Mechanisms Of Neurotrophin Signaling Through The P75 Receptor
Bruce D Carter, Professor
Biochemistryvanderbilt University
Grant 5R01NS038220-11 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Project start date: 1998-12-03
Project end date: 2011-06-30
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Mechanisms Of Neurotrophin Signaling Through The P75 Receptor
Bruce D Carter, Assistant Professor
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 5R01NS038220-09 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The p75 neurotrophin receptor plays a key role in regulating the delicate balance between neuronal survival and death during the development of the mammalian nervous system. In addition, it has been implicated in a number of neuropathologies involving abnormal apoptosis, such as Alzheimer s disease, spinal cord lesion and excitotoxic injury. Like other members of the TNF receptor superfamily, p75 activates a bifurcating signal that promotes both cell survival and cell death. How these pathways are activated and differentially regulated to determine the ultimate fate of a cell is not well understood. The overall objective of this proposal is to elucidate the molecular mechanisms by which the p75 neurotrophin receptor mediates cell death. We previously demonstrated that TRAF6 and NRIF are two receptor interacting proteins essential for p75- mediated apoptosis. TRAF6 is an E3 ubiquitin ligase known to regulate activation of NF-kB and JNK by many members of the TNF and Toll-like receptor superfamilies. In contrast, NRIF is a novel DMA binding protein that associates with the intracellular domain (ICD) of p75, thus raising the question as to how this interactor gets to the nucleus and whether p75 regulates NRIF s transcriptional activity. Our preliminary data indicate that NRIF is ubiquitinated by TRAF6 and this promotes its nuclear translocation. In addition, p75 was recently shown to undergo intramembrane proteolysis by gamma-secretase, leading to the release of the ICD. How this processing affects signaling is not clear; however, our preliminary data indicate that this is required for the receptor to induce apoptosis. A similar cleavage has been shown to mediate nuclear signaling for a number of transmembrane proteins (e.g. Notch and APR). Therefore, we hypothesize that p75 cleavage releases NRIF, which is targeted to the nucleus by TRAF6-mediated ubiquitination, resulting in the regulation of gene transcription and subsequently apoptosis. To test this hypothesis we propose to determine (1) the relationship between p75 cleavage and its signal transduction, (2) the role of receptor proteolysis in the nuclear shuttling of NRIF, (3) the functional consequence of NRIF ubiquitination, and (4) the change in NRIF s transcriptional activity in response to p75 activation. Given the ever-growing number of pathological conditions where p75 has been implicated, it is imperative that we begin to understand how this receptor mediates cell death in order to develop intervention strategies.
Keywords: apoptosis, biological signal transduction, cytokine receptor, growth factor receptor, neurotrophic factor, protein structure function, protein tyrosine kinase, receptor binding, binding protein, developmental neurobiology, nerve growth factor, neuroregulation, protein binding, protein protein interaction
Project start date: 1998-12-03
Project end date: 2011-06-30
5R01NS038220-09 (2007): $301726
5R01NS038220-10 (2008): $301726
NEUROTROPHIN SIGNALING THROUGH THE P75 RECEPTOR
Bruce D Carter, Assistant Professor
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 5R01NS038220-07 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Programmed cell death in the nervous system is a naturally occurring process in mammalian development; however, abnormal apoptosis is the basis for many neuropathologies, e.g. Alzheimer s and Parkinson s disease and ischemic injury. The delicate balance between neuronal survival and death is regulated, in part, by the neurotrophins, which promote survival through binding to the Trks, a family of tyrosine kinase receptors, and induce apoptosis through a 75kDa receptor, p75. While significant progress has been made in elucidating the mechanisms by which the Trks promote survival, much less is known about how p75 induces cell death. Nerve Growth Factor binding to p75 activates a bifurcating signal, promoting both cell survival through the transcription factor NF-kB, and cell death through the stress-activated kinase, JNK. Thus the ultimate fate of a cell in the developing or lesioned nervous system can only be understood by elucidating how these signals are generated and regulated. To meet this goal, we identified a novel zinc finger protein, NRIF, which binds to the intracellular domain (lCD) of p75. Our findings suggested that NRIF plays a role in neurotrophin mediated apoptosis in vivo. Recently, we found that NRIF binds to a member of the cytokine receptor associated factor family, TRAF6, which can also bind to the lCD of p75, and that this interaction directed NRIF translocation to a subnuclear domain. Therefore, we hypothesize that both NRIF and TRAF6 are part of the p75-mediated apoptotic signaling pathway and that their interaction and nuclear translocation are required for this signal. To address this hypothesis, we propose three integrated specific aims I) Determine the ability of p75 to induce apoptosis in the absence of NRIF and TRAF6 binding to the receptor. This will include analysis of the nrzf-I- and traf6-/- mice. II) Establish the functional significance of TRAF6-NRIF interaction. By mutating the sequences on each protein required for their association and nuclear translocation, we will assess how the interaction of these signaling proteins affects their ability to regulate NF-kB, JNK, and apoptosis. III) Determine the mechanisms by which NRIF regulates cellular viability. We will investigate whether NRIF promotes apoptosis through repression of transcription. These studies will reveal the molecular mechanisms by which neurotrophins regulate cellular viability in the nervous system. In particular, they will elucidate how the apoptotic response is generated through the p75 receptor, which has potential clinical applications in developing therapeutic strategies for a variety of neuropathologies.
