Erik W. Dent, Ph.D. - US grants

DESCRIPTION: A functional nervous system requires both the appropriate development of dendritic spines and their functional plasticity throughout life. Because dendritic spines are the primary sites of contact with presynaptic axons in excitatory neurons of hippocampus and cortex, their structure and function have been studied in great detail. Actin filaments (f-actin) play prominent roles in the formation, maintenance and plasticity of dendritic spine structure. However, the role of MTs in spine architecture has been studied little because spines are thought to be devoid of MTs. Prominent in dendrite shafts, MTs are assumed to function exclusively as stable railways for intracellular transport. However, MTs exhibit bouts of rapid polymerization and depolymerization, termed dynamic instability. Surprisingly, we discovered that MTs remain dynamic throughout neuronal development and are capable of rapidly extending into and out of dendritic filopodia and spines of cultured cortical and hippocampal neurons. Using total internal reflection fluorescence microscopy (TIRFM), we show that MT invasion of dendritic spines can be associated with rapid morphological changes of the spine head. These findings suggest that dynamic MTs may be playing an important role in spine structure and function. Indeed, many of the components that are either transported on MTs (lipids, proteins, RNA, organelles) or are associated with their growing tips would be capable of directly entering spines via MTs themselves. In this proposal we will test the hypothesis that dynamic MT entry into dendritic spines occurs in a regulated fashion and is required for spine development and plasticity. Specifically, we will: 1) Characterize the role of MT dynamics in spine morphology and maturation, 2) Determine how MTs affect synaptic activity and spine plasticity, and 3) Investigate MT-based targeting of synaptic components to dendritic spines. This work will provide fundamental insights into synaptogenesis and synaptic plasticity. Furthermore, because dendritic spines are the sites that are affected in numerous psychiatric and neurological diseases these studies hold promise for novel cytoskeletal-based therapies for synaptic dysfunction. PUBLIC HEALTH RELEVANCE: There are many developmental and adult onset neurological diseases, including mental retardation, autism, epilepsy, and Alzheimer's disease, that affect neuronal shape and therefore communication between neurons in the brain. This research will identify and characterize a novel intracellular mechanism that regulates directed movement of components to specific regions of the neuron and controls neuronal shape, thereby providing potential targets for therapies directed at ameliorating these diseases.

DESCRIPTION (provided by applicant): Both membrane protrusion and invagination are fundamental cellular processes and are therefore tightly regulated. Importantly, these apparently antagonistic processes control the size and molecular composition of the plasma membrane, are essential for cellular migration and require actin polymerization. However, there is little dat on how membrane protrusion and invagination are integrated in cellular function, especially in the nervous system. F-BAR proteins are a superfamily of proteins involved in membrane curvature sensing and deformation through their F-BAR domain, positioning them as potentially important players in both of these processes. Structurally, they form a curved dimer that self-multimerizes around endocytic vesicles, causing their elongation into tubules. The CIP4 family of proteins (TOCA1, FBP17 and CIP4) is one family of F-BAR proteins that also bind actin-associated proteins. Like other F-BAR proteins, the CIP4 family is thought to function primarily in membrane invagination and endocytosis, but our recent work has implicated CIP4 in neuronal membrane protrusion as well. We have recently discovered that CIP4 transfection induces actin-based ribs and veils around the periphery of cortical neurons. These ribs and veils are similar to filopodia and lamellipodia, respectively, and result in an scalloped lamellipodia, fille with thin actin bundles connected by actin-rich veils of membrane. In primary cortical neurons CIP4 family proteins are specifically associated with the protruding edges of ribs and veils, positioning them at the nexus of membrane deformation and actin polymerization. In this proposal we will test the following novel hypotheses: 1) F-BAR proteins of the CIP4 family act in a context- specific manner in neurons and non-neuronal cells by interacting with a distinct subset of proteins and 2) CIP4 functions in neurons by inhibiting axon/dendrite outgrowth during migration. This work will provide fundamental insights into how proteins may serve context-specific functions in different cell types and how neurons coordinate membrane protrusion and invagination during migration and axon formation. CIP4 has been implicated in Huntington's disease and several forms of cancer, underscoring the importance of understanding how this family of proteins may function in a context-specific fashion in different cell types.

