Previous experiments have suggested that the neural cell adhesion molecule (N-CAM) may have a role in initial nerve-muscle adhesion. To determine whether N-CAM might be involved in synaptic differentiation, we grew ciliary ganglion neurons and embryonic myotubes together in the presence and absence of monovalent antibodies to N-CAM. In normal cultures, undifferentiated neurites contact myotubes, and the nerve at some of these neurite-myotube contacts acquires concentrations of synaptic vesicle antigens. Most of these vesicle antigen-positive contacts become associated with patches of acetylcholine receptor (AChR) on the surface of the underlying myotube. Contacts without concentrations of vesicle antigens do not become associated with AChR patches. In the presence of antibodies to N-CAM, adhesion between neuronal somata and myotubes was reduced, but neurites contacted myotubes with near-normal frequency. The subsequent differentiation of nerve and muscle at these contacts, as assayed by the localization of vesicle antigens and AChR, proceeded normally in the presence of anti-N-CAM antibodies. The results suggest that N-CAM-mediated adhesion between neurite and myotube is not required for synaptic differentiation.

Two different monoclonal antibodies, characterized initially as binding synaptic terminal regions of rat brain, bind a 65,000-dalton protein, which is exposed on the outer surface of brain synaptic vesicles. Immunocytochemical experiments at the electron microscope level demonstrate that these antibodies bind the vesicles in many different types of nerve terminals. The antibodies have been used successfully to purify synaptic vesicles from crude brain homogenates by immunoprecipitation onto the surface of polyacrylamide beads. The profiles of the structures precipitated by these beads are almost exclusively vesicular, confirming the vesicle-specificity of the antibodies. In SDS gels, the antibodies bind a single protein of 65,000 daltons. The two antibodies are not identical, but compete for binding sites on this protein. Immune competition experiments also demonstrate that the antigenic components on the 65,000-dalton protein are widely distributed in neuronal and neural secretory tissues. Detectable antigen is not found in uninnervated tissue--blood cells and extrajunctional muscle. Low levels are found in nonneural secretory tissues; it is not certain whether this reflects the presence of low amounts of the antigen on all the exocytotic vesicles in these tissues or whether the antigen is found only in neuronal fibers within these tissues. The molecular weight and at least two antigenic determinants of the 65,000-dalton protein are highly conserved throughout vertebrate phylogeny. The two antibodies recognize a 65,000-dalton protein present in shark, amphibia, birds, and mammals. The highly conserved nature of the determinants on this protein and their specific localization on secretory vesicles of many different types suggest that this protein may be essential for the normal function of neuronal secretory vesicles.

Brain-derived neurotrophic factor (BDNF), a neurotrophin, enhances the survival and differentiation of several classes of neurons in vitro. To determine its essential functions, we have mutated the BDNF gene. Most homozygote mutants die within 2 days after birth, but a fraction live for 2-4 weeks. These develop symptoms of nervous system dysfunction, including ataxia. The BDNF mutant homozygotes have substantially reduced numbers of cranial and spinal sensory neurons. Although their central nervous systems show no gross structural abnormalities, expression of neuropeptide Y and calcium-binding proteins is altered in many neurons, suggesting they do not function normally. In contrast with mice lacking the BDNF receptor TrkB, motor neurons appear normal in the BDNF mutant.

The epithelial-mesenchymal interactions required for kidney organogenesis are disrupted in mice lacking the integrin alpha8beta1. None of this integrin's known ligands, however, appears to account for this phenotype. To identify a more relevant ligand, a soluble integrin alpha8beta1 heterodimer fused to alkaline phosphatase (AP) has been used to probe blots and cDNA libraries. In newborn mouse kidney extracts, alpha8beta1-AP detects a novel ligand of 70-90 kD. This protein, named nephronectin, is an extracellular matrix protein with five EGF-like repeats, a mucin region containing a RGD sequence, and a COOH-terminal MAM domain. Integrin alpha8beta1 and several additional RGD-binding integrins bind nephronectin. Nephronectin mRNA is expressed in the ureteric bud epithelium, whereas alpha8beta1 is expressed in the metanephric mesenchyme. Nephronectin is localized in the extracellular matrix in the same distribution as the ligand detected by alpha8beta1-AP and forms a complex with alpha8beta1 in vivo. Thus, these results strongly suggest that nephronectin is a relevant ligand mediating alpha8beta1 function in the kidney. Nephronectin is expressed at numerous sites outside the kidney, so it may also have wider roles in development. The approaches used here should be generally useful for characterizing the interactions of novel extracellular matrix proteins identified through genomic sequencing projects.

Here, we describe a novel mechanism for the rapid regulation of surface levels of the neurotrophin receptor TrkB. Unlike nodose ganglion neurons, both retinal ganglion cells (RGCs) and spinal motor neurons (SMNs) in culture display only low levels of surface TrkB, though high levels are present intracellularly. Within minutes of depolarization or cAMP elevation, surface TrkB levels increase by nearly 4-fold, and this increase is not blocked by cycloheximide. These findings suggest that activity and cAMP elevation rapidly recruit TrkB to the plasma membrane by translocation from intracellular stores. We propose that a fundamental difference between peripheral nervous system (PNS) and central nervous system (CNS) neurons is the activity dependence of CNS neurons for responsiveness to their peptide trophic factors and that differences in membrane compartmentalization of the receptors underlie this difference.

