Signaling Mechanisms Of Retinal Axon Guidance
Christopher William Cowan
Psychiatryuniversity Of Texas Sw Med Ctr/dallas
Grant 5R01EY018207-02 from National Eye Institute, IRG: NDPR
Abstract: During formation of the mammalian visual system, newly born neurons extend axons to make stereotyped connections with specific target field cells within the retina and brain. Eph receptors and their ephrin ligands play critical roles during visual system development in the proper guidance of retinal axons to the brain. However, the intracellular signaling events that mediate Eph-dependent axon targeting events has remained unclear. The main goal of this research proposal is to identify and understand the critical intracellular signaling events that convert the extracellular axon guidance cue into an appropriate axon turning event. Our preliminary studies have revealed that Vav and Ephexin family RhoGEFs (Rho family GTPase guanine nucleotide exchange factors) are essential regulators of Eph-dependent axon repulsion in vitro and in vivo. We seek to explore the precise role of Vav GEFs in the proper targeting of retinal axons to the brain, and to test the hypothesis that Vav GEFs regulate a switch from attractive to repulsive signaling. To address these ideas and to begin to understand the role and regulation of Vav GEFs in retinal development and axoon targeting, we propose the following specific aims 1) to determine the role of Vav GEFs in retinogeniculate axon targeting in vivo by analyzing the behavior of Vav-deficient, EphB1 -positive axons at the optic chiasm during development, 2) to determine the role of Vav GEFs in regulating attractive vs. repulsive Eph signaling in vitro and in vivo, and 3) to determine the regulation of Vav GEFs during retinal axon guidance by studying the Eph kinase-dependent degradation of Vav2. In this proposal, in vivo axon tracing techniques, biochemical and molecular biology approaches combined with cell culture techniques will be employed to explore the role of Vav GEFs in retinal development. Moreover we have developed a chemically-regulated, cell culture-based approach to more easily test the role of Eph receptor signaling events. The results of these studies will provide valuable new insights into the cell signaling processes that control proper visual system development. Understanding these retinal axon guidance mechanisms might provide important new insights into therapeutic approaches to retinal development disorders, axon regeneration following retinal nerve injury and neurodegenerative diseases that lead to partial or total blindness
Project start date: 2007-08-01
Project end date: 2012-07-31
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Grants awarded to Christopher William Cowan
ANALYSIS OF FKHRL1 INDUCED APOPTOSIS IN NEURONS
Christopher William Cowan
Children´s Hospital Boston
300 Longwood Ave
boston, Ma 021155737
Grant 5F32AG005870-02 from National Institute On Aging, IRG: ZRG1
Abstract: The overall goal of the proposed research is to elucidate the cellular mechanisms that govern decisions of neuronal survival and death. The proposal outlines experiments that will dissect the mechanisms of FKHRL1-neuronal survival and death. The proposal outlines experiments that will dissect the mechanisms of FKHRL1-induced apoptosis in neurons. The aims of the proposal are 1) To determine the role of FKHRL1 in normal neuronal apoptosis by analyzing the effects of dominant negative isoforms on FH on apoptosis. 2) To determine the role of 14-3-3 proteins in the cytoplasmic localization of FKHRL1 by assaying for interactions between endogenous FN and 14-3-3 proteins and by analyzing the subcellular localization of mutant FH proteins that are unable to interact with 14-3-3 proteins. 3) To determine the molecular mechanisms for nuclear import and export of FH. Elucidating the involvement of FKHRL1 in neuronal apoptosis and understanding the mechanisms that regulate FKHRL1-induced apoptosis could lead to development of new therapeutics for neurodegenerative diseases or cancer-diseases characterized by abnormal regulation of growth, survival and apoptosis
Keywords: apoptosis, granule cell, neural degeneration, phosphoprotein, transcription factor enzyme inhibitor, gene mutation, green fluorescent protein, intermolecular interaction, phospholipase A2 confocal scanning microscopy, immunocytochemistry, laboratory rat, transfection, yeast two hybrid system
5F32AG005870-02 (2001): $40196
1F32AG005870-01 (2000): $32416
Signaling Mechanisms Of Retinal Axon Guidance
Christopher William Cowan
Psychiatryuniversity Of Texas Sw Med Ctr/dallas
Grant 5R01EY018207-02 from National Eye Institute, IRG: NDPR
Abstract: During formation of the mammalian visual system, newly born neurons extend axons to make stereotyped connections with specific target field cells within the retina and brain. Eph receptors and their ephrin ligands play critical roles during visual system development in the proper guidance of retinal axons to the brain. However, the intracellular signaling events that mediate Eph-dependent axon targeting events has remained unclear. The main goal of this research proposal is to identify and understand the critical intracellular signaling events that convert the extracellular axon guidance cue into an appropriate axon turning event. Our preliminary studies have revealed that Vav and Ephexin family RhoGEFs (Rho family GTPase guanine nucleotide exchange factors) are essential regulators of Eph-dependent axon repulsion in vitro and in vivo. We seek to explore the precise role of Vav GEFs in the proper targeting of retinal axons to the brain, and to test the hypothesis that Vav GEFs regulate a switch from attractive to repulsive signaling. To address these ideas and to begin to understand the role and regulation of Vav GEFs in retinal development and axoon targeting, we propose the following specific aims 1) to determine the role of Vav GEFs in retinogeniculate axon targeting in vivo by analyzing the behavior of Vav-deficient, EphB1 -positive axons at the optic chiasm during development, 2) to determine the role of Vav GEFs in regulating attractive vs. repulsive Eph signaling in vitro and in vivo, and 3) to determine the regulation of Vav GEFs during retinal axon guidance by studying the Eph kinase-dependent degradation of Vav2. In this proposal, in vivo axon tracing techniques, biochemical and molecular biology approaches combined with cell culture techniques will be employed to explore the role of Vav GEFs in retinal development. Moreover we have developed a chemically-regulated, cell culture-based approach to more easily test the role of Eph receptor signaling events. The results of these studies will provide valuable new insights into the cell signaling processes that control proper visual system development. Understanding these retinal axon guidance mechanisms might provide important new insights into therapeutic approaches to retinal development disorders, axon regeneration following retinal nerve injury and neurodegenerative diseases that lead to partial or total blindness
Project start date: 2007-08-01
Project end date: 2012-07-31
1R01EY018207-01 (2007): $353250