A. Beate Oestreicher

Ultrastructural immunocytochemical localization of B-50/GAP43, a protein kinase C substrate, in isolated presynaptic nerve terminals and neuronal growth cones

SummaryAccumulating evidence indicates that the neuron-specific B-50/GAP43, a substrate for protein kinase C, plays a role in neuronal differentiation and neuritogenesis during nervous tissue development and axonal regeneration. An ultrastructural immunocytochemical study on the localization of B-50 in presynaptic terminals (synaptosomes) and neuronal growth cones was carried out by means of cryoultramicrotomy with affinity-purified B-50 antibodies. Detection was accomplished with colloidal gold, conjugated either to protein-A or goat anti-rabbit immunoglobulins. In synaptosomes, isolated from the frontal cortex of 6-week-old rats, and in neuronal growth cones, isolated from forebrains of 5-day-old rats, the majority of B-50 is detected at the surrounding neuronal plasma membrane. In both neuronal growth cones and synaptosomes, a relatively small fraction of B-50 in the cytoplasm was not evidently associated with internal membranes. Our results indicate that B-50 is mainly located at the cytoplasmic face of the synaptosomal and neuronal growth cone plasma membrane. The similar B-50 localization in neuronal growth cones and synaptosomes suggests that, both in extending axons and mature synaptic terminals, B-50 may exert identical functions as a protein kinase C substrate at the plasma membrane.

Noradrenaline Release from Streptolysin O‐Permeated Rat Cortical Synaptosomes: Effects of Calcium, Phorbol Esters, Protein Kinase Inhibitors, and Antibodies to the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP‐43)

Abstract: We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B‐50 (GAP‐43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration‐dependent manner by elevating the free Ca2+ concentration from 10−8 to 10−5M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2+‐induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2+‐induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+‐ and phorbol ester‐induced release were not additive at higher phorbol ester concentrations (>10−7M). We compared the sensitivities of Ca2+‐ and phorbol ester‐induced release of noradrenaline to the protein kinase inhibitors H‐7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B‐50. Ca2+‐induced release was inhibited by B‐50 antibodies and polymyxin B, but not by H‐7; phorbol ester‐induced release was inhibited by polymyxin B and by H‐7, but only marginally by antibodies to B‐50. We suggest that phorbol esters and Ca2+ stimulate noradrenaline release through different mechanisms and that the essential role of B‐50 in Ca2+‐induced noradrenaline release may involve other properties of B‐50 besides protein kinase C–mediated phosphorylation.

Kindling induces a long-lasting change in the activity of a hippocampal membrane calmodulin-dependent protein kinase system

Septal kindling has been shown to produce a long-lasting decrease in endogenous calcium/calmodulin-dependent phosphorylation of hippocampal synaptic plasma membrane proteins, including two major bands of approximately 50,000 and 60,000 Daltons. These two proteins differ from the B-50 protein and tubulin, as evidenced by differences in migration in SDS-PAGE gels and by lack of cross-immunoreactivity with specific antibodies. Identity of these two proteins with the rho and sigma subunits of purified calmodulin-dependent kinase (CaM Kinase II) is suggested by similar migration in SDS-PAGE and two-dimensional gels, by similar calmodulin binding in two-dimensional gels, and similar 125I-peptide mapping of the 50,000 Dalton protein. These results demonstrate that septal kindling is associated with changes in the activity of a major Ca2+/calmodulin-dependent kinase system in hippocampal synaptic plasma membrane. This long-lasting modulation of kinase activity may provide a molecular insight into some aspects of neuronal plasticity.

N‐Terminal‐Specific Anti‐B‐50 (GAP‐43) Antibodies Inhibit Ca2+‐Induced Noradrenaline Release, B‐50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B‐50 (GAP‐43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin‐binding domain of B‐50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B‐50 into streptolysin O‐permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N‐terminal residues 39–43 of rat B‐50 dose‐dependently inhibited Ca2+‐induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C‐terminal‐directed (residues 132–213) anti‐B‐50 antibodies were without effect in the same dose range. NM2 inhibited PKC‐mediated B‐50 phosphorylation at Ser41 in synaptosomal plasma membranes and permeated synaptosomes, inhibited 32P‐B‐50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B‐50 in the absence of Ca2+. Similar concentrations of NM6 did not affect B‐50 phosphorylation or dephosphorylation or B‐50/calmodulin binding. We conclude that the N‐terminal residues 39–43 of the rat B‐50 protein play an important role in the process of Ca2+‐induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca2+‐ and phosphorylation‐dependent fashion.

