Biomedical subjects

J D Sweatt

Publications and source records attributed to J D Sweatt.

At least 37 records · Page 2Linked to original sources

PubMed2000

MAPK regulation of gene expression in the central nervous system.

Long-term potentiation (LTP), a cellular model for long-term memory, is generally acknowledged to consist of both a short-term phase that is characterized by a dependence on autonomous protein kinase activity, and a long-term phase that is characterized by a dependence on changes in gene expression and new protein synthesis. Similarly, long-term memory exhibits a dependence on gene expression and altered protein synthesis. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) cascade plays a role in both LTP and long-term memory. The MAPK cascade has heretofore largely been studied in the context of cell division and proliferation and as such, mechanisms for the regulation of gene expression by the MAPK cascade have received considerable attention. Given the possible role of altered gene expression in the late phase of LTP and in long-term memory, we evaluated the capacity of the MAPK ERK (extracellular signal-regulated kinase) to regulate phosphorylation of the transcription factor cAMP response element binding protein (CREB) in hippocampal area CA1. Our studies indicate a critical role for the MAPK cascade in the regulation of CREB phosphorylation in the hippocampus.

Animals↗

PubMed1999

Reactive oxygen species mediate activity-dependent neuron-glia signaling in output fibers of the hippocampus.

Nonsynaptic signaling is becoming increasingly appreciated in studies of activity-dependent changes in the nervous system. We investigated the types of neuronal activity that elicit nonsynaptic communication between neurons and glial cells in hippocampal output fibers. High-frequency, but not low-frequency, action potential firing in myelinated CA1 axons of the hippocampus resulted in increased phosphorylation of the oligodendrocyte-specific protein myelin basic protein (MBP). This change was blocked by tetrodotoxin, indicating that axonally generated action potentials were necessary to regulate the phosphorylation state of MBP. Furthermore, scavengers of the reactive oxygen species superoxide and hydrogen peroxide and nitric oxide synthase inhibitors prevented activation of this neuron-glia signaling pathway. These results indicate that, during periods of increased neuronal activity in area CA1 of the hippocampus, reactive oxygen and nitrogen species are generated, which diffuse to neighboring oligodendrocytes and result in post-translational modifications of MBP, a key structural protein in myelin. Thus, in addition to their well-known capacity for activity-dependent neuron-neuron signaling, hippocampal pyramidal neurons possess a mechanism for activity-dependent neuron-glia signaling.

Action Potentials↗

PubMed1999

The mitogen-activated protein kinase cascade couples PKA and PKC to cAMP response element binding protein phosphorylation in area CA1 of hippocampus.

Activation of the mitogen-activated protein kinase (MAPK) cascade recently was discovered to play an important role in synaptic plasticity in area CA1 of rat hippocampus. However, the upstream mechanisms regulating MAPK activity and the downstream effectors of MAPK in the hippocampus are uncharacterized. In the present studies we observed that hippocampal MAPK activation is regulated by both the PKA and PKC systems; moreover, we found that a wide variety of neuromodulatory neurotransmitter receptors (metabotropic glutamate receptors, muscarinic acetylcholine receptors, dopamine receptors, and beta-adrenergic receptors) couple to MAPK activation via these two cascades. In additional studies we observed that PKC is a powerful regulator of CREB phosphorylation in area CA1. MAPK plays a critical role in transcriptional regulation by PKC, because MAPK activation is a necessary component for increased CREB phosphorylation in response to the activation of this kinase. Surprisingly, we also observed that MAPK activation is necessary for PKA coupling to CREB phosphorylation in area CA1. Overall, these studies indicate an unexpected richness of diversity in the regulation of MAPK in the hippocampus and suggest the possibility of a broad role for the MAPK cascade in regulating gene expression in long-term forms of hippocampal synaptic plasticity.

Animals↗

PubMed1999

Regulation of myelin basic protein phosphorylation by mitogen-activated protein kinase during increased action potential firing in the hippocampus.

