Phosphorylation Of GAP-43 Research Articles

Amphetamine-induced neurite injury in PC12 cells through inhibiting GAP-43 pathway

Amphetamine (AMPH) causes the degeneration of dopamine terminals in the central nervous system. The mechanisms for this damage are unclear. We found AMPH reduced level of GAP-43 in the striatum of rats that receives rich dopaminergic terminals. Using PC12 cells as dopaminergic neuronal models, we further found that AMPH inhibited GAP-43 and GAP-43 phosphorylation in PC12 cells. The reduced GAP-43 was correlated with neurite injury of PC12 cells. The PKCβ1, an upstream molecule of GAP-43, was also inhibited by AMPH. Phorbol 12-myristate 13-acetate (PMA) as a specific activator of PKC increased levels of PKCβ1 and GAP-43, and efficiently prevented neurite degeneration of PC12 cells induced by AMPH. On the other side, enzastuarin, an inhibitor of PKC, decreased levels of PKCβ1 and GAP-43, and caused neurite injury of PC12 cells. Together, our results suggest that AMPH induces neurite injury in PC12 cells through inhibiting PKCβ1/GAP-43 pathway.

PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.

Protein Kinase C-Dependent Growth-Associated Protein 43 Phosphorylation Regulates Gephyrin Aggregation at Developing GABAergic Synapses

Growth-associated protein 43 (GAP43) is known to regulate axon growth, but whether it also plays a role in synaptogenesis remains unclear. Here, we found that GAP43 regulates the aggregation of gephyrin, a pivotal protein for clustering postsynaptic GABA(A) receptors (GABA(A)Rs), in developing cortical neurons. Pharmacological blockade of either protein kinase C (PKC) or neuronal activity increased both GAP43-gephyrin association and gephyrin misfolding-induced aggregation, suggesting the importance of PKC-dependent regulation of GABAergic synapses. Furthermore, we found that PKC phosphorylation-resistant GAP43(S41A), but not PKC phosphorylation-mimicking GAP43(S41D), interacted with cytosolic gephyrin to trigger gephyrin misfolding and its sequestration into aggresomes. In contrast, GAP43(S41D), but not GAP43(S41A), inhibited the physiological aggregation/clustering of gephyrin, reduced surface GABA(A)Rs under physiological conditions, and attenuated gephyrin misfolding under transient oxygen-glucose deprivation (tOGD) that mimics pathological neonatal hypoxia. Calcineurin-mediated GAP43 dephosphorylation that accompanied tOGD also led to GAP43-gephyrin association and gephyrin misfolding. Thus, PKC-dependent phosphorylation of GAP43 plays a critical role in regulating postsynaptic gephyrin aggregation in developing GABAergic synapses.

Mouse intermittent hypoxia mimicking apnoea of prematurity: effects on myelinogenesis and axonal maturation

Premature babies are at high risk for both infantile apnoea and long-term neurobehavioural deficits. Recent studies suggest that diffuse structural changes in brain white matter are a positive predictor of poor cognitive outcomes. Since oligodendrocyte maturation, myelination, axon development, and synapse formation mainly occur in the third trimester of gestation and first postnatal year, infantile apnoea could lead to and/or exaggerate white matter impairments in preterm neonates. Therefore, we investigated oligodendroglia and axon development in a neonatal mouse model of intermittent hypoxia between postnatal days 2 and 10. During critical phases of central nervous system development, intermittent hypoxia induced hypomyelination in the corpus callosum, striatum, fornix, and cerebellum, but not in the pons or spinal cord. Intermittent hypoxia-elicited alterations in myelin-forming processes were reflected by decreased expression of myelin proteins, including MBP, PLP, MAG, and CNPase, possibly due to arrested maturation of oligodendrocytes. Ultrastructural abnormalities were apparent in the myelin sheath and axon. Immature oligodendrocytes were more vulnerable to neonatal intermittent hypoxia exposures than developing axons, suggesting that hypomyelination may contribute, at least partially, to axonal deficits. Insufficient neurofilament synthesis with anomalous components of neurofilament subunits, β-tubulin, and MAP2 isoforms indicated immaturity of axons in intermittent hypoxia-exposed mouse brains. In addition, down-regulation of synapsin I, synaptophysin, and Gap-43 phosphorylation suggested a potential stunt in axonogenesis and synaptogenesis. The region-selective and complex impairment in brain white matter induced by intermittent hypoxia was further associated with electrophysiological changes that may underlie long-term neurobehavioural sequelae.

