Graeme W. Davis - US grants

neurogenetics; arthropod genetics; neural plasticity; neuronal transport; synaptogenesis; neural transmission; genetic screening; gene mutation; Drosophilidae;

[unreadable] DESCRIPTION (provided by applicant): The advent of green-fluorescent protein (GFP) as a means to visualize proteins in living cells has begun a revolution in most fields of cellular biology. Three major drawbacks currently hamper experiments using GFP-protein tagging. First, proteins of interest, once tagged, must generally be over-expressed and this over-expression can generate novel phenotypes that disrupt the processes being studied. In addition, over-expression will disrupt protein localization. Finally, to tag a protein, a full-length cDNA must be available. We have developed a GFP-protein trap in Drosophila that is capable of generating full-length GFP-fusion proteins, at random, throughout the genome. The GFP-trap is nearly random, and independent of protein size. The expression of the resulting GFP-fusion proteins will be controlled by endogenous genomic regulatory sequences. As a result, the correct spatial and temporal expression patterns will be achieved, as well as the endogenous protein expression levels. We propose develop a database of GFP-fusion proteins encompassing more than 50% of the Drosophila genome that will dramatically facilitate experiments aimed at understanding protein dynamics in living cells, in vivo. We anticipate that this technology will enable a new generation of experiments. Protein localization, trafficking, turnover, concentration and translation will be able to be studied in an in vivo genetic system without the caveat of protein over-expression that often precludes reliable experimental interpretation. Ultimately, the database that we will develop will also enable the development of new assays for cell and developmental studies based on GFP-expression in subsets of cells, determined by the expression of single genes. Our database will also facilitate the identification of new genes that function at discrete times and places involved in processes such as cell fate and neuro-development. Finally, we anticipate that this database will enable future proteomic approaches in an in vivo model genetic organism.

Dr. Davis will combine live imaging techniques with Drosophila genetics to visualize synapse remodeling in vivo and analyze the underlying molecular mechanisms that control synapse remodeling in real time. Synaptic connections are the sites of communication between neurons. The precise modification of synapses is essential to the correct wiring of neuronal circuitry during development and to the modification of neural circuits during adult plasticity. Despite the identification of myriad synaptic proteins, the mechanisms that control synaptic bouton formation, stabilization and retraction are still poorly understood. Dr. Davis will characterize, with fine temporal resolution, the sequence of events by which new synapses are assembled, stabilized and disassembled. Combined with a genetic analysis, these studies aim to identify important molecular mechanisms underlying the events of synapse remodeling. In previously published studies, Dr. Davis has demonstrated that mutations in the cell adhesion molecule Fasciclin II and the microtubule associated protein Futsch impair synaptic growth. Dr. Davis will now examine how these and other molecules affect synapse growth in real time. Preliminary data from Dr. Davis demonstrate that new presynaptic varicosities can be added to the nerve terminal as rapidly as 2 to 5 minutes and can be eliminated during normal development. These data challenge previous hypotheses regarding synaptic growth at this synapse, the Drosophila NMJ, that suggest that synaptic boutons are added slowly over the course of hours and, once added, are not eliminated. Dr. Davis now hypothesizes that presynaptic varicosities can form very rapidly, but only a subset of the newly formed varicosities become stabilized to form functional boutons. Dr. Davis further hypothesizes that, once stabilized, synaptic boutons are rarely eliminated. Thus, an essential growth regulatory event may be the determination of whether a new presynaptic varicosity is stabilized. The live imaging experiments proposed in this study will address this possibility in detail. Dr. Davis will also investigate the molecular mechanisms that determine whether newly formed synaptic boutons are stabilized or retracted. Preliminary data implicate the dynactin protein complex as an important regulator of synapse stabilization. Inhibition the dynactin complex causes the elimination of synaptic terminals at the NMJ. Live imaging combined with genetic analysis will precisely define how synapse elimination occurs in the dynactin mutations. Finally, Dr. Davis will use well characterized mutations that alter nerve and muscle activity to determine the roles of pre- and postsynaptic activity in the process of synaptic growth and elimination using live, in vivo imaging.

