Mechanisms of Axonal Pathfinding

Ryan S. Instrum as part of the Neurodevelopment group Neurowiki

Growth_Cone.jpg
Credit: Yimin Zou, University of California San Diego

Contents [hide]
1 Overview
2 Growth cone
2.1 Structure and dynamics
3 Chemotropic cues
3.1 Secreted factors
3.2 Contact-mediated signaling
4 Ligand-receptor complex-mediated responses
4.1 Cytoskeletal dynamics
5 Role of morphogens in axon guidance
6 Pathophysiological relevance
6.1 Disorders and disease states
6.2 Traumatic CNS injury
6.3 Future treatment
7 References


Overview


Following neuronal differentiation and migration, neurons in the developing central nervous system must extend projections to form proper synaptic connections with target regions throughout the body. Interactions between the growth cone on the leading edge of growing axons and its extracellular environment serve to guide these projections to their appropriate destinations. Specifically, this process is facilitated through long and short-range chemotropic signaling by way of extracellular secreted factors, as well as contact-mediated communication at the level of the growth cone[1] .

The response elicited by a given chemotropic factor (i.e. acting as a chemoattractant or chemorepellent) is not dictated by the molecule itself, but rather by the receptors expressed on the growth cone with which the ligand binds. Many of the responses mediated by specific ligand-receptor interactions have been elucidated by recent research, and this has aided in our understanding of neural development. Furthermore, this knowledge has allowed for greater comprehension of the mechanisms underlying a host of neurodevelopmental and trauma-related pathologies, as well as assisting in our efforts to treat them clinically [2] .

Growth cone


Located at the leading edge of developing axonal projections is a highly specialized structure known as the growth cone. This region is responsible for surveying its external environment and responding to extracellular cues which serve to guide neurite growth toward its appropriate target [3] . In order to understand the mechanisms by which accurate synaptic connectivity is achieved in the nervous system, it is essential to first consider the morphology of the growth cone as well as the dynamic processes that allow it to dictate directionality on a moment-to-moment basis.

Structure and dynamics

GrowthCones_structure.jpg
Fluorescence microscopy overlay showing F-actin rich peripheral zone (green) and microtubule-rich central core (red). Credit University of Wisconsin-Madison.

The unique structure of the growth cone is fundamental in its capacity to navigate great distances in search of cellular partners, and is governed by its underlying cytoskeletal configuration. Finger-like protuberances of membrane, known as filopodia (filopodium: singular), radiate out from the body of the growth cone around bundles of filamentous (F) actin. These regions function to probe the surrounding environment in search of growth signals and are separated by broad sheets of membrane known as lamellipodia (lamellipodium: singular) composed of a meshwork of branched actin [4] . The lamellipodia sense the extracellular milieu as well as acting to push the growth cone forward in the direction of growth. Beneath the peripheral (P) zone lies a central (C) domain comprised of a stable bundle microtubules extending into the growth cone from the distal regions of the axon, as well as the necessary organelles and machinery to power directional growth. Myosin II motors link the dense microtubule-rich core to actin in the periphery, and powers protrusive growth based on the intracellular state of the growth cone, substrate adhesion and the cytoskeletal polymerization state [5] (discussed in detail in sections 2 and 3).

Chemotropic cues


As axons traverse the developmental landscape in search of their downstream targets, they are aided by a series of chemotropic growth signals along the way. These cues can take the form of soluble secreted factors, as well as cellular or extracellular adhesion molecules. The response a particular molecule elicits on axonal growth can be either inhibitory or facilitative, with respect to the locus of signaling, and is determined by the receptor to which it binds. Molecular gradients carry both spatial and temporal information which the growth cone must differentiate and transduce in a sea of simultaneous cues [6] . The highly complex process of axon guidance is regulated by a surprisingly limited number of extracellular molecules, but done so with an incredible level of accuracy.

Secreted factors

These molecules are released from targets or intermediate structures and function through long-range chemotactic mechanisms to direct axonal elongation. Growth cones detect signal gradients, rather than recognizing presence or absence of a given cue, which are typically higher in the region from which they were secreted. These molecules generally have an attractive or repulsive effect on growth, and multiple signals are used in conjunction to define the boundaries and proper path for elongation [7] . Netrins, slits and neurotrophins are a few of the diffusible factors in this category, each exhibiting bifunctionality depending on the receptor types with which they interact.

Contact-mediated signaling

Another chemotropic mechanism involved in axonal pathfinding utilizes substrate bound molecules to regulate neurite growth. In contrast to diffusible factors, these insoluble cues dictate directionality through direct cell-cell or cell-substrate interactions over short distances. These adhesive signaling molecules serve as intermediate targets spread along the route to its synaptic partner by marking the extracellular space with attractive and repulsive cues [8] . Chemoattractive signals direct axon extension through the creation of a favorable environment and substrate on which the growth cone can protrude. These signals include extracellular matrix and cellular adhesion molecules (CAMs) such as ephrins and some semaphorins.



