Cui-wei Xie - US grants

Tolerance and dependence are two major problems which interfere with the clinical utility & opiate drugs and promote their abuse. The cellular mechanisms underlying either process have not been well understood. Opiates reportedly induce dramatic changes in the activity of intracellular cAMP cascade in rat brain, but it remains unclear how these biochemical changes affect synaptic transmission in different brain regions and if they play an important role in the development of tolerance and dependence. In hippocampal dentate gyrus, our recent studies have shown that mu agonists inhibit NMDA receptor-mediated synaptic currents in granule cells through a G protein-mediated mechanism. The present project proposes to further examine the modulatory effect of acute and chronic opiate on NMDA currents in the dentate, and to determine the role of cAMP cascade in transducing opiate effects. The hypothesis to be evaluated is that acute opiates suppress NMDA currents by inhibiting cAMP-dependent phosphorylation in granule cells, while the upregulation of cAMP cascade by chronic opiates may profoundly enhance NMDA currents, which in turn facilitates the development of tolerance and dependence. To test this hypothesis, evoked and spontaneous NMDA currents will be recorded from dentate granule cells in the hippocampal slice using whole-cell recording techniques. Experiments will focus on following specific aims: 1) To determine the type(s) and location (presynaptic vs. postsynaptic) of opiate receptors that inhibit NMDA currents. 2) To examine how NMDA currents are modulated by manipulation of the cAMP cascade, and if acute opioid effects are mediated by inhibiting cAMP cascade. 3) and 4) To evaluate how chronic morphine alters NMDA currents and the activity of cAMP cascade in granule cells, and if these alterations contribute to the development of tolerance and dependence.

DESCRIPTION (adapted from applicant's abstract): Although it is generally agreed that accumulation of amyloid 13 (A,B) peptides in the brain contributes to the pathogenesis of Alzheimer's disease (AD), the cellular mechanisms that link A13 to AD dementia remain unresolved. Our studies showed that brief perfusion with A,B or its active fragment A,B2s 35 at subneurotoxic concentrations strongly inhibited the early and late phase of long-term potentiation (LTP) in the dentate gyrus of rat hippocampal slices, and impairment of late-phase LTP by A,13 could be rescued by calcineurin inhibitors. Furthermore, acute application of A132s 35 resulted in rapid but transient intracellular Ca2+ rises and enhanced Ca2+ oscillations in cultured hippocampal neurons. We have proposed here to further investigate the link between A, about induced alterations in Ca2+ signaling and inhibition of hippocampal LTP. It is hypothesized that A, about induced intracellular Ca2+ rises and subsequent activation of calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, play the key role in LTP impairment. The Ca2+ transient may facilitate Ca2+-dependent inactivation or desensitization of NMDA receptor channels, suppressing induction of LTP. Activation of calcineurin may also shift the balance between a phosphatase cascade and several protein kinase systems, leading to changes in a LTP gating mechanism and/or alterations in the phosphorylation state of cAMP-response element binding protein (CREB). These signaling changes can in turn suppress the later components of LTP. Thus, via altered Ca2+ signaling A,13 interferes with both early and late components of LTP, which forms a possible cellular basis for memory deficits in Alzheimer's disease. Electrophysiological, immunocytochemical and neurochemical approaches will be used to test this hypothesis. The proposed Specific Aims are: 1) To further characterize the inhibitory action of A,B on both early and late components of dentate LTP; 2) To examine whether A,13 inhibits NMDA receptor channels of postsynaptic neurons to suppress LTP induction; 3) To determine whether A, about induced Ca2+ rises and calcineurin activation are responsible for inhibition of NMDA channels; 4) To deter aboutnine whether Ap activates the calcineurin/PP1 cascade to impair the inter aboutnediate phase of LTP; and 5) To determine whether A,13 alters CREB phosphorylation via a calcineurin-dependent mechanism, thus suppressing the late-phase LTP.

