human brain is highly organized and contains a myriad of axon tracts that follow precise pathways and make predictable connections. Model organism research has provided tremendous advances in our understanding of the principles and molecules governing axon growth and guidance. Remarkably, however, only a handful of human disorders resulting from primary errors in these processes have been identified.Traditional tools of the physician have limited sensitivity and specificity to detect human disorders of axon guidance. In particular, congenital synkinesis may be the only physical examination finding that has been attributed to such disorders. Synkinesis is the involuntary and pathological contraction of a muscle simultaneously with contraction of the intended muscle, and is typically reported with hand/finger or eye/eyelid movements and confirmed by electrophysiological studies. Mirror movement synkinesis refers to the contraction of homologous hand/finger muscles bilaterally when one attempts to move only one hand (Schott and Wyke 1981). In humans, 75%–90% of corticospinal tract (CST) fibers normally decussate in the lower medulla. Mirror movement synkinesis occurs in several human disorders with pathological, neuroimaging, and/or electrophysiological evidence of reduced CST decussation, including Joubert, Kallmann, and Klippel-Feil syndromes (Vulliemoz et al. 2005; Cincotta and Ziemann 2008). In some individuals with mirror movements, electrophysiological data are also consistent with bilateral engagement of the motor corticies (Leinsinger et al. 1997). Ocular synkinesis refers to aberrant patterns of eye movement and accompanies various congenital cranial dysinnervation disorders (CCDDs) (Gutowski et al. 2003; Engle 2007), including CFEOM, Duane syndrome, and Marcus Gunn jaw-winking phenomenon (Fig. 1). Finger and ocular movements require precise motor control, and errors in innervation of these muscles may be more easily detected than errors in the wiring of larger muscle groups. If true, this suggests that the clinical exam could fail to recognize many guidance errors in both the peripheral and central nervous system.Open in a separate windowFigure 1.Ocular synkinesis. (A) Child with CFEOM1 and Marcus Gunn jaw-winking phenomenon harboring a KIF21A mutation. His superior branch of the oculomotor nerve is hypoplastic/absent, resulting in bilateral ptosis from lack of appropriate innervation of the levator palpebrae superioris (LPS) muscle, and a downward position of each eye from absent innervation of the superior rectus muscle (left). Marcus Gunn phenomenon (right) is seen as the synkinetic elevation of the left eyelid with a subtle change in jaw position associated with a volitional increase in pterygoid muscle tension. This results from aberrant innervation of the LPS by axons from the motor branch of the trigeminal nerve that also innervates the intended ipsilateral pterygoid muscle. (B) Adult with Duane retraction syndrome harboring a CHN1 mutation. Central gaze reveals mild exotropia (middle). On attempted right gaze (left) and left gaze (right), there is limited horizontal excursion with globe retraction and secondary palpebral fissure narrowing of the adducting eye. Globe retraction results from synkinesis of the medial and lateral recti muscles. (A) Modified with permission from Yamada et al. 2005. Copyright © (2005) American Medial Association. All rights reserved. (B) Modified from Demer et al. 2007. Copyright © (2007) Association for Research in Vision and Ophthalmology. All rights reserved.The physician’s ability to detect disorders of axon guidance has been augmented by classical pathological, radiological, and electrophysiological techniques. Diagnostic radiologic and postmortem neuropathological studies detect overall changes in white matter volume and major abnormalities of axon tracts demarcated from the background such as the corpus callosum, anterior and posterior commissures, optic chiasm, and cerebellar peduncles. Neuropathological studies can also detect absence of axons that normally cross the midline at many points in the brain stem and spinal cord, which are more difficult to visualize by standard magnetic resonance imaging (MRI). Electrophysiological studies such as evoked potentials can reveal aberrant central connections of peripheral sensory or motor nerves.The genetic disorders with aberrant axon connectivity presented in this article have been defined primarily using traditional approaches described above. Exciting advances in neuroimaging and genetics, however, are revolutionizing the ability to define axon guidance disorders, and it is likely that these syndromes are only the first of an important new category of such human neurodevelopmental disorders. Detailed fiber tract anatomy can now be visualized using noninvasive tractography such as diffusion tensor imaging (DTI) and diffusion spectrum imaging (DSI). These techniques provide tract orientation by determining the anisotropic properties of water diffusion, and can be used to reconstruct the trajectories of fiber systems in three-dimensional space (Tovar-Moll et al. 2007; Wahl et al. 2009). Tractography has successfully confirmed aberrant projections in several of the disorders discussed below (Fig. 2). At the same time, human genetics now provides an unbiased approach to identify the etiologies of disorders with aberrant axon tracts. For some syndromes, animal and in vitro studies have confirmed that the encoded protein has a primary role in axon guidance. For others, such studies reveal a primary role in neuronal specification and/or migration rather than, or in addition to, a role in axon guidance. Finally, some neurodevelopmental disorders without clinical, pathologic, or radiologic evidence of aberrant axon tracts have been found to result from mutations in genes that contribute to axon guidance in animal models.Open in a separate windowFigure 2.Tractography studies in patients with partial agenesis of the corpus callosum (pACC). T1-weighted anatomic images and DTI tractography of six subjects with pACC (top panels) and two representative controls (bottom panel). Axial (left) and midline sagittal (middle) T1 sections are shown for each subject. Callosal fragments are identified with yellow arrows, and heterotopic fibers visible on T1-weighted images are denoted by red arrows. Midline sagittal DTI color maps are shown with segmented callosal fibers (right). For subjects with pACC, connectivity ranged from anterior frontal connections (subject 3) to only posterior frontal and occipitotemporal connections (subject 4). One individual (subject 5) displayed a discontinuous set of homotopic callosal connections, with anterior frontal and occipitotemporal connectivity without any posterior frontal or parietal connections. Control subjects (bottom panel) display normal callosal morphology and tractography results. Tracts are segmented and colored according to their cortical projections: homotopic anterior frontal, blue; homotopic posterior frontal, orange; homotopic parietal, pink; homotopic occipitotemporal, green; heterotopic left anterior-right posterior, yellow; heterotopic right anterior-left posterior, red. (Reprinted, with permission, from Wahl et al. 2009 [© AJNR].)The major human genetic disorders that result, or are proposed to result, from defective axon guidance are ordered below from rostral to caudal based on the location of the aberrant axons tracts. These include genetic mutations that alter axon growth cone ligands and receptors, downstream signaling molecules, and axon transport, as well as proteins without currently recognized roles in axon guidance (Fig. 3) (Open in a separate windowFigure 3.Schematic representation of gene products implicated in human disorders of axon guidance. KAL1 (anosmin) and PROK2 are shown as secreted ligands. ROBO3, L1, and PROKR2 are shown as transmembrane receptors on the growth cone. CHN1 is depicted with 3 green domains (SH2, C1, RacGAP), responding to an unknown activated receptor and altering a microtubule, which is depicted as a brown line. KIF21A dimers are depicted walking down MTs. The OCA/OA and JSRD gene products are not depicted. Note: these gene products are not necessarily expressed in the same neurons or function in the same pathways.

