Axon Guidance Cues Research Articles

Restoration of the Topological Organization of the Trigeminal System Following Trigeminal Nerve Root Injury in the Lamprey

Two issues were examined regarding the trigeminal system in larval lampreys: (1) for normal animals, double labeling was used to confirm that the trigeminal system has a topological organization; (2) following trigeminal nerve root transections, double labeling was used to test whether the topological organization of the trigeminal system is restored. First, for normal animals, Alexa 488 dextran amine applied to the medial oral hood (anterior head) labeled trigeminal motoneurons (MNs) in the ventromedial part of the trigeminal motor nuclei (nVm) and axons of trigeminal sensory neurons (SNs) in the ventromedial part of the trigeminal descending tracts (dV). Also, Texas red dextran amine (TRDA) applied to the lateral oral hood labeled trigeminal MNs in the dorsolateral nVm and sensory axons in the dorsolateral dV. These results confirm the topological organization of the trigeminal system of normal lampreys. Second, following trigeminal nerve root transections, the physical integrity of the nerves was restored during growth of trigeminal sensory and motor axons. In addition, double labeling indicated a restoration and refinement of the topological organization of the trigeminal system with increasing recovery times, but mainly for nVm. Despite the paucity of growth of trigeminal sensory axons in dV even at long recovery times (12–16 wks), a substantial percentage of experimental animals recovered trigeminal-evoked swimming responses and trigeminal-evoked synaptic responses in reticulospinal (RS) neurons. Following trigeminal nerve root injury, several mechanisms, including axonal guidance cues, probably contribute to the substantial restoration of the topological organization of the lamprey trigeminal system.

Reduced gene expression of netrin family members in skin and sural nerve specimens of patients with painful peripheral neuropathies.

To investigate the expression of axon guidance cues in skin and sural nerve biopsies of patients with polyneuropathies (PNP) as potential markers of nerve de- and regeneration and inflammation. We prospectively recruited 88 patients with PNP and compared data between patient subgroups and healthy controls. All patients underwent skin punch and/or sural nerve biopsy at the lower leg and proximal thigh. We characterized gene expression profiles of netrin family members as target genes involved in neuronal de- and regeneration [netrin 1, deleted in colorectal cancer (DCC), uncoordinated5H2, neogenin 1 (NEO1), netrin G1, netrin G2] using quantitative real-time PCR. Gene expression of netrin 1 (p < 0.05 in proximal skin), DCC (p < 0.001 in distal skin), NEO1 (p < 0.05 in distal skin), netrin G1 (p < 0.05 in proximal and p < 0.01 in distal skin), and netrin G2 (p < 0.001 in distal skin) was lower in skin biopsies of patients with neuropathy compared to healthy controls. Gene expression of NEO1 (p < 0.05 in distal skin), netrin G2 (p < 0.05 in distal skin), and DCC (p < 0.05 in sural nerve) was lower in samples of patients with painful compared to painless PNP and also correlated positively with intraepidermal nerve fiber density. Skin and sural nerve gene expression of the investigated target genes did not differ between neuropathies of different etiologies. We show reduced cutaneous and neural axon guide expression, which may contribute to a dysregulation of nerve fiber de- and regeneration.

Ex Vivo Oculomotor Slice Culture from Embryonic GFP-Expressing Mice for Time-Lapse Imaging of Oculomotor Nerve Outgrowth.

Accurate eye movements are crucial for vision, but the development of the ocular motor system, especially the molecular pathways controlling axon guidance, has not been fully elucidated. This is partly due to technical limitations of traditional axon guidance assays. To identify additional axon guidance cues influencing the oculomotor nerve, an ex vivo slice assay to image the oculomotor nerve in real-time as it grows towards the eye was developed. E10.5 IslMN-GFP embryos are used to generate ex vivo slices by embedding them in agarose, slicing on a vibratome, then growing them in a microscope stage-top incubator with time-lapse photomicroscopy for 24-72 h. Control slices recapitulate the in vivo timing of outgrowth of axons from the nucleus to the orbit. Small molecule inhibitors or recombinant proteins can be added to the culture media to assess the role of different axon guidance pathways. This method has the advantages of maintaining more of the local microenvironment through which axons traverse, not axotomizing the growing axons, and assessing the axons at multiple points along their trajectory. It can also identify effects on specific subsets of axons. For example, inhibition of CXCR4 causes axons still within the midbrain to grow dorsally rather than ventrally, but axons that have already exited ventrally are not affected.

