Robert A. Nichols - US grants

DESCRIPTION (Applicant's Abstract): The role of protein dephosphorylation as mediated by Ca2+/calmodulin-dependent protein phosphatase (calcineurin), in the regulation of neurotransmitter release from nerve terminals in mammalian brain will be studied. The proposed studies will offer insight into Ca2+-regulated processes at the synapse, contributing to the long-term objective of describing the molecular mechanisms involved in neurosecretion. Specifically, the physiological and pharmacological regulation of calcineurin in isolated brain nerve terminals will be examined. These studies will examine the Ca2+- dependent dephosphorylation of exogenous and endogenous protein phosphosubstrates in isolated brain nerve terminal fractions. Exogenous substrate dephosphorylation will be measured to assess the extent to which calcineurin in nerve terminals can be activated by Ca2+ and calmodulin.Endogenous substrates for Ca2+-dependent dephosphorylation will be studied in intact terminals after standard radiolabeling of endogenous ATP, focusing on two phosphoproteins termed P96 and P139, proteins known to dramatically dephosphorylate upon depolarization-induced Ca2+ entry. Physiological regulation of these dephosphorylation events will be explored by comparing the kinetics of depolarization-induced changes in intraterminal Ca2+ levels with the kinetics of calcineurin activation, and by determining the nature and source of the Ca2+ changes essential for dephosphorylation (e.g., Ca channels, endoplasmic reticulum, Na/Ca antiporter). Pharmacological regulation will be studied using a variety of agents known to change intraterminal Ca2+ levels (e.g. carbachol). The function of calcineurin in neurotransmitter uptake and release will be investigated by separately introducing activated calcineurin, inhibitory synthetic peptides (derived from the regulatory region of calcineurin), and antibodies to calcineurin into isolated nerve terminals, using transient freeze/thaw permeabilization, and examining subsequent effects of neurotransmitter uptake and release. Purification and characterization of the endogenous protein substrates P96 and P139 will be undertaken. Utilizing antibodies raised against the purified proteins and/or partial knowledge of their amino acid sequences, isolation of the cDNAs encoding P96 and P139 from rat brain cDNA libraries will be attempted. The roles that P96 and P139 play in nerve terminal function will be addressed by separately introducing these protein substrates and antibodies against these proteins into isolated terminals, and assessing subsequent effects on neurotransmitter uptake and release.Because calcineurin is differentially distributed throughout brain, being predominantly in corpus striatum and hippocampus, determining the role of calcineurin in nerve terminals may provide new insights into altered function in neuropathological disease states involving these brain regions.

DESCRIPTION (Applicant's Abstract): The role of protein dephosphorylation as mediated by Ca2+/calmodulin-dependent protein phosphatase (calcineurin), in the regulation of neurotransmitter release from nerve terminals in mammalian brain will be studied. The proposed studies will offer insight into Ca2+-regulated processes at the synapse, contributing to the long-term objective of describing the molecular mechanisms involved in neurosecretion. Specifically, the physiological and pharmacological regulation of calcineurin in isolated brain nerve terminals will be examined. These studies will examine the Ca2+- dependent dephosphorylation of exogenous and endogenous protein phosphosubstrates in isolated brain nerve terminal fractions. Exogenous substrate dephosphorylation will be measured to assess the extent to which calcineurin in nerve terminals can be activated by Ca2+ and calmodulin.Endogenous substrates for Ca2+-dependent dephosphorylation will be studied in intact terminals after standard radiolabeling of endogenous ATP, focusing on two phosphoproteins termed P96 and P139, proteins known to dramatically dephosphorylate upon depolarization-induced Ca2+ entry. Physiological regulation of these dephosphorylation events will be explored by comparing the kinetics of depolarization-induced changes in intraterminal Ca2+ levels with the kinetics of calcineurin activation, and by determining the nature and source of the Ca2+ changes essential for dephosphorylation (e.g., Ca channels, endoplasmic reticulum, Na/Ca antiporter). Pharmacological regulation will be studied using a variety of agents known to change intraterminal Ca2+ levels (e.g. carbachol). The function of calcineurin in neurotransmitter uptake and release will be investigated by separately introducing activated calcineurin, inhibitory synthetic peptides (derived from the regulatory region of calcineurin), and antibodies to calcineurin into isolated nerve terminals, using transient freeze/thaw permeabilization, and examining subsequent effects of neurotransmitter uptake and release. Purification and characterization of the endogenous protein substrates P96 and P139 will be undertaken. Utilizing antibodies raised against the purified proteins and/or partial knowledge of their amino acid sequences, isolation of the cDNAs encoding P96 and P139 from rat brain cDNA libraries will be attempted. The roles that P96 and P139 play in nerve terminal function will be addressed by separately introducing these protein substrates and antibodies against these proteins into isolated terminals, and assessing subsequent effects on neurotransmitter uptake and release.Because calcineurin is differentially distributed throughout brain, being predominantly in corpus striatum and hippocampus, determining the role of calcineurin in nerve terminals may provide new insights into altered function in neuropathological disease states involving these brain regions.