Evidence for the Binding of Calmodulin to Endogenous B‐50 (GAP‐43) in Native Synaptosomal Plasma Membranes

Abstract: The neuron‐specific protein B‐50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B‐50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B‐50/CaM binding, we used the cross‐linker disuccimidyl suberate (DSS) to form a covalent B‐50/CaM complex, which is stable on SDS‐PAGE. Upon addition of DSS, purified B‐50 and calmodulin form a 70‐kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti‐B‐50 and anti‐CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70‐kDa B‐50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B‐50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B‐50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.

Studies on the Role of B‐50 (GAP‐43) in the Mechanism of Ca2+‐Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B‐50, protein kinase C (PKC), and PKC‐mediated B‐50 phosphorylation in the mechanism of Ca2+‐induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin‐O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP‐dependent manner as a result of an elevation of the free Ca2+ concentration from 10−8 to 10−5M Ca2+ The Ca2+ sensitivity in the micromolar range is identical for [3H]NA and endogenous NA release, indicating that Ca2+‐induced [3H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca2+‐induced NA release was inhibited by either N‐ or C‐terminal‐directed anti‐B‐50 antibodies, confirming a role of B‐50 in the process of exocytosis. In addition, both anti‐B‐50 antibodies inhibited PKC‐mediated B‐50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC‐mediated B‐50 phosphorylation in Ca2+‐induced NA release. PKC pseudosubstrate PKC19‐36, which inhibited B‐50 phosphorylation (IC50 value, 10−5M), failed to inhibit Ca2+‐induced NA release, even when added before the Ca2+ trigger. Similar results were obtained with PKC inhibitor H‐7, whereas polymyxin B inhibited B‐50 phosphorylation as well as Ca2+‐induced NA release. Concerning the Ca2+ sensitivity, we demonstrate that PKC‐mediated B‐50 phosphorylation is initiated at a slightly higher Ca2+ concentration than NA release. Moreover, phorbol ester‐induced PKC down‐regulation was not paralleled by a decrease in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Finally, the Ca2+‐ and phorbol ester‐induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B‐50 is involved in Ca2+‐induced NA release from streptolysin‐O‐permeated synaptosomes. Evidence is presented challenging a role of PKC‐mediated B‐50 phosphorylation in the mechanism of NA exocytosis after Ca2+ influx. An involvement of PKC or PKC‐mediated B‐50 phosphorylation before the Ca2+ trigger is not ruled out. We suggest that the degree of B‐50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca2+ influx resulting in exocytosis of NA.

B-50/GAP-43 expression by the olfactory receptor cells and the neurons migrating from the olfactory placode in embryonic rats

B-50/GAP-43 is a growth-associated phosphoprotein that is commonly expressed in all developing neuronal systems. Using an immunocytochemistry approach, we have investigated the expression of this protein in the rat olfactory system during embryogenesis and neonatal development with a particular emphasis on the early developmental stages of the olfactory placode. Data show that already at embryonic day 12 (E12), a strong B-50/GAP-43 immunoreactivity was detected in few olfactory receptor cells well-recognizable by their positive short neuritic processes. The B-50/GAP-43 expression in the placodal epithelium thus appeared to coincide with the onset of neurite outgrowth. From E13 onwards, there was a rapid increase in the number of B-50/GAP-43-positive olfactory neurons and from E18, the protein was strongly expressed by nearly all neurons. In addition, results clearly demonstrate that as early as E13, B-50/GAP-43 was strongly expressed by many migrating cells which were seen leaving the pit epithelium in association with the first olfactory axons that penetrated the nasal mesenchyme. Many immunoreactive cells were also observed in the presumptive olfactory nerve layer. Experiments of double-labeling showed that B-50/GAP-43-immunostained migrating cells were also stained with anti-neuron-specific enolase (NSE). This confirms the neuronal nature of these early labeled migrating cells. The progressive disappearance of migrating neurons noted during the late stages of embryonic development is discussed in relation with their possible function in the early stages of development of the peripheral olfactory system.

