Presynaptic localization of phosphoprotein B-50

A major phosphoprotein of synaptic membranes, the phosphorylation of which is stimulated by Ca2+ and inhibited by ACTh, appears to be identical with protein B-50 described by Zwiers, Schotman and Gispen [40]. We have investigated its subsynaptic localization by means of a variety of subfractionation techniques and compared it with that of a number of other phosphoproteins found in synaptic membranes. It appears to be predominantly, if not exclusively, associated with presynaptic membranes of low bouyant density. This localization pattern is similar to, but somewhat more extreme than that exhibited by Protein I, as a brain specific phosphoprotein studied by Greengard and his collaborators [11].     

Localization of the growth-associated phosphoprotein GAP-43 (B-50, F1) in the human cerebral cortex

The growth-associated phosphoprotein GAP-43 is a component of the presynaptic membrane that has been linked to the development and functional modulation of neuronal connections. A monospecific antibody raised against rat GAP-43 was used here to study the distribution of the protein in cortical and subcortical areas of the human brain. On Western blots, the antibody recognized a synaptosomal plasma membrane protein that had an apparent molecular weight and isoelectric point similar to GAP-43 of other species. In brain tissue reacted with the antibody, the heaviest immunoreactivity was found in associative areas of the neocortex, particularly within layers 1 and 6, in the molecular layer of the dentate gyrus, the caudate putamen, and the amygdala. In contrast, primary sensory or motor regions of the cortex, portions of dorsal thalamus, and cerebellum showed only light staining. Staining was generally confined to the neuropil, which showed punctate labeling, whereas most neuronal somata and fiber bundles were unreactive. The pronounced variations in GAP-43 immunostaining among various areas of the human brain may reflect different potentials for functional and/or structural remodeling.     

The neuronal growth-associated protein GAP-43 (B-50, F1): neuronal specificity, developmental regulation and regional distribution of the human and rat mRNAs

The protein that has been designated as GAP-43, B-50, F1 or pp46 is associated with the growth and modulation of neuronal connections. cDNA clones for the rat and human genes were isolated and used to demonstrate that the messenger RNA for the protein is expressed only in neurons, that its overall level is highest in the developing brain, and that in the adult human brain levels of the mRNA are highest in the associative neocortex.     

Presynaptic localization of B-50 phosphoprotein: the (ACTH)-sensitive protein kinase substrate involved in rat brain polyphosphoinositide metabolism

This study describes the ultrastructural localization in rat hippocampal tissue in situ and in isolated synaptosomes of the brain-specific phosphoprotein B-50, using affinity purified anti-B-50 immunoglobulins (IgGs). Evidence is presented for the presynaptic localization of B-50 in rat brain. Given this specific localization a model is presented outlining the presumed function of the B-50 protein in the membrane and describing possible neuromodulation by adrenocorticotropin hormone (ACTH)-like peptides.     

Quantitation of the growth-associated protein B-50/GAP-43 and neurite outgrowth in PC12 cells

A combined assay to measure neurite outgrowth and B-50/GAP-43 levels in PC12 cells is reported. During NGF-induced neuritogenesis, B-50/GAP-43 expression was monitored by enzyme-linked immunosorbent assay (ELISA). Neurite outgrowth was quantified at the same time by the use of video image analysis. Sensitivity and reliability of the methods are shown with a dose-response and time curve of beta-NGF-induced neuritogenesis. A linear increase in total length of neurites was induced by concentrations of beta-NGF greater than or equal to 5 ng/ml and was accompanied by a linear increase in the amount of B-50/GAP-43. The combined methods presented here can conveniently and reliably establish subtle changes in neurite outgrowth and intracellular protein contents.     

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: content and phosphorylation of B-50.

The content and phosphorylation of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of normal development and development in the presence of tetrodotoxin (TTX), a blocker of bioelectric activity (BEA). The observations were correlated with previous morphological findings on neurite outgrowth and B-50 immunolocalization in the same cultures. In control cultures, the concentration of B-50 reached a maximum at 7 days in vitro (DIV) and decreased thereafter, whereas the concentration of neuron specific enolase (NSE), which was used as a neuronal reference marker, rose till 28 DIV and leveled off towards 42 DIV. The degree of basal phosphorylation of B-50 (relative to that of total protein) decreased after the first week in vitro. Stimulation of B-50 phosphorylation by phorbol ester also decreased with age in vitro, indicating that changes in B-50 phosphorylation were mainly due to changes in protein kinase C (PKC) activity. The chronic presence of TTX led to a reduced content of B-50 and NSE after 14 DIV. The basal phosphorylation of B-50 was neither affected by acute nor chronic TTX treatment. However, upon stimulation of PKC with phorbol esters, some alterations of B-50 phosphorylation were revealed in cultures grown in TTX. These biochemical observations are in line with the absence of effects of TTX on neurite outgrowth during the first 2 weeks in culture, and later effects of TTX on neuronal survival. The developmental changes in B-50 concentration and phosphorylation largely correlate with previous morphological observations on axonal outgrowth and growth cone shape in the same cultures. We suggest that B-50 phosphorylation plays an important role in transducing extracellular signals into directed neurite outgrowth.     

