Axon guidance: growth cones make an unexpected turn

Axonal growth cones can turn in response to minute concentration differences in extracellular guidance cues. Surprising new work suggests that these cues might steer the growth cone by inducing rapid local changes in protein levels.     

Local protein synthesis in neuronal axons: why and how we study

Adaptive brain function and synaptic plasticity rely on dynamic regulation of local proteome. One way for the neuron to introduce new proteins to the axon terminal is to transport those from the cell body, which had long been thought as the only source of axonal proteins. Another way, which is the topic of this review, is synthesizing proteins on site by local mRNA translation. Recent evidence indicates that the axon stores a reservoir of translationally silent mRNAs and regulates their expression solely by translational control. Different stimuli to axons, such as guidance cues, growth factors, and nerve injury, promote translation of selective mRNAs, a process required for the axon’s ability to respond to these cues. One of the critical questions in the field of axonal protein synthesis is how mRNA-specific local translation is regulated by extracellular cues. Here, we review current experimental techniques that can be used to answer this question. Furthermore, we discuss how new technologies can help us understand what biological processes are regulated by axonal protein synthesis in vivo. [BMB Reports 2015; 48(3): 139-146]     

Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. We show here that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. Our findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion.     

Local translation and directional steering in axons

The assembly of functional neural circuits in the developing brain requires neurons to extend axons to the correct targets. This in turn requires the navigating tips of axons to respond appropriately to guidance cues present along the axonal pathway, despite being cellular 'outposts' far from the soma. Work over the past few years has demonstrated a critical role for local translation within the axon in this process in vitro, making axon guidance another process that requires spatially localized translation, among others such as synaptic plasticity, cell migration, and cell polarity. This article reviews recent findings in local axonal translation and discusses how new protein synthesis may function in growth cone guidance, with a comparative view toward models of local translation in other systems.     

Regulation of proto-oncogene expression and deoxyribonucleic acid synthesis in granulosa cells of perifused immature rat ovaries.

The present series of experiments examined the effects of follicle-stimulating hormone (FSH) and insulin (IN) on granulosa cell (GC) proto-oncogene expression and DNA synthesis. In the first study, GCs were harvested from immature rat ovaries after 15, 30, or 60 min of perifusion and DNA synthesis (3H-thymidine incorporation) and proto-oncogene mRNA levels were determined. The presence of c-myc and c-fos proteins was localized within GCs immunocytochemically. GCs of control ovaries exhibited modest levels of DNA synthesis and proto-oncogene expression. FSH/IN not only stimulated DNA synthesis but also increased c-myc, c-fos, and c-jun mRNA levels and the percentage of cells staining for c-fos and c-myc proteins. The protein kinase inhibitor, 2-aminopurine (2-AP), inhibited the FSH/IN-induced increases in c-myc and c-fos mRNA levels, the percentage of cells staining for Myc and Fos protein, and DNA and protein synthesis. The effects of 48 h of perifusion with FSH in the presence or absence of IN were also examined. These treatments were selected because after 48 h of continuous exposure to FSH alone, estradiol-17 beta (E2) secretion is enhanced and 3H-thymidine incorporation is inhibited. Conversely, FSH/IN maintains 3H-thymidine incorporation for up to 48 h of perifusion culture without stimulating E2 (Peluso et al., Endocrinology 1991; 128:191-196). After 48 h of perifusion, both FSH and FSH/IN stimulated c-fos mRNA and protein levels. However, high levels of c-jun mRNA and protein were detected only within GCs of FSH/IN-treated ovaries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Local protein synthesis during axon guidance and synaptic plasticity

mRNA localization and regulated translation take central roles in axon guidance and synaptic plasticity. By spatially restricting gene expression within neurons, local protein synthesis provides growth cones and synapses with the capacity to autonomously regulate their structure and function. Studies in a variety of systems have provided insight into the specific roles of local protein synthesis during axonal navigation and during synaptic plasticity, and have begun to delineate the mechanisms underlying mRNA localization and regulated translation. Several powerful new tools have recently been developed to visualize each of these processes.     

Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target

As axons grow past intermediate targets, they change their responsiveness to guidance cues. Local upregulation of receptor expression is involved, but the mechanisms for this are not clear. Here protein synthesis is traced within individual axons by introducing RNAs encoding visualizable reporters. Individual severed axons and growth cones can translate proteins and also export them to the cell surface. As axons reach the spinal cord midline, EphA2 is among the receptors upregulated on at least some distal axon segments. Midline reporter upregulation is recapitulated by part of the EphA2 mRNA 3' untranslated region, which is highly conserved and includes known translational control sequences. These results show axons contain all the machinery for protein translation and cell surface expression, and they reveal a potentially general and flexible RNA-based mechanism for regulation localized within a subregion of the axon.     

Axon guidance: proteins turnover in turning growth cones

Accurate navigation by a neuronal growth cone requires the modulation of the growth cone's responsiveness to spatial and temporal changes in expression of guidance cues. These adaptations involve local protein synthesis and turnover in growth cones and distal axons.     

mRNA translation: regulating an out of soma experience

The regulation of protein synthesis in discrete cellular subdomains, or local protein synthesis, has important roles in development as well as brain function. This review will discuss some recent findings that shed new light on mRNA translation regulation and how these layers of regulation may work together to elicit tissue specific and spatially restricted gene expression.     

Axonal growth: where neurotrophins meet Wnts

Axonal guidance is influenced by many cues, including polypeptide trophic factors, cytokines, diffusible attractants and repellents and changes in calcium. How these signals are conveyed and integrated is not well defined. Recent data suggest that molecules of the canonical Wnt signaling pathway may have direct actions on axonal growth through neurotrophin signaling. This surprising mechanism is supported by local inactivation of glycogen synthase kinase 3beta (GSK-3beta) by nerve growth factor through the integrin-linked kinase. Inhibition of GSK-3beta provides a positive regulatory signal for the cytoskeleton re-arrangement involved in axon extension. Moreover, microtubule stabilization is stimulated by adenomatous polyposis coli protein, a downstream target of GSK3, in response to neurotrophins. Therefore, components of the Wnt signaling pathway are downstream of trophic factors, providing new insights into cytoskeletal regulatory events during axonal growth.     

Local Protein Synthesis in Axonal Growth Cones

While initially thought to be essentially a developmental characteristic, observed in artificial in vitro models, local protein synthesis in growth cones has been described in the adult, and more interestingly, during nerve regeneration. This emerging field is under intense investigation, revealing new functions of localized protein synthesis that include axon guidance, growth cone adaptation and sensitivity modulation at intermediate targets, or axon regeneration. Here, we will review these functions and provide a short survey of the current knowledge on mechanisms of mRNA transport and regulation of localized protein synthesis. In addition, we will consider what lessons can be learned from localized protein synthesis in dendrites, and what developments can be expected next in the field. This latter question relates to the crucial point of which technical strategy to adopt for an ideal and pertinent analysis of the phenomenon.     

DSCR1 is required for both axonal growth cone extension and steering

Local information processing in the growth cone is essential for correct wiring of the nervous system. As an axon navigates through the developing nervous system, the growth cone responds to extrinsic guidance cues by coordinating axon outgrowth with growth cone steering. It has become increasingly clear that axon extension requires proper actin polymerization dynamics, whereas growth cone steering involves local protein synthesis. However, molecular components integrating these two processes have not been identified. Here, we show that Down syndrome critical region 1 protein (DSCR1) controls axon outgrowth by modulating growth cone actin dynamics through regulation of cofilin activity (phospho/dephospho-cofilin). Additionally, DSCR1 mediates brain-derived neurotrophic factor–induced local protein synthesis and growth cone turning. Our study identifies DSCR1 as a key protein that couples axon growth and pathfinding by dually regulating actin dynamics and local protein synthesis.     

