1992 — 1996 |
Colbran, Roger J |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Regulation of Calmodulin-Dependent Protein Kinase Ii |
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1999 — 2002 |
Colbran, Roger J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Targeting of Brain Protein Phosphatase 1
Appropriate regulation of neuronal function, such as synaptic plasticity, depends in part on neurotransmitter receptors and ion channels, especially those localized in synapses. Phosphorylation/ dephosphorylation of serines and/or threonines in the receptor/ion channel subunits is a major mechanism for their regulation; precise modulation of protein kinase and protein phosphatase activities is therefore essential for learning and memory. For example, activation of type 1 protein phosphatase catalytic subunit (PP1C) isoforms localized in dendritic spine sis likely important in the induction of long-term depression. We characterized four rat brain cytoskeletal, isoform- selective, PP1C-binding proteins (PP1bps) and have purified an actin- associated PP1 holoenzyme (PP1A) containing PP1bp134, PP1bp175 and PP1C. We hypothesize that association of PP1C with these PP1bps specifically targets and regulates neuronal PP1 activity. Three complementary Specific Aims are proposed, which will identify PP1bp134 and PP1bp175 and begin to characterize their physiological role: 1. Characterization and cloning of PP1bps. The cDNAs encoding PP1bps present in PP1A (PP1bp134, PP1bp175) will be isolated. The authenticity of cDNAs will be verified by expression of PP1bp activity in heterologous systems, and by immunological identification of the cloned proteins in PP1A. Expression patterns of PP1C isoform and novel PP1bp mRNAs in tissues, brain regions and during development will be compared. 2. Characterization of the interaction of PP1C, with PP1bps. A. Domain interactions in vitro: Deletion, truncation, and site-directed PP1bp mutants (expressed in bacteria) will be used to identify the minimal isoform selective PP1C binding domain using multiple binding assays. B. Interactions in vivo: Interaction of PP1bps with PP1C isoforms in brain region extracts, brain slices and cultured neurons will be elucidated by co-immunoprecipitation, immunoblotting subcellular fractions, and immunofluorescent confocal microscopy. Effects of wild-type and mutated PP1bps on PP1C localization in undifferentiated heterologous cells (e.g., HEK293, NG108, PC12) and during neuronal differentiation, and on neuronal differentiation itself, will be investigated. 3. Regulation of PP1A. The regulation of PP1bp binding to PP1C and actin by Ca2+/calmodulin-dependent protein kinase II will be investigated and specific phosphorylation sites will be identified. Phosphorylation of PP1bps will be examine din intact cells/neurons and the effects on PP1C/PP1bp localization will be elucidated. These Aims provide novel insights into the physiological regulation of PP1, a key modulator of synaptic function.
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2001 — 2015 |
Colbran, Roger J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Cam Kinase Ii Signal Transduction
DESCRIPTION(From applicant's abstract): Detailed characterizations of neuronal signal transduction and protein phosphorylation/dephosphorylation are critical for understanding many brain functions. For example, activation of NMDA-type glutamate receptors stimulates both protein kinases and protein phosphatases, which feedback to modulate AMPA- and NMDA-type glutamate receptors. Ca-+/calmodulin-dependent protein kinase II (CaMKII) is a major dendritic kinase activated by NMDA receptor stimulation, resulting in Thr286-autophosphorylation and phosphorylation of AMPA- and NMDA-receptors and several other proteins including densin-180, an O-sialoglycoprotein with a PDZ domain. We showed that CaMKII autophosphorylation promotes its translocation to postsynaptic densities (PSDs), submembranous cytoskeletal specializations, and identified the NR2B subunit of NMDA receptors and densin- 180 as two proteins that likely contribute to translocation. Five Specific Aims address our hypothesis that binding to NR2B and densin-180 modulates CaMKII, resulting in synapse-specific regulation of glutamate receptors. 1. Neuronal interaction of CaMKII and dens in- 180 will be verified by colocalization using immunofluorescent confocal microscopy and by coimmunoprecipitation assays. Relative contributions of NR2B and densin- 180 to CaMKII binding activities in PSDs will be determined. 