Vivian Budnik - US grants

The development and plasticity of the neuromuscular junction in Drosophila will be studied using mutations that disrupt several aspects of neuronal function. The proposed research will test the hypothesis that neural activity, presumably by alterations in the level of second messengers, is a major factor involved in regulating synaptogenesis. Activity levels will be modified to different extents by mutations affecting ion channels and neurotransmitter release, whereas CAMP levels will be altered by using mutations that affect CAMP metabolic enzymes. We will use immunocytochemical staining of motor axon terminals to determine if changes in the levels of activity of motorneurons or changes in CAMP levels during development generate morphological changes. Ultrastructural reconstructions and macropatch recordings of single synaptic boutons will allow us to assess the structural and functional nature of these changes. We will also determine the sequence of events during larval development that lead to the final pattern of axon terminals, and if there are critical stages of development during which this morphology is amenable to change. We have already found that hyperactivity throughout development leads to morphological and physiological abnormalities at the Drosophila larval neuromuscular junction. The combination of conventional electrophysiological and anatomical techniques, and the powerful genetic and molecular tools developed in Drosophila, should allow us to gain much insight into the mechanisms underlying synaptic plasticity. Furthermore, this study may be valuable for the understanding of neuropathologies that result from abnormal states of neuronal activity.

9451791 Budnik A new undergraduate laboratory course in neurosciences will expose students to the fields of neuroanatomy and neural development, and will emphasize recent advances in these two disciplines that have resulted from genetic approaches. Students will be introduced to state-of-the-art technologies that currently play such an important role in neuroscience research. The main idea underlying this course is to develop these techniques around interesting scientific questions that will teach the students about the fundamental organization of the CNS, and about the process of doing science. This course will become one of the focal points for an undergraduate neuroscience and behavior "concentration" within the university. In recent years, the philosophy of life science education at the University of Massachusetts is shifting from a "textbook" science education for undergraduates to a more hands-on approach. The proposed laboratory course will be part of a more extensive research experience for undergraduates. Our long range goal is to create an integrated training curriculum that would attract undergraduates to careers in biology in general and to neuroscience in particular. The requested equipment will serve the formal laboratory in one semester, will serve other courses in the neuroscience concentration, and will be the center of an honors laboratory and Research Experience for Undergraduates during the summer and intersession.

In this application, the investigator proposes to screen for new mutations that affect NMJ development in Drosophila. Once new mutations are isolated, the investigator will characterize their phenotypic defects, then characterize the corresponding genes using standard methods available for Drosophila. The investigator will also target expression of an already characterized gene, dlg. Mutations of dlg cause an alteration of a postsynaptic specialization called the subsynaptic reticulum. The research plan of this RCDA application reflects work that will be carried out as part of two grant proposals: a project in a Program Project Grant and an R01 application, the latter is also being reviewed by this study section. In this proposal, the investigator continues work she initiated in her postdoctoral training and first several years as Assistant Professor. That is to use the Drosophila neuromuscular junction as a preparation to examine fundamental features of synaptogenesis via mutations which affect normal development. The preparation appears to be a nice one based on some of the mutations which have already been identified (bpd, wwj, wwf, and paw). Unfortunately, the bulk of the proposed analyses do not deal with any of these mutations, but rather will deal with some new mutations which have not yet been identified. The investigator argues that she would like to gain additional experience in genetic and molecular genetic analysis, presently not possible due to a heavy teaching and administrative load. The main strengths is that synaptogenesis is an excellent problem to approach genetically. The basic biology of the neuromuscular junction preparation is excellent and will get better with analyses proposed elsewhere. The preliminary results are excellent. The main weakness is that the proposal does not build at all on the presented preliminary results on already identified mutations.