The effect of systemic injection of cyclosporin A on the phosphorylation of the PKC substrates MARCKS and GAP43 in the rat hippocampus

Cyclosporin A (CsA) is an inhibitor of calcineurin, a calcium/calmodulin dependent serine/threonine phosphatase. Protein kinase C (PKC) is a family of serine/threonine kinases. Both calcineurin and PKC are implicated in psychiatric diseases and the therapeutic mechanisms of treatment agents. It has been reported that calcineurin interacts with components of PKC signaling pathways. We administrated 50mg/kg CsA into rats by intraperitoneal injection and examined the acute effect of single systemic CsA on the locomotor activity of rats and the phosphorylation of PKC and its substrates GAP43 and MARCKS. Systemic CsA increased locomotor activity beginning 1h after injection. The immunoreactivity of p-MARCKS(S152/156) was higher in the CsA group 1h after injection, whereas p-GAP43(S41) immunoreactivity was increased by CsA after 5h. The immunoreactivity of p-PKC pan was increased by CsA at both 1 and 5h after administration. Our data suggest that activation of the PKC pathway might be related to CsA-induced hyperlocomotion.

GAP43 phosphorylation is critical for growth and branching of retinotectal arbors in zebrafish

Visual activity acts via NMDA Receptors to refine developing retinotectal maps by shaping retinal arbors. Retinal axons add and delete transient branches, and the dynamic rates increase when MK801 blocks NMDARs, as if this prevents release of a stabilizing signal. Ca(++) entry through NMDARs activates phospholipase A2 (cPLA2) to release arachidonic acid (AA), which taps into a presynaptic growth control mechanism. NCAM, L1, N-cadherin, and FGF all stimulate axon growth via AA activation of protein kinase C to phosphorylate GAP43 and polymerize/stabilize F-actin. Our previous results show that blocking cPLA2 mimics NMDAR blockers, whereas exogenous AA reverses the increased dynamics, and PKC inhibitors also arrest growth. To test whether this activity-driven F-actin control mechanism shapes retinotectal arbors in zebrafish, we used the alpha-1-tubulin promoter to express GAP43-GFP fusion proteins in retinal ganglion cells, and imaged arbors in time-lapse to test for effects of GAP43 levels and its phosphorylation. Overexpressing wildtype GAP43 gave faster growth and larger arbors (#branches, spatial extent, total length of branches) at three days and especially four days. Surprisingly, the N-terminal 20 amino acid segment alone caused the same increase in branching, but no increase in growth. Earlier studies implicate this region in activating G(o) resulting in collapse of growth cones, which is now known to precede branch initiation. In contrast, GAP43 with ser41 mutated to ala (S41A) to prevent phosphorylation did not increase either branching or growth but resulted in immature, elongated arbors even at four to five days. In support of this atrophic effect, only half of brain/spinal neurons expressing S41A successfully initiated axonal outgrowth (vs. nearly 100% for wtGAP43). These results suggest that the region around the ser41 phosphorylation site, which binds CaM and PIP2, promotes growth only when phosphorylated, and also activates the branching control region in the first 10-20 amino acids. Whereas phosphorylation introduces a bulky negative charge group, mutation of serine to arginine introduces a bulky positive charge. But this also produced the same growth and branching as phosphorylation, suggesting that the effect of phosphorylation is through hydrophilic bulk rather than negative charge, in agreement with other IQ motifs. The results implicate the cPLA2-AA-PKC-GAP43 pathway as part of an F-actin based mechanism that both stabilizes new synapses and initiates new branches near effective synapses.

