Miri K. VanHoven, PhD Genetics UCSF - US grants

[unreadable] DESCRIPTION (provided by applicant): The nervous system is composed of highly complex circuits that govern thought and behavior. To ensure correct circuit formation, neurons must identify appropriate synaptic targets among the many neurites they contact before forming synaptic connections. Altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. Despite its central role in circuit formation, synaptic specificity is poorly understood. I have developed a novel way to visualize synapses between specific neurons in vivo. I propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie this fundamental process. I then propose to extend this my findings to mammalian neurons. This proposal is relevant to the NINDS mission to support basic research in fields related to the causes of neurological disorders, and the NIMH mission to support research on mental disorders and the underlying basic science of brain and behavior. My specific aims are: 1) to visualize changes in synaptic connectivity between a specific pair of pre- and postsynaptic neurons in C. elegans, 2) to discover the molecular determinants of synaptic specificity in this system, and 3) to adapt this new labeling method to mammalian neurons. To visualize changes in synaptic connectivity, I have developed an intersynaptic marker. I have fused complementary fragments of a split GFP to pre- and postsynaptically localized proteins, expressed under cell-specific promoters. I have successfully visualized changes in connectivity in two well-characterized circuits in C. elegans using known synaptic specificity mutants. I am interested in how synaptic specificity is regulated in complex environments, such as nerve bundles, where parallel neurites must distinguish among multiple potential targets to form appropriate connections. Therefore I am now adapting the intersynaptic marker to label synaptic connections between a specific neuron pair in a posterior nerve bundle. I will take an unbiased genetic approach to discover molecular mechanisms guiding synaptic specificity in this system, utilizing this marker to detect defects in connectivity. Finally, I am developing the intersynaptic marker for use in cultured mammalian cortical neurons. Insights gained from this study will aid in understanding synaptic specificity in the complex environments of the vertebrate nervous system. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The human central nervous system is composed of 100 billion neurons interconnected into precise circuits by 100 trillion synaptic connections. These circuits are required for nervous system functions including perception, thought and behavior. Much is known about the early steps in circuit formation in which neurites extend to target regions containing the correct synaptic partners. Much less is known about how individual neurons choose the correct synaptic partner when they reach a target region with many neurons. We have developed a novel method to visualize contact and synaptogenesis between specific neurons in vivo. We propose to use this method and take advantage of the simple, well-characterized nervous system of C. elegans to elucidate molecular mechanisms that underlie synaptic partner choice. This is an important area of study, as altered synaptogenesis is thought to play a role in disorders such as schizophrenia and autism. This proposal is relevant to the NIGMS mission to support research that increases understanding of life processes including the field of developmental biology, that lay the foundation for advances in disease diagnosis, treatment, and prevention, and to train the next generation of scientists. To understand how correct synaptic partner choice is mediated, we have developed a genetically encoded fluorescent trans-synaptic marker to visually label synaptic contacts between individual neurons of interest in complex environments called NLG-1 GRASP, for Neuroligin-1-mediated GFP Reconstitution Across Synaptic Partners. We have also labeled pre- and postsynaptic neurites with the red mCherry fluorophore. Together, these markers enable us to instantly assess correct synaptic partner choice by visualizing neurite contact and synaptogenesis between pre- and postsynaptic neurons of interest in live animals, making it feasible to use genetic methods to discover genes mediating this fundamental process. In addition, we have developed these markers in C. elegans, the only model organism for which there is a complete synaptic map, making it ideal for the study of synaptic partner choice. Using this marker, we have found that two proteins previously studied for their role in cell migration and axon guidance in other systems, UNC-40/DCC (deleted in colorectal cancer) and UNC-6/Netrin, have a novel role in mediating synaptic partner choice between sensory neurons and interneurons in the C. elegans ventral nerve cord. Our research will further characterize this role and define the pathway(s) that mediate synaptic partner choice. Our specific aims are: 1) to characterize the role of the UNC-40/DCC receptor and the UNC-6/Netrin ligand in synaptic partner choice, 2) to investigate genes that transduce UNC-40/DCC-mediated axon guidance and cell migration signals for roles in synaptic partner choice, and 3) to identify new genes that mediate synaptic partner choice by conducting a forward genetic screen. Understanding the mechanisms that regulate circuit formation will bring us closer to understanding and treating neurological diseases. PUBLIC HEALTH RELEVANCE: For the nervous system to function correctly, neurons must faithfully identify cellular partners with which to form synapses. Altered synapse formation is thought to underlie neurological diseases, such as schizophrenia and autism. We seek to identify the molecular mechanisms that underlie synaptic partner choice, as understanding these mechanisms will bring us closer to understanding and treating neurological diseases.