Retinoic acid enhances progesterone production via the cAMP/PKA signaling pathway in immature rat granulosa cells

Retinoic acid (RA) is a metabolite of vitamin A and has important roles in development, differentiation, and reproduction. Activin has been shown to regulate the RA pathway and affect granulosa cell (GC) proliferation, suggesting that RA is important for early follicle development. However, little is known about the effects of RA on GC functions, particularly steroidogenesis, during the early follicle stage. The aim of this study was to investigate the effects of all-trans-RA (atRA) on progesterone production in immature rat GCs cultured without gonadotropin. Our results demonstrated that atRA enhanced progesterone production by upregulating the levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450scc (Cyp11a1) mRNAs, but not 3β-hydroxysteroid dehydrogenase mRNA in immature rat GCs. Additionally, analysis of the mechanisms through which atRA upregulated StAR and Cyp11a1 mRNAs revealed that atRA enhanced intracellular cAMP accumulation and phosphorylation of cAMP response-element binding protein (CREB). In addition, H-89, an inhibitor of protein kinase A (PKA), abolished the stimulatory effects of atRA, indicating that atRA enhanced progesterone synthesis through cAMP/PKA signaling. In conclusion, our data demonstrated that atRA has a crucial role in progesterone synthesis in rat GCs during the early follicle stage.     

Cell adhesion molecules regulate Ca2+-mediated steering of growth cones via cyclic AMP and ryanodine receptor type 3

Axonal growth cones migrate along the correct paths during development, not only directed by guidance cues but also contacted by local environment via cell adhesion molecules (CAMs). Asymmetric Ca2+ elevations in the growth cone cytosol induce both attractive and repulsive turning in response to the guidance cues (Zheng, J.Q. 2000. Nature. 403:89-93; Henley, J.R., K.H. Huang, D. Wang, and M.M. Poo. 2004. Neuron. 44:909-916). Here, we show that CAMs regulate the activity of ryanodine receptor type 3 (RyR3) via cAMP and protein kinase A in dorsal root ganglion neurons. The activated RyR3 mediates Ca2+-induced Ca2+ release (CICR) into the cytosol, leading to attractive turning of the growth cone. In contrast, the growth cone exhibits repulsion when Ca2+ signals are not accompanied by RyR3-mediated CICR. We also propose that the source of Ca2+ influx, rather than its amplitude or the baseline Ca2+ level, is the primary determinant of the turning direction. In this way, axon-guiding and CAM-derived signals are integrated by RyR3, which serves as a key regulator of growth cone navigation.     

Trafficking Guidance Receptors

Wiring of the brain relies initially on the correct outgrowth of axons to reach the appropriate target area for innervation. A large number of guidance receptors present in the plasma membrane of axonal growth cones and elsewhere on the neuron read and execute directional cues present in the extracellular environment of the navigating growth cone. The exact timing, levels, and localization of expression of the guidance receptors in the plasma membrane therefore determine the outcome of guidance decisions. Many guidance receptors are localized in exquisitely precise spatial and temporal patterns. The cellular mechanisms ensuring these localization patterns include spatially accurate sorting after synthesis in the secretory pathway, retrieval of inappropriately expressed receptors by endocytosis followed by degradation or recycling, and restriction of diffusion. This article will discuss the machinery and regulation underlying the restricted distribution of membrane receptors, focusing on the currently best-studied example, the L1 cell adhesion molecule. In addition to the long-range mechanisms ensuring appropriate localization, the same mechanisms can act locally to adjust levels and localization of receptors. These local mechanisms are regulated by ligand binding and subsequent activation of local signaling cascades. It is likely that the localization of all guidance receptors is regulated by a combination of sorting, retrieval, recycling and retention, similar to the ones we discuss here for L1.