Electroconvulsive seizure increases phosphorylation of PKC substrates, including GAP-43, MARCKS, and neurogranin, in rat brain

Protein kinase C (PKC) has been suggested as a molecular target related to the pathogenetic and therapeutic mechanisms of mood disorders in which electroconvulsive seizure (ECS) is effective. However, the reports concerning the effects of ECS on PKC are anecdotal and need further clarification. In this study, we examined the effects of ECS treatment on the phosphorylation of PKC substrates, including GAP-43, MARCKS, and neurogranin. Immunoblot using anti-p-PKC substrate antibodies revealed that a single ECS treatment induced temporal changes in the phosphorylation level of PKC substrates in rat brain, reflecting the effects on PKC activity. Phosphorylation of GAP-43 and MARCKS, representative PKC substrates related to synaptic remodeling, increased from 5 to 30 min, after a transient decrease at 0 min immediately after ECS, and returned to basal levels at 60 min in rat frontal cortex, hippocampus, and cerebellum. Phosphorylation of neurogranin, another PKC substrate, showed a similar pattern of temporal changes in the frontal cortex and hippocampus. Immunohistochemical analysis revealed that p-GAP-43 and p-MARCKS were densely stained throughout the neuronal cells of the prefrontal cortex and hippocampus, and the Purkinje cells of cerebellum, after ECS treatment. Brief and transient activation of PKC may be translated into long-term biochemical changes, resulting in synaptic plasticity. Taken together, the acute effects of ECS on PKC activity, which could be an underpinning of long-term biochemical changes induced by ECS, may contribute to understand the molecular mechanism of ECS.

Alterations in mossy fiber physiology and GAP‐43 expression and function in transgenic mice overexpressing HuD

HuD is a neuronal RNA-binding protein associated with the stabilization of mRNAs for GAP-43 and other neuronal proteins that are important for nervous system development and learning and memory mechanisms. To better understand the function of this protein, we generated transgenic mice expressing human HuD (HuD-Tg) in adult forebrain neurons. We have previously shown that expression of HuD in adult dentate granule cells results in an abnormal accumulation of GAP-43 mRNA via posttranscriptional mechanisms. Here we show that this mRNA accumulation leads to the ectopic expression of GAP-43 protein in mossy fibers. Electrophysiological analyses of the mossy fiber to CA3 synapse of HuD-Tg mice revealed increases in paired-pulse facilitation (PPF) at short interpulse intervals and no change in long-term potentiation (LTP). Presynaptic calcium transients at the same synapses exhibited faster time constants of decay, suggesting a decrease in the endogenous Ca(2+) buffer capacity of mossy fiber terminals of HuD-Tg mice. Under resting conditions, GAP-43 binds very tightly to calmodulin sequestering it and then releasing it upon PKC-dependent phosphorylation. Therefore, subsequent studies examined the extent of GAP-43 phosphorylation and its association to calmodulin. We found that despite the increased GAP-43 expression in HuD-Tg mice, the levels of PKC-phosphorylated GAP-43 were decreased in these animals. Furthermore, in agreement with the increased proportion of nonphosphorylated GAP-43, HuD-Tg mice showed increased binding of calmodulin to this protein. These results suggest that a significant amount of calmodulin may be trapped in an inactive state, unable to bind free calcium, and activate downstream signaling pathways. In conclusion, we propose that an unregulated expression of HuD disrupts mossy fiber physiology in adult mice in part by altering the expression and phosphorylation of GAP-43 and the amount of free calmodulin available at the synaptic terminal.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.