Myelin basic protein (MBP) phosphorylation is a complex regulatory process that modulates the contribution of MBP to the stability of the myelin sheath. Recent research has demonstrated the modulation of MBP phosphorylation by mitogen-activated protein kinase (MAPK) during myelinogenesis and in the demyelinating disease multiple sclerosis. Here we investigated the physiological regulation of MBP phosphorylation by MAPK during neuronal activity in the alveus, the myelinated output fibers of the hippocampus. Using a phosphospecific antibody that recognizes the predominant MAPK phosphorylation site in MBP, Thr95, we found that MBP phosphorylation is regulated by high-frequency stimulation but not low-frequency stimulation of the alveus. This change was blocked by application of tetrodotoxin, indicating that action potential propagation in axons is required. It is interesting that the change in MBP phosphorylation was attenuated by the reactive oxygen species scavengers superoxide dismutase and catalase and the nitric oxide synthase inhibitor N-nitro-L-arginine. Removal of extracellular calcium also blocked the changes in MBP phosphorylation. Thus, we propose that during periods of increased neuronal activity, calcium activates axonal nitric oxide synthase, which generates the intercellular messengers nitric oxide and superoxide and regulates the phosphorylation state of MBP by MAPK.

Action Potentials↗

PubMed1999

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes.

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Amobarbital↗

PubMed1998

Mice lacking ataxin-1 display learning deficits and decreased hippocampal paired-pulse facilitation.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder characterized by ataxia, progressive motor deterioration, and loss of cerebellar Purkinje cells. To investigate SCA1 pathogenesis and to gain insight into the function of the SCA1 gene product ataxin-1, a novel protein without homology to previously described proteins, we generated mice with a targeted deletion in the murine Sca1 gene. Mice lacking ataxin-1 are viable, fertile, and do not show any evidence of ataxia or neurodegeneration. However, Sca1 null mice demonstrate decreased exploratory behavior, pronounced deficits in the spatial version of the Morris water maze test, and impaired performance on the rotating rod apparatus. Furthermore, neurophysiological studies performed in area CA1 of the hippocampus reveal decreased paired-pulse facilitation in Sca1 null mice, whereas long-term and post-tetanic potentiations are normal. These findings demonstrate that SCA1 is not caused by loss of function of ataxin-1 and point to the possible role of ataxin-1 in learning and memory.

Analysis of Variance↗

PubMed1998

A role for superoxide in protein kinase C activation and induction of long-term potentiation.

The induction of several forms of long-term potentiation (LTP) of synaptic transmission in the CA1 region of the mammalian hippocampus is dependent on N-methyl-D-aspartate receptor activation and the subsequent activation of protein kinase C (PKC), but the mechanisms that underlie the regulation of PKC in this context are largely unknown. It is known that reactive oxygen species, including superoxide, are produced by N-methyl-D-aspartate receptor activation in neurons, and recent studies have suggested that some reactive oxygen species can modulate PKC in vitro. Thus, we have investigated the role of superoxide in both the induction of LTP and the activation of PKC during LTP. We found that incubation of hippocampal slices with superoxide scavengers inhibited the induction of LTP. The effects of superoxide on LTP induction may involve PKC, as we observed that superoxide was required for appropriate modulation of PKC activation during the induction of LTP. In this respect, superoxide appears to work in conjunction with nitric oxide, which was required for a portion of the LTP-associated changes in PKC activity as well. Our observations indicate that superoxide and nitric oxide together regulate PKC in a physiologic context and that this type of regulation occurs during the induction of LTP in the hippocampus.

Animals↗

PubMed1998

Mutation of the Angelman ubiquitin ligase in mice causes increased cytoplasmic p53 and deficits of contextual learning and long-term potentiation.

The E6-AP ubiquitin ligase (human/mouse gene UBE3A/Ube3a) promotes the degradation of p53 in association with papilloma E6 protein, and maternal deficiency causes human Angelman syndrome (AS). Ube3a is imprinted with silencing of the paternal allele in hippocampus and cerebellum in mice. We found that the phenotype of mice with maternal deficiency (m-/p+) for Ube3a resembles human AS with motor dysfunction, inducible seizures, and a context-dependent learning deficit. Long-term potentiation (LTP) was severely impaired in m-/p+ mice despite normal baseline synaptic transmission and neuroanatomy, indicating that ubiquitination may play a role in mammalian LTP and that LTP may be abnormal in AS. The cytoplasmic abundance of p53 was increased in postmitotic neurons in m-/p+ mice and in AS, providing a potential biochemical basis for the phenotype through failure to ubiquitinate and degrade various effectors.

