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Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. In this study, we report the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. We demonstrate that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon--DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material.

Members of the bone morphogenetic protein family of secreted protein signals have been implicated as axon guidance cues for specific neurons in Caenorhabditis elegans and in mammals. We have examined axonal pathfinding in mice lacking the secreted bone morphogenetic protein antagonist Noggin. We have found defects in projection of several groups of neurons, including the initial ascending projections from the dorsal root ganglia, motor axons innervating the distal forelimb, and cranial nerve VII. The case of the dorsal root ganglion defect is especially interesting: initial projections from the dorsal root ganglion enter the dorsal root entry zone, as normal, but then project directly into the gray matter of the spinal cord, rather than turning rostrally and caudally. Explant experiments suggest that the defect lies within the spinal cord and not the dorsal root ganglion itself. However, exogenous bone morphogenetic proteins are unable to attract or repel these axons, and the spinal cord shows only very subtle alterations in dorsal-ventral pattern in Noggin mutants. We suggest that the defect in projection into the spinal cord is likely the result of bone morphogenetic proteins disrupting the transduction of some unidentified repulsive signal from the spinal cord gray matter. Key Words: Noggin; BMP; axon guidance.

Growth cone motility and guidance depend on the dynamic reorganization of filamentous actin (F-actin). In the growth cone, F-actin undergoes turnover, which is the exchange of actin subunits from existing filaments. However, the function of F-actin turnover is not clear. We used jasplakinolide (jasp), a cell-permeable macrocyclic peptide that inhibits F-actin turnover, to study the role of F-actin turnover in axon extension. Treatment with jasp caused axon retraction, demonstrating that axon extension requires F-actin turnover. The retraction of axons in response to the inhibition of F-actin turnover was dependent on myosin activity and regulated by RhoA and myosin light chain kinase. Significantly, the endogenous myosin-based contractility was sufficient to cause axon retraction, because jasp did not alter myosin activity. Based on these observations, we asked whether guidance cues that cause axon retraction (ephrin-A2) inhibit F-actin turnover. Axon retraction in response to ephrin-A2 correlated with decreased F-actin turnover and required RhoA activity. These observations demonstrate that axon extension depends on an interaction between endogenous myosin-driven contractility and F-actin turnover, and that guidance cues that cause axon retraction inhibit F-actin turnover.

Results show that axons possess complete machinery for protein synthesis and cell surface expression. Data suggest that individual isolated axons and growth cones can effect protein translation as well as export of the proteins to the cell surface, indicating that an RNA-based mechanism is localized within a specific region of the axons.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TENT [H.sub.c]). Morphological and kinetic analysis of TeNT [H.sub.c] retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT [H.sub.c] carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT [H.sub.c] and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor [p7.sup.5NTR]. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated [K.sup.+] (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 [alpha] subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A[delta] and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or [Na.sup.+] channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 [alpha] subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.

Results demonstrate that sunday driver protein transports the axonal transport kinesin driven vesicles by interacting with the kinesin light chain. Data reveal that sunday driver protein is localized to tubulovesicular structures and forms complex with kinesin-1 in in vivo.

A study was conducted to identify the molecular mechanism that guide axons along their stereotypical pathways and to determine how axons respond to bifunctional guidance cues. Experiments with the Xenopus axon turning assay revealed that transmembrane receptors determine whether the growth cone along the axons' pathway see guidance cues as attractive or repulsive. It was also found that heteromeric receptors control the response of growth cones to bifunctional guidance cues.

Autosomal dominant hereditary spastic paraplegia (HSP) has been shown, through analysis of a Caucasian kindred, to have a novel locus on chromosome 8q23-24. The locus takes in 6.2 cM between D8S1804 and D8S1774, and several potential candidate genes exist. Genetic loci exist on chromosomes 2p, 14q and 15q, but those loci are not a fit in 45% of autosomal dominant HSP kindreds.

Neurons use molecular motors to power the transport of cargoes along their axonal extensions. Fresh evidence challenges the view that cellular organelles called mitochondria are the main energy providers for [...]

