Takuro Tojima

Second messengers and membrane trafficking direct and organize growth cone steering

Graded distributions of extracellular cues guide developing axons toward their targets. A network of second messengers — Ca2+ and cyclic nucleotides — shapes cue-derived information into either attractive or repulsive signals that steer growth cones bidirectionally. Emerging evidence suggests that such guidance signals create a localized imbalance between exocytosis and endocytosis, which in turn redirects membrane, adhesion and cytoskeletal components asymmetrically across the growth cone to bias the direction of axon extension. These recent advances allow us to propose a unifying model of how the growth cone translates shallow gradients of environmental information into polarized activity of the steering machinery for axon guidance.

Attractive axon guidance involves asymmetric membrane transport and exocytosis in the growth cone

Asymmetric elevation of the Ca2+ concentration in the growth cone can mediate both attractive and repulsive axon guidance. Ca2+ signals that are accompanied by Ca2+-induced Ca2+ release (CICR) trigger attraction, whereas Ca2+ signals that are not accompanied by CICR trigger repulsion. The molecular machinery downstream of Ca2+ signals, however, remains largely unknown. Here we report that asymmetric membrane trafficking mediates growth cone attraction. Local photolysis of caged Ca2+, together with CICR, on one side of the growth cone of a chick dorsal root ganglion neuron facilitated the microtubule-dependent centrifugal transport of vesicles towards the leading edge and their subsequent vesicle-associated membrane-protein 2 (VAMP2)–mediated exocytosis on the side with an elevated Ca2+ concentration. In contrast, Ca2+ signals without CICR had no effect on the vesicle transport. Furthermore, pharmacological inhibition of VAMP2-mediated exocytosis prevented growth cone attraction, but not repulsion. These results strongly suggest that growth cone attraction and repulsion are driven by distinct mechanisms, rather than using the same molecular machinery with opposing polarities.

L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow

The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin–actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD–ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

Asymmetric Clathrin-Mediated Endocytosis Drives Repulsive Growth Cone Guidance

Asymmetric Ca(2+) elevations across the axonal growth cone mediate its turning responses to attractive and repulsive guidance cues. Here we show that clathrin-mediated endocytosis acts downstream of Ca(2+) signals as driving machinery for growth cone turning. In dorsal root ganglion neurons, the formation of clathrin-coated pits is facilitated asymmetrically across the growth cone by a directionally applied chemorepellent, semaphorin 3A, or by Ca(2+) signals that mediate repulsive guidance. In contrast, coated pit formation remains symmetric in the presence of attractive Ca(2+) signals. Inhibition of clathrin-mediated endocytosis abolishes growth cone repulsion, but not attraction, induced by Ca(2+) or extracellular physiological cues. Furthermore, asymmetric perturbation of the balance of endocytosis and exocytosis in the growth cone is sufficient to initiate its turning toward the side with less endocytosis or more exocytosis. With our previous finding that growth cone attraction involves asymmetric exocytosis, we propose that the balance between membrane addition and removal dictates bidirectional axon guidance.

The nitric oxide-cGMP pathway controls the directional polarity of growth cone guidance via modulating cytosolic Ca2+ signals.

Asymmetric Ca2+ signals across the growth cone mediate attractive or repulsive axon guidance depending on the occurrence of Ca2+-induced Ca2+ release (CICR) through ryanodine receptors (RyRs). Although the neuronal isoform of nitric oxide (NO) synthase (nNOS) is highly expressed in developing dorsal root ganglion (DRG) neurons, the role of NO in axon guidance remains essentially unknown. Here we report that the NO–cGMP pathway negatively regulates CICR to control the directional polarity of DRG axon guidance. Intracellular levels of NO and cGMP depend on extracellular substrates: laminin activates the NO–cGMP pathway, whereas the adhesion molecule L1 does not. The activity of NO and cGMP determines the turning direction of growth cones with respect to asymmetric Ca2+ signals that are produced by photolysing caged Ca2+. The Ca2+ signals cause growth cone repulsion on a laminin substrate, which is converted to attraction by pharmacological blockade of the NO–cGMP pathway or genetic deletion of nNOS. Conversely, Ca2+-induced growth cone attraction on an L1 substrate is converted to repulsion by increasing NO levels. Such NO-mediated switching of turning direction involves the regulation of CICR through RyRs. Furthermore, growth cone repulsion induced by an extracellular gradient of a physiological cue, neurotrophin-4, is dependent on Ca2+ signals and converted to attraction by inhibiting the NO–cGMP pathway. These results suggest that, on contact with different adhesive environments, growth cones can change their turning responses to axon guidance cues by modulating CICR via endogenous NO and cGMP.