DESCRIPTION (Applicant's Abstract): The role of protein dephosphorylation as mediated by Ca2+/calmodulin-dependent protein phosphatase (calcineurin), in the regulation of neurotransmitter release from nerve terminals in mammalian brain will be studied. The proposed studies will offer insight into Ca2+-regulated processes at the synapse, contributing to the long-term objective of describing the molecular mechanisms involved in neurosecretion. Specifically, the physiological and pharmacological regulation of calcineurin in isolated brain nerve terminals will be examined. These studies will examine the Ca2+- dependent dephosphorylation of exogenous and endogenous protein phosphosubstrates in isolated brain nerve terminal fractions. Exogenous substrate dephosphorylation will be measured to assess the extent to which calcineurin in nerve terminals can be activated by Ca2+ and calmodulin.Endogenous substrates for Ca2+-dependent dephosphorylation will be studied in intact terminals after standard radiolabeling of endogenous ATP, focusing on two phosphoproteins termed P96 and P139, proteins known to dramatically dephosphorylate upon depolarization-induced Ca2+ entry. Physiological regulation of these dephosphorylation events will be explored by comparing the kinetics of depolarization-induced changes in intraterminal Ca2+ levels with the kinetics of calcineurin activation, and by determining the nature and source of the Ca2+ changes essential for dephosphorylation (e.g., Ca channels, endoplasmic reticulum, Na/Ca antiporter). Pharmacological regulation will be studied using a variety of agents known to change intraterminal Ca2+ levels (e.g. carbachol). The function of calcineurin in neurotransmitter uptake and release will be investigated by separately introducing activated calcineurin, inhibitory synthetic peptides (derived from the regulatory region of calcineurin), and antibodies to calcineurin into isolated nerve terminals, using transient freeze/thaw permeabilization, and examining subsequent effects of neurotransmitter uptake and release. Purification and characterization of the endogenous protein substrates P96 and P139 will be undertaken. Utilizing antibodies raised against the purified proteins and/or partial knowledge of their amino acid sequences, isolation of the cDNAs encoding P96 and P139 from rat brain cDNA libraries will be attempted. The roles that P96 and P139 play in nerve terminal function will be addressed by separately introducing these protein substrates and antibodies against these proteins into isolated terminals, and assessing subsequent effects on neurotransmitter uptake and release.Because calcineurin is differentially distributed throughout brain, being predominantly in corpus striatum and hippocampus, determining the role of calcineurin in nerve terminals may provide new insights into altered function in neuropathological disease states involving these brain regions.

DESCRIPTION (provided by applicant): A prominent characteristic of Alzheimer's disease is the loss of brain nerve cells that employ the neurotransmitter acetylcholine. Cholinergic neurons may be lost as a consequence of the formation of toxic deposits, known as neuritic plaques, which contain beta amyloid peptides as a major component. However, the correlation between amyloid deposition and cognitive impairment appears to be poor. On the other hand, target receptors for acetylcholine may be affected, indicating an action of soluble beta amyloid. Indeed, beta amyloid has recently been shown to interact directly with nicotinic receptors on neuronal cell bodies. In brain, a substantial portion of the nicotinic receptors are localized to presynaptic nerve endings. Preliminarily, it was found that beta amyloid first activates and then occludes presynaptic nicotinic receptor-induced functional responses in isolated hippocampal nerve endings, but does not affect responses mediated by the closely related 5-HT3 serotonin receptors co-localized with the nicotinic receptors on the same nerve endings. Thus, it is hypothesized that soluble beta amyloid selectively alters neuronal signaling via disruption of postsynaptic and presynaptic nicotinic receptors in the brain. The specific aims of this proposal are to characterize the effects of beta amyloid peptides on 1) nicotine-induced calcium responses and neurosecretion in individual nerve endings using confocal microscopy; 2) neurotransmitter release in vivo using microdialysis; and 3) initiation of nerve terminal degenerative responses to prolonged beta amyloid treatment, including mitochondria depolarization and calcium dysregulation, and the possible role of protein phosphorylation in these events. In each case, mouse hippocampus, cortex, and, on a limited basis as a control, striatum will be examined. The use of inbred mice will allow studies with transgenic mouse models, including the APP mutant APPswe/PS1 and nicotinic receptor null mutants, allowing definite determination of which particular presynaptic nicotinic receptor subunits are targets for beta amyloid and what effect chronic beta amyloid has on presynaptic nicotinic receptor function. Nicotinic receptors have been strongly implicated in memory mechanisms in the brain. Their disruption by elevated soluble beta amyloid may cause alterations in neuronal signaling in early Alzheimer's disease leading to reduced cognitive function. The role of presynaptic nicotinic receptors may be key, as these receptors are prominently localized to nerve endings in the brain. Characterizing the functional effects of beta amyloid on presynaptic nicotinic receptors may thus yield important information related to early events in Alzheimer's disease.

