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In the present study, the influence of topographic and mechanical cues on neuronal growth cones (NGCs) and network directionality in 3D‐engineered cell culture models is explored. Two‐photon polymerization (2PP) is employed to fabricate nanopillar arrays featuring tunable effective shear modulus. Large variations in mechanical properties are obtained by altering the aspect ratio of the nanostructures. The nanopillar arrays are seeded with different neuronal cell lines, including neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs), I3Neurons, and primary hippocampal neurons. All cell types exhibit preferential orientations according to the nanopillar topology, as shown by neurites creating a high number of oriented orthogonal networks. Furthermore, the differentiation and maturation of NPCs are affected by the topographic and mechanical properties of the nanopillars, as shown by the expression of the mature neuronal marker Synapsin I. Lastly, NGCs are influenced by effective shear modulus in terms of spreading area, and stochastic optical reconstruction microscopy (STORM) is employed to assess the cytoskeleton organization at nanometric resolution. The developed approach, involving laser‐assisted 3D microfabrication, neuro‐mechanobiology, and super‐resolution microscopy, paves the way for prospective comparative studies on the evolution of neuronal networks and NGCs in healthy and diseased (e.g., neurodegenerative) conditions. [ABSTRACT FROM AUTHOR]

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three‐dimensional‐structured illumination microscopy, a type of super‐resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z‐axis ("z‐filopodia") within the growth cone. Z‐filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z‐filopodia formation and dynamics were regulated by actin‐regulatory proteins, such as vasodilator‐stimulated phosphoprotein, fascin, and cofilin. Chromophore‐assisted laser inactivation of cofilin induced the rapid turnover of z‐filopodia. An axon guidance receptor, neuropilin‐1, was concentrated in z‐filopodia and was transported together with them, whereas its ligand, semaphorin‐3A, was selectively bound to them. Membrane domains associated with z‐filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin‐1. The results suggest that z‐filopodia have unique turnover properties, and unlike xy‐filopodia, do not function as force‐generating structures for axon extension. [ABSTRACT FROM AUTHOR]

Spatially and temporally accurate termination of axon outgrowth, a process called axon termination, is required for efficient, precise nervous system construction and wiring. The mechanosensory neurons that sense low-threshold mechanical stimulation or gentle touch have proven exceptionally valuable for studying axon termination over the past 40 years. In this Review, we discuss progress made in deciphering the molecular and genetic mechanisms that govern axon termination in touch receptor neurons. Findings across model organisms, including Caenorhabditis elegans, Drosophila, zebrafish and mice, have revealed that complex signaling is required for termination with conserved principles and players beginning to surface. A key emerging theme is that axon termination is mediated by complex signaling networks that include ubiquitin ligase signaling hubs, kinase cascades, transcription factors, guidance/adhesion receptors and growth factors. Here, we begin a discussion about how these signaling networks could represent termination codes that trigger cessation of axon outgrowth in different species and types of mechanosensory neurons. [ABSTRACT FROM AUTHOR]

RhoA plays a crucial role in neuronal polarization, where its action restraining axon outgrowth has been thoroughly studied. We now report that RhoA has not only an inhibitory but also a stimulatory effect on axon development depending on when and where exerts its action and the downstream effectors involved. In cultured hippocampal neurons, FRET imaging revealed that RhoA activity selectively localized in growth cones of undifferentiated neurites, whereas in developing axons it displayed a biphasic pattern, being low in nascent axons and high in elongating ones. RhoA–Rho kinase (ROCK) signaling prevented axon initiation but had no effect on elongation, whereas formin inhibition reduced axon extension without significantly altering initial outgrowth. In addition, RhoA–mDia signaling promoted axon elongation by stimulating growth cone microtubule stability and assembly, as opposed to RhoA–ROCK signaling, which restrained growth cone microtubule assembly and protrusion. [ABSTRACT FROM AUTHOR]

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development. [ABSTRACT FROM AUTHOR]

Filopodia are narrow actin-rich protrusions with important roles in neuronal development where membrane-binding adaptor proteins, such as I-BAR- and F-BAR-domain-containing proteins, have emerged as upstream regulators that link membrane interactions to actin regulators such as formins and proteins of the Ena/VASP family. Both the adaptors and their binding partners are part of diverse and redundant protein networks that can functionally compensate for each other. To explore the significance of the F-BAR domain-containing neuronal membrane adaptor TOCA-1 (also known as FNBP1L) in filopodia we performed a quantitative analysis of TOCA-1 and filopodial dynamics in Xenopus retinal ganglion cells, where Ena/VASP proteins have a native role in filopodial extension. Increasing the density of TOCA-1 enhances Ena/VASP protein binding in vitro, and an accumulation of TOCA-1, as well as its coincidence with Ena, correlates with filopodial protrusion in vivo. Two-colour single-molecule localisation microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters promote filopodial protrusion and this depends on a functional TOCA-1 SH3 domain and activation of Cdc42, which we perturbed using the small-molecule inhibitor CASIN. We propose that TOCA-1 clusters act independently of membrane curvature to recruit and promote Ena activity for filopodial protrusion. [ABSTRACT FROM AUTHOR]

