PolyADP-ribose polymerase-1 (PARP-1) and the evolution of learning and memory

PARP-1 is a multifunctional enzyme that can modulate gene expression. Cohen-Armon et al.(1) found that a homologue of PARP-1 is activated in the Aplysia nervous system as the animal responds to an aversive stimulus, which leads to sensitization, and during a more complex form of learning that involves feeding behavior. Significantly, inhibiting PARP-1 activation blocked the learning. Several key pathways in Aplysia neurons are activated both during learning and after injury, suggesting that mechanisms of learning evolved from primitive responses to injury. Since PARP-1 is evolutionarily conserved as a responder to various forms of stress, the finding that PARP-1 is activated during learning supports this idea.     

Mnk is a negative regulator of cap-dependent translation in Aplysia neurons

To investigate the mechanisms underlying regulation of eukaryotic initiation factor 4E (eIF4E) phosphorylation in Aplysia neurons, we have cloned the Aplysia homolog of the vertebrate eIF4E kinases, Mnk1 and -2. Aplysia Mnk shares many conserved regions with vertebrate Mnk, including putative eukaryotic initiation factor 4G binding regions, activation loop phosphorylation sites, and a carboxy-terminal anchoring site for MAP kinases. As expected, purified Aplysia Mnk phosphorylated Aplysia eIF4E at a conserved carboxy-terminal serine and over-expression of Aplysia Mnk in sensory neurons led to increased phosphorylation of endogenous eIF4E. Over-expression of Aplysia Mnk led to strong decreases in cap-dependent translation, while generally sparing internal ribosomal entry site (IRES)-dependent translation. However, decreases in cap-dependent translation seen after expression of Aplysia Mnk could only be partly explained by increases in eIF4E phosphorylation. In Aplysia sensory neurons, phosphorylation of eIF4E is reduced during intermediate memory formation. However, we found that this physiological regulation of eIF4E phosphorylation was independent of changes in Aplysia Mnk phosphorylation. We propose that changes in eIF4E phosphorylation in Aplysia neurons are a consequence of changes in cap-dependent translation that are independent of regulation of Aplysia Mnk.     

Attenuation of ischemia/reperfusion induced MAP kinases by N-acetyl cysteine,sodium nitroprusside and phosphoramidon

Ischemia followed by reperfusion has a number of clinically significant consequences. A number of pathophysiological processes appear to be involved in ischemia/reperfusion (I/R) injury. The mitogen activated protein kinases (MAPK) are integral components of the parallel MAP kinase cascades activated in response to a variety of cellular stress inducing ischemia/ATP depletion and inflammatory cytokines. Many studies suggest that members of the MAP kinase family in particular Jun N-terminal kinase (JNK) are activated in kidney following ischemia/reperfusion of this tissue. The present study underlines the therapeutic potential of the combination of N-acetyl cysteine (NAC), a potent antioxidant, sodium nitroprusside (SNP), a nitric oxide donor and phosphoramidon (P), an endothelin-1 converting enzyme inhibitor in ameliorating the MAPK induced damage during renal ischemia/reperfusion injury. Our previous results showed that 90 min of ischemia followed by reperfusion caused very severe injury and that the untreated animals had 100% mortality after the 3rd day whereas there was improved renal function and 100% survival of animals in the three drug combination treatment group. The present study, mainly on tissue sections, further supports the protection provided by the triple drug therapy. A higher degree of expression of all the three classes of MAPK, i.e. JNK, P³⁸ MAP kinases and P-extracellular signal regulated kinases (ERKs) can be seen in kidneys subjected to ischemia/reperfusion insult. Pretreatment with a combination of N-acetyl cysteine, sodium nitroprusside, and phosphoramidon completely inhibits all three classes of MAPK and ameliorates AP-1 whereas individual or a combination of any two drugs is not as effective.     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.     

NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals

Target-derived NGF promotes the phenotypic maintenance of mature dorsal root ganglion (DRG) nociceptive neurons. Here, we provide in vivo and in vitro evidence for the presence within DRG neurons of endosomes containing NGF, activated TrkA, and signaling proteins of the Rap1/Erk1/2, p38MAPK, and PI3K/Akt pathways. Signaling endosomes were shown to be retrogradely transported in the isolated sciatic nerve in vitro. NGF injection in the peripheral target of DRG neurons increased the retrograde transport of p-Erk1/2, p-p38, and pAkt in these membranes. Conversely, NGF antibody injections decreased the retrograde transport of p-Erk1/2 and p-p38. Our results are evidence that signaling endosomes, with the characteristics of early endosomes, convey NGF signals from the target of nociceptive neurons to their cell bodies.     

