Avital A. Rodal, Ph.D. - US grants

Structural plasticity contributes to long-lasting alterations in neuronal function during development, as well as in learning and memory, and occurs in response to environmental and activity-dependent signaling cascades. The receptors that transduce these synaptic growth signals are regulated by endocytic recycling, but we do not understand what internal compartments they signal from, what special properties of those compartments enable signaling to occur, and ultimately how the membrane traffic machinery itself can be regulated to control synaptic growth. The intersection of signaling and membrane traffic is particularly intriguing in the presynaptic compartment of neurons, because synapses are highly specialized for both exocytic and endocytic traffic of synaptic vesicles in response to activity. Signaling receptor internalization and the synaptic vesicle cycle use a highly overlapping set of trafficking machinery, but little is understood about cross-talk between these processes and how activity-dependent modes of regulation of trafficking machinery might be used to control signal transduction. This proposal uses a combination of biochemical, genetic, and cell biological approaches in the Drosophila larval neuromuscular junction (NMJ) to unravel the molecular mechanisms by which conserved membraneremodeling proteins respond to extrinsic and intrinsic cues to modify signal transduction, leading to changes in synaptic architecture. The aims of this proposal are (1) to characterize the biochemical activities and interactions of lipid-deforming proteins that control receptor traffic at early endosomes and to evaluate how these proteins work together in vivo;and (2) to obtain 3-dimenslonal high-resolution ultrastructures of presynaptic endosomes at the NMJ, and to determine their relationship to other cellular structures in wildtype and mutant animals. These receptor trafficking events are implicated in neuronal diseases ranging from mental retardation to neurodegenerative disease and addiction, underlining the health importance of understanding how signal transduction is modulated by intracellular membrane traffic in neurons.

DESCRIPTION (Provided by the applicant) Abstract: Neurons grow elaborate structures tailored to send and receive electrical signals over large distances and through complex networks of connections. Neuronal morphology in these networks changes dynamically in response to both neuronal firing and external growth cues, during development as well as learning and memory, and recedes in the absence of positive growth cues. These growth cues are transduced by receptors that exhibit subcellular location-dependent signaling properties as they are internalized from the plasma membrane and trafficked through endosomal compartments, and therefore regulation of membrane traffic can profoundly alter growth signaling. The intersection of signaling and membrane traffic is particularly intriguing in the presynaptic compartment of neurons, because synapses are highly specialized for both exocytic and endocytic traffic of synaptic vesicles in response to activity. Signaling receptor internalization and the synaptic vesicle cycle use a highly overlapping set of trafficking machinery, but little is understood about cross-talk between these processes and how activity-dependent modes of regulation of trafficking machinery might be used to control signal transduction. These membrane trafficking events are deeply implicated in neurodegenerative diseases, and understanding how they are regulated by neuron-specific mechanisms may lead to new routes for intervention. We are using the Drosophila larval neuromuscular junction (NMJ) to unravel the molecular mechanisms by which activity-dependent changes in receptor traffic lead to changes in synaptic growth signaling and synaptic architecture. This proposal combines two approaches to determining how receptor traffic in this system is regulated by activity: (1) live imaging of signaling receptor traffic and in response to chronic and acute changes in activity, and (2) proteomic analyses of activity-dependent changes in the membrane traffic machinery. We will then combine our findings about molecular mechanisms of activity-dependent regulation of endocytic traffic with in vivo tests of traffic of synaptic growth receptor, leading to models for how activity works through the membrane traffic machinery to tune signaling up or down for structural remodeling of neurons, and for how mis-regulation of these cellular trafficking events might contribute to neurodegenerative disease. Public Health Relevance: Neurons undergo dynamic structural changes in response to external growth signals during development as well as learning and memory. Growth signals are trafficked into the cell via membrane-bound compartments, and defects in these events are a hallmark of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease. We propose to investigate how neurons control the formation of membrane compartments and load them with specific signaling cargoes, and identify ways to manipulate these events to treat neurological disease.

This Major Research Instrumentation (MRI) award supports the acquisition by Brandeis University of an integrated live fluorescence imaging system that greatly increases the speed and resolution at which dynamic events can be imaged in cells and in solution. The system will be equipped with both spinning disk confocal and epifluorescence modes, and will be tailored for photoactivation microscopy, an elegant approach to label small populations of fluorescent molecules acutely in space and/or time. The instrument will use a newly developed setup in which the spinning disk confocal modality is equipped with two cameras, enabling maximum speed for simultaneous detection of multiple fluorophores. The epifluorescence modality will push the limits of fast fluorescence imaging by taking advantage of the most recent developments in LED-based illumination and rapid sCMOS camera acquisition. These new technologies will permit the study of extremely rapid biological events (with durations of a few seconds and velocities of micrometers per second) that until now have been extremely difficult to measure directly. Thus, the proposed instrument will allow a new level of spatial (by labeling a set of molecules starting at a particular location) and temporal (by labeling a sparse population of molecules at any given time) investigation into research problems ranging from cytoskeletal dynamics, membrane traffic and signal transduction to sensory processing and synapse formation.