Binocular vision requires the segregation of retinal ganglion cell (RGC) axons extending from the retina into the ipsilateral and contralateral optic tracts. RGC axon segregation occurs at the optic chiasm, which forms at the ventral diencephalon midline. Using expression analyses, retinal explants and genetically modified mice, we demonstrate that CXCL12 (SDF1) is required for axon segregation at the optic chiasm. CXCL12 is expressed by the meninges bordering the optic pathway, and CXCR4 by both ipsilaterally and contralaterally projecting RGCs. CXCL12 or ventral diencephalon meninges potently promoted axon outgrowth from both ipsilaterally and contralaterally projecting RGCs. Further, a higher proportion of axons projected ipsilaterally in mice lacking CXCL12 or its receptor CXCR4 compared with wild-type mice as a result of misrouting of presumptive contralaterally specified RGC axons. Although RGCs also expressed the alternative CXCL12 receptor ACKR3, the optic chiasm developed normally in mice lacking ACKR3. Our data support a model whereby meningeal-derived CXCL12 helps drive axon growth from CXCR4-expressing RGCs towards the diencephalon midline, enabling contralateral axon growth. These findings further our understanding of the molecular and cellular mechanisms controlling optic pathway development. [ABSTRACT FROM AUTHOR]

NADPH oxidase (Nox), a major source of reactive oxygen species (ROS), is involved in neurodegeneration after injury and disease. Nox is expressed in both neuronal and non‐neuronal cells and contributes to an elevated ROS level after injury. Contrary to the well‐known damaging effect of Nox‐derived ROS in neurodegeneration, recently a physiological role of Nox in nervous system development including neurogenesis, neuronal polarity, and axonal growth has been revealed. Here, we tested a role for neuronal Nox in neurite regeneration following mechanical transection in cultured Aplysia bag cell neurons. Using a novel hydrogen peroxide (H2O2)‐sensing dye, 5′‐(p‐borophenyl)‐2′‐pyridylthiazole pinacol ester (BPPT), we found that H2O2 levels are elevated in regenerating growth cones following injury. Redistribution of Nox2 and p40phox in the growth cone central domain suggests Nox2 activation after injury. Inhibiting Nox with the pan‐Nox inhibitor celastrol reduced neurite regeneration rate. Pharmacological inhibition of Nox is correlated with reduced activation of Src2 tyrosine kinase and F‐actin content in the growth cone. Taken together, these findings suggest that Nox‐derived ROS regulate neurite regeneration following injury through Src2‐mediated regulation of actin organization in Aplysia growth cones. [ABSTRACT FROM AUTHOR]

Axon regeneration is essential for successful recovery after peripheral nerve injury. Although growth cone reformation and axonal extension are crucial steps in axonal regeneration, the regulatory mechanisms underlying these dynamic processes are poorly understood. Here, we identify βPix (Arhgef7), the guanine nucleotide exchange factor for Rac1 GTPase, as a regulator of axonal regeneration. After sciatic nerve injury in mice, the expression levels of βPix increase significantly in nerve segments containing regenerating axons. In regrowing axons, βPix is localized in the peripheral domain of the growth cone. Using βPix neuronal isoform knockout (NIKO) mice in which the neuronal isoforms of βPix are specifically removed, we demonstrate that βPix promotes neurite outgrowth in cultured dorsal root ganglion neurons and in vivo axon regeneration after sciatic nerve crush injury. Activation of cJun and STAT3 in the cell bodies is not affected in βPix NIKO mice, supporting the local action of βPix in regenerating axons. Finally, inhibiting Src, a kinase previously identified as an activator of the βPix neuronal isoform, causes axon outgrowth defects in vitro, like those found in the βPix NIKO neurons. Altogether, these data indicate that βPix plays an important role in axonal regrowth during peripheral nerve regeneration. [ABSTRACT FROM AUTHOR]

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Simple Summary: Neurons are biological wires that send signals not only across the brain, but throughout the body. Like almost all cells in the human body, neurons adhere to their surroundings through receptors known as integrins. As the nervous system develops, integrins become essential for neuronal wires, known as axons, to adhere within their environment and pull themselves forward as they grow towards their target. Following injury to the nervous system, the damaged axons must regrow towards their target to restore communication. The integrin receptor therefore plays an important role in this process of regeneration. Integrins mediate their effects through an intricate scaffold of proteins inside the cell, which must be coordinated by a specific central protein, called focal adhesion kinase. This review discusses the signalling pathway of integrin receptors and focal adhesion kinase, specifically in neurons, and how this signalling pathway mediates neuronal growth. We also discuss how integrins and focal adhesion kinase could contribute to neuronal repair following nervous system injury. Integrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin–FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury. [ABSTRACT FROM AUTHOR]

Aim: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules (the soluble cue netrin/UNC-6 or two receptors, DCC/UNC-40 and UNC-5H). Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. Summary of results: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon. [ABSTRACT FROM AUTHOR]

mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression and mRNA degradation. How mRNA decay pathways regulate cellular function in vivo with specificity is poorly understood. Here, we show that C. elegans mRNA decay factors, including the translational repressors CAR-1/LSM14 and CGH-1/DDX6, and the decapping enzymes DCAP-1/DCP1, function in neurons to differentially regulate axon development, maintenance, and regrowth following injury. In neuronal cell bodies, CAR-1 fully colocalizes with CGH-1 and partially colocalizes with DCAP-1, suggesting that mRNA decay components form at least two types of cytoplasmic granules. Following axon injury in adult neurons, loss of CAR-1 or CGH-1 results in increased axon regrowth and growth cone formation, whereas loss of DCAP-1 or DCAP-2 results in reduced regrowth. To determine how CAR-1 inhibits regrowth, we analyzed mRNAs bound to pan-neuronally expressed GFP::CAR-1 using a crosslinking and immunoprecipitation-based approach. Among the putative mRNA targets of CAR-1, we characterized the roles of micu-1, a regulator of the mitochondrial calcium uniporter MCU-1, in axon injury. We show that loss of car-1 results increased MICU-1 protein levels, and that enhanced axon regrowth in car-1 mutants is dependent on micu-1 and mcu-1. Moreover, axon injury induces transient calcium influx into axonal mitochondria, dependent on MCU-1. In car-1 loss-of-function mutants and in micu-1 overexpressing animals, the axonal mitochondrial calcium influx is more sustained, which likely underlies enhanced axon regrowth. Our data uncover a novel pathway that controls axon regrowth through axonal mitochondrial calcium uptake.

