Coupling of cAMP/PKA and MAPK signaling in neuronal cells is dependent on developmental stage.

Neurite formation, an essential feature of neuronal development, is believed to involve participation of the ras-mitogen-activated protein kinase (MAPK) and cAMP-dependent protein kinase A (cAMP/PKA)-mediated signaling pathways. These pathways have been studied extensively in the rat pheochromocytoma cell line PC12, and current hypotheses suggest a single effector mechanism resulting from the convergence of cAMP/PKA and MAPK signaling. However, based on observations using a central neuronal progenitor cell line, AS583-8, there also exists evidence that the two signaling pathways may act independently resulting in neurites with differing dynamic features. In the present study, the upstream components of cAMP/PKA signaling were examined in AS583-8 cells as well as possible interactions with the MAPK pathway. We found that activation of PKA is both necessary and sufficient for the elaboration of rapidly forming processes, typical of the cAMP response. In addition, blockade of the MAPK pathway has no effect on the cAMP response, suggesting that activation of the cAMP/PKA pathway can stimulate neurite formation independent of the MAPK pathway. In order to evaluate which cell line model, PC12 vs AS583-8, best reflects the signaling features of developing central neurons, we examined interactions between cAMP/PKA and MAPK signaling in primary neuronal cultures from several brain regions. In immature cultures (1-day-old), at a point where the initiation of neurite formation is maximal, no interaction was observed. In more mature cultures (7 days old), where synaptic contacts have been established, we found a weak but reproducible activation of MAPK following stimulation of the cAMP/PKA pathway. These results suggest that cAMP/PKA and MAPK signaling act independently at the initiation of neuritogenesis but become coupled during later stages of neuronal development. Therefore, the interaction of the two pathways may be cell stage (younger vs older) specific and may participate in cellular functions that take place after initial neurite formation.

Nerve growth factor, but not epidermal growth factor, increases Fra-2 expression and alters Fra-2/JunD binding to AP-1 and CREB binding elements in pheochromocytoma (PC12) cells.

In pheochromocytoma (PC12) cells nerve growth factor (NGF) and epidermal growth factor (EGF) activate similar receptor tyrosine kinase signaling pathways but evoke strikingly different biological outcomes: NGF induces differentiation and EGF acts as a mitogen. A novel approach was developed for identifying transcription factor activities associated with NGF-activated, but not EGF-activated, signaling, using random oligonucleotide clones from a DNA recognition library to isolate specific DNA binding proteins from PC12 nuclear extracts. A protein complex from NGF-treated, but not EGF-treated, cells was identified that exhibits increased mobility and DNA binding activity in gel mobility shift assays. The binding complex was identified in supershift assays as Fra-2/JunD. The clones used as probes contain either AP-1 or cAMP response element binding (CREB) recognition elements. Time course experiments revealed further differences in NGF and EGF signaling in PC12 cells. NGF elicits a more delayed and sustained ERK phosphorylation than EGF, consistent with previous reports. Both growth factors transiently induce c-fos, but NGF evokes a greater response than EGF. NGF specifically increases Fra-1 and Fra-2 levels at 4 and 24 hr. The latter is represented in Western blots by bands in the 40-46 kDa range. NGF, but not EGF, enhances the upper bands, corresponding to phosphorylated Fra-2. These findings suggest that prolonged alterations in Fra-2 and subsequent increases in Fra-2/JunD binding to AP-1 and CREB response elements common among many gene promoters could serve to trigger broadly an NGF-specific program of gene expression.

Beta-adrenergic and fibroblast growth factor receptors induce neuronal process outgrowth through different mechanisms.

The mechanisms that initiate and direct neuronal process formation remain poorly understood. We have recently described a neuronal progenitor cell line, AS583-8.E4.22 (AS583-8) which undergoes neurite formation in response to beta2-adrenergic and basic fibroblast growth factor (bFGF) receptor activation [Kwon, J.H. et al., (1996) Eur. J. Neurosci., 8, 2042-2055]. In the present study, a comparison of these responses revealed that isoproterenol (ISO), a beta-adrenergic receptor agonist, induces multiple, highly branched processes within 30 min while bFGF induces fewer, unbranched processes within 24 h. In contrast to the ISO response, bFGF induces mitogen-activated protein kinase activation and c-fos expression in the cell line and results in neurite outgrowth that is dependent on new mRNA and protein synthesis. Two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cytoskeletal preparations revealed different patterns following ISO vs. bFGF exposure suggesting selective changes in protein expression and/or post-translational modifications. Immunoblot analysis of these preparations for beta-tubulin, tyrosinated alpha-tubulin and acetylated alpha-tubulin also revealed different patterns following each type of treatment. Follow-up confocal microscopy revealed that following ISO, the distribution of tyrosinated tubulin extends to the distal ends of processes whereas acetylated alpha-tubulin is diminished within distal ends. This pattern has been reported to be associated with enhanced microtubule dynamics, a state in which process outgrowth is facilitated. In contrast, following bFGF treatment the distributions of tyrosinated and acetylated alpha-tubulin were identical, a state associated with a diminution of microtubule dynamics. These results, a different time course of neurite formation, dependency on new gene expression and differential expression and cellular distribution of major cytoskeleton proteins suggest that neurite outgrowth induced by ISO vs. bFGF is mediated by two distinct intracellular effector mechanisms in AS583-8 cells. In addition, studies, using the differential distribution of post-translational modified alpha-tubulins in neurites of primary neuronal cultures as marker for the two distinct processes of neurite formation suggest, that similar mechanisms are present in vivo. Therefore, the AS583-8 cell line provides a useful model to study these signalling mechanisms that couple neurotransmitter and growth factor receptor activation to the cytoskeletal changes that mediate neurite formation.

Neuronal abnormalities in microtubule-associated protein 1B mutant mice.

Microtubules play an important role in establishing cellular architecture. Neuronal microtubules are considered to have a role in dendrite and axon formation. Different portions of the developing and adult brain microtubules are associated with different microtubule-associated proteins (MAPs). The roles of each of the different MAPs are not well understood. One of these proteins, MAP1B, is expressed in different portions of the brain and has been postulated to have a role in neuronal plasticity and brain development. To ascertain the role of MAP1B, we generated mice which carry an insertion in the gene by gene-targeting methods. Mice which are homozygous for the modification die during embryogenesis. The heterozygotes exhibit a spectrum of phenotypes including slower growth rates, lack of visual acuity in one or both eyes, and motor system abnormalities. Histochemical analysis of the severely affected mice revealed that their Purkinje cell dendritic processes are abnormal, do not react with MAP1B antibodies, and show reduced staining with MAP1A antibodies. Similar histological and immunochemical changes were observed in the olfactory bulb, hippocampus, and retina, providing a basis for the observed phenotypes.