DESCRIPTION (provided by applicant): Summary Homeostatic signaling systems are believed to interface with the mechanisms of learning-related plasticity to achieve stable, yet flexible, neural function and animal behavior. The loss or disruption of homeostatic signaling is believed to participate in the cause or progression of many neurological diseases including autism spectrum disorders and epilepsy. Ultimately, clear links between homeostatic signaling and disease will require a detailed molecular understanding of homeostatic signaling systems. Here we identify several novel homeostatic plasticity genes that dramatically extend our understanding of how homeostatic signaling systems stabilize neural function.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We propose experiments that have significance at two levels. First we will continue to develop and prove the effectiveness of our recently developed, transgenically encoded technique for protein photoinactivation in vivo. If we continue to be successful in this arena, it will substantiate this technique and encourage the use of this approach in other transgenic systems. This technique has the potential to be broadly applicable to diverse areas of basic research in cell biology, neuroscience, cancer and stem cell biology. Second, we propose to use our technique of protein photoinactivation to further dissect the molecular mechanisms that control synaptic vesicle endocytosis in vivo. Synaptic vesicle endocytosis is essential for the maintenance of appropriate neural function in the nervous system. It has been hypothesized that rapid, clathrin-independent endocytosis occurs at the neuronal synapse and that this form of endocytosis is necessary to maintain a releasable pool of synaptic vesicles. However, because clathrin mutations are early embryonic lethal it has not been possib]e to eliminate clathrin assembly at a mature neuronal synapse and directly test whether clathrin-independent endocytosis exists. Dominant interfering approaches have been attempted but in no case has it been possible to eliminate clathrin function at a mature synapse. We propose to photoinactivate clathrin heavy chain (Chc) and block clathrin assembly over a timecourse of seconds to minutes. If we observe clathrin-independent endocytosis following photoinactivation we will then be able to study this form of synaptic vesicle endocytosis in isolation, and we will do so in vivo. Regardless of the experimental outcome, we will also be able to use our new tools to pursue a new generation of experiments examining the molecular mechanisms of clathrin-dependent endocytosis in vivo. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): There has been remarkable progress in identifying the genetic basis for many types of inherited neurodegenerative disease. Despite this progress, genetic factors that contribute to the cause and progression of sporadic neurodegenerative disease remain generally unknown. Furthermore, in most instances, the genetic factors that influence the age of onset and rate of progression for many forms of inherited neurodegenerative disease remain to be identified. A better understanding of the genetic factors that are involved in neurodegenerative disease may not only help elucidate disease cause and progression, but could greatly expand the repertoire of molecular targets that are available for generating novel therapeutic interventions. We have performed an unbiased forward genetic screen for genes that cause neuromuscular degeneration when deleted, and for genes that slow the progression of neurodegeneration caused by neuronal stress or injury. In so doing, we have identified a novel, neuronally expressed, transcription factor that causes neuromuscular degeneration when deleted. Additional preliminary data demonstrate that this transcription factor regulates two downstream genes that are directly involved in neuromuscular stabilization versus degeneration: a secreted proteinase and a previously uncharacterized cell adhesion molecule. The proteinase is over-expressed and increased proteinase levels drive neurodegeneration. The cell adhesion molecule, when knocked down, causes neuromuscular degeneration. Thus, we have the potential to define the function of several new genes in the mechanisms of neuromuscular degeneration. We hypothesize that these molecules, taken together, represent a transcriptional program that determines whether motoneurons will remain stable or whether they will be destined to degenerate. As such, this system could be directly relevant to the factors that contribute to the cause and rate of progression of diverse neurodegenerative disorders.

The Molecular Biosciences (Biochemistry, Cell Biology, Genetics and Genomics) REU program at the University of California -- San Francisco (UCSF) combines an outstanding laboratory research experience with activities designed to foster scientific communication and facilitate a transition to graduate school. The 9- or 10-week program will take place at Mission Bay, a brand new state-of-the-art research facility housing more than 200 research laboratories. The centerpiece of the UCSF summer research program is an intensive laboratory research experience in which students are paired, based on their research interest, with UCSF faculty mentors. Students receive hands-on training using cutting edge biological techniques to creatively solve problems in modern biology. The research program includes daily interaction with a graduate/PhD level research co-mentor, faculty research presentations, a mentored journal club component, and oral/poster research presentations. The second component of the REU program is a co-curriculum designed to foster scientific communication skills, increase scientific breadth, and prepare students for the graduate school admissions process. The co-curriculum includes faculty led sessions focused on preparing a graduate school application, interviewing skills, scientific oral and poster presentation skills, and a workshop focused on issues related to diversity in the biological sciences. An optional GRE preparation course is available. In addition, students will have the opportunity to share their research experiences and mentoring skills with high school-level summer interns at UCSF. The program culminates with widely attended student poster and oral research presentations. Students will be recruited nationally and applications from under-represented minority and disadvantaged (economically or first in college) students are strongly encouraged. Students receive a stipend, dormitory-style housing, travel to and from the program, passes for local public transit and access to free campus transportation services. Further information and application materials are available on the program website: http://saawww.ucsf.edu/diversity/REU. Contact Carol Gross or Kalai Diamond, Program Coordinator at (415) 514-0840, Kalai.Diamond@ucsf.edu.