DESCRIPTION (provided by applicant): Our long-term goal is to understand how the molecular constituents of synapses are organized during development, and to determine the mechanisms that regulate them during plasticity. During more than a decade, our studies have centered on the role of Discs-Large, a scaffolding protein of the PSD95 family. These studies have been instrumental to understanding the dynamics of synapse growth, how scaffolding proteins are regulated, and what additional proteins are required for synaptic protein assembly. More recently, our studies have centered on the regulation of the synaptic cytoskeleton during synapse growth. We have found that a conserved cassette, consisting of the scaffolding proteins Bazooka/Par-3, Par6, and aPKC, plays a primary role in cytoskeletal rearrangements required for new synapse formation. In epithelial cells this complex is necessary to establish cell polarity, and alterations in these proteins result in loss of cell polarity and cancer. In the present proposal we will center on the molecular mechanism by which microtubule dynamics are regulated during synapse growth. In aim 1 we use a genetic analysis in fixed and living preparations to test the hypothesis that aPKC regulates pre- and postsynaptic microtubule dynamics, and that this is required for new synapse formation. In Aim 2 we will investigate the role of Putsch, a microtubule associated protein regulated by aPKC. Finally, in aim 3 we will investigate the role of Baz in synaptic development. We expect that the proposed studies will substantially contribute to an understanding of the role of the cytoskeleton during new synapse formation. Because many of these proteins are highly conserved across diverse phylogenies, our studies in Drosophila will also be relevant to vertebrates and mammals, as we have demonstrated in our previous work. An understanding of the factors involved in building a functional synapse will be essential to decipher the mechanisms underlying a number of neural disorders, as well as to design strategies to repair damage after stroke, trauma, or disease.

DESCRIPTION (Investigator's Abstract): The long term goal is to understand the mechanisms by which synaptic components are assembled into a fully functional synapse. In this project it is proposed to carry out molecular, genetic, and physiological analyses of a group of proteins, MAGUKs, which are involved in the clustering and targeting of channels and receptors to their synaptic locations. This family of proteins is highly conserved between mammals and flies, from both structural and functional perspectives. The investigators propose to perform a deletion analysis of a fly MAGUK member (DLG) to determine the exact domains with which it targets ShakerK+ channels to glutamatergic neuromuscular synapses. Analysis of the DLG mutant proteins generated in this project will be performed in an in vivo model system, the fly neuromuscular junction, using targeted expression of these mutant proteins to postsynaptic muscle cells. They will also use the yeast two-hybrid system, coimmunoprecipitation, and genetic experiments to identify the proteins that directly interact with this DLG targeting site. This investigation will allow them to ascertain how ion channels are anchored to synapses, and to uncover additional functions of the proteins identified. Studies in mammals have identified a novel protein, GKAP, which interacts with the guanylate kinase domain of the vertebrate MAGUK, PSD-95. However, the function of this protein is unknown. The investigators will clone the Drosophila GKAP homolog to initiate a genetic analysis of its synaptic function. Finally, they propose to use anatomical and electrophysiological techniques to determine the ontogeny of Shaker K+ channel clustering, whether glutamate receptor clustering is also dependent on DLG, and if the clustering of Shaker channels is dependent on motor neuron innervation. These studies will contribute in a fundamental way to our knowledge of the mechanisms of synapse formation. This knowledge will be essential to understand the etiology of many neural disorders, as well as to design strategies to repair damage after stroke, trauma, or disease.

DESCRIPTION (provided by applicant): The mechanisms which organize synaptic proteins at neurotransmitter release sites illustrate how a panoply of interacting molecules can orchestrate cell-cell communication. The knowledge of how synapses are assembled and how these molecular components are regulated is central to understanding synapse function and synapse plasticity. In recent years we have contributed to the understanding of synapse assembly and synapse plasticity by the genetic analysis of Discs-Large (DLG), a tumor suppressor and PDZ-containing protein that is essential for proper synapse assembly and function. We demonstrated that DLG is crucial for the in vivo clustering of two synaptic proteins- the Shaker K+ channel and the cell adhesion molecule Fasciclin II (Fasli). Moreover, we demonstrated that the clustering function of DLG can be dynamically regulated by neuronal activity, through Ca++/calmodulin- protein kinase II (CaMKII)-dependent phosphorylation of DLG. Although these studies have established a foundation for understanding the mechanisms of synapse development many elements responsible for this process remain to be discovered. We have now obtained evidence for two additional proteins, GUK-holder (GUKH) and SCRIBBLE (SCRIB), that are likely to function in the formation of a cytoskeletal scaffold that organizes protein complexes at the synapse. The present proposal employs Drosophila genetics to probe the mechanisms of action of these proteins at the synapse. Three sets of experiments are proposed. The first uses a genetic and molecular approach to understand the mechanism for the interactions between DLG, GUKH, and SCRIB as well as for how these interactions might be regulated. The second focuses on the functional analysis of GUKH and SCRIB, by analyzing mutations in the genes. Finally, the third approach is designed to identify synaptic SCRIB and GUKH binding partners that are likely to interface with the synaptic cytoskeleton. We expect that the proposed studies will substantially contribute to what is rapidly becoming a complex but coherent picture of how synapses are formed and are modified. Because many of these proteins are highly conserved across diverse phylogenies, our studies in Drosophila will also be relevant to vertebrates and mammals. An understanding of the factors involved in organizing a synapse will be essential to decipher the mechanisms underlying a number of neuropathologies, as well as to design strategies to repair nervous system damage after stroke, trauma, or disease.