Acoustic Stimulation↗

PubMed1998

The MAPK cascade is required for mammalian associative learning.

Mitogen-activated protein kinase (MAPK) is an integral component of cellular signaling during mitogenesis and differentiation of mitotic cells. Recently MAPK activation in post-mitotic cells has been implicated in hippocampal long-term potentiation (LTP), a potential cellular mechanism of learning and memory. Here we investigate the involvement of MAPK in learning and memory in behaving animals. MAPK activation increased in the rat hippocampus after an associative learning task, contextual fear conditioning. Two other protein kinases known to be activated during hippocampal LTP, protein kinase C and alpha-calcium/calmodulin protein kinase II, also were activated in the hippocampus after learning. Inhibition of the specific upstream activator of MAPK, MAPK kinase (MEK), blocked fear conditioning. Thus, classical conditioning in mammals activates MAPK, which is necessary for consolidation of the resultant learning.

Animals↗

PubMed1998

Protected-site phosphorylation of protein kinase C in hippocampal long-term potentiation.

One important aspect of synaptic plasticity is that transient stimulation of neuronal cell surface receptors can lead to long-lasting biochemical and physiological effects in neurons. In long-term potentiation (LTP), generation of autonomously active protein kinase C (PKC) is one biochemical effect persisting beyond the NMDA receptor activation that triggers plasticity. We previously observed that the expression of early LTP is associated with a phosphatase-reversible alteration in PKC immunoreactivity, suggesting that autophosphorylation of PKC might be elevated in LTP. In the present studies we tested the hypothesis that PKC phosphorylation is persistently increased in the early maintenance of LTP. We generated an antiserum that selectively recognizes the alpha and betaII isoforms of PKC autophosphorylated in the C-terminal domain. Using western blotting with this antiserum we observed an NMDA receptor-mediated increase in phosphorylation of PKC 1 h after LTP was induced. How is the increased phosphorylation maintained in the cell in the face of ongoing phosphatase activity? We observed that dephosphorylation of PKC in vitro requires the presence of cofactors normally serving to activate PKC, i.e., Ca2+, phosphatidylserine, and diacylglycerol. Based on these observations and computer modeling of the three-dimensional structure of the PKC catalytic core, we propose a "protected site" model of PKC autophosphorylation, whereby the conformation of PKC regulates accessibility of the phosphates to phosphatase. Although we have proposed the protected site model based on our studies of PKC phosphorylation in LTP, phosphorylation of protected sites might be a general biochemical mechanism for the generation of stable, long-lasting physiologic changes.

Amino Acid Sequence↗

PubMed1997

A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation.

The mitogen-activated protein kinase (MAPK) cascade has been intensely studied as a primary biochemical pathway through which a variety of extracellular stimuli initiate and regulate processes of cellular transformation. That MAPKs are abundantly expressed in postmitotic neurons, however, suggests different yet currently unknown functions for this cascade in the mature nervous system. Here we report that the MAPK cascade is required for hippocampal long term potentiation (LTP), a robust and widely studied form of synaptic plasticity. We observed that PD 098059, a selective inhibitor of the MAPK cascade, blocked MAPK activation in response to direct stimulation of the NMDA receptor as well as to LTP-inducing stimuli. Furthermore, inhibition of the MAPK cascade markedly attenuated the induction of LTP. PD 098059, however, had no effect on the expression of established LTP, and the MAPK cascade was not persistently activated during LTP expression. Our observations provide the first demonstration of a role for the MAPK cascade in the activity-dependent modification of synaptic connections between neurons in the adult mammalian nervous system.

Animals↗

PubMed1997

Enhanced phosphorylation of the postsynaptic protein kinase C substrate RC3/neurogranin during long-term potentiation.