DESCRIPTION (provided by applicant): Although NF-kB has been studied intensively in the context of immunity, inflammation and cancer, far less is understood about the function of NF-?B in the nervous system. In the central nervous system, NF-kB signaling system has been implicated in neurodegenerative disease, epilepsy, and neuronal plasticity. At the neuromuscular junction, activation of NF-kB has been implicated in the mechanisms of muscle wasting associated with neurodegenerative disease (dystrophies and cachexia) and denervation. Despite these observations, the cellular and molecular mechanisms that activate NF-kB signaling within the nervous system remains to be clearly defined. We recently demonstrated that NF- kB/Dorsal, IkB/Cactus and IRAK/Pelle kinase function within postsynaptic muscle to control glutamate receptor density at the Drosophila NMJ, a process relevant to the function of NF-kB at the vertebrate NMJ (Heckscher and Davis, in review). We now have preliminary data identifying a trans-synaptic signaling system that could control NF-kB at the NMJ. In a forward genetic screen we identified mutations in a secreted ligand (TNFa) and its postsynaptic receptor (TNFR2) that impair GluR abundance at the Drosophila NMJ. We present preliminary data that the TNF-alpha gene is expressed in peripheral glia that reside near the NMJ, and that this source of TNF-alpha is necessary and sufficient to control GluR levels. In addition, it has been established that the TNFR2 receptor is expressed in Drosophila muscle. Thus, we hypothesize that the TNF-a and TNFR2 genes define a new, glia-to-muscle, trans-synaptic signaling system at the NMJ. Importantly, it has been demonstrated that the TNFR2 receptor can activate downstream NFkB signaling in other Drosophila tissues. Therefore, we hypothesize the existence of a conserved, glial-to-muscle signaling system that controls GluR levels and neuromuscular function during postembryonic development. We propose experiments to define and elaborate upon this signaling system at the Drosophila NMJ. Given that these signaling molecules are highly evolutionarily conserved, we predict that our data will have direct relevance to the function of NF-kB during neural disease and injury in mammals. PUBLIC HEALTH RELEVANCE: In the nervous system, NF-kB signaling system has been implicated in the mechanisms of neurodegenerative disease, epilepsy, and the response to neuronal injury. Despite the importance of this evolutionarily conserved signaling system, very little is known about how NF- kB participates in these diverse processes. We propose experiments that will not only define how NF-kB is activated in the nervous system, but will also define an output for NF-kB signaling that may be directly relevant to the role of NF-kB during injury and disease.

DESCRIPTION (provided by applicant): There is increasing evidence that neural function is stabilized by homeostatic signaling systems in organisms ranging from Drosophila to mouse and human. In each example, homeostatic signaling systems were identified following an experimental perturbation of neuronal or muscle excitability. In each experiment, the cells responded to the experimental perturbation by modulating ion channel abundance or synaptic transmission to counteract the perturbation and re-establish normal activity levels. It is now widely hypothesized that defective homeostatic signaling will contribute to the cause or progression of neurological disease. However, clear links between homeostatic signaling and disease will require a detailed cellular and molecular understanding of the underlying signaling systems. Currently, the molecular basis of homeostatic signaling remains largely unknown. Over the past ten years, we have established a model system for the rapid identification and characterization of genes involved in homeostatic signaling in the nervous system of Drosophila melanogaster. Among our recent successes has been the demonstration that a schizophrenia associated gene in human, dysbindin, is critical for homeostatic signaling. In preliminary data we identified two novel proteins that control homeostatic signaling within the presynaptic nerve terminal including a novel protein kinase and protein phosphatase. We propose to characterize these new genes and define how they function during homeostatic signaling. Both genes are highly conserved in human.