Ligand-receptor complex-mediated responses


The growth cone is responsible not only for reception of environmental guidance cues, but also for the transduction of said information into an integrated cellular response. These responses rely on asymmetrical activation of membrane receptors by a given guidance cue, initiating growth toward the region of highest activation if the activated receptor signals attraction, and away if the signal is deemed inhibitory [9] . Receptors bound to their respective ligand initiate second messenger cascades which ultimately drive growth cone orientation and turning. The identity and concentration of a given second messenger (e.g. Ca2+ or cAMP), and the effector proteins optimally activated by a given response profile, determine whether a signal will be attractive or act as a repellant. Regardless of the signal, the subsequent growth cone motility involves large-scale remodeling of the growth cone cytoskeleton.
Netrin_1_turning.png
Netrin-1 induction of growth cone turning via cAMP-dependent guidance mechanism. Ming, G (1997).

Cytoskeletal dynamics

Transduction of guidance information into the modification of growth cone morphology necessary for directional locomotion requires dynamic changes in cytoskeletal configuration, as well as membrane trafficking. Growth cone motility relies on remodeling of F-actin in the filopodia and lamellipodia of the peripheral zone, while axon elongation involves changes in microtubules of the central domain [10] . Protrusive growth mechanisms require asymmetrical alterations of the rates of cytoskeletal polymerization and depolymerization by effector proteins downstream of guidance signals (e.g. Rho GTPase regulation of actin assembly). Recent data indicate that myosin II motor protein linkage of growth cone actin and microtubules is vital for neurite extension and retraction around focal adhesions to its substrate [11] .

Role of morphogens in axon guidance


Morphogens have long been known to play a significant role in embryonic development through concentration-dependent tissue patterning (including cortical patterning) and cell fate determination. Recent evidence suggests they may also function in axonal pathfinding both through direct chemotactic mechanisms, as well as indirectly through alteration of growth cone receptivity for the many simultaneous signals present at a given time [12] . Morphogenic influence appears particularly important in aiding commissural axon midline crosses in the brain and spinal cord. Three particular families of morphogens have been implicated in the literature:

  • Hedgehog family- (specifically Sonic Hedgehog) induce sensitivity of spinal commissural axons to repulsion by Semaphorin following midline cross [13]
  • Wnt family- repulsion of cortical axons and promotion of axon outgrowth via calcium signaling in developing corpus callosum [14]
  • TGFβ- Unc-129 serves as a chemoattractant required for motor axon guidance

Pathophysiological relevance


Numerous developmental and neurological pathologies have been linked to disruption of the processes underlying axonal pathfinding. Furthermore, the inability of the mammalian central nervous system to regenerate following traumatic injury can be in part attributed to guidance cues present in the extracellular environment [15] . An accurate understanding of the guidance mechanisms involved in the generation of normal synaptic connectivity is vital for the comprehension of pathophysiological processes and generation of future treatment protocols.

Disorders and disease states

Appropriate axon guidance is essential for the development of proper neural connectivity and therefore for all higher-order cognitive and motor functioning, as well as characteristic developmental states of the brain (e.g. the so-called 'Teenage Brain'). Disruption of these processes can result in a variety of neurodevelopmental and neurodegenerative disorders depending on the extent of the problem and the modalities that are affected. Genetic mutation and/or transcriptional alterations of chemotropic molecules as well as the receptors they bind have been implicated in a host of developmental and neurological disorders. These include dyslexia (Nogo1 receptor) [16] , autism (ROBO receptor) [17] , Alzheimer’s disease (semaphorin3a) [18] , epilepsy (semaphorin3f) [19] , and Parkinson’s disease [20] , to name just a few.

Traumatic CNS injury

Unlike most invertebrates or the peripheral nervous system, the mammalian central nervous system is incapable of regeneration following injury. It was originally hypothesized that mature neurons in the CNS lacked the programs to regenerate altogether, aspects which were thought to be spared in the PNS. However, further research demonstrated that isolated neurons from the CNS could grow if cultured under appropriate conditions. This eventually led to the conclusion that oligodendrocytes created an hostile environment that was not conducive to neural regeneration, due in part to glial scarring as well as the production of inhibitory guidance molecules. Morphogens such as Wnt and chemotropic molecules including semaphorins, netrins and ephrins have been identified as some of the main inhibitory signals that render the CNS unable to renew itself [21] .

Future treatment

An understanding of the role axonal guidance mechanisms play in neuropathological states has proven valuable in our efforts to treat them clinically. Numerous putative treatment methods have been proposed using these principles with promising results. Recent data suggest that treatments using the guidance molecule neurotrophin-3 have allowed for successful reinnervation of lesioned cervical spinal cord neurons to their respective midbrain targets [22] . This is just one example of the potential therapeutic power chemotropic molecules hold, and for that reason it is presently an area receiving a large amount of attention.


Spinal_lesion.jpg
Representation of adult rat spinal cord with dorsal cervical lesion following traumatic injury. Giger, R.J. (2010).