The effects of netrin-1 on commisural axions of chicks are mediated by the Deleted in Colorectal Cancer (DCC) gene via cellular signal transduction pathway. The DCC gene is a netrin-1 binding protein that is expressed on the axons and growth cones of spinal commissural neurons during their migration to the floor plate. The binding protein also acts as a receptor during the guidance of commissural axons from the dorsal to the ventral midline region of the spinal cord.

Experiments testing if synaptic stimulation of excitatory amino acid receptors in rostral ventrolateral medulla (RVLM) is vital for the regulation of central pressor responses reveal that axonal conduction and synaptic transmission collectively regulate central pressor responses inside and outside the RVLM region and that synaptic neuronal activation alone is not a uniform regulator of the responses. Simultaneous inhibition of synaptic transmission and axonal conduction result in marked decreases of pressor responses.

A unique combination of genes maintained by polyclonal cell lineage restrictions specifies the segmental identities of diencephalic units. A stereotyped sequence of neuromeric subdivisions organizes the highly complex and intricate diencephalon. The same genes appear to pattern units in the head's anterior region, and set up positional cues and pathways for growth cones in the embryonic central nervous system, for both vertebrates and arthropods.

The proportion of tubulin contained in mature motor axons that were transported in a slow component wave was determined. Class II or class III beta-tubulin were immunoprecipitated with specific monoclonal antibodies and immunohistochemistry revealed that most of the class II or class III beta-tubulins were localized within the axons. Axotomized nerve fibers revealedgreater amounts of radioactivity per millimeter of nerve in both isotypes than in control nerves. However, levels of the two isotypes in both axotomized and control nerves were similar. These results indicated that only a small fractionof the tubulin was represented by the slow component wave.

Research shows that neurons expressing different odorant receptors express ephrin-A protein at different concentrations on their axons. Results demonstrate that ephrin-A molecules, together with odorant receptors, are involved in the formation of a sensory map in the olfactory bulb.

Netrins and its receptors are involved in axon guidance in diverse animals from worms to mammals. The UNC-5 family of Netrin receptors predominantly mediates axon repulsion, and the UNC-40/DCC family of Netrin receptors mediates axon attraction. The signaling events downstream of these Netrin receptors are largely unknown. We report here the Caenorhabditis elegans max-1 gene that functions to modulate and transduce the UNC-5 signal. In wild-type worms, the ventral cord type D motor neurons grow circumferential commissures using the guidance signal of UNC-6/Netrin and UNC-5 receptor, max-1 loss of function mutations cause fully penetrant but variable defects in the D neuron commissural guidance. Weak alleles of unc-5 and unc-6 strongly enhance Max-1 phenotypes and null alleles of unc-5 and unc-6 act as dominant enhancers. In contrast, unc-40 mutations, which affect another receptor of UNC-6, do not dominantly enhance Max-1 phenotypes, max-1 encodes a novel conserved protein that has two PH, one Myth4 and one FERM domains. PH domains are known to interact with lipid membrane; FERM domains are also membrane-anchoring domains, originally found in Band4.1 protein and Ezrin/Radixin/Moesin (ERM) proteins and can interact with PDZ domains and the cytoplasmic tails of several transmembrane proteins. MAX-1 functions in motor neurons and is localized to neuronal processes. Yeast two-hybrid analysis supports a physical association of UNC-5 and MAX-1. We propose that MAX-1 may modulate UNC-5 activity by binding to UNC-5 and also acting as a scaffold to transduce UNC-5 signals in axon repulsion.

