Continuous phosphorylation of GAP-43 and MARCKS by long-term TPA treatment in SK-N-SH human neuroblastoma cells.
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Long-term treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) down-regulates select protein kinase C (PKC) isozymes and may differentially affect PKC substrates. We investigated the role of PKC down-regulation on phosphorylation of two PKC substrates, the 43 kDa growth-associated protein (GAP-43) and the myristoylated alanine-rich C-kinase substrate (MARCKS) in SK-N-SH human neuroblastoma cells. Cells were treated with 70 nM TPA for 15 min, 17 or 72 h. Phosphorylation of MARCKS and GAP-43 was elevated throughout 72 h of TPA. The magnitude and peptidic sites of phosphorylation in GAP-43 and MARCKS were similar after all TPA treatments. GAP-43, but not MARCKS, content was increased after 17 and 72 h of TPA. The ratio of GAP-43 phosphorylation to content was elevated throughout 17 h but returned to control by 72 h as content increased. PKC epsilon and alpha isozyme content was greatly reduced after 72 h of TPA but membranes retained 23% of PKC activity. Only PKC epsilon translocated to membranes after 15 min TPA. GAP-43 content after 72 h of TPA was increased in subcellular fractions in which significant PKC epsilon isozyme concentration remained. These results demonstrate that continuous TPA differentially affected phosphorylation of PKC substrate proteins and regulation of PKC isozyme content in SK-N-SH cells. (+info)
Quantitative non-radioactive in situ hybridization study of GAP-43 and SCG10 mRNAs in the cerebral cortex of adult and infant macaque monkeys.
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We performed non-radioactive in situ hybridization histochemistry in several areas that include both the association areas and the lower sensory areas of monkey cerebral cortex, and investigated the localization of neurons expressing two growth-associated proteins: GAP-43 and SCG10. Both GAP-43 and SCG10 mRNAs were observed in both pyramidal and non-pyramidal neurons. Prominent hybridization signals for GAP-43 mRNA were observed in layers II-VI of the adult association areas: the prefrontal areas (FD), the temporal (TE) and the parietal (PG) association areas. The signals for GAP-43 mRNA were weak in layers I-III of the adult primary somatosensory area (PB) and primary (OC) and secondary (OB) visual areas, and cells with prominent signals were observed in layers IV-VI. The prominent signals for SCG10 mRNA were observed in layers IV-VI of all areas examined. These results suggest that the expression pattern of GAP-43 mRNA, but not that of SCG10 mRNA, may be related to the functional difference between association and lower sensory areas of adult cortex. In the infant cortex (postnatal days 2, 8 and 31), the signals for both mRNAs were intense in layers II-VI of all areas. Therefore, layer-specific expressions of GAP-43 and SCG10 mRNAs are established after the first postnatal month. (+info)
Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice.
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There is strong evidence that growth-associated protein (GAP-43), a protein found only in the nervous system, regulates the response of neurons to axonal guidance signals. However, its role in complex spatial patterning in cerebral cortex has not been explored. We show that mice lacking GAP-43 expression (-/-) fail to establish the ordered whisker representation (barrel array) normally found in layer IV of rodent primary somatosensory cortex. Thalamocortical afferents to -/- cortex form irregular patches in layer IV within a poorly defined cortical field, which varies between hemispheres, rather than the stereotypic, whisker-specific, segregated map seen in normal animals. Furthermore, many thalamocortical afferents project abnormally to widely separated cortical targets. Taken together, our findings indicate a loss of identifiable whisker territories in the GAP-43 -/- mouse cortex. Here, we present a disrupted somatotopic map phenotype in cortex, in clear contrast to the blurring of boundaries within an ordered whisker map in other barrelless mutants. Our results indicate that GAP-43 expression is critical for the normal establishment of ordered topography in barrel cortex. (+info)
Divergent cellular differentiation pathways during the invasive stage of cutaneous malignant melanoma progression.
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Melanocytic nevus cells in the dermis adopt many morphological features of Schwann cells. These differentiation-related changes typically are not observed in melanomas. However, nevus cells do not fully recapitulate a Schwann cell phenotype, because they lack expression of mature myelin-associated proteins. In this study, melanocytic nevi and malignant melanomas were examined by immunohistochemistry for expression of low-affinity nerve growth factor receptor (p75NGFR), neural cell adhesion molecule (CD56/N-CAM), and growth-associated phosphoprotein-43 (GAP-43). These three proteins define the earliest stages of Schwann cell development but are not expressed in myelinated Schwann cells or normal melanocytes. p75NGFR was expressed in 25 of 25 (100%) and CD56/N-CAM and GAP-43 in 23 of 25 (92%) nevi, predominantly in type C nevus cells and nevic corpuscles. Most (84%) of the nevi expressed all three proteins. In primary invasive and metastatic melanoma, expression of each of the three proteins was limited to +info)
Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.