Protein kinase C phosphorylated at a conserved threonine is retained in the cytoplasm.

Phosphorylation of calcium-activated protein kinase Cs (PKCs) at threonine 634 and/or threonine 641 increases during long term potentiation or associative learning in rodents. In the marine mollusk Aplysia, persistent activation of the calcium-activated PKC Apl I occurs during long term facilitation. We have raised an antibody to a peptide from PKC Apl I phosphorylated at threonines 613 and 620 (sites homologous to threonines 634 and 641). This antibody recognizes PKC Apl I only when it is phosphorylated at threonine 613. Both phorbol esters and serotonin increase the percentage of kinase phosphorylated at threonine 613 in Aplysia neurons. Furthermore, the pool of PKC that is phosphorylated at threonine 613 in neurons is resistant to both membrane translocation and down-regulation. Replacement of threonine 613 with alanine increased the affinity of PKC Apl I for calcium, suggesting that phosphorylation of this site may reduce the ability of PKC Apl I to translocate to membranes in the presence of calcium. We propose that phosphorylation of this site is important for removal of PKC from the membrane and may be a mechanism for negative feedback of PKC activation.     

Characterization of Neuronal Protein Phosphatases in Aplysia californica

Biochemical properties of neuronal protein phosphatases from Aplysia californica were characterized. Dephosphorylation of phosphorylase alpha by extracts of abdominal ganglia and clusters of sensory neurons from pleural ganglia was demonstrated. Type-1 protein phosphatase (PrP-1) was identified in these extracts by the dephosphorylation of the beta-subunit of phosphorylase kinase and its inhibition by the protein, inhibitor-2. Type-2A protein phosphatase (PrP-2A) was demonstrated by the dephosphorylation of the alpha-subunit of phosphorylase kinase, which was insensitive to inhibitor-2. As in vertebrate tissues, only four enzymes, PrP-1 (47%), PrP-2A (42%), PrP-2B (11%), and PrP-2C (less than 1%), accounted for all the cellular protein phosphatase activity dephosphorylating phosphorylase kinase. Aplysia PrP-1 and PrP-2A were potently inhibited by okadaic acid, with PrP-1 being approximately 20-fold more sensitive than PrP-2A. By comparison, purified PrP-2A from rabbit skeletal muscle was 15- to 20-fold more sensitive to okadaic acid than PrP-1 from the same source. Only PrP-1 was associated with the particulate fractions from Aplysia neurons, whereas PrP-1 and PrP-2A, -2B, and -2C were all present in the cytosol. Extraction of the particulate PrP-1 decreased its sensitivity to okadaic acid by sixfold, suggesting that cellular factor(s) affect its sensitivity to this inhibitor. In most respects, protein phosphatases from Aplysia neurons resemble their mammalian counterparts, and their biochemical characterization sets the stage for examining the role of these enzymes in neuronal plasticity, and in learning and memory.     

NGF-stimulated retrograde transport of trkA in the mammalian nervous system

The present study was designed to clarify the in vivo function of trkA as an NGF receptor in mammalian neurons. Using the rat sciatic nerve as a model system, we examined whether trkA is retrogradely transported and whether transport is influenced by physiological manipulations. Following nerve ligation, trkA protein accumulates distal to the ligation site as shown by Western blot analysis. The distally accumulating trkA species were tyrosine phosphorylated. The trkA retrograde transport and phosphorylation were enhanced by injecting an excess of NGF in the footpad and were abolished by blocking endogenous NGF with specific antibodies. These results provide evidence that, upon NGF binding, trkA is internalized and retrogradely transported in a phosphorylated state, possibly together with the neurotrophin. Furthermore, our results suggest that trkA is a primary retrograde NGF signal in mammalian neurons in vivo.     

Aplysia synapse associated protein (APSAP): identification, characterization, and selective interactions with Shaker-type potassium channels

