2011 |
Liu, Guofa |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
The Coordination of Netrin Signal Transduction
DESCRIPTION (provided by applicant): Netrins, a family of secreted proteins, are not only required for axon guidance and neuronal migration during development, but are also involved in angiogenesis and tumorigenesis. Recent studies including ours indicate that DSCAM (Down syndrome cell adhesion molecule), a transmembrane protein of the immunoglobulin superfamily, functions as a new netrin receptor required for spinal cord commissural axon outgrowth and pathfinding. This application proposes to investigate the molecular mechanisms mediating neuronal responses to netrin-1 with a focus on studying the coordination of signal transduction cascades downstream of different netrin receptors, for instance, DCC, DSCAM and UNC5. Aim 1. Determine the coordinating role of JNK1 in netrin signaling. Our preliminary results suggest that JNK1 is involved in netrin signaling. To further characterize the coordinating role of JNK1 in DCC and DSCAM downstream signaling, we plan to examine the regulation of JNK1 activity by netrin-1 in HEK293 cells and primary neurons, as well as the functional importance of JNK1 in netrin-induced axon outgrowth and attraction. Aim 2. Untangle the coordination of protein phosphorylation in netrin signaling. Protein phosphorylation plays a critical role in netrin signaling during embryonic development. To study the coordination of signal transduction cascades downstream of DSCAM and DCC, we will examine the regulation of protein phosphorylation in netrin/DSCAM signaling, the coordination of protein phosphorylation in netrin/DSCAM and netrin/DCC signaling, relationship of Src family kinases and JNK1 in netrin signal transduction cascades and the functional coordination of DSCAM and DCC in netrin-induced axon outgrowth and attraction. Aim 3. Investigate the coordination of DSCAM and UNC5 in netrin-mediated repulsion. Netrin-1 can act as an attractant or repellent in axon guidance and neuronal migration, depending on when it binds to UNC5. DSCAM forms a receptor complex with UNC5C in HEK293 cells and primary neurons, indicating that they may coordinate in netrin-mediated neuronal repulsion. We plan to examine the distribution and co-localization of DSCAM and UNC5C in the developing nervous system, the coordination of netrin/DSCAM and netrin/UNC5C downstream signaling and the functional role of DSCAM in netrin-mediating axon repulsion. These experiments will shed new light on the coordination of netrin downstream signal transduction cascades and help us better understand how the brain establishes its complex and precise wiring during development. PUBLIC HEALTH RELEVANCE: Coordination of signal transduction cascades downstream of guidance cues is crucial for neurons to find their targets and establish neural circuitry in the developing nervous system. The secreted proteins netrins are prototypical guidance cues for projecting axons and migrating neurons. Studying signal transduction of netrins will not only help us understand how the brain establishes its complex and precise wiring during development, but also provide a basis to design new strategies for injury-related diseases, such as stroke and spinal cord injuries.
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2014 |
Liu, Guofa |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Modulation of Microtubule Dynamics in Axon Guidance
DESCRIPTION (provided by applicant): Proper axon outgrowth and path finding are essential for the formation of functional neuronal circuits in the developing nervous system. Coordination of different guidance cues, their receptors and intracellular signal transduction cascades eventually converges on orchestrating cytoskeleton dynamics to maneuver growth cone navigation. Netrins, a conserved family of canonical guidance cues, play an important role in axon outgrowth and guidance in the developing nervous system. The coupling of axon guidance cues, such as netrin-1, to microtubule (MT) dynamics is crucial for growth cone (GC) navigation. However, whether axon guidance signaling regulates MT dynamics directly or indirectly is unclear. Our preliminary data indicate that DCC and DSCAM interact directly with TUBB3, the most dynamic ?-tubulin isoform in neurons, and netrin-1 induces these interactions. TUBB3 is required for netrin-1- induced axon outgrowth and path finding in the developing nervous system. These results suggest that DCC and DSCAM directly couple MT dynamics in netrin-1-mediated axon attraction. Interestingly, UNC5 also interacts with TUBB3. In this study, we will determine how netrin-1 modulates microtubule dynamics through coupling of its receptors DCC, DSCAM and UNC5C with dynamic TUBB3 in attractive and repulsive signaling. Aim1. Untangle the role of TUBB3 in coordination of netrin-1 attractive signaling. To further investigate the coordinating role of TUBB3 in netrin/DCC/DSCAM-mediated axon attractive signaling, we will examine: 1) Netrin-1 regulation of the interaction of TUBB3 with DCC and DSCAM; 2) Modulation of MT dynamics by netrin-1 via the interaction of dynamic TUBB3 with DCC and DSCAM; 3) The functional importance of TUBB3 in netrin attractive signaling. Aim 2. Determine the role of TUBB3 in coordination of netrin-1 repulsive signaling. UNC5 interacts with DCC and DSCAM and these interactions mediate repulsive responses to netrin-1. Our preliminary data show that TUBB3 interacts with UNC5C, DCC and DSCAM, suggesting it may play a role in coordinating netrin-1 repulsive signaling. To further characterize this hypothesis, we will focus on studying: 1) Netrin-1 regulation of the interaction of TUBB3 with UNC5C, DCC and DSCAM; 2) Modulation of MT dynamics by netrin-1 via coordination of the interaction of dynamic TUBB3 with UNC5C, DCC and DSCAM; 3) The functional importance of TUBB3 in netrin-1-mediated axon repulsion. Aim 3. Investigate the mechanistic link between netrin signaling and MT dynamics. To examine how netrin-1 directly regulates MT dynamics via TUBB3 in axon turning, we will focus on: 1) assessing tyrosine phosphorylation of TUBB3 in netrin signaling; 2) tracking modulation of MT dynamics in the GC during netrin-1-mediated attraction using live cell imaging; 3) tracking netrin-1- mediated MT dynamics in the GC during axon repulsion. This study should reveal how netrin-1 directly modulates MT dynamics via differential coupling of its receptors with dynamic TUBB3 in axon turning.