Keywords: apoptosis, biological signal transduction, cytokine receptor, growth factor receptor, neurotrophic factor, protein structure function, protein tyrosine kinase, receptor binding, binding protein, developmental neurobiology, nerve growth factor, neuroregulation, protein binding, protein protein interaction
Project start date: 1998-12-03
Project end date: 2006-06-30
5R01NS038220-07 (2005): $286900
5R01NS038220-06 (2004): $286900
MECHANISMS NEUROTROPHIN SIGNALING THROUGH P75 RECEPTOR
Bruce D Carter, Assistant Professor
Biochemistryvanderbilt University
medical Center
nashville, Tn 372036869
Grant 5R01NS038220-03 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Adapted from applicant´s ) During the normal development of the mammalian nervous system nearly half of the neurons undergo apoptosis as part of a natural pruning process. Dysfunctional apoptosis has been associated with a variety of neurological diseases such as Alzheimer´s, Huntington´s and Parkinson´s diseases, ALS and multiple system atrophy. The critical balance between necessary cell death and maintenance of essential neurons is controlled by the neurotrophins. Their effects are mediated through binding to a family of tyrosine kinase receptors, the Trks, and a 75-kD receptor, p75. It has been shown that the Trks activate canonical growth factor receptor signaling pathways. In contrast, the function of p75 remains largely unexplored. Recent evidence suggests that ligand binding to p75 in specific cell types can activate the transcription factor NFkappaB and induce apoptosis through a mechanism involving jun kinase. The activation of programmed cell death by classical trophic factors is somewhat surprising, especially given the widespread expression of both p75 and the neurotrophins throughout the nervous system. Clearly, the activation of apoptosis must be highly regulated such that it only occurs in specific contexts. The overall goal of this research proposal is to understand the molecular mechanisms by which the neurotrophins regulate neuronal survival, in particular, signaling through the p75 receptor. Currently there are no known receptor-associated proteins that transduce p75´s signal. This proposal describes a novel zinc finger protein, NRIF, isolated using the yeast two hybrid system, which binds to the cytoplasmic domain of p75 in a ligand-dependent manner. This proposal will test the hypothesis that neurotrophin binding to p75 activates a signal transduction pathway that promotes both cell survival and programmed cell death mediated in part by the novel p75 interactor NRIF. To test this hypothesis the following specific aims are proposed (1) Define the domains of NRIF and p75 responsible for their interaction; (2) Determine the molecular components of p75 signaling; (3) Assess the mechanisms by which p75 regulates cell survival. Understanding neurotrophin signaling mechanisms will provide insight into how these essential factors regulate the development of the vertebrate nervous system and could suggest novel strategies for therapeutic intervention in neurological disorders
Keywords: biological signal transduction, growth factor receptor, neurotrophic factor, programmed cell death, protein structure /function, protein tyrosine kinase, receptor binding binding protein, developmental neurobiology, neuroregulation, protein protein interaction
Project start date: 1998-12-03
Project end date: 2001-11-30
5R01NS038220-03 (2001): $260673
5R01NS038220-02 (2000): $245314
Grants awarded to Bruce D Carter
Mechanisms Of Neurotrophin Signaling Through The P75 Receptor
Bruce D Carter, Assistant Professor
Vanderbilt University Medical Center Nashville, Tn 372036869
Grant 2R01NS038220-08A1 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: The p75 neurotrophin receptor plays a key role in regulating the delicate balance between neuronal survival and death during the development of the mammalian nervous system. In addition, it has been implicated in a number of neuropathologies involving abnormal apoptosis, such as Alzheimer s disease, spinal cord lesion and excitotoxic injury. Like other members of the TNF receptor superfamily, p75 activates a bifurcating signal that promotes both cell survival and cell death. How these pathways are activated and differentially regulated to determine the ultimate fate of a cell is not well understood. The overall objective of this proposal is to elucidate the molecular mechanisms by which the p75 neurotrophin receptor mediates cell