Cellular processes essential for life are driven by complex molecular machines that move through the cell, interact with each other, and execute their functions in seconds. Recent technological breakthroughs in microscopy have greatly improved the speed and resolution at which we can directly see these dynamic molecular events. Researchers at Brandeis across the Biology, Biochemistry, and Physics departments and at the University of Massachusetts, Boston will use the new imaging system to tackle a broad range of scientific problems, ranging from how a neuron forms synapses to how simple biological molecules self-assemble into complex force-generating machines. This instrument will train undergraduates, graduate students and postdoctoral fellows, fostering interdisciplinary collaborations across science departments at Brandeis and in the Boston science community. As part of this training, we will take advantage of the instrument to establish a new project laboratory in live-cell imaging at Brandeis for our undergraduate and Masters students who may not be members of a research laboratory and therefore would not otherwise have access to an advanced instrument. This instrument will integrate education, training and research to provide new fundamental insights into the dynamics and interactions of molecules in cells and in the test tube.

The goal of this project is to understand the molecular mechanisms of extracellular vesicle (EV) trafficking in the nervous system in vivo. EVs are small vesicles secreted from donor cells that can carry a diverse array of cargoes, and have recently come to the forefront as a novel mode of intercellular traffic and communication in the brain. EVs are thought to contribute to many human health conditions, including the spread of pathological proteins in neurodegenerative disease. However, because most studies of EVs are conducted with cells in culture, little is known about the release, uptake and fate of EVs in the diverse interacting cell types of the intact nervous system. We have developed a system in which to study traffic of endogenous neuronal EV cargoes in a living animal, using cutting edge genetic and cell biological tools available in Drosophila. Using this system, we discovered an unexpected role for synaptic periactive zone (PAZ) membrane remodeling machinery in EV cargo sorting, stability and release from axon terminals at the Drosophila larval neuromuscular junction. We also found that endogenous EV cargo release is dynamically regulated by neuronal activity. Finally, we made the surprising discovery that released EVs are partially protected from the target cell cytoplasm, suggesting the possibility that additional regulatory steps may govern their exposure and/or degradation upon internalization to recipient cells. Our proposed research will elucidate how cellular membrane traffic machinery controls the release, uptake, and fate of EV cargoes. To achieve these goals we will use advanced Drosophila genetics, live cell imaging techniques, structured illumination microscopy, electron microscopy, and tissue-specific detection and manipulation of EV cargoes in donor and recipient cells. Specifically, we propose: 1) To elucidate in vivo mechanisms of EV traffic and release by PAZ proteins. 2) to determine how activity regulates neuronal traffic of endogenous EV cargoes and 3) to determine the fate of NMJ EV cargoes, at rest and in response to neuronal activity. Given the conserved nature of synaptic membrane trafficking machinery, our findings and tools will lay the foundation for new insights into endogenous EV traffic in many aspects of nervous system function, including in human neurological disease.

PROJECT SUMMARY The goal of this proposal is to understand the spatial and functional organization of the presynaptic periactive zone (PAZ), which is found adjacent to sites of synaptic vesicle release. The PAZ is a micron-scale structure, occupied by dozens of proteins that work together in multivalent assemblies to couple membrane remodeling to force-generating actin polymerization. Studies of PAZ proteins in many systems have suggested that these proteins act at multiple steps of the synaptic vesicle cycle as well as in other synaptic membrane functions (e.g. synaptic morphogenesis and receptor traffic). It remains unknown how the micron-scale organization and regulation of PAZ proteins direct their membrane and cytoskeleton remodeling activities to these different neuron-specific functions. We will use the Drosophila larval neuromuscular junction (NMJ), a powerful model synapse, to decipher how PAZ protein assemblies, activities, and cellular functions are linked. Using high- resolution imaging, we recently found that PAZ proteins occupy both overlapping and distinct domains within the PAZ, and that proper segregation of PAZ proteins between these domains depends on their multivalent interactions with each other. We have also recently described PAZ-dependent dynamic actin filament structures, which represent a direct readout of PAZ protein activities in these different domains. Using these tools, we will ask how synapses regulate PAZ protein activities and interactions in space and time, and how PAZ organization underlies its diverse neuron-specific functions, in response to synaptic activity and transmission. In Aim 1, we will determine how PAZ proteins are organized at resting and active synapses using complementary fixed, live, and super-resolution imaging methods, and develop new quantitative methods to describe their geometric relationships in PAZ domains. In Aim 2, we will test the hypothesis that synaptic actin patches represent clathrin-dependent synaptic vesicle recycling events, and identify the determinants of synaptic actin patch assembly and dynamics. In Aim 3, we will ask how PAZ organization and synaptic activity control diverse cell biological PAZ functions, including release site clearance and organization of cell adhesion complexes. Overall, our experiments will explain how organization of the PAZ into distinct subdomains underlies multiple functional and structural properties of synapses. PAZ proteins are implicated in multiple neurological disorders, so deciphering their in vivo functions will be critical for understanding the etiology of these human diseases.