Developmental changes in B-50 (GAP-43) in primary cultures of cerebral cortex: B-50 immunolocalization, axonal elongation rate and growth cone morphology

Changes in neurite outgrowth parameters and in the immunolocalization of the neuronal growth-associated protein B-50 (GAP-43) were studied in cultured neocortex as a function of development. In addition, we studied the effects of chronic blockade of bioelectric activity (BEA) with tetrodotoxin (TTX) on these parameters. Axonal outgrowth rate in control cultures reached a maximum at 8 days in vitro (DIV) and declined to a low level at 21 DIV. B-50 staining shifted from the perikaryon to the axons and growth cones during the first 3 DIV. In axons the intensity of B-50 staining increased towards the growth cone. Within growth cones, the central/basal region and filopodia were intensely stained, whereas lamellipodia showed only marginal staining. Growth cone size gradually decreased after 3 DIV, due to the successive loss of lamellipodia and filopodia, and became club-shaped during the second week, until by 21 DIV growth cones were completely lost, and axons started retracting and degenerated. In the central area of the cultures, growth cones also decreased in size with time, but became stabilized as presynaptic elements onto other neurons. Acute addition of TTX did not affect the outgrowth rate at 6 DIV. Chronic TTX treatment led to an earlier retraction and degeneration of axons than in control cultures and to a loss of B-50-stained cells and varicosities during the third week, but did not affect growth cone morphology or B-50 staining. The regressive phenomena are probably due to an increased neuronal cell death shown to occur after chronic TTX treatment. The developmental changes in axonal elongation rate and growth cone morphology may be related to developmental changes in the content and/or phosphorylation of B-50 (GAP-43, which are studied in the same cultures in the following paper (Ramakers et al. (1991) Int. J. Devl Neurosci. 9, 231-241].     

Light- and electron-microscopical study of phosphoprotein B-50 following denervation and reinnervation of the rat soleus muscle

The neuron-specific phosphoprotein B-50 was originally identified as a phosphoprotein in synaptic plasma membranes isolated from adult brain tissue. In this paper we study the reinnervation of the soleus muscle, a target muscle of sciatic nerve axons, using affinity-purified anti-B-50 antibodies. Light-microscopical evaluation of the reinnervation process revealed that the period of muscle fiber reinnervation corresponds closely with the time in which high B-50 immunoreactivity was observed in the nerve fibers that invade the muscle and in the newly formed neuromuscular junctions. Upon completion of reinnervation, B-50 immunoreactivity decreased. In the newly innervating terminals, B-50 was associated with presynaptic vesicular structures and with the presynaptic plasma membrane. In intact mature neuromuscular junctions, virtually no B-50 immunoreactivity could be detected with either light- or electron-microscopic procedures. These observations corroborate the association of high levels of B-50/GAP43 during axon outgrowth and support the concept that B-50 may be a key molecule in the reconstruction of axonal structures. We also observed an unexpected transient increase in B-50 immunoreactivity in the degenerating neuromuscular junctions. This observation cannot be explained in terms of increased neuronal synthesis of B-50, since the degenerating axon processes have been completely disconnected from their cell bodies. Thus, our evidence implies that a rise of B-50 immunoreactivity can be associated with stages of neuronal degeneration as well as with those of neuronal differentiation and axon outgrowth.     

Lesion-specific pattern of immunocytochemical distribution of growth-associated protein B-50 (GAP-43) in the cerebellum of weaver and PCD-mutant mice: Lack of B-50 involvement in neuroplasticity of Purkinje cell terminals?

The growth-associated protein B-50 (GAP-43) is thought to play a major role in the development and regeneration of neurons. The participation of B-50 in neuronal plasticity is well documented, especially for monoaminergic systems. However, such an important role for B-50 in GABAergic systems has not been substantiated to date. This study was performed to obtain detailed information about the identity of B-50 immunopositive axons and terminals in the cerebellum and to test the involvement of this protein during plastic changes as observed in the projections of GABAergic Purkinje cells to the lateral vestibular nucleus (LVN). For this purpose mutant mice with specific cerebellar cell loss were used. Weaver mutants (B6CBA wv/wv), PCD-mutants (B6C3Fe pcd/pcd), and their corresponding wild-type mice were investigated with immunocytochemical and immunoblot procedures at the age of 8--23 days and 5--6 months using polyclonal and monoclonal antibodies to B-50. Substantial differences in B-50 distribution wee detected between normals and mutants and between young and adult animals. These results demonstrate that the labeling of B-50 is mainly related to the outgrowth of parallel fibers and to a minor degree on the ingrowth of non-GABAergic cerebellar afferents. There was no immunocytochemical indication that B-50 is related to Purkinje cells or accompanies the plasticity of the GABAergic innervation of the LVN. © 1994 Wiley-Liss, Inc.     