DSCAM Deficiency Causes Loss of Pre-Inspiratory Neuron Synchroneity and Perinatal Death

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam−/−) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla–spinal cord preparation of Dscam−/− mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam−/− mice. Dscam+/− mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam−/− mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

Steering Neuronal Growth Cones by Shifting the Imbalance between Exocytosis and Endocytosis

Extracellular molecular cues guide migrating growth cones along specific routes during development of axon tracts. Such processes rely on asymmetric elevation of cytosolic Ca2+ concentrations across the growth cone that mediates its attractive or repulsive turning toward or away from the side with Ca2+ elevation, respectively. Downstream of these Ca2+ signals, localized activation of membrane trafficking steers the growth cone bidirectionally, with endocytosis driving repulsion and exocytosis causing attraction. However, it remains unclear how Ca2+ can differentially regulate these opposite membrane-trafficking events. Here, we show that growth cone turning depends on localized imbalance between exocytosis and endocytosis and identify Ca2+-dependent signaling pathways mediating such imbalance. In embryonic chicken dorsal root ganglion neurons, repulsive Ca2+ signals promote clathrin-mediated endocytosis through a 90 kDa splice variant of phosphatidylinositol-4-phosphate 5-kinase type-1γ (PIPKIγ90). In contrast, attractive Ca2+ signals facilitate exocytosis but suppress endocytosis via Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cyclin-dependent kinase 5 (Cdk5) that can inactivate PIPKIγ90. Blocking CaMKII or Cdk5 leads to balanced activation of both exocytosis and endocytosis that causes straight growth cone migration even in the presence of guidance signals, whereas experimentally perturbing the balance restores the growth cones turning response. Remarkably, the direction of this resumed turning depends on relative activities of exocytosis and endocytosis, but not on the type of guidance signals. Our results suggest that navigating growth cones can be redirected by shifting the imbalance between exocytosis and endocytosis, highlighting the importance of membrane-trafficking imbalance for axon guidance and, possibly, for polarized cell migration in general.

Exocytic and endocytic membrane trafficking in axon development.

In the complex neuronal circuits in the nervous systems, billions of neurons are precisely interconnected by long, thin processes called the axons. The growth cone, a highly motile structure at the tip of an extending axon, navigates by responding to a variety of extracellular molecular cues toward their distant target cells and make synaptic connections. Emerging evidence indicates that exocytic and endocytic membrane trafficking systems play multiple important roles in the regulation of such axonal morphogenetic processes. Exocytosis and endocytosis organize the subcellular distribution of membrane‐associated molecules, such as receptors, cell adhesion molecules, and cytoskeletal regulators, to control intracellular signaling and driving machineries. Furthermore, the exocytosis of trophic factors and extracellular proteinases act on surrounding microenvironments to affect growth cone motility. In this Review Article, we summarize our current understanding of the regulation and function of exocytic and endocytic membrane trafficking in axon morphogenesis during development, and discuss potential mechanisms of how the membrane trafficking systems exert such morphological changes.

Intracellular signaling and membrane trafficking control bidirectional growth cone guidance

The formation of precise neuronal networks is critically dependent on the motility of axonal growth cones. Extracellular gradients of guidance cues evoke localized Ca(2+) elevations to attract or repel the growth cone. Recent studies strongly suggest that the polarity of growth cone guidance, with respect to the localization of Ca(2+) signals, is determined by Ca(2+) release from the endoplasmic reticulum (ER) in the following manner: Ca(2+) signals containing ER Ca(2+) release cause growth cone attraction, while Ca(2+) signals without ER Ca(2+) release cause growth cone repulsion. Recent studies have also shown that exocytic and endocytic membrane trafficking can drive growth cone attraction and repulsion, respectively, downstream of Ca(2+) signals. Most likely, these two mechanisms underlie cue-induced axon guidance, in which a localized imbalance between exocytosis and endocytosis dictates bidirectional growth cone steering. In this Update Article, I summarize recent advances in growth cone research and propose that polarized membrane trafficking plays an instructive role to spatially localize steering machineries, such as cytoskeletal components and adhesion molecules.

Paxillin phosphorylation counteracts proteoglycan-mediated inhibition of axon regeneration

In the adult central nervous system, the tips of axons severed by injury are commonly transformed into dystrophic endballs and cease migration upon encountering a rising concentration gradient of inhibitory proteoglycans. However, intracellular signaling networks mediating endball migration failure remain largely unknown. Here we show that manipulation of protein kinase A (PKA) or its downstream adhesion component paxillin can reactivate the locomotive machinery of endballs in vitro and facilitate axon growth after injury in vivo. In dissociated cultures of adult rat dorsal root ganglion neurons, PKA is activated in endballs formed on gradients of the inhibitory proteoglycan aggrecan, and pharmacological inhibition of PKA promotes axon growth on aggrecan gradients most likely through phosphorylation of paxillin at serine 301. Remarkably, pre-formed endballs on aggrecan gradients resume forward migration in response to PKA inhibition. This resumption of endball migration is associated with increased turnover of adhesive point contacts dependent upon paxillin phosphorylation. Furthermore, expression of phosphomimetic paxillin overcomes aggrecan-mediated growth arrest of endballs, and facilitates axon growth after optic nerve crush in vivo. These results point to the importance of adhesion dynamics in restoring endball migration and suggest a potential therapeutic target for axon tract repair.