DESCRIPTION (provided by applicant): This application seeks continuation funding for a Specialized Neuroscience Research Program (SNRP) at this minority serving institution. Whereas the goals of the SNRP during the previous funding period were to increase Neuroscience research capacity on this campus, the present application seeks to build on that previous success by establishing a focused and coordinated, team research, program. This program will address "Emotion and cognition on gene, cell and systems levels", with a primary focus on the effects of stress and anxiety on emotion and cognition, recognizing that increased understanding of these interactions is of major importance to human health. The proposal brings together a wide range of interdisciplinary experience (including psychiatry, psychology, molecular biology, neuroimaging, genetics, neuroanatomy, neuropharmacology, electrophysiology, biochemistry and animal behavior) to address four specific aspects of this overall problem, utilizing both human subjects and animal models (Aim 1). Nevertheless, the demands of team research will be balanced by the need to provide a carefully constructed program that fosters individual academic development of SNRP participants at all levels (Aim 2). Additionally, this proposal will attempt to demonstrate the effectiveness of team research in the biomedical sciences, such as to stimulate additional team research efforts in this area (Aim 3). Finally, this proposal seeks to generate a strong and cohesive academic and research training "center" for the Neurosciences at this University (Aim 4), such that this University will become known over time as a national and international resource for Neuroscience research and training. Titles of the specific projects are: Stress-induced CRF actions on fear and anxiety;Stress effects on amygdalar CRF and emotion;Methamphetamine psychosis: a model in man and mouse;CRF systems and defensive behavior. The proposal provides a Peptide and DNA Technology Core to support these projects.

DESCRIPTION (provided by applicant): The Hawaii State Research & Education Partnership (HISREP) consists of a network of 10 Universities and community colleges encompassing nearly all of the institutions of higher learning in the State of Hawaii. As in past cycles, HISREP will continue to expand biomedical research capacity by providing resources to a wide spectrum of promising undergraduate students, graduate students, post-docs and junior faculty members residing at partner institutions. HISREP will spark the imagination and inspire the interest of students at all stages of development to pursue careers in biomedical research with a robust portfolio of hands-on-laboratory research, and a diverse menu of education and training sessions. Through a well-crafted Research Development Activity (RDA), HISREP will support a cadre of promising junior faculty members at the PUI's to conduct research projects with a thematic focus of either Natural Products or Health Disparities, with the goal of formulating competitive research applications to secure extramural funding on a path to research independence. A Bioinformatics Core will provide an expanded menu of bioinformatics, informatics, biostatistical, and computational tools, assistance and advice, as well as training and education activities for HISREP investigators and students alike. An established spirit of cooperation has evolved over time among HISREP partners to increase collaborative research opportunities and share resources. Moreover, students at all stages of development benefiting from HISREP will be encouraged to assume key roles in the future by participating in HISREP training and education activities, and offering research laboratory rotations and mentoring to the next generation of HISREP student, thus creating a self-perpetuating cycle of research career development. Finally, HISREP will be led by a responsible and experienced administration team with support from a Steering Committee as well as expertise and oversight from an External Advisory Committee. Significant support to the UH Hilo College of Pharmacy will be provided to share administrative responsibilities as they mature and expand into an independent administrative campus. Overall, HISREP has matured into a well-developed alliance of partners participating in a broad range of research opportunities to expand research capacity in the state of Hawaii.