Protein Kinase C Inhibition by Tamoxifen Antagonizes Manic-Like Behavior in Rats: Implications for the Development of Novel Therapeutics for Bipolar Disorder

Rationale: In the context of bipolar disorder (BPD) research it was demonstrated that administration of the structurally dissimilar mood stabilizers lithium and valproate produced a striking reduction in protein kinase C (PKC) in rat brain. In a small clinical study, tamoxifen (a PKC inhibitor) had antimanic efficacy. However, both lithium and valproate exert many biochemical changes and attribution of therapeutic relevance to any molecular findings needs to be based on linking them to behavioral effects. Objectives: The present study was designed to explore such relationship by studying the effects of PKC inhibition in amphetamine-induced behavioral animal models of mania and changes in GAP-43. Methods: The effects of two daily tamoxifen (1 mg/kg) i.p. injections on acute or chronic (7 injections) amphetamine (0.5 mg/kg) -induced behaviors and GAP-43 phosphorylation were tested. Results: The study demonstrates that tamoxifen significantly reduced amphetamine-induced hyperactivity in a large open field without affecting spontaneous activity levels and normalized amphetamine-induced increase in visits to the center of an open field (representing risk-taking behavior). Tamoxifen also attenuated amphetamine-induced phosphorylation of GAP-43, a result that is consistent with the behavioral findings. Conclusions: These results support the possibility that PKC signaling may play an important role in the pathophysiology and treatment of BPD. These findings may have direct clinical implications as they offer a new avenue for attempts to develop more specific drugs for the disorder.

GAP‐43 regulates NCAM‐180‐mediated neurite outgrowth

The neural cell adhesion molecule (NCAM), and the growth-associated protein (GAP-43), play pivotal roles in neuronal development and plasticity and possess interdependent functions. However, the mechanisms underlying the functional association of GAP-43 and NCAM have not been elucidated. In this study we show that (over)expression of GAP-43 in PC12E2 cells and hippocampal neurons strongly potentiates neurite extension, both in the absence and in the presence of homophilic NCAM binding. This potentiation is crucially dependent on the membrane association of GAP-43. We demonstrate that phosphorylation of GAP-43 by protein kinase C (PKC) as well as by casein kinase II (CKII) is important for the NCAM-induced neurite outgrowth. Moreover, our results indicate that in the presence of GAP-43, NCAM-induced neurite outgrowth requires functional association of NCAM-180/spectrin/GAP-43, whereas in the absence of GAP-43, the NCAM-140/non-receptor tyrosine kinase (Fyn)-associated signaling pathway is pivotal. Thus, expression of GAP-43 presumably acts as a functional switch for NCAM-180-induced signaling. This suggests that under physiological conditions, spatial and/or temporal changes of the localization of GAP-43 and NCAM on the cell membrane may determine the predominant signaling mechanism triggered by homophilic NCAM binding: NCAM-180/spectrin-mediated modulation of the actin cytoskeleton, NCAM-140-mediated activation of Fyn, or both.

Changes in the composition of detergent‐resistant membrane domains of cultured neurons following protein kinase C activation

Changes in the composition of cell fractions, and in particular of detergent-resistant membranes (DRM) isolated from cultured rat cerebellar granule cells, were taken as possible changes in lipid raft composition during a signal transduction event. After activation of protein kinase C (PKC) with phorbol esters (PMA) or glutamate, the content of PKC and of proteins highly enriched (GAP43, Fyn, and PrP(c)) or not (MARCKS) in DRM was followed. PKC activation strongly increased its association with membranes (from 2% to 75%), causing its enrichment within DRM; the substrate GAP43, enriched in DRM, remained membrane associated, but its proportion in DRM dramatically decreased (from about 40% to 2.5%), suggesting its shift from raft to nonraft membranes, possibly as a consequence of phosphorylation by PKC. The distribution of Fyn and PrP(c) (DRM-enriched) and of MARCKS (present mainly outside DRM) did not change. PKC activation was followed by an increase of GAP43 and MARCKS phosphorylation (about 7- and 8-fold, respectively). Noteworthy was that, after cell treatment with the lipid raft-disrupting drug methyl-beta-cyclodextrin, PKC activation occurred normally, followed by MARCKS phosphorylation, but GAP43 phosphorylation did not occur. Taken altogether, these data suggest that the integrity of lipid rafts is necessary for PKC to affect GAP43 and catalyze its phosphorylation.