Evidence for a role of protein kinase C substrate B 50 (GAP 43) in Ca2+-induced neuropeptide cholecystokinin-8 release from permeated synaptosomes

Abstract: To study the involvement of the protein kinase C (PKC) substrate B‐50 [also known as growth‐associated protein‐43 (GAP‐43), neuromodulin, and F1] in presynaptic cholecystokinin‐8 (CCK‐8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL‐O). CCK‐8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca2+ in a concentration‐dependent manner (EC50 of ∼10‐5M). Ca2+‐induced CCK‐8 release was maximal at 104M Ca2+, amounting to ∼10% of the initial 6,000 ± 550 fmol of CCK‐8 content/mg of synaptosomal protein. Only 30% of the Caa+‐induced CCK‐8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti‐B‐50 antibodies were introduced into permeated synaptosomes to study their effect on Ca2+‐induced CCK‐8 release. The N‐terminally directed antibodies (NM2), which inhibited PKC‐mediated B‐50 phosphorylation, inhibited Ca2+‐induced CCK‐8 release in a dose‐dependent manner, whereas the C‐terminally directed antibodies (NM6) affected neither B‐50 phosphorylation nor CCK‐8 release. The PKC inhibitors PKC19–36 and 1 −(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7), which inhibited B‐50 phosphorylation in permeated synaptosomes, had no effect on Ca2+‐induced CCK‐8 release. Our data strongly indicate that B‐50 is involved in the mechanism of presynaptic CCK‐8 release, at a step downstream of the Ca2+ trigger. As CCK‐8 is stored in large densecored vesicles, we conclude that B‐50 is an essential factor in the exocytosis from this type of neuropeptide‐containing vesicle. The differential effects of the monoclonal antibodies indicate that this B‐50 property is localized in the N‐terminal region of the B‐50 molecule, which contains the PKC phosphorylation site and calmodulin‐binding domain.

Immunocytochemical study of the differentiation process of the septal organ of Masera in developing rats

The septal organ of Masera is a small patch of olfactory epithelium located near the base of the nasal septum. Using the growth-associated protein B-50/GAP-43 as neuronal marker, we have studied the differentiation process of this organ from the olfactory sheet in embryonic and newborn rats. Results show that the septal organ first appeared at embryonic day 16. Even though it was included in the olfactory sheet, the presumptive septal organ could be distinguished by a higher density of B-50/GAP-43-positive neurons. Concomitantly to its morphological development, the septal organ progressively isolated from the main olfactory epithelium. This isolation resulted from the extension of a transitional area which progressively lost its typical features of olfactory epithelium to become a putative respiratory epithelium in late embryonic stages. Results strongly suggest that the septal organ should be a proper chemosensory system with its own time-course of development.

Monoclonal Antibody NM2 Recognizes the Protein Kinase C Phosphorylation Site in B-50 (GAP-43) and in Neurogranin (BICKS)

Abstract: Mouse monoclonal B‐50 antibodies (Mabs) were screened to select a Mab that may interfere with suggested functions of B‐50 (GAP‐43), such as involvement in neurotransmitter release. Because the Mab NM2 reacted with peptide fragments of rat B‐50 containing the unique protein kinase C (PKC) phosphorylation site at serine‐41, it was selected and characterized in comparison with another Mab NM6 unreactive with these fragments. NM2, but not NM6, recognized neurogranin (BICKS), another PKC substrate, containing a homologous sequence to rat B‐50 (34–52). To narrow down the epitope domain, synthetic B‐50 peptides were tested in ELISAs. In contrast to NM6, NM2 immunoreacted with B‐50 (39–51) peptide, but not with B‐50 (43–51) peptide or a C‐terminal B‐50 peptide. Preabsorption by B‐50 (39–51) peptide of NM2 inhibited the binding of NM2 to rat B‐50 in contrast to NM6. NM2 selectively inhibited phosphorylation of B‐50 during endogenous phosphorylation of synaptosomal plasma membrane proteins. Preabsorption of NM2 by B‐50 (39–51) peptide abolished this inhibition. In conclusion, NM2 recognizes the QASFR peptide in B‐50 and neurogranin. Therefore, NM2 may be a useful tool in physiological studies of the role of PKC‐mediated phosphorylation and calmodulin binding of B‐50 and neurogranin.