Abstract 1725: Mutationally activated KRAS(G12D) upregulates the expression of axonal guidance cue Semaphorin-5A in pancreatic cancer

Abstract Mutation of KRAS is commonly found in different human cancers and is generally responsible for driving tumorigenesis. Unfortunately, direct inhibitors of KRAS are not available. Thus, scientists are in search of alternative strategies that can repress the oncogenic properties of KRAS and can benefit a large number of cancer patients. Drugs targeting KRAS downstream effectors are under investigation but are facing numerous challenges in their clinical implementation. Accumulating evidence suggests that the genetic status of KRAS can modulate the role of axonal guidance cue family members in tumorigenesis. Previous reports from our laboratory have identified upregulation of one such molecule Semaphorin-5A (SEMA5A) in murine model based on KRAS gene mutation in pancreatic progenitor cells that bear a striking similarity to the human PDAC condition. We utilized PDX-cre-LSL-Kras(G12D) (KC) for studying Sema5A expression and observed no expression of Sema5A in the normal pancreas, derived from the control PDX-cre mice, however, we observed Sema5A expression in PDX-cre-LSL-Kras(G12D) mice beginning at ten weeks of age. Furthermore, we observed an increase in SEMA5A expression in the tumors of mice aged at twenty-five and fifty weeks, representing a fully developed PDAC condition. To investigate the relationship between KRAS status and SEMA5A, we assessed whether KRAS(G12D) alters the expression of SEMA5A using immortalized human pancreatic ductal cells having endogenous wild-type KRAS but an exogenous expression of KRAS(G12D) [HPNE-KRAS]. We detected higher expression of SEMA5A in the KRAS(G12D) bearing HPNE-KRAS cells in comparison with their control counterpart HPNE-Tert. Stable clones of CD18/HPAF cell line knocked-down for KRAS(G12D) demonstrated inhibition of SEMA5A expression in comparison with their respective Control CD18/HPAF cells bearing an endogenous expression of mutant KRAS. We also analyzed SEMA5A expression in the cells lines derived from the serial passage of COLO 357 cells through mice to achieve highly invasive/metastatic variants also leading to the selection of cells that express mutationally activated KRAS. L3.3 cells, with no point mutations in the KRAS gene, expressed lower SEMA5A in comparison with highly metastatic L3.6pl cells bearing a point mutation in codon 12, thereby encoding a mutationally activated KRAS(G12D). Together, our data suggest that KRAS(G12D) mutation directly upregulates the expression of axon guidance cue molecule-SEMA5A in the PDAC cells. Our findings open an avenue of molecules that can serve as secondary targets to repress KRAS activity in pancreatic cancer. Citation Format: Sugandha Saxena, Babita Tomar, Lingyun Wu, Mohammad Awaji, Satyanarayan Rachagani, Michel J. Ouellette, Surinder K. Batra, Bhavana J. Dave, Rakesh K. Singh. Mutationally activated KRAS(G12D) upregulates the expression of axonal guidance cue Semaphorin-5A in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1725.

RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in Caenorhabditis elegans.

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.

Pain-associated Mediators and Axon Pathfinders in Fibromyalgia Skin Cells

To investigate whether the expression of cytokine, nociception-associated ion channel, and axon guidance genes in patients with skin cell fibromyalgia syndrome (FMS) differs from healthy controls, potentially contributing to pain and small-fiber degeneration in FMS. We prospectively recruited 128 patients and 26 healthy controls. All study participants underwent neurological examination, and a skin punch biopsy was obtained from the lateral calf and thigh. Skin samples were processed to histologically determine intraepidermal nerve fiber density (IENFD) and for primary fibroblast and keratinocyte cell cultures. Gene expression of selected pro- and antiinflammatory cytokines, nociception-associated ion channels, and axon guidance cues was assessed with quantitative real-time PCR. In fibroblasts, transforming growth factor-ß1 (TGF-ß1) gene expression was higher in patients with FMS compared to controls (calf and thigh: p < 0.001). Also, expression was higher in patients than in controls for these variables: hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (calf: p < 0.01), ephrin-A4 (EFNA4; calf: p < 0.05, thigh: p < 0.001), and ephrin receptor-A4 (EPHA4; thigh: p < 0.05). In keratinocytes, interleukin 10 gene expression was higher in patients with FMS than in controls (thigh: p < 0.05). While no intergroup difference was found for nociception-associated ion channels, EFNA4 and EPHA4 (calf: p < 0.01 each) expression was higher in patients with FMS than in controls. Axon guide expression did not correlate with IENFD. In FMS, skin cells may contribute to cutaneous nociception by differentially expressing membrane-bound and soluble pain mediators and axon pathfinders.