DESCRIPTION (provided by applicant): The research proposed in this application will be primarily performed in Santiago, Chile at the Universidad de Chile in collaboration with Dr. Jimena Sierralta as an extension of NIH grant RO1 NS42629.The long-term goal of the PI's research is to understand the molecular mechanisms by which synapses are assembled using a genetic approach in the glutamatergic neuromuscular junction of Drosophila. An important finding emerging from this investigation is that a PDZ-containing scaffolding protein of the PSD-95 family, DLG, is essentially required to properly localize a number of synaptic proteins. The central aim of the parent grant is to characterize the role of two DLG interacting proteins, Scribble and GUK-holder, in the process of synapse assembly. Previous studies of the dig locus identified the presence of a single transcript, dig-A, which is present both in synapses and epithelial cells. However, recent studies by Dr. Sierralta demonstrate extensive alternative processing of transcripts originated from the dig locus. Very interestingly, a group of splice variants are excluded from epithelial tissue and are specifically expressed in the nervous system. Moreover, a subset of isoforms contains an N-terminal extension similar to mammalian SAP97, which regulates SAP97 localization as well as intramolecular interactions that control the binding between SAP97 and its partners. The main goal of the proposed collaborative research is to investigate the role of these novel splice variants in different aspects of synapse formation and maturation. The work involves the study of the expression of DLG isoforms containing the $97N domain during the development of synapses, the selective disruption of dig gene products containing the $97N domain during synapse development, and the study of the role of this domain in Dig binding to its partners. We envision that the novel dimension provided by the observations in the foreign lab will dramatically enhance our understanding of DLG during synapse formation, and therefore will increase the knowledge obtained during the execution of the parent grant. [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate signaling mechanisms underlying synapse development and plasticity. Our studies in the previous funding cycle demonstrate that Wnts, pivotal and phylogenetically conserved synaptic organizers, are released via exosomes, extracellular vesicles containing proteins and RNAs, at synaptic boutons of the Drosophila larval neuromuscular junction (NMJ). Our studies also provide evidence that a retrograde signal mediated by Synaptotagmin 4 conveys Wnt signals, and is also controlled by exosome release from presynaptic boutons. These studies place trans-synaptic exosome communication as a key coordinator of pre- and postsynaptic modifications. Cell-cell signaling through exosomes is just beginning to be documented during immunity, the spread of cancer, and intercellular prion transmission in the CNS. In addition, exosomes are emerging as promising vectors for the delivery of targeted therapies. However, most studies of exosomes have been carried out in cell culture, and exosome function in the nervous system is virtually unknown. Our demonstration that exosomes mediate trans-synaptic signaling in vivo, establish Drosophila as a powerful model system to efficiently unravel mechanisms of exosome release and trans-synaptic transfer. Wnt misregulation is associated with a number of cognitive disorders, such as Schizophrenia and Alzheimer's disease. Thus, understanding the mechanisms of Wnt signaling in the nervous system has important implications for the design of clinical strategies to treat these conditions. In this project our experimental strategies will mak extensive use of genetics and state-of the art cellular approaches in vivo, to elucidate the mechanisms of exosome release at synaptic sites and the principles underlying the exosome regulation of synapse development and plasticity. We will (1) identify the molecular machinery mediating exosome release by synaptic boutons, (2) determine the synapse specificity or global nature of retrograde signaling through exosomes, and (3) characterize the role of a Wnt protein in Synaptotagmin 4-mediated retrograde signaling. The outcomes of this project will constitute a significant advancement in our understanding of Wnt signaling at synapses, and promises to accelerate the development of exosomes for targeted therapies.