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2019 |
Liu, Guofa |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Microrna Regulation of Guidance Receptors in Axonal Pathfinding
Project Summary Proper axon outgrowth and pathfinding is essential for establishing precise neural networks of the nervous system during development. Defects in axon guidance are implicated in a variety of neurological disorders. Spatiotemporal regulation of guidance receptor expression facilitates differential responses of neurons to guidance cues in order to form accurate neuronal wiring. In vertebrates, levels of the guidance receptor Robo1 are low in precrossing and high in postcrossing commissural axons (CAs), functioning as a ?molecular switch? to regulate sensitivity to Slit repulsion and guide CA midline crossing. However, the mechanism underlying the fine-tuned spatiotemporal regulation of Robo1 expression remains largely unknown. MicroRNAs (miRNAs) regulate target gene expression by binding specifically to the 3?untranslated region (3?UTR) of target mRNAs, thus repressing translation and/or inducing mRNA degradation. Our preliminary studies indicate that chicken Robo1 (cRobo1) 3?UTR is required for regulation of protein expression in developing chicken spinal cords. miR-92, a highly conserved miRNA, suppresses cRobo1 expression in a miR-92 miRNA response element (MRE)-dependent manner. miR-92 and cRobo1 are differentially expressed in the developing chicken spinal cord with a mutually exclusive expression pattern. Ectopic expression of miR-92 in postcrossing commissural neurons results in CAs stalling in the floor plate. Therefore, we propose that miR-92 is a negative regulator of Robo1 expression in CAs by targeting its mRNA at the 3?UTR, thereby regulating Slit sensitivity to control CA projection and midline crossing. To test this hypothesis, we will first determine whether endogenous miR-92 specifically regulates cRobo1 expression in commissural neurons of embryonic chicken spinal cords during midline crossing (Aim 1): we will (1) examine the activity of endogenous miR-92 in precrossing commissural neurons of chicken spinal cords, (2) determine the subcellular expression patterns of miR-92 and cRobo1 in commissural neurons, and (3) untangle the mechanisms underlying miR-92-mediated repression of cRobo1. Secondly, we will focus on studying the functional importance of miR-92 in Slit/Robo1-mediated CA outgrowth and turning in vitro and CA projection and pathfinding in vivo (Aim 2). Finally, we will identify novel miRNAs targeting to cRobo1 in commissural neurons and examine their roles in Slit/Robo1-mediated CA guidance during midline crossing (Aim 3). These proposed experiments will support a model that specific miRNAs suppress Robo1 expression, thereby modulating Slit sensitivity to control Slit/Robo1- mediated CA guidance during embryonic spinal cord development.
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2019 — 2020 |
Liu, Guofa |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
The Role of Micrornas in Axon Pathfinding
Project Summary In the developing nervous system, proper axon outgrowth and pathfinding are regulated by multiple guidance molecules, their receptors and intracellular signaling pathways. Spatiotemporal regulation of guidance receptor expression facilitates differential responses of neurons to guidance cues in order to form accurate neuronal wiring. In vertebrates, levels of the guidance receptor Robo1 are low in precrossing and high in postcrossing commissural axons (CAs), a pattern functioning as a ?molecular switch? to regulate sensitivity to Slit repulsion and guide CA midline crossing. However, the mechanism underlying the fine- tuned spatiotemporal regulation of Robo1 expression remains largely unknown. MicroRNAs (miRNAs) regulate gene expression by binding specifically to the 3?untranslated region (3?UTR) of target mRNAs, thus repressing translation and/or inducing mRNA degradation. Our preliminary studies indicate that the Robo1 3?UTR is required for regulation of protein expression in the developing spinal cord. Gga-miR-92, a highly conserved miRNA, is differentially expressed in the developing chicken spinal cord, and regulates Slit sensitivity via suppression of cRobo1 expression in commissural neurons, thereby controlling CA projection and midline crossing. The mature gga-miR-92 has the same sequence as mmu-miR-92a and mmu-miR-92b except the tenth base at the 5? end of mmu-miR-92b. Mmu-miR-92b can also repress mRobo1 expression by targeting its 3?UTR. Therefore, we hypothesize that mmu-miR-92a and mmu-miR-92b, like gga-miR-92, specifically regulate mRobo1 expression in commissural neurons to control Slit/Robo1-mediated CA guidance in the developing mouse spinal cord. We will examine the role of mmu-miR-92a and mmu-miR- 92b in (1) suppressing mRobo1 expression in developing commissural neurons (Aim 1) and (2) Slit/mRobo1-mediated spinal CA guidance (Aim 2). Studies in this proposal will advance our understanding of molecular mechanisms underlying Slit/Robo-mediated axon guidance.
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