DESCRIPTION (provided by applicant): Neuronal cell biology has emerged as one of the most exciting and rapidly moving fields in contemporary biology. This field is experiencing a revolution in both imaging technology and reporters for the analysis of cellular structure and physiology. There is an unprecedented ability to interrogate the intricate design and function of individual neurons and neural circuitry. The Cell Biology of the Neuron Gordon Research Conference will be held on June 22-27, 2014 at Waterville Valley Resort, NH. A stated goal of this meeting is to bring together leading scientists to present the most recent advances in this rapidly moving field. Relevance to human neurological disease will be addressed throughout the program, including presentations by prominent cell biologists working at the academic-industry interface, tackling the challenge of translating cell biological advances into therapeutics. The conference format is based on 25-minute talks with 15 minutes of discussion following each talk. Topics will focus on synapse formation, synaptic transmission, neural plasticity, membrane trafficking, neural regeneration, and degenerative disease. Specific areas of disease relevance include talks on neural degeneration, regeneration, ALS, prion-disease, Parkinson's, and diseases that can be traced to early neurodevelopmental errors including schizophrenia and autism-spectrum disorders. Speakers were selected as world leaders in their respective areas. Eleven out of the 29 invited speakers, including a plenary lecturer, are women. Nine of the speakers are from outside the US including Europe and Asia. Six speakers are early career scientists and we have reserved 8 short talks for graduate students, postdoctoral fellows or junior faculty to be selected from the abstracts submitted to the meeting. Most participants will present posters, ensuring maximal participation and discussion among meeting participants. This also ensures that the conference will provide ample opportunities for junior scientists and graduate students to present their work. Members of under-represented minority groups are especially encouraged to attend. The collegial atmosphere of this conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings, provides an avenue for scientists from different disciplines to brainstorm and promotes cross-disciplinary collaborations in the various research areas represented. The conference should be of great interest to the missions of the National Institutes of Health including NINDS.

PROJECT SUMMARY/ABSTRACT The brain is astonishing in its complexity and capacity for change. It seems certain that the plasticity that drives our ability to learn and remember can only be meaningful in the context of otherwise stable, reproducible, and predictable baseline neural function. It is now clear that homeostatic signaling systems function throughout the central and peripheral nervous systems to stabilize neural function throughout life. As a consequence, it is widely believed that impaired or maladaptive homeostatic signaling will be directly relevant to the cause and progression of neurological diseases that include epilepsy, autism and neurodegeneration. However, despite widespread evidence for the homeostatic control of neural function throughout the animal kingdom and implicit relevance to disease and aging, very little is known about the underlying mechanisms. The field of homeostatic plasticity is wide open for exploration and the potential for transformative advancement in cellular and molecular neuroscience is tremendous. We are leading the rapidly emerging field of homeostatic plasticity, harnessing the power of unbiased model system genetics to identify and characterize fundamentally new cellular and molecular mechanisms of homeostatic signaling in the nervous system. Our experiments will define many of the first signaling pathways identified to participate in the homeostatic signaling systems that control presynaptic neurotransmitter release and intrinsic neural excitability. Our approaches have uncovered a novel activity of the innate immune signaling system, new trans-synaptic signaling pathways, novel calcium sensors, novel neuronal kinase signaling systems, new roles for the presynaptic endoplasmic reticulum and tangible links to neurological disease. As such, our data will provide a foundation for exploring the impact homeostatic plasticity in mammalian models of neurological disease including epilepsy, autism and neurodegeneration. Our data will also directly impact current theories and models of homeostatic signaling. Current theoretical models have captured widespread interest. Molecular insight will provide important new ideas and new constraints for the next generation of theoretical models of homeostatic plasticity, learning and memory.

ABSTRACT The identification of gene mutations that cause autism and syndromes associated with severe intellectual disability has greatly advanced both research and new therapeutic development. Yet, the mechanisms that link gene loss of function to impaired brain function remain poorly understood in almost every instance. There is a pressing need to elucidate how individual gene mutations lead to deficits in brain function, with the potential to identify common grounds for therapeutic intervention that will benefit the largest patient population. Recently, homeostatic plasticity was demonstrated to have a potent ability to counteract neurological disease onset and progression. This is referred to as Homeostatic Neuroprotection. Based on preliminary data, we propose that the mechanisms of homeostatic neuroprotection also apply to the onset and progression of impaired hippocampal function, based on work examining a new mouse model of severe intellectual disability. This work has the potential to open a new approach toward therapeutic development in psychiatric disease.

ABSTRACT In humans, the age-related decline in neuromuscular function is associated with loss of muscle mass (sarcopenia), increased frailty and a degradation of both health and quality of life. The only known treatments are life-style based, including exercise and diet. Rodent models have been used to demonstrate age-related changes at the neuromuscular junction (NMJ) including the fragmentation, remodeling and eventual denervation of muscle. We have recently demonstrated the power of homeostatic plasticity to preserve neuromuscular anatomy and function, with dramatic effects on organismal health, behavior and lifespan in rodent models of neurodegenerative disease. We refer to this as ?Homeostatic Neuroprotection?. We propose that homeostatic neuroprotection also functions at the aged synapse, normally counteracting the insidious effects of age-related neuromuscular decline. We will determine whether homeostatic neuroprotection can be potentiated to combat sarcopenia and age-related frailty in rodent models, paving the way to new therapeutic approaches in human.