Robert S. Freeman - US grants

DESCRIPTION: Programmed cell death (PCD) is a necessary developmental phenomenon that is widespread in the nervous systems. Recent evidence suggests that PCD also occur in several pathological conditions. We and other have hypothesized that neurotrophic factor deprivation induced PCD is mediated by key cell cycle regulators activated by the removal of survival promoting factors. Recently, we provided the first evidence for the increased expression of a specific gene, cyclin D1, in neurons undergoing PCD. The only previously described function for cyclin D1 is its role in progression through the G1-phase of the cell cycle. We shall investigate the specific hypothesis that cyclin D1 functions as a necessary part of the death program in neurons. Toward this objective, we shall assess directly the role of cyclin D1 in the death of nerve growth factor (NGF) deprived sympathetic neurons. Using intracellular microinjections, we shall express (1) inhibitors of cyclin D1 function, (2) antisense cyclin D1 sequences, or (3) neutralizing cyclin D1 antibodies to examine whether cyclin D1 expression is required for NGF deprivation-induced PCD. We shall also examine whether overexpression of cyclin D1 ectopically is sufficient to induce PCD in neurons maintained in the presence of NGF. Biochemical approaches, including protein kinase assays, immunoprecipitations, and immunoblotting, will be used to identify and characterize the molecules that interact with cyclin D1 during PCD. These experiments will address whether a cyclin-dependent protein kinase is activated in dying neurons or whether cyclin D1 interacts with the retinoblastoma protein as part of a mechanism for cell death. Lastly, we shall use reverse transcription-polymerase chain reaction technology to continue to catalog cell cycle gene expression in dying neurons. These studies will determine the significance of the increased expression of cyclin D1 during PCD and will test the general hypothesis that neuronal cell death involves the activation of cell cycle events. This work should further our long-term goals of elucidating the molecular mechanism of neuronal PCD and of developing the means to manipulate the process pharmacologically.

DESCRIPTION: Programmed cell death (PCD) is a necessary developmental phenomenon that is widespread in the nervous systems. Recent evidence suggests that PCD also occur in several pathological conditions. We and other have hypothesized that neurotrophic factor deprivation induced PCD is mediated by key cell cycle regulators activated by the removal of survival promoting factors. Recently, we provided the first evidence for the increased expression of a specific gene, cyclin D1, in neurons undergoing PCD. The only previously described function for cyclin D1 is its role in progression through the G1-phase of the cell cycle. We shall investigate the specific hypothesis that cyclin D1 functions as a necessary part of the death program in neurons. Toward this objective, we shall assess directly the role of cyclin D1 in the death of nerve growth factor (NGF) deprived sympathetic neurons. Using intracellular microinjections, we shall express (1) inhibitors of cyclin D1 function, (2) antisense cyclin D1 sequences, or (3) neutralizing cyclin D1 antibodies to examine whether cyclin D1 expression is required for NGF deprivation-induced PCD. We shall also examine whether overexpression of cyclin D1 ectopically is sufficient to induce PCD in neurons maintained in the presence of NGF. Biochemical approaches, including protein kinase assays, immunoprecipitations, and immunoblotting, will be used to identify and characterize the molecules that interact with cyclin D1 during PCD. These experiments will address whether a cyclin-dependent protein kinase is activated in dying neurons or whether cyclin D1 interacts with the retinoblastoma protein as part of a mechanism for cell death. Lastly, we shall use reverse transcription-polymerase chain reaction technology to continue to catalog cell cycle gene expression in dying neurons. These studies will determine the significance of the increased expression of cyclin D1 during PCD and will test the general hypothesis that neuronal cell death involves the activation of cell cycle events. This work should further our long-term goals of elucidating the molecular mechanism of neuronal PCD and of developing the means to manipulate the process pharmacologically.