Axon growth enables the rapid wiring of the central nervous system. Understanding this process is a prerequisite to retriggering it under pathological conditions, such as a spinal cord injury, to elicit axon regeneration. The last decades saw progress in understanding the mechanisms underlying axon growth. Most of these studies employed cultured neurons grown on flat surfaces. Only recently studies on axon growth were performed in 3D. In these studies, physiological environments exposed more complex and dynamic aspects of axon development. Here, we describe current views on axon growth and highlight gaps in our knowledge. We discuss how axons interact with the extracellular matrix during development and the role of the growth cone and its cytoskeleton within. Finally, we propose that the time is ripe to study axon growth in a more physiological setting. This will help us uncover the physiologically relevant mechanisms underlying axon growth, and how they can be reactivated to induce axon regeneration. [ABSTRACT FROM AUTHOR]

The establishment and maintenance of neuronal polarity are important for neural development and function. Abnormal neuronal polarity establishment commonly leads to a variety of neurodevelopmental disorders. Over the past three decades, with the continuous development and improvement of biological research methods and techniques, we have made tremendous progress in the understanding of the molecular mechanisms of neuronal polarity establishment. The activity of positive and negative feedback signals and actin waves are both essential in this process. They drive the directional transport and aggregation of key molecules of neuronal polarity, promote the spatiotemporal regulation of ordered and coordinated interactions of actin filaments and microtubules, stimulate the specialization and growth of axons, and inhibit the formation of multiple axons. In this review, we focus on recent advances in these areas, in particular the important findings about neuronal polarity in two classical models, in vitro primary hippocampal/cortical neurons and in vivo cortical pyramidal neurons, and discuss our current understanding of neuronal polarity.​ [ABSTRACT FROM AUTHOR]

In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca2+ transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands. [ABSTRACT FROM AUTHOR]

In the polarity/protrusion model of growth cone repulsion from UNC-6/ netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plusend entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion. [ABSTRACT FROM AUTHOR]

Introduction: UNC-6/Netrin is a conserved bi-functional guidance cue which regulates dorsal-ventral axon guidance in C. elegans. In the Polarity/Protrusion model of UNC-6/Netrin mediated dorsal growth away from UNC-6/Netrin, The UNC-5 receptor first polarizes the VD growth cone such that filopodial protrusions are biased dorsally. Based on this polarity, the UNC-40/DCC receptor stimulates growth cone lamellipodial and filopodial protrusion dorsally. The UNC-5 receptor maintains dorsal polarity of protrusion, and inhibits growth cone protrusion ventrally, resulting in net dorsal growth cone advance.Methods: Growth cone imaging in mutants, combined with Cas9 genome editing and genetic analysis, were used to analyze the role of a novel short isoform on unc-5 in growth cone polarity and protrusion.Results: Work presented here demonstrates a novel role of a previously undescribed, conserved short isoform of UNC-5 (UNC-5B). UNC-5B lacks the cytoplasmic domains of UNC-5 long, including the DEATH domain, the UPA/DB domain, and most of the ZU5 domain. Mutations that specifically affect only the unc-5 long isoforms were hypomorphic, suggesting a role of unc-5B short. A mutation specifically affecting unc-5B caused loss of dorsal polarity of protrusion and reduced growth cone filopodial protrusion, the opposite of unc-5 long mutations. Transgenic expression of unc-5B partially rescued unc-5 axon guidance defects, and resulted in large growth cones. Tyrosine 482 (Y482) in the cytoplasmic juxtamembrane region has been shown to be important for UNC-5 function, and is present in both UNC-5 long and UNC-5B short. Results reported here show that Y482 is required for the function of UNC-5 long and for some functions of UNC-5B short. Finally, genetic interactions with unc-40 and unc-6 suggest that UNC-5B short acts in parallel to UNC-6/Netrin to ensure robust growth cone lamellipodial protrusion.Discussion: These results demonstrate a previously-undescribed role for the UNC-5B short isoform, which is required for dorsal polarity of growth cone filopodial protrusion and to stimulate growth cone protrusion, in contrast to the previously-described role of UNC-5 long in inhibiting growth cone protrusion.

The mechanism of axon growth and guidance is a core, unsolved problem in neuroscience and cell biology. For nearly three decades, our view of this process has largely been based on deterministic models of motility derived from studies of neurons cultured in vitro on rigid substrates. Here, we suggest a fundamentally different, inherently probabilistic model of axon growth, one that is grounded in the stochastic dynamics of actin networks. This perspective is motivated and supported by a synthesis of results from live imaging of a specific axon growing in its native tissue in vivo, together with single-molecule computational simulations of actin dynamics. In particular, we show how axon growth arises from a small spatial bias in the intrinsic fluctuations of the axonal actin cytoskeleton, one that produces net translocation of the axonal actin network by differentially modulating local probabilities of network expansion versus compaction. We discuss the relationship between this model and current views of axon growth and guidance mechanism and demonstrate how it offers explanations for various longstanding puzzles in this field. We further point out the implications of the probabilistic nature of actin dynamics for many other processes of cell morphology and motility.