death. We previously demonstrated that TRAF6 and NRIF are two receptor interacting proteins essential for p75- mediated apoptosis. TRAF6 is an E3 ubiquitin ligase known to regulate activation of NF-kB and JNK by many members of the TNF and Toll-like receptor superfamilies. In contrast, NRIF is a novel DMA binding protein that associates with the intracellular domain (ICD) of p75, thus raising the question as to how this interactor gets to the nucleus and whether p75 regulates NRIF s transcriptional activity. Our preliminary data indicate that NRIF is ubiquitinated by TRAF6 and this promotes its nuclear translocation. In addition, p75 was recently shown to undergo intramembrane proteolysis by gamma-secretase, leading to the release of the ICD. How this processing affects signaling is not clear; however, our preliminary data indicate that this is required for the receptor to induce apoptosis. A similar cleavage has been shown to mediate nuclear signaling for a number of transmembrane proteins (e.g. Notch and APR). Therefore, we hypothesize that p75 cleavage releases NRIF, which is targeted to the nucleus by TRAF6-mediated ubiquitination, resulting in the regulation of gene transcription and subsequently apoptosis. To test this hypothesis we propose to determine (1) the relationship between p75 cleavage and its signal transduction, (2) the role of receptor proteolysis in the nuclear shuttling of NRIF, (3) the functional consequence of NRIF ubiquitination, and (4) the change in NRIF s transcriptional activity in response to p75 activation. Given the ever-growing number of pathological conditions where p75 has been implicated, it is imperative that we begin to understand how this receptor mediates cell death in order to develop intervention strategies.
Keywords: apoptosis, biological signal transduction, cytokine receptor, growth factor receptor, neurotrophic factor, protein structure function, protein tyrosine kinase, receptor binding, binding protein, developmental neurobiology, nerve growth factor, neuroregulation, protein binding, protein protein interaction
Project start date: 1998-12-03
Project end date: 2011-06-30
2R01NS038220-08A1 (2006): $309725
3R01NS038220-10S1 (2008): $76750
The Regulation Of Myelination By The P75-NFkB Pathway
Bruce D Carter, Assistant Professor
Biochemistryvanderbilt University
medical Center
nashville, Tn 372036869
Grant 5R01NS048249-05 from National Institute Of Neurological Disorders And Stroke IRG: NDBG
Abstract: Myelin is necessary for the rapid conduction of nerve impulses, the protection of axons from damaging agents, providing trophic support to neurons and regulating the ability of injured axons to regenerate. Hence, pathological conditions that cause demyelination such as Multiple Sclerosis, Guillian Barre Syndrome or nerve injury can have disastrous consequences for the afflicted individuals. Myelin is multilamellar structure produced by Schwann cells in the peripheral nervous system and oligodendrocytes in the CNS in response to axonal signals that have yet to be defined. Our overall objective is to elucidate the mechanisms regulating the formation of this essential neural structure. We recently demonstrated that activation of the transcription factor NFkB in pre-myelinating Schwann cells is essential for their differentiation into a myelinating phenotype. Inhibition or genetic deletion of NFkB in Schwann cells prevented myelin formation. The up stream activator of NFkB remains to be determined; however, the p75 neurotrophin receptor, which can activate this transcription factor, was recently shown to have an essential role in the process of myelination. During the development of the peripheral nervous system, p75 is highly expressed in pre-myelinating Schwann cells, but begins to decline as myelin forms and is difficult to detect in the mature, myelinating glia. Nevertheless, p75 is also expressed in the sensory neurons that become myelinated, thus it is unclear whether p75´s critical function is specifically in Schwann cells, neurons or both. It is our overall hypothesis that p75 mediated activation of NFkB is required for Schwann cells to form myelin. We will investigate this proposal using transgenic animals and an in vitro myelination system of Schwann cells and sensory neurons in co-culture. Our specific objectives are (1) Define the mechanisms of NFkB activation. (2) Investigate where p75 expression is required in order for myelin to form by use of a conditional gene deletion. (3) Determine the contribution of p75 to the endogenous NFkB activity in pre-myelinating Schwann cells. (4) Define the target genes of NFkB in Schwann cells through gene profiling by microarray and the analysis of promoters of myelin genes we have already identified as regulated by the transcription factor
