A.B. Oestreicher

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.

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.

Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices

Abstract: Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B‐50. To investigate whether B‐50 mediates the actions of PKC in neurotransmitter release, we have studied B‐50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B‐50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B‐50 was determined. Chemical depolarization (30 μM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B‐50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization‐induced stimulation of B‐50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B‐50 may mediate the action of PKC in neurotransmitter release.

Affinity‐Purified Anti‐B‐50 Protein Antibody: Interference with the Function of the Phosphoprotein B‐50 in Synaptic Plasma Membranes

Abstract: Affinity‐purified anti‐B‐50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B‐50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B‐50 protein and its adrenocorticotropin‐sensitive protein kinase, obtained from rat brain. Anti‐B‐50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B‐50 protein isolated by an improved procedure. The purified antibodies reacted only with the B‐50 and B‐60 protein, a proteolysis derivative (of B‐50), as assessed by the sodium dodecyl sulfate‐gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B‐50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5‐diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4‐phosphate was not altered. Inhibition by ACTH 1–24 of the endogenous phosphorylation of B‐50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4‐phosphate to phosphatidylinositol 4,5‐diphosphate. These data support our hypothesis on the functional interaction of B‐50 protein and phosphatidylinositol 4‐phosphate kinase in rat brain membranes. The evidence shows that purified anti‐B‐50 protein antibodies can be used to probe specifically the function of B‐50 protein in membranes.

Role of the growth-associated protein B 50/GAP 43 in neuronal plasticity

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.

Phosphoprotein B-50 in nerve growth cones from fetal rat brain

The presynaptic, nervous tissue-specific phosphoprotein B-50 is present in infant and adult rat brain. In the present study we demonstrate that B-50 is a major phosphoprotein in nerve growth cones obtained from fetal rat brain. As this protein is an endogenous substrate for protein kinase C, an enzyme linked to cell growth and proliferation, a role for B-50 in nerve growth cone function is suggested.

Evidence That the Synaptic Phosphoprotein B-50 Is Localized Exclusively in Nerve Tissue

Abstract: The localization of the phosphoprotein B‐50 (molecular weight 48,000, isoelectric point 4.5) in the rat has been studied. Inspection of endogenous phosphorylation patterns of the particulate as well as the cytosolic subcellular fractions from a variety of peripheral organs failed to demonstrate phosphorylation of a molecular weight 48,000 protein. Only in the particulate fractions from brain tissue was there endogenous phosphorylation of the B‐50 protein. Two‐dimensional analysis (isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis) and an immunochemical detection method employing an anti‐B‐50 antiserum revealed the presence of B‐50 in particulate material from brain, but not in that of other tissues. Therefore the data were interpreted as pointing to the localization of B‐50 in nervous tissue. In addition, the regional distribution of endogenous B‐50 phosphorylation was studied using synaptosomal plasma membranes (SPM) obtained from individual rat brain regions. The highest value was found in SPM of septal origin, the lowest in SPM from the medulla spinalis. The relationship of the high value for B‐50 phosphorylation in the septum to the sensitivity of that brain area to ACTH1–24 is discussed.

Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.

We examined the suitability of freeze-substitution and Lowicryl HM20 embedding of aldehyde-fixed rat brain to localize several neural antigens at the ultrastructural level. The following rabbit polyclonal and mouse monoclonal antibodies were used: affinity-purified polyclonal immunoglobulins G raised to B-50/GAP43 (a membrane-anchored, growth-associated protein); affinity-purified polyclonal immunoglobulins G to human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a neuropeptide present in dense-core granules); a polyclonal antiserum raised to myelin basic protein (a protein present in compact myelin of the central nervous system); and mouse monoclonal antibodies to synaptophysin (an integral membrane protein of small synaptic vesicles). Rat mesencephalon was fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde, cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees C. Semi-thin Lowicryl sections were used for light microscopic visualization of B-50 in the ventromedial mesencephalic central gray substance. The procedure preserves well the ultrastructure of this region and the immunoreactivity of the selected antigens. This study shows that dehydration by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero temperature, provides a successful method of preparation of fixed brain tissue for ultrastructural studies, allowing immunogold localization of several neural antigens by double labeling in the same section and in serial sections.

Activation of dopaminergic D1 receptors promotes morphogenesis of developing striatal neurons

The early dopaminergic input from the midbrain may play an important role in the development of the basal ganglia. We therefore investigated whether and how dopamine affects the morphogenesis of striatal target neurons. Dissociated cell cultures of embryonic day 17 rat striatum were raised for seven days. Cells were then incubated with dopamine or various receptor-specific ligands for 1 h. At various times after termination of the treatment, cells were immunostained for growth-associated protein-43. Morphological parameters including numbers of growth cones, length of neurites, number of bifurcations, and neuronal soma size were assessed by means of a computer-based morphometric device. Treatment with dopamine in low concentrations as well as with the D1-like receptor agonist SKF 38393 increased the numbers of growth cones and neurite length and arborization. The morphogenetic effect took several hours to evolve and remained stable for at least 24 h. It could be blocked by the D1-like receptor antagonist SCH 23390 or by cycloheximide but not by pretreatment of the cultures with tetrodotoxin. The D2-like receptor agonist quinpirole had no effect on the morphological parameters and did not contribute to that of SKF 38393. Dopamine and SKF 38393 but not quinpirole also induced an increase in the number of neurons immunoreactive for Fos-like proteins. However, this effect was restricted to growth-associated protein-43-negative neurons. This is the first observation of a positive regulatory effect of D1-like receptors on neuronal morphogenesis. We conclude that the changes reflect true differentiation rather than short-term modulation of cellular properties and that c-fos induction is not an obligatory step in the transduction pathway coupling D1-like receptors to neurite outgrowth. Our results suggest that the differentiation of embryonic striatal neurons is promoted by the dopaminergic nigrostriatal projection through D1-like receptors.

The Kinase C Substrate Protein B-50 and Axonal Regeneration

As reported previously the prominent protein kinase C substrate protein B-50 is present in growth cones isolated from fetal rat brain and in outgrowing hippocampal neurites. These findings suggest that B-50 plays a role in axonal growth during development of the nervous system. In the present paper the fate of B-50 is investigated in regenerating rat sciatic nerve. Using affinity-purified anti-B-50 antibodies B-50 levels have been compared in crushed and contralateral intact nerves by means of immunoblotting and radioimmunoassay. B-50 levels in the crushed nerve increased 5.3-fold as compared to non-crushed controls. Furthermore, the cellular localization of B-50 has been assessed by immunohistochemistry. Virtually no B-50 immunoreactivity was seen in control nerves, but bright immunofluorescence appeared in regenerating sprouts. Our data are in line with current evidence from several laboratories that B-50 is a member of a small family of growth-associated proteins and support the hypothesis that B-50 is involved in axonal growth.