Neuron navigators (Navigators) are cytoskeletal-associated proteins important for neuron migration, neurite growth, and axon guidance, but they also function more widely in other tissues. Recent studies have revealed novel cellular functions of Navigators such as macropinocytosis, and have implicated Navigators in human disorders of axon growth. Navigators are present in most or all bilaterian animals: vertebrates have three Navigators (NAV1-3), Drosophila has one (Sickie), and Caenorhabditis elegans has one (Unc-53). Structurally, Navigators have conserved N- and C-terminal regions each containing specific domains. The N-terminal region contains a calponin homology (CH) domain and one or more SxIP motifs, thought to interact with the actin cytoskeleton and mediate localization to microtubule plus-end binding proteins, respectively. The C-terminal region contains two coiled-coil domains, followed by a AAA+ family nucleoside triphosphatase domain of unknown activity. The Navigators appear to have evolved by fusion of N- and C-terminal region homologs present in simpler organisms. Overall, Navigators participate in the cytoskeletal response to extracellular cues via microtubules and actin filaments, in conjunction with membrane trafficking. We propose that uptake of fluid-phase cues and nutrients and/or downregulation of cell surface receptors could represent general mechanisms that explain Navigator functions. Future studies developing new models, such as conditional knockout mice or human cerebral organoids may reveal new insights into Navigator function. Importantly, further biochemical studies are needed to define the activities of the Navigator AAA+ domain, and to study potential interactions among different Navigators and their binding partners. [ABSTRACT FROM AUTHOR]

Much research has focused on the PI3-kinase and PTEN signaling pathway with the aim to stimulate repair of the injured central nervous system. Axons in the central nervous system fail to regenerate, meaning that injuries or diseases that cause loss of axonal connectivity have life-changing consequences. In 2008, genetic deletion of PTEN was identified as a means of stimulating robust regeneration in the optic nerve. PTEN is a phosphatase that opposes the actions of PI3-kinase, a family of enzymes that function to generate the membrane phospholipid PIP3 from PIP2 (phosphatidylinositol (3,4,5)-trisphosphate from phosphatidylinositol (4,5)-bisphosphate). Deletion of PTEN therefore allows elevated signaling downstream of PI3-kinase, and was initially demonstrated to promote axon regeneration by signaling through mTOR. More recently, additional mechanisms have been identified that contribute to the neuron-intrinsic control of regenerative ability. This review describes neuronal signaling pathways downstream of PI3-kinase and PIP3, and considers them in relation to both developmental and regenerative axon growth. We briefly discuss the key neuron-intrinsic mechanisms that govern regenerative ability, and describe how these are affected by signaling through PI3-kinase. We highlight the recent finding of a developmental decline in the generation of PIP3 as a key reason for regenerative failure, and summarize the studies that target an increase in signaling downstream of PI3-kinase to facilitate regeneration in the adult central nervous system. Finally, we discuss obstacles that remain to be overcome in order to generate a robust strategy for repairing the injured central nervous system through manipulation of PI3-kinase signaling.

A challenge in analyzing dynamic intracellular cell biological processes is the dearth of methodologies that are sufficiently fast and specific to perturb intracellular protein activities. We previously developed a light-sensitive variant of the microtubule plus end-tracking protein EB1 by inserting a blue light-controlled protein dimerization module between functional domains. Here, we describe an advanced method to replace endogenous EB1 with this light-sensitive variant in a single genome editing step, thereby enabling this approach in human induced pluripotent stem cells (hiPSCs) and hiPSC-derived neurons. We demonstrate that acute and local optogenetic EB1 inactivation in developing cortical neurons induces microtubule depolymerization in the growth cone periphery and subsequent neurite retraction. In addition, advancing growth cones are repelled from areas of blue light exposure. These phenotypes were independent of the neuronal EB1 homolog EB3, revealing a direct dynamic role of EB1-mediated microtubule plus end interactions in neuron morphogenesis and neurite guidance.

Neuron navigators (Navigators) are cytoskeletal-associated proteins important for neuron migration, neurite growth, and axon guidance, but they also function more widely in other tissues. Recent studies have revealed novel cellular functions of Navigators such as macropinocytosis, and have implicated Navigators in human disorders of axon growth. Navigators are present in most or all bilaterian animals: vertebrates have three Navigators (NAV1-3), Drosophila has one (Sickie), and Caenorhabditis elegans has one (Unc-53). Structurally, Navigators have conserved N- and C-terminal regions each containing specific domains. The N-terminal region contains a calponin homology (CH) domain and one or more SxIP motifs, thought to interact with the actin cytoskeleton and mediate localization to microtubule plus-end binding proteins, respectively. The C-terminal region contains two coiled-coil domains, followed by a AAA+ family nucleoside triphosphatase domain of unknown activity. The Navigators appear to have evolved by fusion of N- and C-terminal region homologs present in simpler organisms. Overall, Navigators participate in the cytoskeletal response to extracellular cues via microtubules and actin filaments, in conjunction with membrane trafficking. We propose that uptake of fluid-phase cues and nutrients and/or downregulation of cell surface receptors could represent general mechanisms that explain Navigator functions. Future studies developing new models, such as conditional knockout mice or human cerebral organoids may reveal new insights into Navigator function. Importantly, further biochemical studies are needed to define the activities of the Navigator AAA+ domain, and to study potential interactions among different Navigators and their binding partners.