Drosophila Tet Is Expressed in Midline Glia and Is Required for Proper Axonal Development.

Ten-Eleven Translocation (TET) proteins are important epigenetic regulators that play a key role in development and are frequently deregulated in cancer. Drosophila melanogaster has a single homologous Tet gene (dTet) that is highly expressed in the central nervous system during development. Here, we examined the expression pattern of dTet in the third instar larval CNS and discovered its presence in a specific set of glia cells: midline glia (MG). Moreover, dTet knockdown resulted in significant lethality, locomotor dysfunction, and alterations in axon patterning in the larval ventral nerve cord. Molecular analyses on dTet knockdown larvae showed a downregulation in genes involved in axon guidance and reduced expression of the axon guidance cue Slit. Our findings point toward a potential role for dTet in midline glial function, specifically the regulation of axon patterning during neurodevelopment.

NR4A3 fusion proteins trigger an axon guidance switch that marks the difference between EWSR1 and TAF15 translocated extraskeletal myxoid chondrosarcomas.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma histotype with uncertain differentiation. EMC is hallmarked by the rearrangement of the NR4A3 gene, which in most cases fuses with EWSR1 or TAF15. TAF15‐translocated EMC seem to feature a more aggressive course compared to EWSR1‐positive EMCs, but whether the type of NR4A3 chimera impinges upon EMC biology is still largely undefined. To gain insights on this issue, a series of EMC samples (7 EWSR1‐NR4A3 and 5 TAF15‐NR4A3) were transcriptionally profiled. Our study unveiled that the two EMC variants display a distinct transcriptional profile and that the axon guidance pathway is a major discriminant. In particular, class 4–6 semaphorins and axonal guidance cues endowed with pro‐tumorigenic activity were more expressed in TAF15‐NR4A3 tumors; vice versa, class 3 semaphorins, considered to convey growth inhibitory signals, were more abundant in EWSR1‐NR4A3 EMC. Intriguingly, the dichotomy in axon guidance signaling observed in the two tumor variants was recapitulated in in vitro cell models engineered to ectopically express EWSR1‐NR4A3 or TAF15‐NR4A3. Moreover, TAF15‐NR4A3 cells displayed a more pronounced tumorigenic potential, as assessed by anchorage‐independent growth. Overall, our results indicate that the type of NR4A3 chimera dictates an axon guidance switch and impacts on tumor cell biology. These findings may provide a framework for interpretation of the different clinical–pathological features of the two EMC variants and lay down the bases for the development of novel patient stratification criteria and therapeutic approaches. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Semaphorins and Their Receptors in Hematological Malignancies.

While semaphorins were initially identified as axonal guidance cues for wiring the neural network, it was then recognized their wide relevance in tissue development and homeostasis. Notably, semaphorin activities were also extensively studied in many types of solid tumors; however, their relevance in hematological malignancies is far from understood. In this mini-review, we surveyed the current knowledge about semaphorins and their receptors in leukemias, lymphomas, and multiple myeloma. Noteworthy, current data support a promoting role for Semaphorin 4D and Neuropilin-1 in these tumors, while Semaphorin 3A seems to consistently act as oncosuppressor in leukemias and multiple myeloma. The expression levels and functional activities of SEMA3B, SEMA3F, and Neuropilin-2 have furthermore been investigated in leukemias and lymphoma cells. Herein, we reviewed the state of the art and highlighted some of the open questions to be addressed in the field.

Multifaceted Functional Role of Semaphorins in Glioblastoma.

Glioblastoma (GBM) is the most malignant tumor type affecting the adult central nervous system. Despite advances in therapy, the prognosis for patients with GBM remains poor, with a median survival of about 15 months. To date, few treatment options are available and recent trials based on the molecular targeting of some of the GBM hallmark pathways (e.g., angiogenesis) have not produced any significant improvement in overall survival. The urgent need to develop more efficacious targeted therapies has led to a better molecular characterization of GBM, revealing an emerging role of semaphorins in GBM progression. Semphorins are a wide group of membrane-bound and secreted proteins, originally identified as axon guidance cues, signaling through their receptors, neuropilins, and plexins. A number of semaphorin signals involved in the control of axonal growth and navigation during development have been found to furthermore participate in crosstalk with different dysfunctional GBM pathways, controlling tumor cell proliferation, migration, and invasion, as well as tumor angiogenesis or immune response. In this review, we summarize the regulatory activities mediated by semaphorins and their receptors on the oncogenic pathways implicated in GBM growth and invasive/metastatic progression.