References

the growth cone
1.1a Structure and dynamics
1.2 Chemotropic cues
1.2a Secreted factors
1.2b Contact-mediated signaling
1.3 Ligand-receptor complex-mediated responses
1.3a Actin dynamics
1.4 Role of morphogens in altering receptivity
1.4a Sonic Hedgehog
2.1 Pathophysiological mechanisms
2.1a Neurodevelopmental
2.1b Traumatic spinal cord injury
2.2 Future treatment
  1. ^
    Murray, A.J., Peace, A.G., Tucker, S.J., & Shewan, D.A. Mammalian growth cone turning assays identify distinct cell signalling mechanisms that underlie axon growth, guidance and regeneration. Methods in Molecular Biology, 846, 167-78 (2012).
  2. ^
    McCormik, A.M., & Leipzig, N.D. Neural regenerative strategies incorporating biomolecular axon guidance signals. Annals of Biomedical Engineering, 40, 578-97 (2012).
  3. ^
    Chilton, J.K. Molecular mechanisms of axon guidance. Developmental Biology, 292, 13-24 (2006).
  4. ^
    Laishram, J. et al. A morphological analysis of growth cones of DRG neurons combining atomic force and confocal microscopy. Journal of Structural Biology, 168, 366-377 (2009).
  5. ^ Betz, T., Koch, D., Stuhrmann, B., Ehrlicher, A., & Kas, J. Statistical analysis of neuronal growth: edge dynamics and the effect of a focused laser on growth cone motility. J. Phys. 9, 426-447 (2007).
  6. ^
    Bonanomi, D. et al. Ret is a multifunctional coreceptor that integrates diffusible- and contact-axon guidance signals. Cell, 148, 568-82 (2012).
  7. ^
    Killeen, M.T., & Sybingco, S.S. Netrin, Slit and Wnt receptors allow axons to choose the axis of migration. Developmental Biology, 323, 143-51 (2008).
  8. ^
    Gibson, N.J. Cell adhesion molecules in context CAM function depends on neighborhood. Cell Adhesion and Migration, 5, 48-51 (2011).
  9. ^
    Tojima, T., Hines, J.H., Henley, J.R., & Kamiguchi, H. Second messengers and membrane trafficking direct and organize growth cone steering. Nature Neuroscience, 12, 191-203 (2011).
  10. ^
    Lee, A.C., & Suter, D.M. Quantitative analysis of microtubule dynamics during adhesion-mediated growth cone guidance. Developmental Neurobiology, 68, 1363-77 (2008).
  11. ^ Ketschek, A.R., Jones, S.L., & Gallo, G. Axon extension in the fast and slow lanes: substratum-dependent engagement of myosin II function. Developmental Neurobiology, 61, 1305-20 (2007).
  12. ^
    Sanchez-Camacho, C., & Bovolenta, P. Emerging mechanisms in morphogen-mediated axon guidance. Bioessays, 31, 1013-25 (2009).
  13. ^ Parra, L.M., & Zou, Y. Sonic hedgehog induces response of commissural axons to Semaphorin repulsion during midline cross. Nature Neuroscience, 13, 29-37 (2010).
  14. ^ Hutchins, B.I., Li, L., & Kalil, K. Wnt-induced calcium signaling mediates axon growth and guidance in the developing corpus callosum. Science Signaling, 5, 1-3 (2012).
  15. ^
    Yaron, A.,& Zheng, B. Emerging roles for axon guidance molecules in neurological disorders and injury. Developmental Neurobiology, 10, 1216-31 (2006).
  16. ^
    Poon, M.W. et al. Dyslexia-associated Kiaa0319-like protein interacts with axon guidance receptor Nogo receptor 1. Cell Mol Neurobiol., 31, 27-35 (2011).
  17. ^ Anitha, A. et al. Genetic analysis of Roundabout (ROBO) axon guidance receptors in autism. American Journal of Medical Genetics, 147, 1019-27 (2008).
  18. ^ Uchida, Y. et al. Semaphorin3a signalling is mediated via sequential cdk5 and gsk3b phosphorylation of crmp2: implication of common phosphorylating mechanism underlying axon guidance and Alzheimer's disease. Genes to Cells, 10, 165-79 (2005).
  19. ^ Barnes, G.N., Li, Y., & Aschner, M. Genetic regulation of murine Semaphorin3f signaling modulates the development of hippocampal GABAergic circuitry. Epilepsia, 49, 325-6 (2008).
  20. ^ Bossers, K. et al. Analysis of gene expression in Parkinson's disease: possible involvement of neurotrophic support and axon guidance in dopaminergic cell death. Brain Pathology, 19, 91-107 (2009).
  21. ^
    Liu, Y. et al. Repulsive Wnt signaling inhibits axon regeneration after CNS injury. Journal of Neuroscience, 28, 8376-82 (2008).
  22. ^
    Alto, L.T. et al. Chemotropic guidance facilitates axonal regeneration and synapse formation after spinal cord injury. Nature Neuroscience, 12, 1106-13 (2009).