Growth cones navigate axonal projection in response to guidance cues. However, it is unclear how they can decide the migratory direction by transducing the local spatial cues into protrusive forces. Here we show that knockout mice of Shootin1 display abnormal projection of the forebrain commissural axons, a phenotype similar to that of the axon guidance molecule netrin-1. Shallow gradients of netrin-1 elicited highly polarized Pak1-mediated phosphorylation of shootin1 within growth cones. We demonstrate that netrin-1-elicited shootin1 phosphorylation increases shootin1 interaction with the cell adhesion molecule L1-CAM; this, in turn, promotes F-actin-adhesion coupling and concomitant generation of forces for growth cone migration. Moreover, the spatially regulated shootin1 phosphorylation within growth cones is required for axon turning induced by netrin-1 gradients. Our study defines a mechano-effector for netrin-1 signaling and demonstrates that shootin1 phosphorylation is a critical readout for netrin-1 gradients that results in a directional mechanoresponse for axon guidance.

BackgroundExposure of the developing brain to propofol results in cognitive deficits. Recent data suggest that inhibition of neuronal apoptosis does not prevent cognitive defects, suggesting mechanisms other than neuronal apoptosis play a role in anaesthetic neurotoxicity. Proper neuronal growth during development is dependent upon growth cone morphology and axonal transport. Propofol modulates actin dynamics in developing neurones, causes RhoA-dependent depolymerisation of actin, and reduces dendritic spines and synapses. We hypothesised that RhoA inhibition prevents synaptic loss and subsequent cognitive deficits. The present study tested whether RhoA inhibition with the botulinum toxin C3 (TAT-C3) prevents propofol-induced synapse and neurite loss, and preserves cognitive function.MethodsRhoA activation, growth cone morphology, and axonal transport were measured in neonatal rat neurones (5-7 days in vitro) exposed to propofol. Synapse counts (electron microscopy), dendritic arborisation (Golgi-Cox), and network connectivity were measured in mice (age 28 days) previously exposed to propofol at postnatal day 5-7. Memory was assessed in adult mice (age 3 months) previously exposed to propofol at postnatal day 5-7.ResultsPropofol increased RhoA activation, collapsed growth cones, and impaired retrograde axonal transport of quantum dot-labelled brain-derived neurotrophic factor, all of which were prevented with TAT-C3. Adult mice previously treated with propofol had decreased numbers of total hippocampal synapses and presynaptic vesicles, reduced hippocampal dendritic arborisation, and infrapyramidal mossy fibres. These mice also exhibited decreased hippocampal-dependent contextual fear memory recall. All anatomical and behavioural changes were prevented with TAT-C3 pre-treatment.ConclusionInhibition of RhoA prevents propofol-mediated hippocampal neurotoxicity and associated cognitive deficits.

Neuronal migration and axon elongation in the developing brain are essential events for neural network formation. Leading processes of migrating neurons and elongating axons have growth cones at their tips. Cytoskeletal machinery for advance of growth cones of the two processes has been thought the same. In this study, we compared axonal-elongating growth cones and leading-process growth cones in the same conditions that manipulated filopodia, lamellipodia, and drebrin, the latter mediates actin filament-microtubule interaction. Cerebral cortex (CX) neurons and medial ganglionic eminence (MGE) neurons from embryonic mice were cultured on less-adhesive cover glasses. Inhibition of filopodia formation by triple knockdown of mammalian-enabled, Ena-VASP-like, and vasodilator-stimulated phosphoprotein or double knockdown of Daam1 and fascin affected axon formation of CX neurons but did not affect the morphology of leading process of MGE neurons. On the other hand, treatment with CK666, to inhibit lamellipodia formation, did not affect axons but destroyed the leading-process growth cones. When drebrin was knocked down, the morphology of CX neurons remained unchanged, but the leading processes of MGE neurons became shorter. In vivo assay of radial migration of CX neurons revealed that drebrin knockdown inhibited migration, while it did not affect axon elongation. These results showed that the filopodia-microtubule system is the main driving machinery in elongating growth cones, while the lamellipodia-drebrin-microtubule system is the main system in leading-process growth cones of migrating neurons. [ABSTRACT FROM AUTHOR]

Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are a group of conserved eukaryotic kinases phosphorylating tyrosine, serine, and threonine residues. The human DYRK family comprises 5 members (DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4). The different DYRKs have been implicated in neurological diseases, cancer, and virus infection. Specifically, DYRK2 has been mainly implicated in cancer progression. However, its role in healthy and pathological nervous system function has been overlooked. In this context, we review current available data on DYRK2 in the nervous system, where the available studies indicate that it has key roles in neuronal development and function. DYRK2 regulates neuronal morphogenesis (e.g., axon growth and branching) by phosphorylating cytoskeletal elements (e.g., doublecortin). Comparative data reveals that it is involved in the development of olfactory and visual systems, the spinal cord and possibly the cortex. DYRK2 also participates in processes such as olfaction, vision and, learning. However, DYRK2 could be involved in other brain functions since available expression data shows that it is expressed across the whole brain. High DYRK2 protein levels have been detected in basal ganglia and cerebellum. In adult nervous system, DYRK2 mRNA expression is highest in the cortex, hippocampus, and retina. Regarding nervous system disease, DYRK2 has been implicated in neuroblastoma, glioma, epilepsy, neuroinflammation, Alzheimer's disease, Parkinson's disease, spinal cord injury and virus infection. DYRK2 upregulation usually has a negative impact in cancer-related conditions and a positive impact in non-malignant conditions. Its role in axon growth makes DYRK2 as a promising target for spinal cord or brain injury and regeneration. [ABSTRACT FROM AUTHOR]