Fetuses that develop without both Myf5 and MyoD genes entirely lack skeletal myogenesis and as a consequent of that their spinal motor and proprioceptive neurons undergo complete apoptosis. Primary motor cortex of these fetuses completely lack giant pyramidal cells, but their absence is not correlated with increased levels of apoptosis. This suggests a failure in competence of progenitor cells. To further investigate how the peripheral axonal inputs influence neuronal precursor cell differentiation in various brain areas involved in motor control wild-type and mutant brains were assessed for structural and molecular differences. Mutant brain anatomical milestones appeared unaffected, but nestinexpressing clusters of neural precursor cells, the calretininexpressing pyramidal neurons, and the calbindin-expressing clusters of differentiating neurons appeared significantly less defined in mutant motor cortex area. Consistently, some areas of the extra-pyramidal tract (e.g., caudoputamen and globus pallidus) were also affected in mutant brains. Surprisingly, we were unable to detect any change in proliferation or cell death in the mutant neuroepithelium. Taken together, it appears that patterning mechanisms intrinsic to the areas for motor control specify the basic organization of its subdivisions. However, external axonal inputs seem to change intrinsic properties of neural progenitors and influence the ratios of the cell types they produce.

Although it is believed that little recovery occurs after adult mammalian spinal cord injury, in fact significant spontaneous functional improvement commonly occurs after spinal cord injury in humans. To investigate potential mechanisms underlying spontaneous recovery, lesions of defined components of the corticospinal motor pathway were made in adult rats in the rostral cervical spinal cord or caudal medulla. Following complete lesions of the dorsal corticospinal motor pathway, which contains more than 95% of all corticospinal axons, spontaneous sprouting from the ventral corticospinal tract occurred onto medial motoneuron pools in the cervical spinal cord; this sprouting was paralleled by functional recovery. Combined lesions of both dorsal and ventral corticospinal tract components eliminated sprouting and functional recovery. In addition, functional recovery was also abolished if dorsal corticospinal tract lesions were followed 5 weeks later by ventral corticospinal tract lesions. We found extensive spontaneous structural plasticity as a mechanism correlating with functional recovery in motor systems in the adult central nervous system. Experimental enhancement of spontaneous plasticity may be useful to promote further recovery after adult central nervous system injury.

Abstract Objectives--To investigate whether accommodation to depolarising and hyperpolarising currents differs for motor axons of human upper and lower limb nerves. Methods--The threshold tracking technique was used to measure threshold [...]

Dbl-homology guanine nucleotide exchange factors (DH-GEFs) regulate actin cytoskeletal reorganization, cell adhesion, and gene transcription via activation of Rho GTPases. However, little is known about the physiological role of mammalian DH-GEFs during development. The DH-GEF family member Trio is of particular interest because it is a multifunctional protein possessing two GEF domains, as well as a protein serine/threonine kinase domain, and trio-like genes in Caenorhabditis elegans and Drosophila were shown to function in neural migration and axon guidance. To determine the role of Trio during mammalian development, we generated a mouse trio loss-of-function mutation ([trio.sup.-/-]). Trio function is essential during late embryonic development as genotype analysis indicated that [trio.sup.-/-] embryos died between embryonic day (E)-15.5 and birth, or shortly thereafter. In the [trio.sup.-/-] embryos, primary skeletal myofibers were relatively normal at E14.5, but by E18.5 highly unusual spherical myofibers accumulated. Trio deficiency may cause a defect in secondary myogenesis, as the appearance of the abnormal [trio.sup.-/-] skeletal myofibers temporally coincided with the onset of secondary myogenesis, and smaller secondary myofibers located adjacent to the primary myofibers were absent. The proliferation of [trio.sup.-/-] secondary myoblasts appeared normal, suggesting that Trio may regulate secondary myoblast alignment or fusion. [trio.sup.-/-] embryos also displayed aberrant organization in several regions within the brain, including the hippocampal formation and olfactory bulb. We thus conclude that Trio is essential for late embryonic development, and that Trio functions in fetal skeletal muscle formation and in the organization of neural tissues. gene targeting | embryonic lethality | Rac | mouse development

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein-tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases. Key words: neurofilament proteins * phosphorylation * amyotrophic lateral sclerosis * Alzheimer's disease

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth. Key words: axon guidance * growth cone * neuronal cell adhesion molecules * immunoglobulin superfamily * signal transduction

INTRODUCTION The recent controversy of botulinum toxin and potential neurotoxicity to the brain has spurred on a tremendous amount of concern on the part of patients seeking this treatment for [...]

