P.N.E. de Graan

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.

Reduced glutamine synthetase in hippocampal areas with neuron loss in temporal lobe epilepsy

Background: Increased levels of glutamate have been reported in the epileptogenic hippocampus of patients with temporal lobe epilepsy (TLE). This sustained increase, which may contribute to the initiation and propagation of seizure activity, indicates impaired clearance of glutamate released by neurons. Glutamate is predominantly cleared by glial cells through the excitatory amino acid transporter 2 (EAAT2) and its subsequent conversion to glutamine by the glial enzyme glutamine synthetase (GS). Methods: The authors examined the hippocampal distribution of GS, EAAT2, and glial fibrillary acidic protein (GFAP) by immunohistochemistry in TLE patients with (HS group) and without hippocampal sclerosis (non-HS group), and in autopsy controls. In hippocampal homogenates the authors measured relative protein amounts by immunoblotting and GS enzyme activity. Results: In the autopsy control and non-HS group GS immunoreactivity (IR) was predominantly found in glia in the neuropil of the subiculum, of the pyramidal cell layer of all CA fields, and in the supragranular layer of the dentate gyrus. In the HS group, GS and EAAT2 IR were markedly reduced in subfields showing neuron loss (CA1 and CA4), whereas GFAP IR was increased. The reduction in GS IR in the HS group was confirmed by immunoblotting and paralleled by decreased GS enzyme activity. Conclusions: Glial glutamine synthetase is downregulated in the hippocampal sclerosis (HS) hippocampus of temporal lobe epilepsy (TLE) patients in areas with severe neuron loss. This downregulation appears to be pathology-related, rather than seizure-related, and may be part of the mechanism underlying impaired glutamate clearance found in the hippocampus of TLE patients with HS.

Modulation of the activity of purified phosphatidylinositol 4-phosphate kinase by phosphorylated and dephosphorylated B-50 protein

To investigate the modulation of phosphatidylinositol 4-phosphate kinase activity by the degree of phosphorylation of the B-50 protein, the enzyme was purified from rat brain cytosol by ammonium sulphate precipitation and DEAE-cellulose column chromatography. Purified rat brain B-50 was phosphorylated with protein kinase C and dephosphorylated with alkaline phosphatase. Incubation of the semi-purified phosphatidylinositol 4-phosphate kinase with 1 microgram of the B-50 preparation enriched in the dephospho-form, resulted in a small reduction of phosphatidylinositol 4-phosphate kinase activity (-16%), whereas incubation with the phospho B-50 preparation inhibited the enzyme activity by 40%. The effect of exogenous B-50 was studied in the presence of 10 micrograms albumin to minimize aspecific protein-protein interactions. The present data on the effect of exogenous B-50 protein on phosphatidylinositol 4-phosphate kinase activity, further support our hypothesis that the phosphorylation state of B-50 may be a regulatory factor in phosphoinositide metabolism in rat brain.

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.

Long-term potentiation and synaptic protein phosphorylation

Long-term potentiation (LTP) is a well known experimental model for studying the activity-dependent enhancement of synaptic plasticity, and because of its long duration and its associative properties, it has been proposed as a system to investigate the molecular mechanisms of memory formation. At present, there are several lines of evidence that indicate that pre- and postsynaptic kinases and their specific substrates are involved in molecular mechanisms underlying LTP. Many studies focus on the involvement of protein kinase C (PKC). One way to investigate the role of PKC in long-term potentiation is to determine the degree of phosphorylation of its substrates after in situ phosphorylation in hippocampal slices. Two possible targets are the presynaptic membrane-associated protein B-50 (a.k.a. GAP 43, neuromodulin and F1), which has been implicated in different forms of synaptical plasticity in the brain such as neurite outgrowth, hippocampal LTP and neurotransmitter release, and the postsynaptic protein neurogranin (a.k.a. RC3, BICKS and p17) which function remains to be determined. This review will focus on the protein kinase C activity in pre- and postsynaptic compartment during the early phase of LTP and the possible involvement of its substrates B-50 and neurogranin.

Ontogeny of hippocampal corticosteroid receptors: effects of antenatal glucocorticoids in human and mouse.