The vertebrate post-synaptic density (PSD) is a region of high molecular complexity in which dynamic protein interactions modulate receptor localization and synaptic function. Members of the membrane-associated guanylate kinase (MAGUK) family of proteins represent a major structural and functional component of the vertebrate PSD. In order to investigate the expression and significance of orthologous PSD components associated with the Aplysia sensory neuron-motor neuron synapse, we have cloned an Aplysia Dlg-MAGUK protein, which we identify as Aplysia synapse associated protein (ApSAP). As revealed by western blot, RT-PCR, and immunocytochemical analyses, ApSAP is predominantly expressed in the CNS and is located in both sensory neuron and motor neurons. The overall amino acid sequence of ApSAP is 55–61% identical to Drosophila Dlg and mammalian Dlg-MAGUK proteins, but is more highly conserved within L27, PDZ, SH3, and guanylate kinase domains. Because these conserved domains mediate salient interactions with receptors and other PSD components of the vertebrate synapse, we performed a series of GST pull-down assays using recombinant C-terminal tail proteins from various Aplysia receptors and channels containing C-terminal PDZ binding sequences. We have found that ApSAP selectively binds to an Aplysia Shaker-type channel AKv1.1, but not to (i) NMDA receptor subunit AcNR1-1, (ii) potassium channel AKv5.1, (iii) receptor tyrosine kinase ApTrkl, (iv) glutamate receptor ApGluR1/4, (v) glutamate receptor ApGluR2/3, or (vi) glutamate receptor ApGluR7. These findings provide preliminary information regarding the expression and interactions of Dlg-MAGUK proteins of the Aplysia CNS, and will inform questions aimed at a functional analysis of how interactions in a protein network such as the PSD may regulate synaptic strength.     

Mitogen-activated protein kinases: A new therapeutic target in cardiac pathology

Eukaryotic cells respond to different external stimuli by activation of mechanisms of cell signaling. One of the major systems participating in the transduction of signal from the cell membrane to nuclear and other intracellular targets is the highly conserved mitogen-activated protein kinase (MAPK) superfamily. The members of MAPK family are involved in the regulation of a large variety of cellular processes such as cell growth, differentiation, development, cell cycle, death and survival. Several MAPK subfamilies, each with apparently unique signaling pathway, have been identified in the mammalian myocardium. These cascades differ in their upstream activation sequence and in downstream substrate specifity. Each pathway follows the same conserved three-kinase module consisting of MAPK, MAPK kinase (MAPKK, MKK or MEK), and MAPK kinase kinase (MAPKKK, MEKK). The major groups of MAPKs found in cardiac tissue include the extracellular signal-regulated kinases (ERKs), the stress-activated/c-Jun NH2-terminal kinases (SAPK/JNKs), p38-MAPK, and ERK5/big MAPK 1 (BMK1). The ERKs are strongly activated by mitogenic and growth factors and by physical stress, whereas SAPK/JNKs and p38-MAPK can be activated by various cell stresses, such as hyperosmotic shock, metabolic stress or protein synthesis inhibitors, UV radiation, heat shock, cytokines, and ischemia. Activation of MAPKs family plays a key role in the pathogenesis of various processes in the heart, e.g. myocardial hypertrophy and its transition to heart failure, in ischemic and reperfusion injury, as well in the cardioprotection conferred by ischemia- or pharmacologically-induced preconditioning. The following approaches are currently utilized to elucidate the role of MAPKs in the myocardium: (i) studies of the effects of myocardial processes on the activity of these kinases; (ii) pharmacological modulations of MAPKs activity and evaluation of their impact on the (patho)physiological processes in the heart; (iii) gene targeting or expression of constitutively active and dominant-negative forms of enzymes (adenovirus-mediated gene transfer).This review is focused on the regulatory role of MAPKs in the myocardium, with particular regard to their involvement in pathophysiological processes, such as myocardial hypertrophy and heart failure, ischemia/reperfusion injury, as well as in the mechanisms of cardioprotection. In addition, it summarizes current information on pharmacological modulations of MAPKs activity and their impact on the cardiac response to pathophysiological processes.     

p38 mitogen-activated protein kinase (MAPK) first regulates filamentous actin at the 8-16-cell stage during preimplantation development