Keywords: Schwann cell, growth factor receptor, myelination, nerve growth factor, neuroregulation, nuclear factor kappa beta I kappa B beta, gene deletion mutation, gene expression, nerve /myelin protein, neurogenetics, posttranslational modification embryo /fetus tissue /cell culture, gel mobility shift assay, genetically modified animal, laboratory mouse, laboratory rat, microarray technology
Project start date: 2004-03-01
Project end date: 2010-02-28
5R01NS048249-05 (2008): $297983
5R01NS048249-04 (2007): $297983
5R01NS048249-03 (2006): $306883
5R01NS048249-02 (2005): $314269
1R01NS048249-01 (2004): $314269
MECHANISMS NEUROTROPHIN SIGNALING THROUGH P75 RECEPTOR
Bruce D Carter, Assistant Professor
Biochemistryvanderbilt University
medical Center
nashville, Tn 372036869
Grant 1R01NS038220-01 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Abstract: Adapted from applicant´s ) During the normal development of the mammalian nervous system nearly half of the neurons undergo apoptosis as part of a natural pruning process. Dysfunctional apoptosis has been associated with a variety of neurological diseases such as Alzheimer´s, Huntington´s and Parkinson´s diseases, ALS and multiple system atrophy. The critical balance between necessary cell death and maintenance of essential neurons is controlled by the neurotrophins. Their effects are mediated through binding to a family of tyrosine kinase receptors, the Trks, and a 75-kD receptor, p75. It has been shown that the Trks activate canonical growth factor receptor signaling pathways. In contrast, the function of p75 remains largely unexplored. Recent evidence suggests that ligand binding to p75 in specific cell types can activate the transcription factor NFkappaB and induce apoptosis through a mechanism involving jun kinase. The activation of programmed cell death by classical trophic factors is somewhat surprising, especially given the widespread expression of both p75 and the neurotrophins throughout the nervous system. Clearly, the activation of apoptosis must be highly regulated such that it only occurs in specific contexts. The overall goal of this research proposal is to understand the molecular mechanisms by which the neurotrophins regulate neuronal survival, in particular, signaling through the p75 receptor. Currently there are no known receptor-associated proteins that transduce p75´s signal. This proposal describes a novel zinc finger protein, NRIF, isolated using the yeast two hybrid system, which binds to the cytoplasmic domain of p75 in a ligand-dependent manner. This proposal will test the hypothesis that neurotrophin binding to p75 activates a signal transduction pathway that promotes both cell survival and programmed cell death mediated in part by the novel p75 interactor NRIF. To test this hypothesis the following specific aims are proposed (1) Define the domains of NRIF and p75 responsible for their interaction; (2) Determine the molecular components of p75 signaling; (3) Assess the mechanisms by which p75 regulates cell survival. Understanding neurotrophin signaling mechanisms will provide insight into how these essential factors regulate the development of the vertebrate nervous system and could suggest novel strategies for therapeutic intervention in neurological disorders
Keywords: biological signal transduction, growth factor receptor, neurotrophic factor, programmed cell death, protein structure /function, protein tyrosine kinase, receptor binding binding protein, developmental neurobiology, neuroregulation, protein protein interaction
Project start date: 1998-12-03
Project end date: 2001-11-30
1R01NS038220-01 (1999): $247669
MECHANISMS OF P75 NEUROTROPHIN RECEPTOR SIGNALING
Bruce D Carter, Assistant Professor
Cell Biology And Anatomyweill Medical College Of Cornell Univ
1300 York Avenue
new York, Ny 10021
Grant 1F32NS010394-01 from National Institute Of Neurological Disorders And Stroke IRG: ZRG1
Keywords: biological signal transduction, growth factor receptor, neurotrophic factor, protein structure /function, receptor binding gene deletion mutation, plasmid polymerase chain reaction, tissue /cell culture, transfection, western blotting
1F32NS010394-01 (1997): $32200