Molecular Mechanisms, Biological Actions, and Neuropharmacology of the Growth-Associated Protein GAP-43

GAP-43 is an intracellular growth-associated protein that appears to assist neuronal pathfinding and branching during development and regeneration, and may contribute to presynaptic membrane changes in the adult, leading to the phenomena of neurotransmitter release, endocytosis and synaptic vesicle recycling, long-term potentiation, spatial memory formation, and learning. GAP-43 becomes bound via palmitoylation and the presence of three basic residues to membranes of the early secretory pathway. It is then sorted onto vesicles at the late secretory pathway for fast axonal transport to the growth cone or presynaptic plasma membrane. The palmitate chains do not serve as permanent membrane anchors for GAP-43, because at steady-state most of the GAP-43 in a cell is membrane-bound but is not palmitoylated. Filopodial extension and branching take place when GAP-43 is phosphorylated at Ser-41 by protein kinase C, and this occurs following neurotrophin binding and the activation of numerous small GTPases. GAP-43 has been proposed to cluster the acidic phospholipid phosphatidylinositol 4,5-bisphosphate in plasma membrane rafts. Following GAP-43 phosphorylation, this phospholipid is released to promote local actin filament-membrane attachment. The phosphorylation also releases GAP-43 from calmodulin. The released GAP-43 may then act as a lateral stabilizer of actin filaments. N-terminal fragments of GAP-43, containing 10-20 amino acids, will activate heterotrimeric G proteins, direct GAP-43 to the membrane and lipid rafts, and cause the formation of filopodia, possibly by causing a change in membrane tension. This review will focus on new information regarding GAP-43, including its binding to membranes and its incorporation into lipid rafts, its mechanism of action, and how it affects and is affected by extracellular agents.

Leptin Increases Axonal Growth Cone Size in Developing Mouse Cortical Neurons by Convergent Signals Inactivating Glycogen Synthase Kinase-3β

We examined the effects of the adipose hormone leptin on the development of mouse cortical neurons. Treatment of neonatal and adult mice with intraperitoneal leptin (5 mg/kg) induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in pyriform and entorhinal cortex neurons. Stimulation of cultured embryonic cortical neurons with leptin evoked Janus kinase 2 and ERK1/2 phosphorylation and activated the downstream effector 90-kDa ribosomal protein S6 kinase. Moreover, leptin elicited the phosphorylation of the phosphatidylinositol 3-kinase effector Akt and evoked Ser-9 phosphorylation of glycogen synthase kinase-3beta (GSK3beta), an event inactivating this kinase. Leptin-mediated GSK3beta phosphorylation was prevented by the MEK/ERK inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor LY294002, or the protein kinase C inhibitor GF109203X. Exposure of cortical neurons to leptin also induced Ser-41 phosphorylation of the neuronal growth-associated protein GAP-43, an effect prevented by LY294002 and GF109203X but not by PD98059. Ser-41-GAP-43 phosphorylation is usually high in expanding axonal growth cones. Neurons exposed to 100 ng/ml leptin for 72 h displayed reduced rate of growth cone collapse, a shift of growth cone size distribution toward higher values, and a 4-fold increase in mean growth cone surface area compared with control cultures. The leptin-induced growth cone spreading was hampered in cortical neurons from Lepr(db/db) mice lacking functional leptin receptors; it was associated with localized Ser-9-GSK3beta phosphorylation and mimicked by the GSK3beta inhibitor SB216763. At concentrations preventing GSK3beta phosphorylation, PD98059, LY294002, or GF109203X reversed the leptin-induced growth cone surface enlargement. We concluded that the leptin-mediated regulation of growth cone morphogenesis in cortical neurons relies on upstream regulators of GSK3beta activity.