Light microscopy study of low-affinity nerve growth factor receptor and phosphoprotein B-50/neuromodulin in inflammatory myopathies

Phosphoprotein B-50, also termed neuromodulin or growth-associated protein GAP43, is a membrane-bound molecule expressed in neurons. It is particularly abundant during periods of axonal outgrowth in development and regeneration of the central and peripheral nervous systems. Recently it was reported that B-50 plays a role in the growth morphology of regenerating muscle fibers. Moreover, in vitro studies have demonstrated that the expression of B-50 in the pheochromocytoma PC12 cells can be stimulated by the nerve growth factor (NGF). Expression of the low-affinity nerve growth factor receptor (LNGFR) during muscle regeneration has also been reported. Here, we studied the expression of NGF, LNGFR and B-50 in myopathy. To investigate the state of regeneration, we examined serial sections stained to demonstrate neural cell adhesion molecule and desmin. Light microscopy showed that muscle fiber regeneration in idiopathic inflammatory myopathy corresponds closely to NGF, LNGFR and B-50 immunoreactivity. The coexpression of phosphoprotein B-50, NGF and LNGFR in regenerating muscle fiber corroborates the assumption that in muscle there is a trophic pathway concerning phosphorylation or de novo synthesis of B-50 by the NGF via the LNGFR. In conclusion, a simultaneous expression of NGF, LNGFR and B-50 in muscles plays a role in the growth morphology of regenerating muscle fibers. Received: 4 November 1994 / Revised: 4 August 1995 / Revised, accepted: 23 October 1995     

A plasminogen activator from benign ovarian cystadenoma: partial purification and characterization

A plasminogen activator has been partially purified from benign serous cystadenomas by a combination of Sephadex G-200 gel filtration, CM-Sephadex ion exchange, Concanavalin A-Sepharose and arginine-Sepharose affinity chromatographies. Its apparent size was very large and could not penetrate Sepharose 6B or 5% SDS polyacrylamide gel. It hydrolyzed plasminogen in a manner similar to that of urokinase in terms of their apparent Michaelis constants, although a one-minute lag period had to be allowed for this activation before the hydrolysis of plasminogen. It was very sensitive to reducing agent such that 5 mM dithiothreitol could completely destroy its activity. The activator crossreacted with anti-uterine plasminogen activator IgG, but did not react with anti-urokinase at all. It also bound fibrin well. In the absence of plasminogen, the activator was devoid of amidolytic activity towards S-2251, S-2302 and S-2288 but had a small but measurable activity against S-2444.     

Comparison of the immunocytochemical distribution of the phosphoprotein B-50 in the cerebellum and hippocampus of immature and adult rat brain

In this study we compare the distribution of the phosphoprotein B-50 in two regions of immature and adult rat brain using affinity-purified antibodies to B-50. In the cerebellum of the 8-day-old rat we observed distinct patterns of distribution of B-50 immunoreactivity (BIR) in the premigratory zone and the developing molecular layer, likely associated with outgrowing parallel and climbing fibers contacting Purkinje cells in the internal granular layer and in axons coursing through the cerebellar medulla. In contrast, in adult cerebellum, a sparcer distribution of BIR as punctuate deposits is observed in the molecular layer, outlining dendritic trees and the perikarya of neurons. At relatively lower density BIR is found dispersed between the cells of the granular layer and along fibers in the white matter. In the immature hippocampal formation, fibers penetrating between unstained cells of the stratum pyramidale and the subiculum, and neuropil areas are immunostained. In the adult rat a graded immunostaining pattern corresponding to the laminar structure of the hippocampal formation is found with high density of BIR in the strata oriens, radiatum, parts of stratum lacunosum molecular and in the stratum molecular adjoining the field of the proximal apical dendrites of the granule cells. BIR appears to be absent from the proximal part of the mossy fiber pathway. In neuropil areas of adult hippocampus and cerebellum BIR is fairly restricted to dot-like deposits indicating a synaptic localization. This is in correspondence with our previous ultrastructural findings. The present observations in developing brain of B-50-like components in fibers, as well, suggest that B-50 (and/or B-50-like precursors) are involved in neurite outgrowth.     

Chicken growth-associated protein (GAP)-43: primary structure and regulated expression of mRNA during embryogenesis

Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes.     