Long-term potentiation (LTP) is a sustained strengthening of synaptic connections that occurs in the mammalian hippocampus, and is a cellular mechanism likely to contribute to memory formation. One question of current interest is whether the biochemical mechanisms responsible for the maintenance of LTP have a presynaptic or postsynaptic locus. We have determined that the phosphorylation of the postsynaptic protein kinase (PKC) substrate RC3/neurogranin is increased in the maintenance phase of LTP, and that the induction of this effect is dependent on activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. The sustained increase in RC3/neurogranin phosphorylation requires ongoing protein kinase activity, as application of the protein kinase inhibitor H-7 after LTP induction can reverse the increased RC3/neurogranin phosphorylation. Overall, these data are evidence for postsynaptic biochemical changes in the maintenance of LTP. They also implicate RC3/neurogranin as a downstream effector of PKC activity in LTP that could contribute to physiologic expression of LTP.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1997

Increased phosphorylation of myelin basic protein during hippocampal long-term potentiation.

Hippocampal long-term potentiation (LTP) is a long-lasting and rapidly induced increase in synaptic strength. Previous experiments have determined that persistent activation of protein kinase C (PKC) contributes to the early maintenance phase of LTP (E-LTP). Using the back-phosphorylation method, we observed an increase in the phosphorylation of a 21-kDa PKC substrate, termed p21, 45 min after LTP was induced in the CA1 region of the hippocampus. p21 was found to have the same apparent molecular weight as the 18.5-kDa isoform of myelin basic protein (MBP) and was recognized by an antibody to MBP in western blotting and immunoprecipitation. Furthermore, p21 from control and potentiated hippocampal slices and purified MBP have identical phosphopeptide maps when back-phosphorylated and then digested with either endoproteinase Lys-C or endoproteinase Asp-N, suggesting that p21 and MBP are identical proteins. As there was no observed change in the amount of MBP in LTP, the increase in MBP phosphorylation during LTP cannot be explained by a change in the amount of protein. From these experiments, we conclude that the phosphorylation of the 18.5-kDa isoform of MBP is increased during E-LTP.

Animals↗

PubMed1996

Transient activation of cyclic AMP-dependent protein kinase during hippocampal long-term potentiation.

Long-term potentiation (LTP) in the hippocampus is a possible mechanism for mammalian learning and memory in which protein kinases play critical roles. We have investigated the involvement of cyclic AMP-dependent protein kinase (PKA) in LTP by directly studying its activation. We developed an in vitro assay which is useful for selective and accurate measurement of stimulus-induced changes in PKA activity in hippocampal slices. PKA was transiently activated 2 and 10 min after delivery of LTP-inducing stimuli in area CA1 of the hippocampus. This activation did not persist during early or late phases of LTP, suggesting that the role of PKA is in the induction of LTP, not in its expression. LTP was not associated with any change in the total activity of PKA, consistent with activation by cyclic AMP, as opposed to an increase in the amount or efficacy of the enzyme. The LTP-associated activation of PKA required stimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor, and bath application of NMDA was sufficient to activate PKA. Together, these results indicate that at the initiation of LTP, NMDA receptor stimulation leads to transient activation of PKA, and support a role for PKA in the induction of LTP.

Adenylyl Cyclases↗

PubMed1996

Activation of p42 mitogen-activated protein kinase in hippocampal long term potentiation.

Although classically studied as regulators of cell proliferation and differentiation, mitogen-activated protein kinases (MAPKs) are highly expressed in post-mitotic neurons of the adult nervous system. We have begun investigating the potential role of MAPKs in the regulation of synaptic plasticity in mature neurons. In particular, we have studied the regulation of two MAPK isoforms, p44 and p42 MAPK, in hippocampal long term potentiation (LTP), a system widely studied as a model for the cellular basis of learning and memory. We have found that p42 MAPK, but not p44 MAPK, is activated in area CA1 following direct stimulation of two required components of the LTP induction cascades: protein kinase C and the N-methyl--aspartate (NMDA) subtype of glutamate receptor. Furthermore, we have demonstrated that p42 MAPK, but not p44 MAPK, is activated in area CA1 in response to LTP-inducing high frequency stimulation and that this activation requires NMDA receptor stimulation. These data demonstrate that p42 MAPK can be regulated in an activity-dependent manner in the hippocampus and identify it as a potential component of the LTP induction cascades in area CA1. Such observations suggest that p42 MAPK might be an important regulator of synaptic plasticity in post-mitotic neurons.