Neuronal differentiation, maturation, and synapse formation are regulated by various growth factors. Here we show that epidermal growth factor (EGF) negatively regulates presynaptic maturation and synapse formation. In cortical neurons, EGF maintained axon elongation and reduced the sizes of growth cones in culture. Furthermore, EGF decreased the levels of presynaptic molecules and number of presynaptic puncta, suggesting that EGF inhibits neuronal maturation. The reduction of synaptic sites is confirmed by the decreased frequencies of miniature EPSCs. In vivo analysis revealed that while peripherally administrated EGF decreased the levels of presynaptic molecules and numbers of synaptophysin-positive puncta in the prefrontal cortices of neonatal rats, EGF receptor inhibitors upregulated these indexes, suggesting that endogenous EGF receptor ligands suppress presynaptic maturation. Electron microscopy further revealed that EGF decreased the numbers, but not the sizes, of synaptic structures in vivo. These findings suggest that endogenous EGF and/or other EGF receptor ligands negatively modulates presynaptic maturation and synapse formation. [ABSTRACT FROM AUTHOR]

Vertebrate brains have a dual structure, composed of (i) axons that can be well-captured with graph-theoretical methods and (ii) axons that form a dense matrix in which neurons with precise connections operate. A core part of this matrix is formed by axons (fibers) that store and release 5-hydroxytryptamine (5-HT, serotonin), an ancient neurotransmitter that supports neuroplasticity and has profound implications for mental health. The self-organization of the serotonergic matrix is not well understood, despite recent advances in experimental and theoretical approaches. In particular, individual serotonergic axons produce highly stochastic trajectories, fundamental to the construction of regional fiber densities, but further advances in predictive computer simulations require more accurate experimental information. This study examined single serotonergic axons in culture systems (co-cultures and monolayers), by using a set of complementary high-resolution methods: confocal microscopy, holotomography (refractive index-based live imaging), and super-resolution (STED) microscopy. It shows that serotonergic axon walks in neural tissue may strongly reflect the stochastic geometry of this tissue and it also provides new insights into the morphology and branching properties of serotonergic axons. The proposed experimental platform can support next-generation analyses of the serotonergic matrix, including seamless integration with supercomputing approaches.

Dual specificity tyrosine-phosphorylation-regulated kinases (DYRKs) are a group of conserved eukaryotic kinases phosphorylating tyrosine, serine, and threonine residues. The human DYRK family comprises 5 members (DYRK1A, DYRK1B, DYRK2, DYRK3, and DYRK4). The different DYRKs have been implicated in neurological diseases, cancer, and virus infection. Specifically, DYRK2 has been mainly implicated in cancer progression. However, its role in healthy and pathological nervous system function has been overlooked. In this context, we review current available data on DYRK2 in the nervous system, where the available studies indicate that it has key roles in neuronal development and function. DYRK2 regulates neuronal morphogenesis (e.g., axon growth and branching) by phosphorylating cytoskeletal elements (e.g., doublecortin). Comparative data reveals that it is involved in the development of olfactory and visual systems, the spinal cord and possibly the cortex. DYRK2 also participates in processes such as olfaction, vision and, learning. However, DYRK2 could be involved in other brain functions since available expression data shows that it is expressed across the whole brain. High DYRK2 protein levels have been detected in basal ganglia and cerebellum. In adult nervous system, DYRK2 mRNA expression is highest in the cortex, hippocampus, and retina. Regarding nervous system disease, DYRK2 has been implicated in neuroblastoma, glioma, epilepsy, neuroinflammation, Alzheimer’s disease, Parkinson’s disease, spinal cord injury and virus infection. DYRK2 upregulation usually has a negative impact in cancer-related conditions and a positive impact in non-malignant conditions. Its role in axon growth makes DYRK2 as a promising target for spinal cord or brain injury and regeneration.

Aim: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules (the soluble cue netrin/UNC-6 or two receptors, DCC/UNC-40 and UNC-5H). Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. Summary of results: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon.

Computational techniques for analyzing biological images offer a great potential to enhance our knowledge of the biological processes underlying disorders of the nervous system. Friedreich's Ataxia (FRDA) is a rare progressive neurodegenerative inherited disorder caused by the low expression of frataxin, which is a small mitochondrial protein. In FRDA cells, the lack of frataxin promotes primarily mitochondrial dysfunction, an alteration of calcium (Ca2+) homeostasis and the destabilization of the actin cytoskeleton in the neurites and growth cones of sensory neurons. In this paper, a computational multilinear algebra approach was used to analyze the dynamics of the growth cone and its function in control and FRDA neurons. Computational approach, which includes principal component analysis and a multilinear algebra method, is used to quantify the dynamics of the growth cone (GC) morphology of sensory neurons from the dorsal root ganglia (DRG) of the YG8sR humanized murine model for FRDA. It was confirmed that the dynamics and patterns of turning were aberrant in the FRDA growth cones. In addition, our data suggest that other cellular processes dependent on functional GCs such as axonal regeneration might also be affected. Semiautomated computational approaches are presented to quantify differences in GC behaviors in neurodegenerative disease. In summary, the deficiency of frataxin has an adverse effect on the formation and, most importantly, the growth cones' function in adult DRG neurons. As a result, frataxin deficient DRG neurons might lose the intrinsic capability to grow and regenerate axons properly due to the dysfunctional GCs they build. [ABSTRACT FROM AUTHOR]