[unreadable] DESCRIPTION (provided by applicant): Beta-Amyloid (Abeta) is associated with age-related cognitive decline, neurotoxicity and synaptic failure in Alzheimer's disease (AD). Significant progress has been made in understanding the genetic factors and cellular mechanisms contributing to Abeta-induced neurotoxicity. However, the mechanisms responsible for Abeta-induced synaptic dysfunction prior to cell death are largely unknown, and finding safe and effective therapeutic interventions to reverse Abeta-related early AD pathology remains a great challenge. We previously showed that acute application of Abeta inhibited hippocampal long-term potentiation (LTP), a synaptic model of learning and memory, and that this effect was associated with altered intracellular Ca2+ signaling leading to activation of a Ca2+-dependent protein phosphatase calcineurin. Interestingly, we observed in several model systems that neurotrophin 4 and brain-derived neurotrophic factor rescued Abeta- induced deficits in LTP and synaptic transmission. Based on the preliminary data, we hypothesize that a focal point for the neurotrophin (NT)- Abeta interaction is Ca2+ and calmodulin-dependent protein kinase II (CaMKII). Abeta inhibits CaMKII activation via enhanced calcineurin activity, whereas NT counteracts Abeta action by stimulating CaMKII and enhancing the function and synaptic targeting of the AMPA type of glutamate receptors. In addition, NT is known to promote neuronal differentiation, survival and plasticity through two major kinase pathways, mitogen-activated protein kinase (MAPK) and phosphoinositide kinase 3 (PI3K). These two kinase pathways may contribute to the NT rescue by enhancing AMPA receptor function or promoting gene transcription required for both synaptic plasticity and neuronal survival. These hypotheses will be tested using a combination of electrophysiological, biochemical, immunocytochemical and molecular genetic approaches. We will further examine NT- Abeta interactions in regulating multiple forms of hippocampal synaptic plasticity; determine the synaptic locus and mechanisms underlying their opposing effects; and analyze the role of CaMKII, MAPK and PI3K in the NT rescue using various pharmacological, molecular and genetic manipulations. The results may provide new insights into synaptic mechanisms for Abeta action and the therapeutic potentials of trkB-acting NT for early AD. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Beta-Amyloid (Abeta) is associated with age-related cognitive decline, neurotoxicity and synaptic failure in Alzheimer's disease (AD). Significant progress has been made in understanding the genetic factors and cellular mechanisms contributing to Abeta-induced neurotoxicity. However, the mechanisms responsible for Abeta-induced synaptic dysfunction prior to cell death are largely unknown, and finding safe and effective therapeutic interventions to reverse Abeta-related early AD pathology remains a great challenge. We previously showed that acute application of Abeta inhibited hippocampal long-term potentiation (LTP), a synaptic model of learning and memory, and that this effect was associated with altered intracellular Ca2+ signaling leading to activation of a Ca2+-dependent protein phosphatase calcineurin. Interestingly, we observed in several model systems that neurotrophin 4 and brain-derived neurotrophic factor rescued Abeta- induced deficits in LTP and synaptic transmission. Based on the preliminary data, we hypothesize that a focal point for the neurotrophin (NT)- Abeta interaction is Ca2+ and calmodulin-dependent protein kinase II (CaMKII). Abeta inhibits CaMKII activation via enhanced calcineurin activity, whereas NT counteracts Abeta action by stimulating CaMKII and enhancing the function and synaptic targeting of the AMPA type of glutamate receptors. In addition, NT is known to promote neuronal differentiation, survival and plasticity through two major kinase pathways, mitogen-activated protein kinase (MAPK) and phosphoinositide kinase 3 (PI3K). These two kinase pathways may contribute to the NT rescue by enhancing AMPA receptor function or promoting gene transcription required for both synaptic plasticity and neuronal survival. These hypotheses will be tested using a combination of electrophysiological, biochemical, immunocytochemical and molecular genetic approaches. We will further examine NT- Abeta interactions in regulating multiple forms of hippocampal synaptic plasticity; determine the synaptic locus and mechanisms underlying their opposing effects; and analyze the role of CaMKII, MAPK and PI3K in the NT rescue using various pharmacological, molecular and genetic manipulations. The results may provide new insights into synaptic mechanisms for Abeta action and the therapeutic potentials of trkB-acting NT for early AD. [unreadable] [unreadable]