Animals↗

PubMed1994

Autonomously active protein kinase C in the maintenance phase of N-methyl-D-aspartate receptor-independent long term potentiation.

In area CA1 of the hippocampus, the induction of long term potentiation (LTP) requires activation of either N-methyl-D-aspartate receptors (NMDA receptor-dependent LTP) or voltage-gated Ca2+ channels (NMDA receptor-independent LTP). We have investigated biochemical sequelae of NMDA receptor-independent LTP induction. We find that a persistent increase in second messenger-independent protein kinase C activity is associated with the maintenance phase of NMDA receptor-independent LTP. This increase in protein kinase C activity is prevented by blocking LTP with nifedipine, a Ca2+ channel antagonist, or kynurenic acid, a nonselective glutamate receptor antagonist. Additionally, we find an increase in the catalytic fragment of protein kinase C (PKM) in the maintenance phase of NMDA receptor-independent LTP, indicating that proteolytic activation of protein kinase C may account for its autonomous activation. This increase in the catalytic fragment of protein kinase C is also prevented by blocking LTP induction. These results are the first to demonstrate that persistent protein kinase C activation is a possible mechanism for the maintenance of NMDA receptor-independent LTP.

Animals↗

PubMed1993

Mechanism of protein kinase C activation during the induction and maintenance of long-term potentiation probed using a selective peptide substrate.

Previous reports using various protein kinase inhibitors have suggested that protein kinase activity is necessary for both the induction and maintenance of hippocampal long-term potentiation (LTP), a cellular phenomenon likely to contribute to mammalian memory formation. We designed and characterized a selective peptide substrate for protein kinase C (PKC), corresponding to amino acids 28 to 43 of the neuronal protein neurogranin, and used the substrate to obtain direct biochemical evidence for activation of PKC in both the induction and maintenance phases of LTP. As the effect cannot be accounted for by either of two well-known mechanisms for persistent PKC activation, membrane insertion, or proteolysis, the persistent activation of PKC in the maintenance phase of LTP appears to occur via another mechanism. The maintenance phase of LTP is associated with decreased immunoreactivity of PKC, an effect that can be reversed with phosphatase treatment. Thus, PKC appears to be both phosphorylated and persistently activated in the maintenance phase of LTP.

Animals↗

PubMed1993

Studies with synthetic peptide substrates derived from the neuronal protein neurogranin reveal structural determinants of potency and selectivity for protein kinase C.

The neuronal protein neurogranin, also known as RC3, is a selective substrate for protein kinase C (PKC). We synthesized a peptide corresponding to the phosphorylation domain of neurogranin (amino acids 28-43) and characterized its properties as a PKC substrate. Neurogranin(28-43) was phosphorylated by purified PKC with a Km of 150 nM. No significant phosphorylation of the peptide by either cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase II could be detected. Thus, neurogranin(28-43) is a potent and selective substrate for PKC. We tested several peptide analogues of neurogranin(28-43) for their substrate potency and specificity as kinase substrates, in order to help elucidate the structural determinants involved in the phosphorylation of substrates by PKC. Substituting Arg36 with Ile caused a significant reduction in the affinity for PKC. Replacing Lys30 with Arg enhanced the catalytic efficiency (Vmax/Km) for PKC but diminished the selectivity of the substrate for PKC. These results support the generally held model that basic amino acids on both sides of the phosphorylated Ser are important structural determinants in PKC substrates. However, the data also suggest that the presence of particular basic amino acids (Arg vs Lys) can contribute to the degree of selectivity of a substrate for PKC. Replacement with Ala of Phe35, the amino acid adjacent to the Ser34 phosphorylation site, resulted in a peptide with greatly diminished potency as a PKC substrate. This finding indicates a critical role of Phe35 in modulating binding and phosphorylation of neurogranin-derived peptides by PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