This REU Site award to San Jose University, located in San Jose, CA, will support the training of 10 students for 10 weeks during the summers of 2017- 2019. The REU program will focus on the theme of molecular biology applications in biological research. Nine faculty members with expertise in the areas of neuroscience, genetics, molecular environmental microbiology, biochemistry, and cell & molecular biology will mentor REU students. Aside from the research projects, students will participate in: (1) professional development activities, (2) the creation of individual development plans (IDP), (3) journal clubs to discuss relevant papers in the field, (4) ethics and the responsible conduct of research training, and (5) weekly research meetings where the entire cohort and their faculty mentors meet to discuss research progress. The ethics training is a shared experience with the BIO REU sites at San Francisco State University and the University of California at Berkeley. An on-line application is due the first Friday of March. Ten students will be selected each year based on their application and a phone interview. Notification of finalists happens on the third week of March.

It is anticipated that a total of 30 students, primarily from schools with limited research opportunities, will be trained in the program. Rising sophomore and junior students are encouraged to apply. At the end of the program, students will learn how research is conducted, and many will present the results of their work at scientific conferences.

A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. In addition to a final research paper, students will do poster and oral presentations to present the results of their research. Students will be tracked after the program in order to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.biology.sjsu.edu/rumba/NSF-REU_RUMBA/Welcome.html, or by contacting the PI (Dr. Soto at julio.soto@sjsu.edu) or the co-PI (Dr. Miri VanHoven at miri.vanhoven@sjsu.edu).

Funds from this Major Research Instrumentation Program grant will be used for the purchase and support of a Zeiss LSM 700 Confocal Microscope at San José State University. Research groups from the Biology, Chemistry and Civil Engineering departments will be among the major users, as well as a biology professor from Santa Clara University. A common theme to the research that will be enabled and enhanced by this acquisition is to elucidate biomechanistic processes. Faculty members with expertise in the areas of genetics, development, environmental microbiology, cell biology, immunology, biochemistry, and civil engineering will comprise the major users of the confocal microscope. Specifically, the following research projects will utilize this microscope: apoptotic induction by r-disintegrins; molecular mechanisms that mediate neuronal circuit formation and maintenance; mechanisms by which ethanol exposure affects synaptic plasticity; characterization of novel uncultured bacterial groups for ecophysiological studies; immunomodulation of lymphocyte trafficking; cellular localization of inositol glycans; examining angiogenesis during ovarian development in mice, signal transduction resulting from binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to the nuclear vitamin D receptor (VDR); using microbiological dating and confocal microscopy to date structural cracks; and developmental analysis of LIN-1 expression.

San José State University is a minority serving institution in Northern California with a large proportion of students with physical disabilities. Research groups actively incorporate undergraduate researchers and students at the Master?s level in their projects. Four main components of the training infrastructure at the university will be impacted by the purchase of this confocal microscope: 1) increased involvement of undergraduate students in research, 2) promoting active learning, 3) student training in specialized masters programs, and 4) collaborations with faculty and students at other institutions. Faculty that will utilize this confocal microscope are research mentors for several programs that aim to increase the number of minority students who pursue Ph.D. degrees in the sciences, including NIH-MARC, MBRS-RISE, HHMI-SCRIBE, and NSF-REU RUMBA. Learning to use this state of the art microscope in their research will help prepare students for careers in science. In addition, data collected from this microscope will be used in undergraduate courses to bring contemporary research into the classroom. Finally, smaller institutions in the surrounding community including Santa Clara University will have the opportunity to utilize this microscopy to increase their research potential.

