Microtubule-associated protein 2 phosphorylation is decreased in the human epileptic temporal lobe cortex.

Microtubule-associated protein 2 (MAP2) is an abundant component of the neuronal cytoskeleton whose function is related to the outgrowth and stability of neuronal processes, to synaptic plasticity and neuronal cell death. We have sought to study whether abnormal patterns of neuronal activity which are characteristic of epileptic patients are associated to alterations of MAP2 phosphorylation. An antibody (305) that selectively recognizes a phosphorylated epitope in a proline-rich region of the MAP2 molecule has been used to analyze neocortical biopsy samples from temporal lobe epileptic patients, whose electrocorticogram activity had been previously monitored. Immunoblot analysis showed that samples with greater spiking activity displayed significantly diminished MAP2 phosphorylation. Immunocytochemical analysis revealed the occurrence of discrete areas in the neocortex with highly decreased or no immunostaining for antibody 305, which showed a clear, although non-significant, tendency to appear more frequently in areas with greater spiking activity. To further support an association between epileptiform activity and MAP2 dephosphorylation an experimental model of epileptiform activity in cultures of rat hippocampal neurons was used. Neurons were cultured during 15 days in the presence of kynurenic acid, an antagonist of glutamate receptors. At this time, kynurenic acid was removed from the culture medium and neurons developed seizure-like activity. Using antibody 305, we found a decrease of MAP2 phosphorylation that was already visible after 15 min of kynurenic acid withdrawal. We therefore propose that MAP2 phosphorylation is decreased in the neocortex of epileptic patients and that this decrease is a likely consequence of seizure activity. Also, MAP2 dephosphorylation may lead to alterations of the neuronal cytoskeleton and eventually to neuronal damage and loss, which is typical of epileptic patients.

bFGF stimulates GAP-43 phosphorylation at ser41 and modifies its intracellular localization in cultured hippocampal neurons.

Cultured hippocampal neurons have been used to study GAP-43 phosphorylation and subcellular distribution. By immunofluorescence, GAP-43 was found associated with adherent membrane patches that remained attached to the substratum after in situ permeabilization with Nonidet-NP40. This association increases during neuronal development and is stabilized by the actin cytoskeleton. Basic fibroblast growth factor (bFGF) promotes GAP-43 translocation from the cytosol to adherent membrane patches and, at the same time, stimulates GAP-43 phosphorylation, mainly at the protein kinase C (PKC) site (Ser41). Inhibition of PKC prevented bFGF-stimulated GAP-43 phosphorylation and translocation, while activation by phorbol esters mimicked bFGF effects, suggesting that phosphorylation at Ser41 regulates GAP-43 subcellular localization. Using biochemical fractionation and phosphorylation analysis, it was found that Ser41 phosphorylation was highest in cytoskeleton-associated GAP-43 and lowest in membrane-associated GAP-43. It is proposed that GAP-43 is continuously cycling between intracellular compartments depending on its phosphorylation state and could be taking part in initial adhesive complexes assembled during growth cone advance.

Microscale purification of proteins exhibiting anomalous electrophoretic migration: application to the analysis of GAP-43 phosphorylation.

Quite often, in vivo analysis of posttranslational protein modifications is complicated by the lack of specific antibodies or unsatisfactory immunoprecipitation efficiency. Here, we present an alternative method to immunoprecipitation that takes advantage of the anomalous electrophoretic behavior exhibited by GAP-43. This method can be applied to other proteins which show similar characteristics, such as myristoylated alanine-rich C kinase, NAP-22, and Neurogranin, among others. All these proteins display relative mobility values that depend on the concentration of polyacrylamide used in the resolving gel. Cell extracts or tissue homogenates are first separated by SDS-PAGE in 15% polyacrylamide gels, and the bands containing GAP-43 are identified, excised from the gel, and rerun on a second SDS-PAGE in 7.5% polyacrylamide/6 M urea gels. To quickly identify the position of GAP-43 in the first gel, a small amount of fluorescein-labeled recombinant GAP-43 was added to the initial extracts. The method, applied to the analysis of GAP-43 phosphorylation in rat hippocampal slices, can be typically completed in less than 4 h. The excellent yields of purification obtained contributed to a greater accuracy and increased reliability of the radioactivity measurements. It also allowed further processing of the samples, including the analysis of the different phosphorylation sites by proteolytic digestion and peptide mapping.

Glutamate stimulates neurogranin phosphorylation in cultured rat hippocampal neurons.

Neurogranin is a calmodulin-binding and a protein kinase C substrate, that is expressed in telencephalic regions of the rat brain and has been associated with signal transduction and long-term potentiation (LTP). We here report that neurogranin is present in cultured hippocampal neurones, although in amounts lower than those present in the adult hippocampus, and that is also phosphorylated 'in vivo'. Glutamate receptor activation rapidly and significantly increases neurogranin phosphorylation, which achieves maximal phosphate labeling after ionotropic receptor stimulation (kainate and N-methyl-D-aspartate) and more moderate one after metabotropic receptor activation. It is proposed that neurogranin phosphorylation responds to changes in intracellular free Ca2+ and, also, that an increase in neurogranin phosphorylation contributes to enhance and extend calmodulin action, and therefore participate in post-synaptic signal transduction and LTP.

Neurogranin in the development of the rat telencephalon.

We have used a novel antibody to map the distribution of the protein kinase C substrate protein RC3/neurogranin during the development of the rat telencephalon. Neurogranin appearance in the rat brain is biphasic: it shows an early stage of anatomically restricted, low-intensity expression, and a juvenile stage of anatomically widespread, high-intensity expression. Most of the structures that express neurogranin during development conserve it in the adult stage. Neurogranin expression starts on embryonic day 18 in two different sites-the amygdalar primordium and in the piriform cortex-and is confined to these structures until the first postnatal day (P1). On P1, neurogranin expression increases dramatically in intensity, and appears in the olfactory cortex, isocortex, subiculum and hippocampus. In the striatum, expression starts on P1 and extends to the caudoputamen and parts of the globus pallidus and septum. Particularly complex patterns of labelling can be seen in the amygdala and cerebral cortex. Cortical layers showing early expression are the presumptive layers 4 and 5 in the somatosensory cortex, and layers 2 and 5 in the anterior cingulate and agranular insular cortices. Immunoreactivity is found mostly in cell bodies during the early and juvenile stages, but by the end of the first postnatal week it starts being more apparent in the neuropil. This phenomenon probably reflects the intracellular translocation of neurogranin to distal parts of the dendrites and dendritic spines. This process culminates by the end of the second postnatal week, when the adult pattern is reached. According to the timing and anatomy of its distribution, expression of neurogranin seems to be independently regulated in each telencephalic region by specific signalling mechanisms. It is proposed, on this basis, that neurogranin could be implicated in neuronal differentiation and synaptogenesis during telencephalic development.

Expression of protein kinase C isozymes in hippocampal neurones in culture.

Several protein kinase C (PKC) isozymes were analyzed by immunoblot and immunocytochemistry in cultures of hippocampal neurones at several stages of differentiation. Our findings reveal the existence of two distinct patterns of expression. Firstly, conventional PKC isozymes alpha, beta and gamma, that are expressed at very low levels during the initial stages and then increase continuously with time of culture. Secondly, novel PKC isozymes delta, epsilon and zeta, whose contents increase very early to reach a maximum after three days of culture and then progressively decline. Specific proteolysis for PKC isozymes beta and gamma was observed throughout the period studied. The developmental profile obtained for the different PKC isozymes is discussed in relation to the differentiation of hippocampal neurones in culture.