Monoclonal antibodies directed against a neuronal cell surface heparan sulfate proteoglycan and against a synaptic vesicle protein were used to study the postnatal development of ganglionic neurons and synapses in the rat superior cervical ganglion. Antigen levels in developing ganglia were quantitated by radioimmune assays. Localization of antigens in adult and developing ganglia was carried out using peroxidase-antiperoxidase immunocytochemistry at the light microscopic level. Ultrastructural staining patterns in adult ganglia also were studied. The time course of antigen increases parallels those in previous reports on the accumulation of neurotransmitter enzymes within the ganglion. Both synaptic and surface antigens increase postnatally, with the most rapid changes occurring during the 2nd week. Antibodies stain adult tissue in patterns consistent with the expected distribution of antigens: antibodies directed against synaptic vesicles stain synaptic terminals and cell cytoplasm and those directed against surface proteoglycan stain the plasma membranes of neuronal cell bodies and processes. Variable staining of the cell cytoplasm also is observed. No apparent changes in antigen distribution are observed with the light microscope during development. Variations in the time course of the development of antigens associated with different portions of the proteoglycan molecule suggest that the intracellular processing of the molecule may vary during development.

In Xenopus laevis development, beta-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell-cell adhesion and can modulate beta-catenin signaling. alpha-catenin links beta-catenin to the actin-based cytoskeleton. To study the role of endogenous alpha-catenin in early development, we have made deletion mutants of alphaN-catenin. The binding domain of beta-catenin has been mapped to the NH2-terminal 210 amino acids of alphaN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length alphaN-catenin or a mutant of beta-catenin that lacks the internal armadillo repeats. We next show that coexpression of alphaN-catenin antagonizes the dorsalizing effects of beta-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, alpha-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular beta-catenin signaling pathway in vivo.

The present investigation used an antibody directed against the extracellular domain of the signal transducing nerve growth factor receptor, trkA, to reveal immunoreactive perikarya or fibers within the olfactory bulb and tubercle, cingulate cortex, nucleus accumbens, striatum, endopiriform nucleus, septal/diagonal band complex, nucleus basalis, hippocampal complex, thalamic paraventricular and reuniens nuclei, periventricular hypothalamus, interpeduncular nucleus, mesencephalic nucleus of the fifth nerve, dorsal nucleus of the lateral lemniscus, prepositus hypoglossal nucleus, ventral cochlear nucleus, ventral lateral tegmentum, medial vestibular nucleus, spinal trigeminal nucleus oralis, nucleus of the solitary tract, raphe nuclei, and spinal cord. Colocalization experiments revealed that virtually all striatal trkA-immunoreactive neurons (> 99%) coexpressed choline acetyltransferase (ChAT) but not p75 nerve growth factor receptor (NGFR). Within the septal/diagonal band complex virtually all trkA neurons (> 95%) coexpressed both ChAT and p75 NGFR. More caudally, dual stained sections revealed numerous trkA/ChAT (> 80%) and trkA/p75 NGFR (> 95%) immunoreactive neurons within the nucleus basalis. In the brainstem, raphe serotonergic neurons (45%) coexpressed trkA. Sections stained with a pan-trk antibody that recognizes primarily trkA, as well as trkB and trkC, labeled neurons within all of these regions as well as within the hypothalamic arcuate, supramammilary, and supraoptic nuclei, hippocampus, inferior and superior colliculus, substantia nigra, ventral tegmental area of T'sai, and cerebellular Purkinje cells. Virtually all of these other regions with the exception of the cerebellum also expressed pan-trk immunoreactivity in the monkey. The widespread expression of trkA throughout the central neural axis suggests that this receptor may play a role in signal transduction mechanisms linked to NGF-related substances in cholinergic basal forebrain and noncholinergic systems. These findings suggest that pharmacological use of ligands for trkA could have beneficial effects on the multiple neuronal systems that are affected in such disorders as Alzheimer's disease.

To test the molecular nature of the NGF receptor responsible for the ability of NGF to rescue septal cholinergic neurons following axotomy, we infused polyclonal antibodies that act as specific agonists of trkA (RTA) into the lateral ventricle of fimbria-fornix lesioned animals. Rats receiving chronic intraventricular infusions of RTA showed significantly more low affinity NGF receptor immunoreactive (p75NGFR-IR) neurons on the lesioned side than did control animals 2 weeks following unilateral fimbria-fornix lesion. RTA also initiated cholinergic sprouting. Infusions of RTA in combination with an antibody that blocks p75NGFR (REX) did not reduce the cell savings effect observed with RTA alone. However, animals infused with RTA plus REX demonstrated significantly less sprouting. These findings suggest that antibody-induced trkA activation is sufficient to mediate NGF-promoted survival of axotomized cholinergic neurons in vivo.