Table 1

Summary of major human genetic disorders resulting, or hypothesized to result, from errors in axon growth and guidance

Mesodermal Guidance of Pioneer Axon Growth

Pioneer axons in insect legs are experimentally accessible model systems for the molecular identification and cellular localization of guidance cues regulating the path of axon growth. A detailed study of the Fe2 pioneer axons in the legs of the cockroach was performed to examine the diversity of guidance mechanisms. A detailed microscopic analysis of the axons at various points in their trajectory indicates that the Fe2 axons grow on a mesodermal substratum which contains the cues guiding their growth along a stereotyped path. An identified pair of muscle pioneer cells (MPC) are likely to play an important role in enabling the Fe2 growth cones to respond to mesodermal guidance cues. The addition of heparan sulfate, heparitinase, and phosphatidylinositol-specific phospholipase C to the medium perturbs thein situpath of growth of the Fe2 axons and the location of the MPC in cultured embryos. This indicates a role for heparan sulfate proteoglycans and glycosylphosphatidylinositol-anchored proteins in axon guidance. When these results are compared to those of similar experiments performed on the well-characterized Ti1 axons, they indicate significant differences in the mechanisms that are used for axon guidance. The Fe2 neurons are a good model for elucidating the mechanisms used to guide axon growth on nonmuscle mesodermal substrates often encountered in the periphery of vertebrate embryos.     

轴突导向分子及受体结构与功能进展

在神经发育过程中,轴突导向分子引导轴突选择正确途径,从而成功到达靶区.近年有关轴突导向分子及其受体的研究进展迅速,主要介绍了Netrin-DCC和UNC5, semaphorin-neuropilins和plexins, Slit-robo, Eph受体酪氨酸激酶及其配体的结构和功能.     