Regulation of subcellular dendritic synapse specificity by axon guidance cues.

Neural circuit assembly occurs with subcellular precision, yet the mechanisms underlying this precision remain largely unknown. Subcellular synaptic specificity could be achieved by molecularly distinct subcellular domains that locally regulate synapse formation, or by axon guidance cues restricting access to one of several acceptable targets. We address these models using two Drosophila neurons: the dbd sensory neuron and the A08a interneuron. In wild-type larvae, dbd synapses with the A08a medial dendrite but not the A08a lateral dendrite. dbd-specific overexpression of the guidance receptors Unc-5 or Robo-2 results in lateralization of the dbd axon, which forms anatomical and functional monosynaptic connections with the A08a lateral dendrite. We conclude that axon guidance cues, not molecularly distinct dendritic arbors, are a major determinant of dbd-A08a subcellular synapse specificity.

Computationally Modeling Axonal Guidance Cue Expression in The Developing Olfactory Bulb

Olfactory sensory neurons (OSNs) in most mammals have cell bodies in the olfactory epithelium (OE). OSNs project axons to glomeruli in the olfactory bulb (OB) of the brain. Developmentally, signal guidance cues direct axons to their target glomeruli. We created a program that displays a model of the OB using a series of sagittal section images based on in situ hybridization (ISH) images from the Allen Brain Atlas (ABA). The ABA's program, Brain Explorer 2, allows for the limited 3D visualization of gene expression data. Our program adds functionality by allowing users to clearly visualize and quantify the expression of several genes simultaneously via superimposition.To optimize visualization of multiple ISH data sets simultaneously, we aimed to improve the signal to noise ration of those ISH data. We use color thresholding and density thresholding algorithms to computationally detect and filter guidance cue expression data. Color thresholds that establish gene expression from the background of the image were determined using ImageJ. The expression data were filtered using a density threshold in a 2D sliding window algorithm. The 2D sliding window algorithm is implemented for every pixel in the image. It distinguishes a window of the image around each pixel. The density of gene expression within that window is calculated. The expression for the current pixel is then decided using a density threshold. Methods for visualization of the superimposed genes were derived using the matplotlib and PIL libraries. Our program provides a tool for investigating olfactory axon targeting, glomeruli formation and glomeruli related pathologies in mammalian models. For example, users can examine the expression of different genes and investigate if guidance cue expression results in unique locations for mapping specific glomeruli. This program can also be adapted to examine guidance cue expression for other regions of nervous tissue.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recurrent Laryngeal Nerve Reinnervation in Rats Posttransection: Neurotrophic Factor Expression over Time.

Recurrent laryngeal nerve (RLN) injury causes vocal fold paralysis from which functional recovery is typically absent due to nonselective reinnervation. This study investigates expression of axon guidance cues and their modulators relative to the chronology of reinnervation by examining the expression of glial-derived neurotrophic factor (GDNF), netrin 1, and laminin 111 (LAMA1) in nonpooled laryngeal muscles. This study is the first to describe the post-RLN injury expression pattern of LAMA1, a target of particular interest as it has been shown to switch netrin 1-mediated growth cone attraction to repulsion. Animal experiment (rat model). Basic science laboratory. The right RLNs of 64 female Sprague-Dawley rats were transected, with sacrifice at 1, 3, 7, 21, 28, and 56 days postinjury (DPI). Single-animal messenger RNA was isolated from the ipsilateral posterior cricoarytenoid (PCA), lateral thyroarytenoid (LTA), and medial thyroarytenoid (MTA) for quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. Immunostaining for LAMA1 expression was performed in the same muscles. LAMA1 was elevated in the PCA at 3 to 56 DPI, LTA at 7 DPI, and MTA at 14 and 28 DPI. This correlates with the chronology of laryngeal reinnervation. Using a new protocol, single-animal muscle qRT-PCR possible and expression results for GDNF and netrin 1 were similar to previous pooled investigations. Reliable qRT-PCR is possible with single rat laryngeal muscles. The expression of netrin 1 and LAMA1 is chronologically coordinated with muscle innervation in the LTA and MTA. This suggests that LAMA1 may influence netrin 1 to repel axons and delay LTA and MTA reinnervation.