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Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation. (+info)
Growth-associated protein 43 is located in type I corticothalamic terminals in the cat visual thalamus.
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Growth-associated protein 43 (GAP 43) is a presynaptic protein that has been proposed to be involved in synaptic plasticity. To determine the location of GAP 43 within the synaptic circuitry of the thalamus, immunocytochemical staining for GAP 43 was examined in a relay nucleus, the dorsal lateral geniculate nucleus (dLGN), and two association nuclei, the pulvinar nucleus and the lateral subdivision of the lateral posterior (LP) nucleus. In the dLGN, moderate neuropil staining was seen in the A laminae, and denser staining was found in the interlaminar zones and the C laminae. Uniform dense staining of the neuropil was found in both the pulvinar and LP nuclei. At the ultrastructural level, the GAP 43 staining was restricted to small-diameter myelinated axons, thin unmyelinated fibers, and small terminals that contained densely packed round vesicles (RS profiles) and made asymmetric synaptic contacts with small-caliber dendrites in the extraglomerular neuropil. The distribution of immunocytochemical label within the visual thalamus suggests that GAP 43 is confined to type I corticothalamic terminals and axons that originate from extrastriate cortical areas. These results also suggest that in both relay and association nuclei GAP 43 may be used to augment the cortical control of thalamic activity. In addition, these results underscore the distinction between the small type I corticothalamic terminals, which appear to contain GAP 43 throughout the visual thalamus, and the large type II corticothalamic terminals that, like the type II retinal terminals in the dLGN, do not contain GAP 43. (+info)
Novel genes expressed in the developing medial cortex.
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Two cDNAs, M1 and M2, recently isolated by the differential display method from embryonic rat cerebral hemisphere were characterized and their patterns of spatiotemporal expression analysed in developing rat forebrain by in situ hybridization histochemistry and correlative immunocytochemistry. Neither gene bears any sequence homology to other known genes. Both genes are particularly expressed in medial regions of the cerebral hemisphere and M2 in the roof of the adjacent diencephalon. M1 expression is highly localized and confined to the neuroepithelium of the hippocampal rudiment from embryonic day (E) 12 onward. Its location corresponds to the fimbrial anlage, and immunocytochemical localization of M1 protein indicates its expression in radial glial cells. M2 expression at E12 is more extensive in the medial cerebral wall, extending into the preoptic region and beyond the hippocampus into dorsal hemisphere and into the dorsal diencephalon, with caudal extension along the dorsal midline and in the zona limitans intrathalamica. Later, M2 expression is found in association with the corpus callosum, hippocampal commissure, fimbria, optic nerve, stria medullaris, mamillothalamic tract and habenulopeduncular tract. M1 and M2 expression domains corresponding to the locations of fiber tracts are present prior to the arrival of the earliest axons, as identified by neuron specific markers. These findings suggest M1 and/or M2 genes are involved in early regional specification of the hippocampus and related structures in paramedian regions of the forebrain, and that cell populations expressing these genes in advance of developing axonal pathways may be involved in the early specification of tract location. (+info)
Selective glutamate receptor antagonists can induce or prevent axonal sprouting in rat hippocampal slice cultures.
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After the transection of the Schaffer collateral pathway in hippocampal slice cultures, reactive sprouting is induced in the CA3 area, and eventually synaptic transmission between areas CA1 and CA3 is restored. Using this model, we have studied the role of ionotropic glutamate receptors in the initiation of axonal sprouting and the regeneration of functional synapses. We show that neither reactive sprouting nor functional recovery of synaptic transmission occur in the presence of the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6-nitro-7-sulfamoylbenzoquinoxaline-2,3-dione (CNQX). In contrast, the NMDA receptor antagonists methyl-10, 11-dihydro-5-H-dibenzocyclohepten-5,10-imine (MK-801) or 3-(RS)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) did not interfere with these processes. Moreover, we observed that the application of NMDA receptor antagonists induced massive axonal sprouting and an increase in the frequency of miniature excitatory postsynaptic currents in unlesioned cultures. Our results thus indicate that NMDA and non-NMDA receptors exert a differential effect on reactive sprouting and the recovery of synaptic transmission after injury in the hippocampus. Activation of non-NMDA receptors appears necessary for these processes to occur, whereas activation of NMDA receptors suppresses growth-associated protein -43 expression and axonal outgrowth. (+info)