PROJECT SUMMARY The Hawaii Statewide Research and Education Partnership (HiSREP) comprises a network of nearly all of the institutions of higher learning in the state of Hawaii, specifically 4 primarily undergraduate institutions (PUIs), 1 University of Hawaii (UH) college and 4 UH community colleges under the management of the lead R1 institution, the University of Hawaii at Manoa. In the previous grant cycle, INBRE III focused on strengthening the research community and infrastructure at the partner institutions outside of the lead institution. This was accomplished through a tightly coordinated leadership team overseeing an integrated program of support for new laboratory construction under Alterations & Renovation funding, acquisition of new instrumentation, funding for junior investigator research and undergraduate student research experiences paired with outreach and multi-level mentoring. This resulted in substantial expansion of the research base, stronger student engagement leading to a near doubling in student participants, and new initiatives in career advancement. In INBRE IV, HiSREP will continue the development of emerging investigators, but will widen the reach of the network to all levels of biomedical research scientists through a new array of competitive granting mechanisms including teaching-postdoctoral fellowships, pilot projects, new initiatives and team-based collaborative grants through a Developmental Research Project Program (DRPP). Research under HiSREP will be guided by two key themes, Natural Products and Molecular Medicine, which emphasize notable strengths in the biomedical research community in Hawaii. In addition, HiSREP will support a reorganized Bioinformatics Core as a centralized resource across the state to provide education on bioinformatics, aid with research design, technical expertise including development of new informatics tools, data management and analysis. The Bioinformatics Core will promote community synergy for researchers and students through one-on-one, group, workshop, course and online interactions, the latter through a real-time research community portal. The student research program under the PATHway to Biomedical Careers will look to embrace a wider range of undergraduates by emphasizing collaborative group projects through volunteer, intern, scholar and returning researcher opportunities, including partnerships with complementary undergraduate research programs, while maintaining focus on individual career development through skill training, practical mentoring sessions, extended resources and workforce development. In addition, PATHway will develop proactive advanced training for laboratory supervisors to increase research sophistication as well as opportunity statewide, leading to increased capacity. It will also ultimately enhance undergraduate education through incorporation of research activity into the basic science curriculum. Overall, HiSREP will serve as a catalyst for advancement of the research scientist pipeline with the ultimate goal of elevating the ability of the biomedical research community to make new and important discoveries for improving health and well-being in the state of Hawaii.

PROJECT SUMMARY The Hawaii Statewide Research and Education Partnership (HiSREP) comprises a network of nearly all of the institutions of higher learning in the state of Hawaii, specifically 4 primarily undergraduate institutions (PUIs), 1 University of Hawaii (UH) college and 4 UH community colleges under the management of the lead R1 institution, the University of Hawaii at Manoa. In the previous grant cycle, INBRE III focused on strengthening the research community and infrastructure at the partner institutions outside of the lead institution. This was accomplished through a tightly coordinated leadership team overseeing an integrated program of support for new laboratory construction under Alterations & Renovation funding, acquisition of new instrumentation, funding for junior investigator research and undergraduate student research experiences paired with outreach and multi-level mentoring. This resulted in substantial expansion of the research base, stronger student engagement leading to a near doubling in student participants, and new initiatives in career advancement. In INBRE IV, HiSREP will continue the development of emerging investigators, but will widen the reach of the network to all levels of biomedical research scientists through a new array of competitive granting mechanisms including teaching-postdoctoral fellowships, pilot projects, new initiatives and team-based collaborative grants through a Developmental Research Project Program (DRPP). Research under HiSREP will be guided by two key themes, Natural Products and Molecular Medicine, which emphasize notable strengths in the biomedical research community in Hawaii. In addition, HiSREP will support a reorganized Bioinformatics Core as a centralized resource across the state to provide education on bioinformatics, aid with research design, technical expertise including development of new informatics tools, data management and analysis. The Bioinformatics Core will promote community synergy for researchers and students through one-on-one, group, workshop, course and online interactions, the latter through a real-time research community portal. The student research program under the PATHway to Biomedical Careers will look to embrace a wider range of undergraduates by emphasizing collaborative group projects through volunteer, intern, scholar and returning researcher opportunities, including partnerships with complementary undergraduate research programs, while maintaining focus on individual career development through skill training, practical mentoring sessions, extended resources and workforce development. In addition, PATHway will develop proactive advanced training for laboratory supervisors to increase research sophistication as well as opportunity statewide, leading to increased capacity. It will also ultimately enhance undergraduate education through incorporation of research activity into the basic science curriculum. Overall, HiSREP will serve as a catalyst for advancement of the research scientist pipeline with the ultimate goal of elevating the ability of the biomedical research community to make new and important discoveries for improving health and well-being in the state of Hawaii.