2. Interaction domains in NR2B, densin-180 and CaMKII will be identified in vitro by truncation/deletion and site-directed mutagenesis, and their importance will be confirmed in HEK293 cells and neurons. This information will be used to develop reagents that specifically manipulate CaMKII localization in cells. 3. Dynamics of CaMKII.densin-180 and CaMKII.NR2B interactions, and regulatory roles of phosphorylation/dephosphorylation of densin-180, NR2B and CaMKII, as well as NMDA receptor activation, will be examined in vitro and in intact cells. 4. Effects of interaction with NR2B or densin-180 on CaMKJI autophosphorylation will be investigated in vitro and in intact cells. AMPA receptor phosphorylation and potentiation in HEK293 cells and neurons will be compared under conditions where NR2B and densin-180 are used to differentially target CaMKJI. 5. Roles of CaMKII-binding and CaMKII-mediated NR2B phosphorylation in regulation of NMDA receptors will be determined. More long-range goals are to establish the roles of CaMKII targeting by NR2B and densin-180 in regulation of synaptic transmission and synapse-specific synaptic plasticity. These studies will provide fundamental insights into signal transduction mechanisms underlying normal brain functions such as learning and memory. Reagents that block these protein.protein interactions also have potential for development as therapeutic compounds to treat mental disorders, such as schizophrenia or depression, and possibly brain injuries.
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2002 |
Colbran, Roger J |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Modulation of Dendritic Camkii by Dopamine
Description (provided by applicant): Dopamine (DA) replacement therapy of Parkinson's Disease (PD) often becomes ineffective late in disease progression due to development of drug resistance and dyskinesias. We hypothesize that this is due to altered morphology of striatal medium spiny neurons arising from changes in expression andlor function of dendritic proteins. Preliminary Studies exploit a rat model of PD generated by unilateral injection of 6-hydroxyzopamine (6-OHDA) into substantia nigra. Total CaMKIIa levels in lesioned striaturn were decreased by 30% relative to contralateral control striaturn 9 months after 6-OHDA-injection; several other proteins were unchanged. CaMKII is critical for hippocampal synaTtic plasticity: NMDA receptor activation induces Thr 286 autophosphorylation, generating a Ca2+- independent form of CaMKII that translocates to postsynaptic densities, phsphorylates AMPA receptors (GluRl) and is required for long term potentiation. Although striatal CaMKII functions remain obscure, preliminary data indicate that Thr 286 autophosphorylation of CaMKII is increased in 6-OHDA lesioned striatum. Together, these exciting findings suggest the specific hypothesis that chronic dopamine deficiency in PD or induced by 6 OHDA perturbs normal physiological regulation of the key CaMKII signaling pathway, which will be tested using three Aims. 1. Characterize CaMKII expression in 6-OHDA-lesioned striaturn. CaMKII expression/localization in 6-OHDA lesioned striaturn will be compared to control striatum from the contralateral hemisphere and from vehicle-injected animals. Possible amelioration of changes by I-DOPA, or glutamate-receptor blockers will be investigated. 2. Determine role(s) of DA in acute regulation of CaMKII in normal striaturn. Isolated striatal slices will be used to investigate roles of D1 and D2 DA receptors in regulating basal and glutamate-stimulated CaMKII autophosphorylation as well as phosphorylation of upstream (DARPP32) and downstream (GluRl, %) components of this signaling pathway. 3. Define effect(s) of chronic DA depletion on CaMKII signaling. Effects of striatal 6-OHDA lesion or chronic administration of D1/D2 DA receptor antagonists on basal CaMKII autophosphorylation and phosphorylation of DARPP32, GluRl and NR2B will be determined. Pathological defects in acute DA signaling will be determined by comparing CaMKII regulation in acutely isolated slices from 6-OHDA lesioned and control striaturn. All data will be correlated with plasticity of spine morphology and synaptic transmission determined in Projects 1 and 3 of this PPG. Chronic disruption of DA signaling is predicted to substantially impact CaMKIi signaling, which may contribute to complications of late-phase PD. These insights may help develop improved PD treatment strategies.