DESCRIPTION (provided by applicant): The research proposed in this application will be primarily performed in Santiago, Chile at the Universidad de Chile in collaboration with Dr. Jimena Sierralta as an extension of NIH grant RO1 NS42629.The long-term goal of the PI's research is to understand the molecular mechanisms by which synapses are assembled using a genetic approach in the glutamatergic neuromuscular junction of Drosophila. An important finding emerging from this investigation is that a PDZ-containing scaffolding protein of the PSD-95 family, DLG, is essentially required to properly localize a number of synaptic proteins. The central aim of the parent grant is to characterize the role of two DLG interacting proteins, Scribble and GUK-holder, in the process of synapse assembly. Previous studies of the dig locus identified the presence of a single transcript, dig-A, which is present both in synapses and epithelial cells. However, recent studies by Dr. Sierralta demonstrate extensive alternative processing of transcripts originated from the dig locus. Very interestingly, a group of splice variants are excluded from epithelial tissue and are specifically expressed in the nervous system. Moreover, a subset of isoforms contains an N-terminal extension similar to mammalian SAP97, which regulates SAP97 localization as well as intramolecular interactions that control the binding between SAP97 and its partners. The main goal of the proposed collaborative research is to investigate the role of these novel splice variants in different aspects of synapse formation and maturation. The work involves the study of the expression of DLG isoforms containing the $97N domain during the development of synapses, the selective disruption of dig gene products containing the $97N domain during synapse development, and the study of the role of this domain in Dig binding to its partners. We envision that the novel dimension provided by the observations in the foreign lab will dramatically enhance our understanding of DLG during synapse formation, and therefore will increase the knowledge obtained during the execution of the parent grant. [unreadable]

DESCRIPTION (provided by applicant): Glial cells are major constituents of the nervous system, and an array of devastating diseases, such as childhood periventricular leukomalacia, Alexanders Disease, and demyelinating diseases such as multiple sclerosis, arise from glial malfunction. Although glial cells have been recently recognized as playing key roles in neuronal function, sculpting synaptic connections, and providing essential trophic factors to neurons, there are major gaps in our understanding of the molecular mechanisms mediating their diverse actions. This proposal employs a highly tractable system, the glutamatergic Drosophila neuromuscular junction (NMJ), to investigate major questions in glial cell biology with exquisite cellular detail. Our preliminary data provides compelling evidence for a role of glial cells in the plasticity of NMJs, regulating an important pathway required for the differentiation of synapses, and sculpting synaptic connections by a process of pruning. In this project we will (Aim 1) test the hypothesis that glial cells play a primary role in the extension and retraction of synaptic boutons during NMJ expansion, (Aim 2) test the hypothesis that glial cells regulate a Wnt pathway at the NMJ and that this regulation is essential for NMJ development, and (Aim 3) determine the role of the Draper signaling pathway in sculpting synaptic connections at the NMJ. We expect that these studies will provide fundamental insights into the cellular interactions between synapses and glia in live animals and unravel molecular mechanisms by which glia actively sculpt synaptic connections. Project Narrative Glial cells, the major cell type in the human brain, have emerged as important regulators of brain development and physiology, and a number of devastating neurological diseases, such as multiple sclerosis or glioma, are associated with glial dysfunction. This proposal will explore how glia (in live animals) modulate the formation and modification of synapses, the basic functional units through which neurons communicate with other cell types. Our work will provide fundamental knowledge regarding how neurons and glia communicate during the modification of synapses, and is expected to provide important insights into how glial dysfunction might cause disease.