Neuronal aggregation and neurotransmitter regulation: partial purification and characterization of a membrane-derived factor

Cell membrane contact induces marked differential changes in neurotransmitter expression. In cultures of virtually pure dissociated sympathetic neurons, when such contact is provided by either high cell densities or addition of membranes derived from specific tissues, there is a marked increase in cell-specific content of substance P and de novo induction of choline acetyltransferase. To identify molecular mechanisms underlying regulation of transmitter expression by neuronal aggregation and membrane contact, we have begun to isolate and characterize a membrane-associated factor responsible for stimulation of choline acetyltransferase activity. The factor was found in substantial quantities in membranes from adult rat spinal cord as well as from sympathetic and sensory ganglia. Ionic mechanisms were employed to extract transmitter-inducing activity from spinal cord membranes in soluble form. The solubilized factor was then partially purified by ion exchange and gel filtration chromatography. It appears to be an extrinsic (non-integral) protein with an apparent molecular weight of 27. It is inactivated by trypsin and chymotrypsin, but is only moderately sensitive to heat inactivation, retaining activity at 60 degrees C but not at 90 degrees C. Neuronal perikaryal contact via aggregation represents a critical mechanism by which neurons themselves may influence phenotypic expression. Membrane localization of the factor provides a means by which cell contact may regulate transmitter expression.     

Subcellular localization of alpha-synuclein in primary neuronal cultures: effect of missense mutations

Numerous recent observations have implicated alpha-synuclein in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, dementia with Lewy bodies and multiple-system atrophy. Two missense mutations in the gene for alpha-synuclein have been identified in some cases of familial Parkinson's disease and it is thought that these may disrupt the normal structure of the protein and thus promote aggregation into Lewy body filaments. Here, we examine the subcellular localization of alpha-synuclein in primary cortical neurons maintained in a monolayer culture. The protein has widespread expression throughout neurons, including the nucleus, and has a discete localization in the neurites of more mature neurons, reminiscent of synaptic specializations. Interestingly, in a subpopulation of cortical neurons transfected at 13 days in vitro, we find that alpha-synuclein appears to aggregate into distinct punctate inclusions in the cytoplasm and proximal neurites. Unlike Lewy bodies, these structures are not ubiquitin positive. These regions of alpha-synuclein accumulation are observed following transfections with wild-type, Ala30Pro or Ala53Thr alpha-synuclein; neither mutation alters their frequency.     

Human spleen insoluble fibrinolytic proteinase acting at neutral pH: its partial purification and characterization

An insoluble fibrinolytic enzyme with a molecular weight of approximately 30,000, was purified from the human spleen. A single protein band possessing fibrinolytic activity was obtained on polyacrylamide gel disk electrophoresis at pH 4.5. The enzyme, tentatively termed spleen fibrinolytic proteinase (SFP), degraded fibrinogen at neutral pH following Michaelis-Menten kinetics. The fibrinogenolytic activity was not inhibited by t-AMCHA, a specific plasmin inhibitor. SFP barely degraded certain synthetic ester or polypeptide substrates for trypsin, chymotrypsin, plasma, Xa, elastase and collagenase. These results indicate a different nature for SFP compared to other enzymes examined. SFP was found to digest no elastin and its fibrinogenolytic activity was strongly inhibited by STI, indicating that it was not an elastase. SFP required neither Zn++ nor CA++ for its fibrinogenolytic activity, indicating that it differed from metal-dependent proteinases such as collagenase. SFP was inhibited by DFP but not by TLCK, suggesting that it contains an active serine residue, but no trypsin type histidine at its active center. These results appear to show that SFP is a unique proteinase in the spleen, which is capable of degrading fibrin and fibrinogen at neutral pH.     

Cloning of cDNA for DARPP-32, a dopamine- and cyclic AMP-regulated neuronal phosphoprotein

A cDNA clone for the mRNA of bovine DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, Mr = 32,000) was isolated from a modified Okayama-Berg plasmid library. Transformed Escherichia coli colonies were screened by in situ colony hybridization with 2 different oligonucleotide probes corresponding to a region unusually rich in glutamate within the protein. Three positive clones were isolated and shown to encode DARPP-32 by an in situ immunoblot assay of their fusion protein products with beta-galactosidase. The results of the sequence analysis of the longest cDNA clone, pTKD7 (1771 nucleotides), revealed a 606-nucleotide-long coding region, in exact agreement with the bovine DARPP-32 amino acid sequence (Williams et al., 1986). Southern blot analysis of total bovine genomic DNA showed that there is a single gene coding for DARPP-32. Northern blot analysis of caudate nucleus RNA using antisense RNA derived from the clone pTKD7 demonstrated the existence of 2 abundant mRNA species, corresponding to 1.8 and 1.65 kilobase in length. The high concentration of DARPP-32 mRNAs in the caudate nucleus is in agreement with the known distribution of this protein.