Cannabinoid Receptor CB2 Modulates Axon Guidance

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB₂R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB₂R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 ^−/−). The CB₂R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB₂R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 ^−/− mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB₂R’s implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB₁R, but also involves CB₂R.     

A Mathematical Framework for Modeling Axon Guidance

In this paper, a simulation tool for modeling axon guidance is presented. A mathematical framework in which a wide range of models can been implemented has been developed together with efficient numerical algorithms. In our framework, models can be defined that consist of concentration fields of guidance molecules in combination with finite-dimensional state vectors. These vectors can characterize migrating growth cones, target neurons that release guidance molecules, or other cells that act as sources of membrane-bound or diffusible guidance molecules. The underlying mathematical framework is presented as well as the numerical methods to solve them. The potential applications of our simulation tool are illustrated with a number of examples, including a model of topographic mapping.     

Axon Guidance at the Midline: From Mutants to Mechanisms

How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Understanding the mechanisms that mediate axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for therapeutic approaches to developmental brain disorders and nerve regeneration. Achieving this understanding will require unraveling the molecular logic that ensures the proper expression and localization of axon guidance cues and receptors, and elucidating the signaling events that regulate the growth cone cytoskeleton in response to guidance receptor activation. Studies of axon guidance at the midline of many experimental systems, from the ventral midline of Drosophila to the vertebrate spinal cord, have led to important mechanistic insights into the complex problem of wiring the nervous system. Here we review recent advances in understanding the regulation of midline axon guidance, with a particular emphasis on the contributions made from molecular genetic studies of invertebrate model systems.     

Repulsion by Slit and Roundabout prevents Shotgun/E-cadherin-mediated cell adhesion during Drosophila heart tube lumen formation

During Drosophila melanogaster heart development, a lumen forms between apical surfaces of contralateral cardioblasts (CBs). We show that Slit and its receptor Roundabout (Robo) are required at CB apical domains for lumen formation. Mislocalization of Slit outside the apical domain causes ectopic lumen formation and the mislocalization of cell junction proteins, E-cadherin (E-Cad) and Enabled, without disrupting overall CB cell polarity. Ectopic lumen formation is suppressed in robo mutants, which indicates robo's requirement for this process. Genetic evidence suggests that Robo and Shotgun (Shg)/E-Cad function together in modulating CB adhesion. robo and shg/E-Cad transheterozygotes have lumen defects. In robo loss-of-function or shg/E-Cad gain-of-function embryos, lumen formation is blocked because of inappropriate CB adhesion and an accumulation of E-Cad at the apical membrane. In contrast, shg/E-Cad loss-of-function or robo gain-of-function blocks lumen formation due to a loss of CB adhesion. Our data show that Slit and Robo pathways function in lumen formation as a repulsive signal to antagonize E-Cad-mediated cell adhesion.     

Blocking Apoptotic Signaling Rescues Axon Guidance in Netrin Mutants

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Netrin-Integrin Signaling in Epithelial Morphogenesis,Axon Guidance and Vascular Patterning

Netrins and their classical receptors - DCC and neogenin - play key roles in neuronal guidance. Recent developments identify new roles for netrins in epithelial and vascular morphogenesis. Netrins accomplish these effects, at least in part, through binding to integrins and/or activating integrin-associated kinases such as FAK and Fyn. Here we discuss these recent findings and propose that integrins and classical netrin receptors cooperate to regulate multiple aspects of development.     

Glypican Is a Modulator of Netrin-Mediated Axon Guidance

Netrin is a key axon guidance cue that orients axon growth during neural circuit formation. However, the mechanisms regulating netrin and its receptors in the extracellular milieu are largely unknown. Here we demonstrate that in Caenorhabditis elegans, LON-2/glypican, a heparan sulfate proteoglycan, modulates UNC-6/netrin signaling and may do this through interactions with the UNC-40/DCC receptor. We show that developing axons misorient in the absence of LON-2/glypican when the SLT-1/slit guidance pathway is compromised and that LON-2/glypican functions in both the attractive and repulsive UNC-6/netrin pathways. We find that the core LON-2/glypican protein, lacking its heparan sulfate chains, and secreted forms of LON-2/glypican are functional in axon guidance. We also find that LON-2/glypican functions from the epidermal substrate cells to guide axons, and we provide evidence that LON-2/glypican associates with UNC-40/DCC receptor–expressing cells. We propose that LON-2/glypican acts as a modulator of UNC-40/DCC-mediated guidance to fine-tune axonal responses to UNC-6/netrin signals during migration.