A Research Experience for Undergraduates (REU) Sites award has been made to San José State University to provide research training for 10 weeks for 10 students, for the summers of 2013-2016. Our proposed REU program at San José State University called Research by Undergraduates using Molecular Biology Applications (RUMBA) will focus its efforts in increasing the number of underrepresented minority doing biological research in the Silicon Valley. Our REU Program will focus on the theme of molecular biology applications in biological research. Ten faculty members with expertise in the areas of genetics, molecular evolution, molecular environmental microbiology, cell & molecular biology, and forensics will mentor students. Regardless of the research laboratory placement, RUMBA students will comprise a cohort with shared thematic training experiences. The cohort experiences will consist of the following: (1) molecular biology techniques training, (2) a bi-weekly formal group research meeting, (3) an ethics seminar, and (4) a culminating research symposium. Students will be recruited from San José State University and other undergraduate institutions with high enrollment of female and underrepresented minority students. Priority will be given to students of underrepresented groups as defined by NSF (African American, Latino, Native American, and Pacific Islander). We will utilize the following selection criteria for RUMBA participants: a GPA of at least 3.0, completion of the first year university biology and chemistry core course sequence, two letters of recommendation, and the quality of a personal statement. Student applicants will be interviewed by RUMBA faculty before a decision is made. A common web-based assessment instrument available to BIO-funded REU PIs will be used to evaluate the effectiveness of our program. Student participants will also be tracked to determine their continued interest in their academic field of study, their career paths, and the lasting influences of the research experience. San José State University (SJSU), the oldest public university in the state of California, is located in downtown San José and in the heart of Silicon Valley. It is a metropolitan university with an enrollment of approximately 27,000 students. Students interested in participating in the RUMBA should contact Dr. Julio G. Soto, Professor, Biological Sciences Department, julio.soto@sjsu.edu. Application forms and deadlines can be found on our web site: http://www.sjsu.edu/depts/Biology/RUMBA/RUMBA.html.

In the last century, a large number of neural circuits in the brain and throughout the body have been described. These neural circuits mediate such critical functions as perception and behavior. However, we understand much less about the genes involved in building these circuits. This project will use a unique fluorescent marker to visualize synaptic contacts between specific neurons in live nematodes, allowing us to advance our understanding of neural circuit formation by investigating the role of a conserved cell surface molecule. The broader impact of this project is to encourage students to develop critical thinking skills through the use of the scientific process. Toward that goal, the project will include direct participation of undergraduate and Master's-level researchers; mentorship of a community college professor in genetics research during the summer; and development of modules for a new course, which will include data
generated by this research project. In addition, the PI has and will continue to actively seek to increase participation of underrepresented minorities and women in science through these activities.

Much is known about early steps in neural circuit formation, including the migration of neuronal cell bodies and axons to the correct regions of the body. Much less is known about how neurons recognize the correct partners from the many potential partners they may encounter in a target region. This project will utilize the trans-synaptic split-GFP based marker, NLG-1 GRASP (Neuroligin-1-mediated GFP Reconstitution Across Synaptic Partners), to visualize synaptic contacts between individual neurons of interest in live animals. NLG-1 GRASP, together with a circuit-specific behavioral assay to assess synaptic function, will be used to characterize the novel role of a conserved receptor protein tyrosine phosphatase (RPTP) in synaptic partner recognition. Further work will determine which domains are required for RPTP function. Finally, genes that function with this RPTP will be identified and studied, with the goal of elucidating a
new synaptic partner recognition pathway. The primary intellectual merit of this research is advancing our understanding of the mechanisms that underlie the specificity of neural circuit formation.