Sleep deprivation‐induced protein changes in basal forebrain: Implications for synaptic plasticity

The need to sleep is universal and lack of sleep often results in decreases in alertness and cognitive function. Data suggest that sleep-related mechanisms and deficits resulting from loss of sleep are associated anatomically with the basal forebrain. Long-term effects of sleep deprivation, those lasting a day or more, likely require transcriptional changes leading to changes in protein synthesis, whereas short-term effects may be mediated by rapid changes in the functional status of proteins. To understand sleep deprivation-induced changes in proteins in the wake-promoting area of the basal forebrain in rat, proteomic analysis was carried out by a combination of 2D gel electrophoresis to separate and visualize proteins and matrix-assisted laser desorption/ionization time-of flight mass spectrometry for protein identification. Among 969 protein spots that were compared, 89 spots showed more than a twofold difference between 6-hr sleep-deprived rats and undisturbed sleeping controls. We have identified 11 of these proteins to be either cytoskeletal or associated with synapses. The changes in four of these proteins were analyzed further by Western blots of 1D and 2D. Two proteins associated with the cytoskeleton, tubulin and GAP43, show posttranslational modifications. Increased tyrosination of alpha tubulin isoforms and increased phosphorylation of GAP43 was observed after 6-hr sleep deprivation when compared to that in sleeping controls. The synaptic protein synaptosomal-associated protein-25 (SNAP25) is decreased whereas amphiphysin is phosphorylated after sleep deprivation. These changes in proteins in the basal forebrain during short-term sleep deprivation are suggestive of changes in the substrate for neuronal transmission and plasticity.

Rho‐associated kinase (ROCK) inhibitor, Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurite extension and retraction are very important processes in the formation of neuronal networks. A strategy for fostering axonal regrowth/regeneration of injured adult neurons is attractive therapeutically for various diseases such as traumatic brain injury, stroke and Alzheimer's disease. The Rho family of small GTPases, including Rac and Cdc42 have been shown to be involved in promoting neurite outgrowth. On the other hand, activation of RhoA induces collapse of growth cone and retraction of neurites. Rho‐associated kinase (ROCK) an effector molecule of RhoA, is downstream of a number of axonal outgrowth and growth cone collapse inhibition mechanisms. In the present study, we sought to identify the role of ROCK in neurite outgrowth in PC12 cells. Y27632, a specific inhibitor of ROCK, induced a robust increase in neurite outgrowth in these cells within 24–48 h as visualized by phase contrast microscopy. Staining with FITC‐tubulin or phalloidin show extended neurites in PC12 cells treated with Y27632, comparable to that with 100 ng/mL of NGF. Assessment of other biochemical markers of neurite outgrowth such as GAP43, neurofilament and tyrosine hydroxylase phosphorylation further indicates that inhibition of ROCK in PC12 cells causes differentiation of these cells to a neuronal phenotype.

Changes in protein kinase C (PKC) activity, isozyme translocation, and GAP-43 phosphorylation in the rat hippocampal formation after a single-trial contextual fear conditioning paradigm.