BACKGROUND INFORMATION: The MAPK (mitogen-activated protein kinase) superfamily of proteins consists of four separate signalling cascades: the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPK); the ERKs (extracellular-signal-regulated kinases); the ERK5 or big MAPK1; and the p38 MAPK group of protein kinases, all of which are highly conserved. To date, our studies have focused on defining the role of the p38 MAPK pathway during preimplantation development. p38 MAPK regulates actin filament formation through the downstream kinases MAPKAPK2/3 (MAPK-activated protein kinase 2/3) or MAPKAPK5 [PRAK (p38 regulated/activated kinase)] and subsequently through HSP25/27 (heat-shock protein 25/27). We recently reported that 2-cell-stage murine embryos treated with cytokine-suppressive anti-inflammatory drugs (CSAIDtrade mark; SB203580 and SB220025) display a reversible blockade of development at the 8-16-cell stage, indicating that p38 (MAPK) activity is required to complete murine preimplantation development. In the present study, we have investigated the stage-specific action and role of p38 MAPK in regulating filamentous actin during murine preimplantation development. RESULTS: Treatment of 8-cell-stage embryos with SB203580 and SB220025 (CSAIDtrade mark) resulted in a blockade of preimplantation development, loss of rhodamine phalloidin fluorescence, MK-p (phosphorylated MAPKAPK2/3), HSP-p (phosphorylated HSP25/27) and a redistribution of alpha-catenin immunofluorescence by 12 h of treatment. In contrast, treatment of 2- and 4-cell-stage embryos with CSAIDtrade mark drugs resulted in a loss of MK-p and HSP-p, but did not result in a loss of rhodamine phalloidin fluorescence. All these effects of p38 MAPK inhibition were reversed upon removal of the inhibitor, and development resumed in a delayed but normal manner to the blastocyst stage. Treatment of 8-cell embryos with PD098059 (ERK pathway inhibitor) did not affect development or fluorescence of MK-p, HSP-p or rhodamine phalloidin. CONCLUSION: Murine preimplantation development becomes dependent on p38 MAPK at the 8-16-cell stage, which corresponds to the stage when p38 MAPK first regulates filamentous actin during early development.     

Regulation of MAPK pathways in response to purinergic stimulation of adult rat cardiac myocytes

We investigated the activation of mitogen-activated protein kinases (MAPKs) pathways by purinergic stimulation in cardiac myocytes from adult rat hearts. ATPS increased the phosphorylation (activation) of the extracellular signal regulated kinase 1 and 2 (ERK1/2) and p38 MAPK. ERK1/2 and p38 MAPK activation was differential, ERK1/2 being rapid and transient while that of p38 MAPK slow and sustained. Using selective inhibitors, activation of ERK1/2 was shown to involve protein kinase C and MEK1/2 while that of p38 MAPK was regulated by both protein kinase C and protein kinase A. Furthermore, we show that purinergic stimulation induces the phosphorylation of the MAPK downstream target, mitogen- and stress-activated protein kinase 1 (MSK1), in cardiac myocytes. The time course of MSK1 phosphorylation closely follows that of ERK activation. Inhibitors of the ERK and p38 MAPK pathways were tested on the phosphorylation of MSK1 at two different time points. The results suggest that ERKs initiate the response but both ERKs and p38 MAPK are required for the maintenance of the complete phosphorylation of MSK1. The temporal relationship of MSK1 phosphorylation and cPLA₂ translocation induced by purinergic stimulation, taken together with previous findings, is an indication that cPLA₂ may be a downstream target of MSK1.     

Differential Activation of Mitogen-Activated Protein Kinase Pathways in PC12 Cells by Closely Related α1-Adrenergic Receptor Subtypes

Coupling of the three known alpha1-adrenergic receptor (alpha1-AR) subtypes to mitogen-activated protein kinase (MAPK) pathways were studied in stably transfected PC12 cells. Subclones stably expressing alpha1A-, alpha1B-, and alpha1D-ARs under control of an inducible promoter, or at high and low receptor density, were isolated and characterized. Radioligand binding showed similar ranges of expression of each subtype. Norepinephrine (NE) increased inositol phosphate formation and intracellular Ca2+ level in these cells in a manner dependent on receptor density. However, alpha1A-ARs activated these second messenger responses more effectively than alpha1B-ARs, whereas alpha1D-ARs were least effective. NE stimulated activation of extracellular signal-regulated kinases (ERKs) in cells expressing all three alpha1-AR subtypes, although alpha1A- and alpha1B-ARs caused larger ERK activation than did alpha1D-ARs. Nerve growth factor (NGF) caused similar levels of ERK activation in all subclones. NE also activated p38 MAPK in alpha1A- and alpha1B- but not alpha1D-transfected cells and activated c-Jun NH2-terminal kinase (JNK) only in alpha1A-transfected cells. NE, but not NGF, strongly stimulated tyrosine phosphorylation of a 70-kDa protein only in alpha1A-transfected PC12 cells. NE caused neurite outgrowth only in alpha1A-expressing PC12 cells, but not in alpha1B- or alpha1D-transfected cells, whereas NGF caused neurite outgrowth in all cells. These studies show that alpha1A-ARs activate all three MAPK pathways, alpha1B-ARs activate ERKs and p38 but not JNKs, and alpha1D-ARs only activate ERKs. Only the alpha1A-AR-expressing cells differentiated in response to NE. The relationship of these responses to second messenger pathways activated by these subtypes is discussed.