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2012 — 2016 |
Colbran, Roger J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Camkii, Endocannabinoids, Synaptic Plasticity and Motor Function
DESCRIPTION (provided by applicant): The striatum plays a key role in motor activity/coordination and goal-directed, habitual learning. Normal striatal drive of motor activity requires precisely balanced opposing outputs from two types of striatal medium spiny neurons (MSNs) that express D1- and D2-dopamine(DA) receptors. A complex signaling cross-talk between glutamate and DA in D1- and D2-MSNs involves on demand Ca2+-dependent endocannabinoid (eCB) synthesis. Thus, DA, glutamate and eCBs collaborate to balance short- and long-term control of the two striatal output pathways by engaging distinct signaling mechanisms in the two MSN subtypes. Disruption of these mechanisms can induce motor deficits (e.g., Parkinson's Disease) or other abnormal striatal-based behaviors. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has diverse bidirectional roles controlling excitatory synapses in hippocampus, cortex and cerebellum. While CaMKII is expressed in both striatal MSN subtypes, suggesting that it regulates excitatory inputs to striatal MSNs and motor activity, the precise functions of striatal CaMKII are poorly understood. Our analyses of knockin mutant mice with the Thr286 autophosphorylation site in CaMKII? replaced by Ala (T286A-KI mice) revealed specific roles for CaMKII in long- and short term eCB- dependent control of excitatory inputs to D1- and D2-MSNs. We also found that CaMKII? associates with and phosphorylates diacylglycerol lipase ? (DGL?), the rate-limiting enzyme for Ca2+-dependent synthesis of the most abundant brain eCB, 2-arachidonyl glycerol (2-AG). In addition, baseline hyperactivity of T286A-KI mice can be rescued by inhibiting 2-AG breakdown. These initial findings strongly support a novel hypothesis that CaMKII is a critical link between postsynaptic Ca2+ and the initiation of 2-AG signaling that controls striatal synapses and striatal based-behaviors. We also created novel transgenic eAC3I mice that selectively express a short CaMKII inhibitor peptide fused to eGFP in striatal MSNs. Three specific aims will exploit unique features of T286A-KI and eAC3I mice to test specific hypotheses about the roles of striatal CaMKII autophosphorylation and activity. 1. Test the hypothesis that Ca2+-dependent 2-AG synthesis is modulated by CaMKII. We will identify sites of phosphorylation and CaMKII-binding domains in DGL?. DGL? phosphorylation, DGL? activity and 2-AG synthesis will be investigated in heterologous cells and in striatal slices from WT, T286A-KI and eAC3I mice. 2. Test the hypothesis that CaMKII modulates eCB-dependent synaptic regulation in striatal MSNs. Short and long-term roles of CaMKII will be determined by comparing the properties of excitatory synaptic inputs to D1- and D2-MSNs in striatal slices from WT, T286A-KI and eAC3I mice, and by using CaMKII inhibitor peptides. 3. Test the hypothesis that CaMKII modulates eCB-dependent motor activity. We will evaluate motor activity and coordination under basal conditions and following pharmacological modulation of 2-AG metabolism in WT, T286A-KI and eAC3I mice.