Nell2 regulates the contralateral-versus-ipsilateral visual projection as a domain-specific positional cue.

In mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific domains in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN), underpinning disparity-based stereopsis. Although domain-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specific retinogeniculate projection, the molecular nature of such cues has remained elusive. Here, we show that the extracellular glycoprotein Nell2 (neural epidermal growth factor-like-like 2) is expressed in the dorsomedial region of the dLGN, which ipsilateral RGC axons terminate in and contralateral axons avoid. In Nell2 mutant mice, contralateral RGC axons abnormally invaded the ipsilateral domain of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon-termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a domain-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in the establishment of the eye-specific retinogeniculate projection.

Inhibition of Semaphorin3A Promotes Ocular Dominance Plasticity in the Adult Rat Visual Cortex.

Perineuronal nets (PNNs) are condensed structures in the extracellular matrix that mainly surround GABA-ergic parvalbumin-positive interneurons in the adult brain. Previous studies revealed a parallel between PNN formation and the closure of the critical period. Moreover, ocular dominance plasticity is enhanced in response to PNN manipulations in adult animals. However, the mechanisms through which perineuronal nets modulate plasticity are still poorly understood. Recent work indicated that perineuronal nets may convey molecular signals by binding and storing proteins with important roles in cellular communication. Here we report that semaphorin3A (Sema3A), a chemorepulsive axon guidance cue known to bind to important perineuronal net components, is necessary to dampen ocular dominance plasticity in adult rats. First, we showed that the accumulation of Sema3A in PNNs in the visual cortex correlates with critical period closure, following the same time course of perineuronal nets maturation. Second, the accumulation of Sema3A in perineuronal nets was significantly reduced by rearing animals in the dark in the absence of any visual experience. Finally, we developed and characterized a tool to interfere with Sema3A signaling by means of AAV-mediated expression of receptor bodies, soluble proteins formed by the extracellular domain of the endogenous Sema3A receptor (neuropilin1) fused to a human IgG Fc fragment. By using this tool to antagonize Sema3A signaling in the adult rat visual cortex, we found that the specific inhibition of Sema3A promoted ocular dominance plasticity. Thus, Sema3A accumulates in perineuronal nets in an experience-dependent manner and its presence in the mature visual cortex inhibits plasticity.

Single-cell transcriptional logic of cell-fate specification and axon guidance in early-born retinal neurons.

Retinal ganglion cells (RGCs), cone photoreceptors (cones), horizontal cells and amacrine cells are the first classes of neurons produced in the retina. However, an important question is how this diversity of cell states is transcriptionally produced. Here, we profiled 6067 single retinal cells to provide a comprehensive transcriptomic atlas showing the diversity of the early developing mouse retina. RNA velocities unveiled the dynamics of cell cycle coordination of early retinogenesis and define the transcriptional sequences at work during the hierarchical production of early cell-fate specification. We show that RGC maturation follows six waves of gene expression, with older-generated RGCs transcribing increasing amounts of guidance cues for young peripheral RGC axons that express the matching receptors. Spatial transcriptionally deduced features in subpopulations of RGCs allowed us to define novel molecular markers that are spatially restricted. Finally, the isolation of such a spatially restricted population, ipsilateral RGCs, allowed us to identify their molecular identity at the time they execute axon guidance decisions. Together, these data represent a valuable resource shedding light on transcription factor sequences and guidance cue dynamics during mouse retinal development.

Netrin-1-like-immunoreactivity Coexpresses With DCC and Has a Differential Level in the Myenteric Cholinergic and Nitrergic Neurons of the Adult Mouse Colon.

Netrin-1 is a potent axonal and neuronal guidance cue in the developing nervous system. Netrin-1 functions are mediated by its receptors, such as deleted in colorectal cancer (DCC) present on axons and neurons. Localization of DCC and Netrin-1 on various types of enteric neurons and their role in the mature enteric nervous system is unknown. The results of our study revealed that almost all enteric neurons and processes express DCC and Netrin-1 in the adult mice. Netrin-1-like-immunoreactivity (IR) was detected in the cytoplasm of neurons with some showing strong or weak staining. The majority of Netrin-1-like-immunoreactive enteric neurons were choline acetyltransferase (ChAT)-positive. However, ~19% of neurons were strongly Netrin-1-like-positive but ChAT-negative while ~8% of neurons were Netrin-1-like-negative but strongly ChAT-positive. In contrast, almost all nitric oxide synthase (nNOS)-positive enteric neurons displayed strong Netrin-1-like-IR. This differential intensity of Netrin-1 expression in the myenteric neurons might determine major neuronal subtypes regulating intestinal motility, ChAT-IR excitatory, and nNOS-IR inhibitory muscle motor and interneurons. This is the first study demonstrating the localization of DCC and Netrin-1 in the colonic myenteric plexus of the adult mice and their expression level determining two major neuronal subtypes regulating intestinal motility.

