James H. Schwartz - US grants

DESCRIPTION (Adapted from applicant's abstract) : The importance of studying synaptic functions at the molecular level is most obvious for understanding mental and neurological diseases where psychopharmacological therapeutics, modern molecular genetics and biochemically-oriented neuropathology suggest an underlying synaptic malady. Long-term presynaptic facilitation of sensory-to-motor, synapses, which is a form of plasticity underlying behavioral sensitization in the marine mollusk Aplysia and an elementary form of learning, can be produced by the action of the cAMP-dependent protein kinases (PKA). A decrease of about 25% in the regulatory (R) subunits of PKA occurs in sensory neurons when treated to produce long-term facilitation; this molecular change endures only if new protein is made. No change in catalytic (C) subunits occurs. A decreased R/C ratio produces a kinases more sensitive to subsaturating cAMP and sets the baseline extent of protein phosphorylation within the neuron at a higher level for at least 24 h; this change could be the molecular phosphorylation within the neuron at a higher level for at least 24 h; this change could be the molecular mechanism underlying an intermediary form of memory. The fine control of cAMP-dependent phosphorylation is mediated by regulated proteolysis through the ubiquitin-proteasome pathway, which degrades R Subunits selectively. During the development of long-term facilitation, persistent protein phosphorylation results in the enhancement of synaptic strength by increasing the output of neurotransmitter at existing synapses; later, the memory is consolidated by normal output of transmitter at an increased number of new synapses. Our working idea is that signal transduction by a facilitation transmitter (e.g., serotonin) activates PKA, which then triggers a molecular cascade in the nucleus involving cAMP-reactive elements for transcription activator proteins and effector proteins, one or more of which alter the ubiquitin-proteasome pathway in sensory neurons. A ubiquitin carboxyl-terminal hydrolase and elongation factor 1a, two proteins that are induced during long-term facilitation, have been implicated in proteasome function in other system. Our first aim is to show, using specific proteasome inhibitors, that the ubiquitin-proteasome pathway is needed for long-term facilitation. Our second aim is to determine whether the induced hydrolase and elongation factor facilitate proteolysis by proteasomes.

Nicotine-type acetylcholine receptors (nAChRs) are expressed on pre- as well as post synaptic structures within the vertebrate central and peripheral nervous systems as well as being widely distributed in invertebrate systems. There is considerable diversity in neuronal nAChR subtypes evident in distinct biophysical and pharmacological profiles that likely reflect differences in nAChR subunit composition. Our previous studies have elucidated two unusual subsets of nAChRs. The first, in vertebrates, is a pre synaptic nAChR that is gated by minute amounts of nicotine and mediates an alpha-bungarotoxin-sensitive facilitation of synaptic transmission, requiring Ca influx through this ionotropic receptor. The second, in this invertebrate, Aplysia californica, is also activated by nicotine blocked by alpha-bungarotoxin and appears to mediate a previously unrecognized slow chloride current in identified neurons. Using a combination of biochemical and electrophysiological methods, we will investigate the role of arachidonic acid metabolism as a signaling pathway in relation to both receptor subtypes and evaluate the functional consequences of these biologically active lipids in neuronal activity. We will address three specific aims: 1) What is the role of lipid metabolites in pre synaptic facilitation by nicotine in vertebrates? 2) What are the roles of the lipoxygenase pathways in neural signaling in Aplysia? 3) How closely related are the 8- and 12-lipoxygenases? Where are they located? Do both exist in invertebrate neurons? Do both exist in vertebrate neurons? In view of the emergent role of specific lipid metabolites in synaptic plasticity, the proposed studies should provide important insights into the molecular mechanisms of eicosanoid action by analyzing excitability at well characterized interneuronal synapses.

DESCRIPTION (provided by applicant): Gene expression is thought to be the molecular basis of long term synaptic plasticity. Identified sensory neurons mediate well-characterized defensive reflexes in Aplysia. Plasticity of sensory-to-motor synapses underlies both sensitization and habituation of these reflexes. Much work has elucidated the cell- and molecular mechanisms underlying long term sensitization. A facilitatory neurotransmitter, eg serotonin, activates the cAMP-dependent protein kinase, which then is imported into the sensory neuron's nucleus to initiate a cascade of gene expression by phosphorylating the constitutive transcription activator, CREB1. The proteins synthesized are the structural basis for the enhanced transmitter release (facilitation) that underlies the alteration of behavior. We now plan to examine the molecular basis of long term depression (LTD), the physiological process underlying habituation. We showed that short term depression, which lasts for minutes, is produced by brief application of the inhibitory neuropeptide transmitter, FMRFamide; prolonged application results in LTD lasting days or weeks. The decrease in excitatory post-synaptic potentials that characterize synaptic depression is produced by an incompletely analyzed second-message pathway that involves receptor-mediated release of arachidonic acid, which is then converted to active metabolites by 12-lipoxygenase. We now find that the stress MAP kinase, p38, is activated by FMRFa during LTD, as has been reported for depression in pyramidal cells in the vertebrate hippocampus. Once activated, the kinase is imported into the sensory neuron's nucleus. We propose to characterize the signal transduction mechanisms by which p38 kinase operates, both in nucleus and cytoplasm. Since activation of transcription factors is known to induce the synthesis of the proteins needed for the maintenance and consolidation of synaptic plasticity, it is likely that p38 phosphorylates transcription factors. We therefore would identify those factors in order to determine what role p38 kinase plays in producing LTD. Finally we will identify the induced proteins that are required for LTD by differential screening. The mechanisms underlying synaptic plasticity are important because they are likely to be disturbed in mental disorders (schizophrenia and depression), and to play a crucial role in drug addiction. Aplysia neurons offer a convenient experimental system for approaching these processes successfully.