Lumbriculus variegatus, a freshwater oligochaete worm, demonstrates patterned regeneration of lost body parts and morphallaxis of original body parts (neural reflexes and behavior) following segmental amputation. Axial gradients in neurophysiological function of medial and lateral giant fibers provide a model system for the study of the neural plasticity underlying morphallaxis of synaptic function, axonal morphology, and reflexive behavior. Following isolation of anterior and posterior segments, giant fiber sensory fields and conduction velocities are modified during regeneration to reflect new segmental positions (Dev.Bio. 138:94-103). Fluorescence imaging following neurobiotin injections demonstrates strong dye-coupling between segmentally-arranged giant axons in both control and regenerating fragments. Dye-coupling coefficients, based on fluorescence intensity, range from 0.55 to 0.25 depending on segmental level. Dye-coupling between medial axons is increased and the size of lateral axons is decreased during morphallactic plasticity of posterior fragments to more anterior segmental positions. Thus, axonal size and segmental coupling are important loci of changes in giant fiber pathways during neural morphallaxis. VGM is supported by T32MH18882

A study was conducted to determine the effect of the interaction of two families of receptors, namely, the Netrin receptors of the Deleted in Colorectal Cancer (DCC) and the UNC5 family receptors in axon guidance. Experiments with Xenopus spinal cord neurons revealed that the introduction of UNC5 proteins in cells transform the spinal axon-attraction of DCC netrin receptors to axon-repulsion. This is effected by the interaction of netrin-1 to UNC5.

A study was conducted to evaluate the function of a Drosophila homolog labeled, p21-activated kinase (Pak) in R cell axon guidance. Results suggest that the Pak regulates R cell axon guidance and targeting by colocalizing and physically interacting with the R cell axons. It was also observed that a membrane-tethered form of Pak functions as a dominant gain-of-function protein. Moreover, the Pak has been found to act as the downstream effector of the SH2/SH3 adaptor protein dock.

Axonal interactions, which are mediated by cell adhesion molecules (CAMs) as well as other types of membrane proteins, are important for sensory axon pathfinding in the developing chick hindlimb. We have previously shown that injection of antibodies that block the function of either G4/L1 or N-cadherin into the limb, starting when the first sensory axons reach the plexus, alters the segmental pattern of projections along cutaneous nerves. Specific removal of polysialic acid from NCAM using the enzyme endoneuraminidase N (Endo N) also resulted in significant changes in cutaneous projection patterns, while injection of antibodies against NCAM itself had no obvious effect (M. G. Honig and U.S. Rutishauser, 1996, Dev. Biol. 175, 325-337). To help understand the cellular basis for these findings, we developed a tissue culture system in which the axons from dorsal root ganglion explants grow within defined laminin lanes and examined whether the same treatments increased or decreased a growth cone's tendency to be closely associated with neighboring axons. After 2 days in culture, images of the cultures were recorded, antibodies or Endo N was added, and images of the same fields were recaptured an hour later. To quantify the results, growth cones located in defined regions of the laminin lanes were classified, before and after the perturbation, as 'free' (i.e., growing primarily on the laminin substratum), 'fasciculated' (i.e., growing tightly along other neurites), or 'intermediate' (i.e., growing both on the laminin substratum and in contact with other neurites). We found that anti-G4/L1 and anti-N-cadherin, but not anti-NCAM, caused an increase in defasciculated growth cones, whereas Endo N resulted in an increase in fasciculated growth cones. These changes in fasciculation are consistent with the changes in cutaneous projections seen in our previous in ovo perturbations. The results from these tissue culture experiments thus provide strong support for the idea that one mechanism by which CAMs affect sensory axon pathfinding in vivo is by regulating the affinity of sensory growth cones for neighboring axons, which in turn can modulate the growth cone's ability to navigate through the surrounding environment. Key Words: pathfinding; cell adhesion molecules; fasciculation.