Return of function after CNS axon regeneration: Lessons from injury-responsive intrinsically photosensitive and alpha retinal ganglion cells.

This review addresses issues relating to the survival and axon regeneration of both intrinsically photosensitive retinal ganglion cells (ipRGC) and αRGC, and possible ensuing patterns of functional recovery after optic nerve crush, all of which are broadly relevant to recovery from injury in the central nervous system (CNS) as whole. Although much needs to be clarified about the connectivity, function and patterns of myelination of regenerated CNS axons, the results of recent research on activity-induced αRGC axon regeneration associated with functional restitution have highlighted key focal obstacles to recovery including neurotrophic support, axon misguidance, target recognition failure and dysmyelination. Pan RGC survival/axon regeneration requires receptor binding and downstream signalling by a cocktail of growth factors, more generally defined in the CNS by the individual trophic requirements of neuronal subsets within a given disconnected centre. Resolution of the problem of failed axon guidance and target recognition is complicated by a confounding paradox that axon growth inhibitory ligand disinhibition required for axon regeneration may mask axon guidance cues that are essential for accurate re-innervation. The study of the temporal parameters of remyelination of regenerated αRGC axons may become feasible if they establish permanent homologous connections, allowing time for new myelin sheaths to fully form. Unless near complete re-innervation of denervated targets is re-instated in the CNS, debilitating dysfunctional neurological sequelae may ensue from the resulting imbalance in connectivity.

Loss of CXCR4/CXCL12 Signaling Causes Oculomotor Nerve Misrouting and Development of Motor Trigeminal to Oculomotor Synkinesis.

PurposeProper control of eye movements is critical to vision, but relatively little is known about the molecular mechanisms that regulate development and axon guidance in the ocular motor system or cause the abnormal innervation patterns (oculomotor synkinesis) seen in developmental disorders and after oculomotor nerve palsy. We developed an ex vivo slice assay that allows for live imaging and molecular manipulation of the growing oculomotor nerve, which we used to identify axon guidance cues that affect the oculomotor nerve.MethodsEx vivo slices were generated from E10.5 IslMN-GFP embryos and grown for 24 to 72 hours. To assess for CXCR4 function, the specific inhibitor AMD3100 was added to the culture media. Cxcr4cko/cko:Isl-Cre:ISLMN-GFP and Cxcl12KO/KO:ISLMN-GFP embryos were cleared and imaged on a confocal microscope.ResultsWhen AMD3100 was added to the slice cultures, oculomotor axons grew dorsally (away from the eye) rather than ventrally (toward the eye). Axons that had already exited the midbrain continued toward the eye. Loss of Cxcr4 or Cxcl12 in vivo caused misrouting of the oculomotor nerve dorsally and motor axons from the trigeminal motor nerve, which normally innervate the muscles of mastication, aberrantly innervated extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis.ConclusionsCXCR4/CXCL12 signaling is critical for the initial pathfinding decisions of oculomotor axons and their proper exit from the midbrain. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles lacking innervation may secrete cues that attract motor axons.

The Role of Apoptotic Signaling in Axon Guidance.

Navigating growth cones are exposed to multiple signals simultaneously and have to integrate competing cues into a coherent navigational response. Integration of guidance cues is traditionally thought to occur at the level of cytoskeletal dynamics. Drosophila studies indicate that cells exhibit a low level of continuous caspase protease activation, and that axon guidance cues can activate or suppress caspase activity. We base a model for axon guidance on these observations. By analogy with other systems in which caspase signaling has non-apoptotic functions, we propose that caspase signaling can either reinforce repulsion or negate attraction in response to external guidance cues by cleaving cytoskeletal proteins. Over the course of an entire trajectory, incorrectly navigating axons may pass the threshold for apoptosis and be eliminated, whereas axons making correct decisions will survive. These observations would also explain why neurotrophic factors can act as axon guidance cues and why axon guidance systems such as Slit/Robo signaling may act as tumor suppressors in cancer.