DESCRIPTION (provided by applicant): In models of stroke, neurodegenerative disease, and developmental cell death, the delivery of specific neurotrophins can protect neurons from apoptosis. Thus an understanding of the molecular mechanisms that neurotrophins use to promote cell survival and to protect neurons from injury is likely to contribute to the development of novel therapies for neuronal disorders and diseases associated with cell death. Recent studies have firmly established that phosphatidylinositol (PI) 3-kinase and the Akt protein kinase comprise an important cell survival pathway activated by nerve growth factor (NGF) and other neurotrophins. However, it is now becoming clear that neurotrophins utilize additional survival pathways. The overall aim of this application is to test the hypothesis that NGF transduces survival signals through a pathway involving the transcriptional regulator nuclear factor kappa-B (NF-KB). A major theme that will be explored is that NF-kB contributes to NGF-promoted survival by up-regulating anti-apoptotic mechanisms in neurons. Specific aim 1 will investigate the relationship between the PI 3-kinase/Akt and NF-KB survival pathways in neurons. Experiments will test whether inhibiting the PT 3-kinase/Akt pathway effects NGF-induced NF-KB activation in neurons. Additional experiments will examine whether activation of either pathway can protect neurons from cell death caused by inhibiting the other. Experiments in specific aim 2 will determine the cell death events induced after NGF withdrawal that can be blocked by activating NF-kB, focusing on reactive oxygen species, Bax, and cytochrome c. The goal of specific aim 3 is to identify pro-survival transcriptional targets of NF-kB in neurons and to test whether these proteins function as part of a NGF-dependent survival pathway. Experiments in the last specific aim (4) will test the importance of the endogenous c-Rel subunit of NF-iB for neuronal survival using neurons from c-Re! "knockout" mice. Together these experiments will test the importance of NF-kB for neurotrophinregulated survival and they will uncover important new information concerning the mechanisms by which neurotrophins protect neurons from cell death.

DESCRIPTION (provided by applicant): Cell death is widespread during the development of the nervous system, where it helps to ensure that the proper number and types of connections are formed between neurons and their targets. It is often initiated when neurons fail to receive adequate survival promoting signals from trophic factors such as nerve growth factor (NGF). Recent evidence suggests that a similar type of death may occur in human neuronal disorders and degenerative diseases, when neurons gain insufficient access to trophic support. Trophic factor deprivation-induced death requires RNA and protein synthesis and involves complex, incompletely understood mechanisms leading to activation of caspases that dismantle the cell. We and others have hypothesized that genes upregulated after trophic factor withdrawal function in critical cell death pathways. We previously identified SM-20 as one of a select group of genes whose expression increases in NGF-deprived neurons and that promote caspase-dependent death when overexpressed. Very recently, SM-20 and two closely related proteins (collectively called EGLN proteins) were shown to comprise a new family of prolyl hydroxylases involved in regulating the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha). EGLN-catalyzed proline hydroxylation destabilizes HIF-1alpha by increasing its affinity for a ubiquitin ligase. Based on these and other findings, we hypothesize that SM-20/EGLN3-mediated proline hydroxylation of critical target proteins (including but not limited to HIF-1alpha) plays an important role in regulating cell death initiated by trophic factor withdrawal. Here we propose experiments to (1) define the importance of SM-20/EGLN3 for trophic factor deprivation-induced death, and (2) to characterize its mechanism of action in NGF-deprived neurons. In aim 1, we shall assess the importance of SM-20/EGLN3 expression and activity for death caused by NGF withdrawal. For aim 2, we shall use complementary approaches to determine if HIF-1alpha transcription factor activity is regulated by the presence or absence of NGF and if stabilized, SM-20/EGLN3-resistant forms of HIF-1alpha are neuroprotective. Additional experiments will determine the effects of disrupting HIF-1alpha expression on trophic factor deprivation-induced death and NGF-dependent survival. In the third aim, we will examine the functional significance of a recently identified interaction between SM-20/EGLN3 and NRAGE, a protein previously shown to bind to the NGF receptor p75 NTR. Lastly, in aim 4 we propose new approaches for identifying novel substrates for this increasingly important family of enzymes. These studies should help further our understanding of the mechanisms that lead to trophic factor deprivation-induced death. They will also provide new information concerning the function of EGLN-catalyzed proline hydroxylation as a novel mechanism for altering protein function in neurons.