The role of mitochondria within injured neurons is an area of active interest since these organelles are vital for the production of cellular energy in the form of ATP. Using mechanosensory neurons of the nematode Caenorhabditis elegans to test regeneration after neuronal injury in vivo, we surveyed genes related to mitochondrial function for effects on axon regrowth after laser axotomy. Genes involved in mitochondrial transport, calcium uptake, mitophagy, or fission and fusion were largely dispensable for axon regrowth, with the exception of eat-3/Opa1. Surprisingly, many genes encoding components of the electron transport chain were dispensable for regrowth, except for the iron-sulfur proteins gas-1, nduf-2.2, nduf-7, and isp-1, and the putative oxidoreductase rad-8. In these mutants, axonal development was essentially normal and axons responded normally to injury by forming regenerative growth cones, but were impaired in subsequent axon extension. Overexpression of nduf-2.2 or isp-1 was sufficient to enhance regrowth, suggesting that mitochondrial function is rate-limiting in axon regeneration. Moreover, loss of function in isp-1 reduced the enhanced regeneration caused by either a gain-of-function mutation in the calcium channel EGL-19 or overexpression of the MAP kinase DLK-1. While the cellular function of RAD-8 remains unclear, our genetic analyses place rad-8 in the same pathway as other electron transport genes in axon regeneration. Unexpectedly, rad-8 regrowth defects were suppressed by altered function in the ubiquinone biosynthesis gene clk-1. Furthermore, we found that inhibition of the mitochondrial unfolded protein response via deletion of atfs-1 suppressed the defective regrowth in nduf-2.2 mutants. Together, our data indicate that while axon regeneration is not significantly affected by general dysfunction of cellular respiration, it is sensitive to the proper functioning of a select subset of electron transport chain genes, or to the cellular adaptations used by neurons under conditions of injury.

Mechanistic studies of axon growth during development are beneficial to the search for neuron-intrinsic regulators of axon regeneration. Here, we discovered that, in the developing neuron from rat, Akt signaling regulates axon growth and growth cone formation through phosphorylation of serine 14 (S14) on Inhibitor of DNA binding 2 (Id2). This enhances Id2 protein stability by means of escape from proteasomal degradation, and steers its localization to the growth cone, where Id2 interacts with radixin that is critical for growth cone formation. Knockdown of Id2, or abrogation of Id2 phosphorylation at S14, greatly impairs axon growth and the architecture of growth cone. Intriguingly, reinstatement of Akt/Id2 signaling after injury in mouse hippocampal slices redeemed growth promoting ability, leading to obvious axon regeneration. Our results suggest that Akt/Id2 signaling is a key module for growth cone formation and axon growth, and its augmentation plays a potential role in CNS axonal regeneration.

Computational techniques for analyzing biological images offer a great potential to enhance our knowledge of the biological processes underlying disorders of the nervous system. Friedreich’s Ataxia (FRDA) is a rare progressive neurodegenerative inherited disorder caused by the low expression of frataxin, which is a small mitochondrial protein. In FRDA cells, the lack of frataxin promotes primarily mitochondrial dysfunction, an alteration of calcium (Ca2+) homeostasis and the destabilization of the actin cytoskeleton in the neurites and growth cones of sensory neurons. In this paper, a computational multilinear algebra approach was used to analyze the dynamics of the growth cone and its function in control and FRDA neurons. Computational approach, which includes principal component analysis and a multilinear algebra method, is used to quantify the dynamics of the growth cone (GC) morphology of sensory neurons from the dorsal root ganglia (DRG) of the YG8sR humanized murine model for FRDA. It was confirmed that the dynamics and patterns of turning were aberrant in the FRDA growth cones. In addition, our data suggest that other cellular processes dependent on functional GCs such as axonal regeneration might also be affected. Semiautomated computational approaches are presented to quantify differences in GC behaviors in neurodegenerative disease. In summary, the deficiency of frataxin has an adverse effect on the formation and, most importantly, the growth cones’ function in adult DRG neurons. As a result, frataxin deficient DRG neurons might lose the intrinsic capability to grow and regenerate axons properly due to the dysfunctional GCs they build.

UNC-6/Netrin is a secreted conserved guidance cue that regulates dorsal-ventral axon guidance of Caenorhabditis elegans and in the vertebral spinal cord. In the polarity/protrusion model of VD growth cone guidance away from ventrally expressed UNC-6 (repulsion), UNC-6 first polarizes the growth cone via the UNC-5 receptor such that filopodial protrusions are biased dorsally. UNC-6 then regulates a balance of protrusion in the growth cone based upon this polarity. UNC-5 inhibits protrusion ventrally, and the UNC-6 receptor UNC-40/DCC stimulates protrusion dorsally, resulting in net dorsal growth cone outgrowth. UNC-5 inhibits protrusion through the flavin monooxygenases FMO-1, 4, and 5 and possible actin destabilization, and inhibits pro-protrusive microtubule entry into the growth cone utilizing UNC-33/CRMP. The PH/MyTH4/FERM myosin-like protein was previously shown to act with UNC-5 in VD axon guidance utilizing axon guidance endpoint analysis. Here, we analyzed the effects of MAX-1 on VD growth cone morphology during outgrowth. We found that max-1 mutant growth cones were smaller and less protrusive than wild type, the opposite of the unc-5 mutant phenotype. Furthermore, genetic interactions suggest that MAX-1 might normally inhibit UNC-5 activity, such that in a max-1 mutant growth cone, UNC-5 is overactive. Our results, combined with previous studies suggesting that MAX-1 might regulate UNC-5 levels in the cell or plasma membrane localization, suggest that MAX-1 attenuates UNC-5 signaling by regulating UNC-5 stability or trafficking. Alternately, MAX-1 might inhibit UNC-5 independent of this known mechanism. We also show that the effects of MAX-1 in growth cone protrusion are independent of UNC-40/DCC, UNC-33/CRMP, and UNC-34/Enabled. In summary, in the context of growth cone protrusion, MAX-1 inhibits UNC-5, demonstrating the mechanistic insight that can be gained by analyzing growth cones during outgrowth in addition to axon guidance endpoint analysis. [ABSTRACT FROM AUTHOR]