The hippocampus plays an important role in spatial learning and memory. However, the biochemical alterations that subserve this function remain to be fully elucidated. In this study, rats were subjected to a single-trial contextual fear conditioning (CFC) paradigm; the activation of different protein kinase C (PKC) subtypes and the levels and phosphorylation of the plasticity-associated protein GAP-43 were assayed in the hippocampus at varying times after training. We observed a rapid activation of hippocampal PKC (15 min through 24 h), with differential translocation of the PKC isotypes studied. At early times after CFC (15-90 min), PKCalpha and PKCgamma translocated to the membrane, while PKCbetaII and PKCepsilon moved more transiently (15 to 30 min) to the cytosol. These PKC isotypes returned to the membrane at later time points after CFC. Correlating with these changes in PKC translocation and activity, there was an early decrease in GAP-43 phosphorylation followed by a more sustained increase from 1.5-72 h. GAP-43 protein levels were also increased after 3 h, and these levels remained elevated for at least 72 h. These changes in PKC and GAP-43 were specific to the CFC trained animals and no changes were seen in animals exposed to the same stimuli in a non-associative fashion. Comparison of translocation of different PKC isotypes with the changes in GAP-43 phosphorylation suggested that PKCbetaII and PKCepsilon may mediate both the early changes in the phosphorylation of this protein and the increases in GAP-43 expression at later times after CFC.

Effects of nerve growth factor on expression of GAP-43 in right atria after sympathectomy in diabetic rats.

The present study investigated the role of nerve growth factor (NGF) in the regeneration of noradrenergic nerves of right atria (following 6-hydroxydopamine; 6-OHDA, 100 mg/kg, i.p.) from non-diabetic and 8-week diabetic rats. In cryostat sections of the right atria, GAP-43 immunoreactivity was concentrated in nerve terminals, preterminal axons of the endocardium, epicardium and myocardium, as well as in nerve fibres innervating the blood vessels and ganglionic cells. In serial sections, all positive staining for GAP-43 showed immunoreactivity for the neuronal marker PGP-9.5. In untreated non-diabetic rats, the total GAP-43 immunoreactivity was reduced to 60% relative to pretreatment levels, at day 14 after 6-OHDA, as quantified by Western blotting. In diabetic rats, 6-OHDA treatment produced a marked increase in the levels of total GAP-43 at days 28 and 49. NGF treatment (1 mg/kg, s.c., 3 times/week, for 2 weeks) had no effect on the level of total GAP-43 in right atria from non-diabetic and diabetic rats before treatment with 6-OHDA. However, it normalized the reduced GAP-43 immunoreactivity observed in 6-OHDA-treated non-diabetic rats. Interestingly, NGF treatment alone produced an increase in GAP-43 phosphorylation relative to total GAP-43 in right atria from both non-diabetic (44%) and diabetic groups (42%). These findings suggest that nerve terminals of the right atria retain, in the mature adult, the capacity for structural and functional plasticity. The expression of GAP-43 in right atria of control and diabetic rats was differentially affected by 6-OHDA treatment. In injured noradrenergic neurones of the right atria, NGF modified the expression of GAP-43 only in non-diabetic rats and not in diabetic rats.

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.

Nitrous oxide, but not xenon, affects the signaling in the neuronal growth cone

1. 1. Xenon (Xe) is an inert gas with the anesthetic property. To investigate whether Xe affects the neural network formation, the authors examined the biochemical characteristics of growth cones prepared from rat forebrains at different perinatal periods, in comparison with inhalation of N 2O. 2. 2. Fetal or neonatal rats were exposed to an atmosphere containing inhalational anesthetics (70% Xe or N 2O) or the control atmosphere (30% O 2and 70% N 2) for 6 h. After the exposure, isolated growth cone particles (IGCs) were prepared from their forebrains using a subcellular fractionation method. Protein composition, Ca 2+-dependent protein phosphorylation, protein kinase C (PKC) activity, and degradation of PKC in the IGCs were compared among three groups. 3. 3. No apparent change of protein composition in IGCs was observed by electrophoresis. Ca 2+-dependent phosphorylation of GAP-43 and MARCKS protein, and PKC activity in IGCs significantly decreased after exposure to N 2O. The degradation of PKC increased significantly after inhalation of N 2O. 4. 4. The authors concluded that Xe dose not change the above biochemical characteristic of the growth cones, suggesting that Xe is free from the teratogenic effect on the neuronal network formation and that Xe is a safe anesthetics for the perinatal neuronal development.