[unreadable] DESCRIPTION (provided by applicant): Attention to the study of the development of the nervous system is one of the keys in advancing our knowledge base in normal brain function, mental disorders and maladaptive behaviors. To be effective, such an undertaking must be multidisciplinary in its approach and young investigators contemplating research careers in developmental neuroscience must be accordingly trained. This is the goal of the current application, which proposes to establish a program devoted to the training of predoctoral fellows in Developmental Neuroscience. Funding is sought to train predoctoral students with expressed interest in establishing research careers in Developmental Neuroscience. The training will include a firm curricular grounding in the fundamental neurosciences, with emphasis on appreciating the complexity of the developing nervous system, and on extensive continuous participation in laboratory research as well as collateral activities. The Program Faculty consists of 17 members of the Faculty of the Graduate Education of the Biomedical Sciences. They are recruited based on research interests in molecular, cellular and systems aspects of nervous system development, including differentiation, neurogenesis, gliogenesis, function and dynamics of neurotransmitters and receptors, ion channels and related signal transduction processes, synaptic plasticity, emergence of functional architectures, neural and behavioral sensitive periods, and neural processing. It is the primary goal of the training program to ensure that every trainee acquires hands-on research proficiency in at least one of these disciplines, incorporating all facets of state-of-the-art neuroanatomy and image-processing techniques, cell biology, molecular biology and electrophysiology. In assembling the Program Faculty, the goal has been to cross not only research disciplines but also departmental and cluster boundaries. This multidisciplinary nature of the Program favors cross-fertilization of research methodologies, ideas and training opportunities for both preceptors and trainees. Although the emphasis is on training in research, the training program will offer specific journal dubs, seminars and workshops. The program will also incorporate specialized components dealing with issues such as survival skills, grant writing and responsible conduct in scientific research. A plan is also in place that will increase the pool of predoctoral applicants from underrepresented racial and ethnic groups. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Neuronal death is a highly regulated event that occurs throughout the developing nervous system. In many cases, developing neurons die because they fail to receive adequate supplies of target-derived, survival promoting factors such as nerve growth factor (NGF). This type of developmentally programmed cell death is important for ensuring that the proper numbers and types of connections are established between neurons and their targets. Importantly, trophic factor deprivation is also a frequent consequence of injury and disease in the mature nervous system. In such cases, the neuronal death is pathologic and contributes to the functional deficits seen in spinal cord injury, stroke, and Alzheimer's disease. Using a well-characterized and physiologically important model for trophic factor deprivation involving NGF-dependent sympathetic neurons, we recently identified the prolyl hydroxylase EGLN3 as a mediator of cell death. While the mechanism by which EGLN3 promotes death is largely unknown, we have discovered a novel interaction between EGLN3 and the BH3-only Bcl-2 family protein BIMEL, an established regulator of cell death induced by trophic factor withdrawal. Preliminary results reveal that death induced by BIMEL is reduced in EGLN3-deficient neurons, suggesting a functional relationship exists between these two proteins. Co-immunoprecipitation experiments demonstrate that EGLN3 and BIMEL each interact with the von Hippel-Lindau tumor suppressor protein pVHL. Expression of pVHL in sympathetic neurons promotes cell death while other preliminary results suggest that pVHL may enhance EGLN3 and BIMEL protein stability. Based on these novel observations, we hypothesize that pVHL, BIMEL and EGLN3 function coordinately to regulate trophic factor deprivation-induced cell death. In Aim 1 we will use a combination of over-expression and neurons from knockout mice to determine the functional relationship between EGLN3 and BIMEL during NGF deprivation-induced cell death. Experiments in Aim 2 will determine the biochemical significance of the interaction between EGLN3 and BIMEL for cell death. Specific experiments will determine if EGLN3 influences the function of BIMEL, if BIMEL affects the prolyl hydroxylase activity of EGLN3, or if BIMEL is a substrate for prolyl hydroxylation by EGLN3. In Aim 3, we will test the hypothesis that pVHL regulates EGLN3 and BIMEL protein levels during trophic factor deprivation. In addition, we will test if pVHL expression is necessary for trophic factor deprivation-induced cell death. These studies will further our understanding of the mechanisms that lead to neuronal cell death during normal development and in nervous systems disorders where trophic factor deprivation contributes to neuronal loss and dysfunction. PUBLIC HEALTH RELEVANCE: Trophic factor deprivation-induced cell death is not only critical for proper development of the nervous system but it also contributes to the loss and dysfunction of neurons that accompanies stroke, spinal cord injury, and neurodegenerative disease. This project will characterize new mechanisms that regulate cell death caused by neurotrophic factor deprivation. Information gained from this project will further our understanding of normal development and could help identify new targets for therapies aimed at preventing pathological neuronal cell death.