Women at risk for preterm delivery are treated with synthetic glucocorticoids (GCs) to enhance fetal lung maturation. GCs can bind to two intracellular receptors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR), which function as transcription factors. Both are highly expressed in the hippocampus. Several studies have focused on adverse side effects of antenatal GC treatment. However, relatively little is known about the ontogeny of GR and MR, especially in human. Therefore, we studied the ontogeny of both receptors in the human and mouse hippocampus and investigated the effects of antenatal dexamethasone (dex) treatment, a synthetic glucocorticoid, on MR and GR mRNA levels during hippocampal development. The results demonstrate that MR mRNA was first expressed in mouse hippocampus at embryonic day (E)15.5, at the timepoint when dex was administered. In contrast, GR mRNA expression was first observed after birth at postnatal day (P)5. However, in the human hippocampus both receptors are expressed at 24 weeks of gestation, when antenatal GCs are administered in clinical practice. Quantitative in situ hybridization demonstrated that MR mRNA levels were reduced only shortly after dex treatment at E16, but were unaffected from E18 onwards. These findings indicate that a single antenatal dex administration at E15.5 transiently affects MR mRNA levels in the mouse hippocampus. No effect of antenatal dex treatment was found on the human hippocampus at the third trimester of pregnancy. These data on the prenatal ontogeny of both corticosteroid receptors in the human hippocampus is important for understanding the significance of fetal glucocorticoid or stress exposure and its potential effects on health and disease. J. Comp. Neurol. 499:924–932, 2006.

Transmitter release: target of regulation by protein kinase C?

Publisher Summary This chapter evaluates the involvement of the Ca 2+ /phospholipid-dependent protein kinase C (PKC) in transmitter release in the nervous system. It discusses pharmacological and biochemical evidence, indicating the involvement of PKC and its neuron-specific substrate B-50 (GAP-43) in the molecular mechanism of stimulus secretion coupling during synaptic transmitter release. There is ample evidence suggesting an involvement of PKC in stimulus-secretion coupling during synaptic transmitter release. In general, this role seems to be modulatory rather than essential. An intriguing possibility is that PKC affects the ability of calmodulin-binding proteins to bind calmodulin—a property that in itself could be modulated by the concentration of intracellular Ca 2+ in the synaptic terminal. This would imply that the molecular mechanism of synaptic transmitter release involves a coordinated activation of PKC-dependent, Ca 2+ -dependent, and calmodulin-dependent processes.

The impaired long-term potentiation in the CA1 field of the hippocampus of cognitive deficient microencephalic rats is restored by D-serine

Rat embryos exposed on gestational day 15 to methyl-azoxymethanol acetate develop a microencephaly characterized primarily by a hypoplasia of the neocortex and CA fields of the hippocampus that in adulthood is associated with disturbances in learning. In brain slices prepared from microencephalic rats, we have examined the field excitatory postsynaptic potentials and population spike in the CA1 field of the hippocampus evoked by stimulation of the stratum radiatum. These parameters did not differ from those obtained in slices from control rats. High frequency stimulation of the stratum radiatum afferent fibres, which readily induced long-term potentiation of the field excitatory postsynaptic potentials and population spike in the CA1 field of the hippocampus of control rats, failed to induce long-term potentiation in that of microencephalic rats. High frequency stimulation of the perforant path readily elicited long-term potentiation in the dentate gyrus of both control and microencephalic rats. Picrotoxin had no apparent effect on field excitatory postsynaptic potentials and population spike in the CA1 field of the microencephalic rats, indicating that little GABAergic inhibition was present in slices from these rats. D-2-Amino-phosphonovalerate suppressed the field potentials in slices from microencephalic rats by more than 50%, suggesting that N-methyl-D-aspartate receptors contributed markedly to the synaptic responses evoked by single stimuli. D-Serine, but not picrotoxin, restored long-term potentiation in the CA1 field of the microencephalic rats. The D-serine effect was prevented by pretreating the slices with either 7-chloro-kynurenate or D-2-amino-phosphonovalerate. The failure to induce long-term potentiation, if also found in vivo, may be among the factors related to the learning deficits displayed by these rats.

Nerve growth factor enhances the level of the protein kinase C substrate B-50 in pheochromocytoma PC12 cells

Exposure of PC12 cells to nerve growth factor results in arrest of cell growth and induction of differentiation to sympathetic neuron-like cells, bearing neurites. In this study we identify a 48 kDa PC12 phosphoprotein as the neuron-specific protein kinase C substrate B-50 (Mr 48 kDa; IEP 4.5) on basis of comigration with purified B-50, immunoreactivity and phosphopeptide mapping. B-50 is present in both undifferentiated and differentiated PC12 cells. Exposure of PC12 cells to nerve growth factor for two days results in a 2.5-fold increase in the amount of B-50 as measured by RIA. Indirect immunofluorescence microscopy reveals that B-50 is mainly localized at the cell membrane and in growth cones. Our data are in line with the hypothesis that B-50 plays a role in neurite outgrowth and indicate that PC12 cells provide a suitable model to study this hypothesis.