PROJECT SUMMARY/ABSTRACT A functional nervous system requires both the appropriate development of dendritic spines and their functional plasticity throughout life. Because dendritic spines are the primary sites of contact with presynaptic axons in excitatory neurons of hippocampus and cortex, their structure and function have been studied in great detail. During development, spines undergo marked changes in structure, progressing from motile filopodial protrusions to stable mushroom-shaped spines. Activity-driven structural changes in spines of mature neurons also play important roles in learning and memory. It is therefore not surprising that changes in dendritic spines are one of the first harbingers of neuronal dysfunction in many developmental diseases, such as Fragile X syndrome and autism, as well as neurodegenerative diseases, such as Alzheimer's disease. Actin filaments play important roles in the formation, maintenance and plasticity of dendritic spine structure. Prominent in dendrite shafts, microtubules (MTs) function as stable railways for intracellular transport, but also exhibit bouts of rapid polymerization and depolymerization, termed dynamic instability. We discovered that MTs remain dynamic in dendrites throughout neuronal development and are capable of rapidly polymerizing into and out of dendritic spines in an activity-dependent fashion. In this proposal we will test the hypothesis that MT invasion of dendritic spines is a tightly regulated process resulting in motor-driven transport of cargo directly into and out of dendritic spines. Specifically, we will: 1) Determine the molecular mechanism by which MTs target specific spines, 2) Identify motor proteins and cargo that are transported into spines along MTs, and 3) Determine how material is transported out of spines along MTs. This work will provide fundamental insights into synaptogenesis and synaptic plasticity. Furthermore, because dendritic spines play essential roles in learning and memory and are the structures affected in numerous psychiatric and neurological diseases, these studies hold promise for novel cytoskeletal-based therapies for synaptic dysfunction.

Breaking neuronal symmetry is a fundamental process in the formation of a polarized neuron. Neurons in the developing cerebral cortex are born as spherical cells that must extend leading and trailing processes to migrate to their destination in the developing cortical plate. Cortical neurons then extend long axons and dendrites from these processes to create functional circuits. Cortical neuron migration and process extension is critically dependent on the microtubule and actin cytoskeleton, but relatively little is known about how the actin cytoskeleton and plasma membrane are coordinated during these events. Membrane protrusion and invagination are fundamental cellular activities that require coordination of the plasma membrane and underlying actin cytoskeleton. However, there is a dearth of data on how membrane protrusion and invagination are integrated in process outgrowth and neuronal migration. The F-BAR superfamily of proteins are involved in membrane curvature sensing and deformation through their F-BAR domain, positioning them as potentially important players in both membrane invagination and protrusion. Structurally, they form a curved dimer that self-multimerizes around endocytic vesicles, causing their elongation into tubules. The CIP4 family of proteins (CIP4, FBP17 and TOCA1) is a group of F-BAR proteins that bind actin-associated proteins. Like other F-BAR proteins, the CIP4 family is thought to function primarily in membrane invagination and endocytosis, but our recent work has implicated CIP4 in neuronal membrane protrusion as well. Lamellipodial-like protrusions induced by CIP4 strongly inhibit neurite outgrowth in culture. Conversely, we find that a close family member, FBP17, forms endocytic tubules in developing cortical neurons and promotes prominent filopodia formation, resulting in precocious neurite outgrowth. In this proposal we will test the novel hypothesis that protrusion through CIP4 and invagination through FBP17 act in opposing manners to regulate cortical neuron migration and process formation in the developing cortex. Specifically, we will: 1) Determine how CIP4 induces membrane protrusions and FBP17 forms endocytic tubules, 2) Establish how membrane tubulation results in precocious filopodia formation and neurite outgrowth, 3) Reveal the spatial and temporal expression pattern of endogenously-labeled CIP4 and FBP17 in mouse lines and 4) Resolve CIP4 and FBP17 function in cortical development in vivo. This work will provide fundamental insights into how proteins that bridge the membrane and actin cytoskeleton function to regulate process outgrowth and cortical neuron migration in the early developing mammalian brain. CIP4 and FBP17 have been implicated in Huntington's disease and several forms of cancer, underscoring the importance of determining the function of these proteins in the developing cerebral cortex.