DESCRIPTION (provided by applicant): The goal of this project is to elucidate the genetic programs that are regulated during synaptic development and activity-dependent plasticity. Recent advances in the study of nuclear functions reveal an unprecedented dynamics in the organization of nuclear subdomains which coordinate gene expression. However, the physiological pathways that regulate these dynamics are largely unknown. We have identified a critical transduction cascade, involving a member of the Wnt family and its receptor, in the communication between the synapse and the nucleus during activity-dependent synaptic growth. Essential roles of Wnts in synapse development and plasticity have also been uncovered in the mammalian brain, and a number of cognitive disorders, such as schizophrenia, bipolar disorder, and Alzheimer's disease show alterations in Wnt signaling. Thus, understanding how Wnts function in the brain is a highly significant area with important clinical implications. Our studies demonstrate that Wnt signaling at synapses activates a novel signaling pathway, the Frizzled Nuclear Import (FNI) pathway, in which a fragment of the Wg receptor, DFrizzled2 (DFz2), is imported into the nucleus. Within the nucleus, this DFz2 fragment, together with the A-type lamin, Lamin-C, establishes a specialized subdomain, which regulates gene expression by controlling mRNA biogenesis. Importantly, alterations in A-type lamins have been involved in a group of hereditary disorders, the laminopathies, which have devastating impact on the function of the neuromuscular system. In this project we propose to investigate the function of this nuclear subdomain in activity-dependent synaptic plasticity. In particular, we propose to (1) determine the role of the nuclear subdomain in controlling nuclear mRNA polyadenylation and to identify the genes that are regulated by this pathway, (2) determine the dynamics of the nuclear subdomain during motorneuron stimulation, and (3) begin the characterization of an important gene regulated by this transduction cascade. We predict that these studies will be highly significant for our understanding of how synaptic events are communicated to the nucleus to regulate gene expression. Ultimately, we expect that the proposed studies will be highly relevant to our understanding of cognitive disorders associated with the malfunction of Wnt signaling and to identify the cellular events that are altered in laminopathies. PUBLIC HEALTH RELEVANCE: A fundamental property of synaptic connections in the nervous system is their ability to change in response to experiences, a process that is referred to as synaptic plasticity. Studies in many systems show that a mechanism underlying this event relies on the regulation of genes within the nucleus. Thus, significant research efforts have been dedicated to understanding how synaptic connections communicate with the nucleus. In our research, we have uncovered a novel signaling pathway that, at least in part, mediates this communication. This pathway involves a member of a protein family, the Wnts and its receptor, which appears fundamental for the development of synaptic connections. These finding are particularly important, given that in humans alterations in Wnt signaling are associated with cognitive disorders, such as schizophrenia, bipolar disorder, and Alzheimer's disease. Thus, understanding this pathway may provide important insight into the causes of these conditions. We have also found that the Wnt signaling pathway collaborates with a nuclear protein, an A-type lamin, in the regulation of gene expression. Interestingly, alterations in A-type lamins lead to devastating diseases of the neuromuscular system. In this project we will identify the genes that are regulated by Wnts and A-type lamins, examine the properties of a nuclear region where this regulation is accomplished, and begin to characterize the function of the regulated genes in synapse development and plasticity. These studies, which will be conducted in a powerful model system amenable to sophisticated genetic techniques, the fruit fly Drosophila, is expected to provide significant advances to our understanding of the processes by which experiences modify the synaptic connections in the brain. In addition, by elucidating the mechanisms underlying these events, we hope to contribute to the development of clinical strategies to ameliorate or cure laminopathies and cognitive disorders associated with malfunction of Wnt signaling.

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize a novel pathway for the nuclear packaging and export of ultra-large ribonucleoprotein granules (megaRNPs) and its impact on synapse assembly. A long-standing tenet of cellular biology has been that all nucleo-cytoplasmic traffic takes place through the nuclear pore complex (NPC). Using the Drosophila neuromuscular junction (NMJ) as a model system, however, we recently uncovered an alternative pathway for the nuclear export of megaRNPs. This NPC- independent pathway has the potential to fundamentally change our understanding of nucleo-cytoplasmic communication. In this pathway, transcripts are packaged in megaRNPs within the nucleus and are exported to the cytoplasm by budding through nuclear envelope membranes in a process akin to the nuclear egress of Herpes-type viruses. The proposed project represents an incisive approach to understand the impact of this pathway on cellular biology, with a focus on synapse formation. We will investigate the protein and mRNA composition of megaRNPs, and determine if megaRNPs are assemblages of multiple or single mRNAs species. We will use molecular genetic tools in Drosophila to determine the significance of megaRNP composition in transcript localization and proper synapse assembly. We will also determine the relationship between nuclear envelope budding and NPC-dependent modes of mRNA export. Finally, we will begin an analysis of the nuclear envelope budding pathway in mammalian cells. These studies are likely to be paradigm-shifting for our understanding of how and where large RNP transport granules are formed, which is elemental to the understanding of how the precise polarized assembly of cellular macromolecular complexes is achieved. In addition, these studies will elucidate novel biological pathways that will almost certainly lead to new clinical strategies for the treatment of laminophathies such as Emery-Dreifuss muscular dystrophy, movement disorders such as dystonia, and Herpes virus-type infections.