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2016 — 2021 |
Colbran, Roger J |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Postdoctoral Program in Functional Neurogenomics
PROJECT SUMMARY The genetic inheritance of specific risk alleles is widely accepted as a major contributing factor to many, if not all, mental illnesses. Moreover, recent studies found that the precise epigenetic regulation of gene expression is critical for normal learning and memory processes and is often disrupted in the diseased brain. However, despite recent concerted efforts of numerous neuroscientists and physicians, the links between precise molecular and synaptic defects resulting from (epi)genetic variation and specific brain circuit abnormalities that result in particular behavioral disorders remain rather poorly understood. Given the ongoing avalanche of new genetic/genomic data associated with neuropsychiatric disorders, there is a rapidly expanding need to train the next generation of neuroscientists to be facile in both modern molecular genetic approaches in different model systems, as well as in cutting edge molecular bioinformatics techniques that are required to link individual genes to normal brain functions and disease processes. The over-arching goal of this multi-disciplinary postdoctoral Training Program in Functional Neurogenomics is to support a training pipeline that fosters the development of new investigators with these skills. This competing renewal application will demonstrate an advancement and evolution in both the available training mentors and in cutting edge inter-disciplinary technical capabilities that builds on the substantial prior successes of this long-running program. Our efforts are supported by significant ongoing investments by Vanderbilt in new neuroscience leadership, faculty, educational programs, technological expertise and core facilities. Taken together, this provides a robust and rigorous environment for trainees to gain expertise in opportunities afforded by genetic model systems, the translation of human genetic findings into construct-valid animal models, in vivo manipulations of molecules, cells and circuits using advanced approaches, and in capturing the epigenetic, physiological, and behavioral consequences of such manipulations. The Program Director is Roger J. Colbran, Ph.D., Professor and Interim Chair of the Department of Molecular Physiology & Biophysics. Dr. Colbran has a long-standing, well-funded program investigating molecular mechanisms involved in synaptic plasticity using multi-disciplinary approaches from biochemical structure-function to mouse genetics and behavior. The Co-Director, J. David Sweatt, Ph.D., was recently recruited to Vanderbilt as the Chair of the Department of Pharmacology and has previously served on both the NIMH Advisory Council and the NIMH intramural program Board of Scientific Councilors. Dr. Sweatt has made numerous highly-cited contributions to understanding mechanisms underlying learning and memory in previous positions at Baylor College of Medicine and UAB, with a particular recent focus on the role of epigenetics in cognition and neural plasticity. The program leadership has a long-standing commitment to mentoring the next generation of neuroscientists, with a strong track record of facilitating the establishment of enduring and productive research careers for their trainees.
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2020 — 2021 |
Colbran, Roger J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Neuropharmacology and Signaling of Histone H2a.Z
? DESCRIPTION (provided by applicant): Histone subunit exchange represents an entire branch of epigenetics that is the subject of rigorous experimentation in many model systems, including yeast, plants, and cancer, but its role in the nervous system is virtually unknown. We recently conducted the first in vivo experimental investigation of activity-induced histone subunit exchange in the nervous system, focusing specifically on the histone variant H2A.Z in rodent hippocampus and cortex. In these studies we discovered that behavioral experience triggers histone subunit exchange and attendant alterations in gene transcription in the adult CNS. The characterization of experience- dependent histone subunit exchange in the brain represents a significant step forward in our knowledge of activity-regulated epigenetic mechanisms in the nervous system and provides crucial insights into the general function of this process for the field of epigenetics in general. These findings introduce a novel mechanism for regulating the three-dimensional structure of chromatin in neurons, triggering attendant alterations in gene readout, and driving experience-dependent changes in behavior. These discoveries also open up the possibility that targeting histone subunit exchange may be a novel target for therapeutic intervention in a broad range of CNS disorders, including drug addiction, cognitive disorders, and disorders of neural plasticity in general. Given this background of new information concerning a role for histone H2A.Z subunit exchange in the CNS, for this Project we propose to pursue the following four Specific Aims: Aim 1: To test the hypothesis that the SIRT1 histone/lysine de-acetylase signaling cascade regulates H2A.Z subunit exchange in neurons. Aim 2: To test the hypothesis that H2A.Z controls transcription and CpG methylation of plasticity- associated genes using a genome-wide approach. Aim 3: To enable the selective pharmacologic inhibition of H2A.Z by developing antisense oligonucleotide-based constructs that are sufficient to alter H2A.Z mRNA and protein expression and augment the acquisition of long-term behavioral change. Aim 4: To test the hypotheses that H2A.Z regulates neural plasticity via controlling both synaptic plasticity and homeostatic synaptic scaling in neurons. We anticipate that our results will be broadly applicable to understanding experience- and drug-induced neural plasticity involved in the induction and maintenance of lasting behavioral change.
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