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly. [ABSTRACT FROM AUTHOR]

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3,a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5,a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background.We show that ccd-5 acts in a pathway with cdk-5, aex-3,and unc-5.Navigationdefectsof follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons. [ABSTRACT FROM AUTHOR]

Proteases comprise a variety of enzymes defined by their ability to catalytically hydrolyze the peptide bonds of other proteins, resulting in protein lysis. Cathepsins, specifically, encompass a class of at least twenty proteases with potent endopeptidase activity. They are located subcellularly in lysosomes, organelles responsible for the cell’s degradative and autophagic processes, and are vital for normal lysosomal function. Although cathepsins are involved in a multitude of cell signaling activities, this chapter will focus on the role of cathepsins (with a special emphasis on Cathepsin B) in neuronal plasticity. We will broadly define what is known about regulation of cathepsins in the central nervous system and compare this with their dysregulation after injury or disease. Importantly, we will delineate what is currently known about the role of cathepsins in axon regeneration and plasticity after spinal cord injury. It is well established that normal cathepsin activity is integral to the function of lysosomes. Without normal lysosomal function, autophagy and other homeostatic cellular processes become dysregulated resulting in axon dystrophy. Furthermore, controlled activation of cathepsins at specialized neuronal structures such as axonal growth cones and dendritic spines have been positively implicated in their plasticity. This chapter will end with a perspective on the consequences of cathepsin dysregulation versus controlled, localized regulation to clarify how cathepsins can contribute to both neuronal plasticity and neurodegeneration.

Precise wiring of neural circuits is essential for brain connectivity and function. During development, axons respond to diverse cues present in the extracellular matrix or at the surface of other cells to navigate to specific targets, where they establish precise connections with post-synaptic partners. Cell adhesion molecules (CAMs) represent a large group of structurally diverse proteins well known to mediate adhesion for neural circuit assembly. Through their adhesive properties, CAMs act as major regulators of axon navigation, fasciculation, and synapse formation. While the adhesive functions of CAMs have been known for decades, more recent studies have unraveled essential, non-adhesive functions as well. CAMs notably act as guidance cues and modulate guidance signaling pathways for axon pathfinding, initiate contact-mediated repulsion for spatial organization of axonal arbors, and refine neuronal projections during circuit maturation. In this review, we summarize the classical adhesive functions of CAMs in axonal development and further discuss the increasing number of other non-adhesive functions CAMs play in neural circuit assembly.

The correct wiring of neuronal circuits depends on outgrowth and guidance of neuronal processes during development. In the past two decades, great progress has been made in understanding the molecular basis of axon outgrowth and guidance. Genetic analysis in Caenorhabditis elegans has played a key role in elucidating conserved pathways regulating axon guidance, including Netrin signaling, the slit Slit/Robo pathway, Wnt signaling, and others. Axon guidance factors were first identified by screens for mutations affecting animal behavior, and by direct visual screens for axon guidance defects. Genetic analysis of these pathways has revealed the complex and combinatorial nature of guidance cues, and has delineated how cues guide growth cones via receptor activity and cytoskeletal rearrangement. Several axon guidance pathways also affect directed migrations of non-neuronal cells in C. elegans, with implications for normal and pathological cell migrations in situations such as tumor metastasis. The small number of neurons and highly stereotyped axonal architecture of the C. elegans nervous system allow analysis of axon guidance at the level of single identified axons, and permit in vivo tests of prevailing models of axon guidance. C. elegans axons also have a robust capacity to undergo regenerative regrowth after precise laser injury (axotomy). Although such axon regrowth shares some similarities with developmental axon outgrowth, screens for regrowth mutants have revealed regeneration-specific pathways and factors that were not identified in developmental screens. Several areas remain poorly understood, including how major axon tracts are formed in the embryo, and the function of axon regeneration in the natural environment.

The extracellular matrix (ECM) plays a critical role in development, homeostasis, and regeneration of tissue structures and functions. Cell interactions with the ECM are dynamic and cells respond to ECM remodeling by changes in morphology and motility. During nerve regeneration, the ECM facilitates neurite outgrowth and guides axons with target specificity. Decellularized ECMs retain structural, biochemical, and biomechanical cues of native ECM and have the potential to replace damaged matrix to support cell activities during tissue repair. To determine the ECM components that contribute to nerve regeneration, we analyzed neuron-ECM interactions on two types of decellularized ECM. One matrix was composed primarily of fibronectin (FN) fibrils, and the other FN-rich ECM also contained significant numbers of type I collagen (COL I) fibrils. Using primary neurons dissociated from superior cervical ganglion (SCG) explants, we found that neurites were extended on both matrices without a significant difference in average neurite length after 24 h. The most distinctive features of neurites on the FN matrix were numerous short actin-filled protrusions and longer branches extending from neurite shafts. Very few protrusions and branches were detected on FN-COL matrix. Growth cone morphologies also differed with mostly filopodial growth cones on FN matrix whereas on FN-COL matrix, equivalent numbers of filopodial and slender growth cones were formed. Our work provides new information about how changes in major components of the ECM during tissue repair modulate neuron and growth cone morphologies and helps to define the contributions of neuron-ECM interactions to nerve development and regeneration. [ABSTRACT FROM AUTHOR]