DESCRIPTION (provided by applicant): The human central nervous system is composed of 100 billion neurons interconnected into precisely regulated circuits to mediate vital functions such as perception, thought, and behavior. Sensory activity has long been known to have important affects on synaptic plasticity. Understanding the molecular underpinnings of these processes may aid in understanding neurological disorders, such as autism and schizophrenia. However, much remains unknown about the molecular mechanisms by which normal activity and long-term activity affect synaptic plasticity. The discovery of molecules required in these pathways may be accelerated by the study of genetic model organisms, in which large-scale gene discovery techniques are feasible. To address this, we propose to take an innovative approach to studying the affects of sensory activity on synaptic plasticity, investigating all leves of the responses within two defined circuits in the genetic model organism C. elegans. We will assay behavioral output at the highest level, cellular-level calcium responses, specific synaptic connections between the neurons utilizing the trans-synaptic split-GFP based marker NLG-1 GRASP, and the individual molecules required for these events. This integrative proposal is relevant to the NINDS mission to support research that aims to reduce the burden of neurological disease by supporting basic research on the biology of the cells of the nervous system, nervous system development, genetics of the brain, behavior, neurodegeneration, brain plasticity, sensory function, synapses, and circuits. Using this approach, we have discovered a Galpha-olf-dependent pathway by which normal sensory activity maintains appropriate synaptic connections, and an EGL-4/PKG dependent pathway by which animals adapt to long-term sensory signaling. Our research will characterize the mechanism by which these pathways affect structural and functional synaptic plasticity. Our specific aims are to: 1) elucidate the molecular pathway by which normal sensory activity affects structural plasticity, and 2) determine the molecular basis for plasticity induced by long-term sensory activity. The robust prior characterization of these two circuits, in combination with the powerful tools we have available to study them, offers the unique potential to discover new and unexpected signaling pathways that mediate sensory activity dependent synaptic plasticity, which can then be explored in other systems.

Project Summary The proper formation of neural circuits is critical for fundamental processes, such as perception, thought, and behavior. While much is known about the molecules that promote synaptogenesis, far less is known about the molecular pathways that negatively regulate this process. The use of genetic model organisms may accelerate the discovery of molecules required for this process, as large-scale gene discovery techniques are feasible. We propose to take an innovative approach to studying synaptic regulation by investigating a defined neural circuit at multiple levels. We will assay behavioral output at the highest level, specific synaptic connections between the neurons utilizing the trans-synaptic split-GFP based marker NLG-1 GRASP, and the individual molecules required for these events using molecular and genetic manipulations. This proposal is relevant to the NINDS mission to seek fundamental knowledge about the brain and nervous system and to use that knowledge to reduce the burden of neurological disease. Using this approach, we have discovered that Fibroblast Growth Factor (FGF) is required to downregulate synaptogenesis within a defined sensory circuit. In addition, we have discovered that a Receptor Protein Tyrosine Phosphatase (RPTP) is required to promote synaptogenesis within this circuit. Our research will characterize the pathways that transduce the FGF signal, and the means by which these two conserved pathways interact to regulate synaptogenesis. Our specific aims are to: 1) elucidate the pathway by which the FGF signal regulates synaptogenesis, and 2) determine how the FGF and RPTP pathways interact to mediate synaptogenesis. The robust prior characterization of this circuit, in combination with the powerful tools we have to study them, offers the unique potential to discover new and unexpected signaling pathways that negatively regulate synaptogenesis during neural circuit formation, which can then be explored in other systems. FGFs and their receptors have been linked to epilepsy, schizophrenia, bipolar disorder and depression, so understanding this new role for FGF in neural circuit formation may inform our understanding of these disorders. This R15 will fund the research of approximately 18 undergraduates and 3 M.S. students over three years, including many minority trainees at San José State University (SJSU), a primarily undergraduate and Masters-level university committed to training under-represented minority students. Undergraduates will perform the majority of the proposed work, with training and mentorship from M.S. students, a technician, and the PI. This funding would allow the PI to continue to develop a strong track record in research, give meaningful research experiences to undergraduate students, and strengthen the research environment at SJSU.

Project Summary/Abstract A fundamental question in neuroscience and human health is how do different brain states alter neuronal connections and how are these changes carried out at the cellular and molecular levels? We propose to address this question by examining neuronal connections through out the compact, completely described nervous system of the transparent nematode C. elegans as its whole brain activity cycles between wakefulness and sleep. Specifically, we propose to use our ability to modulate the C. elegans brain state to examine how the structure and function of excitatory as well as inhibitory synapses are changed as a function of recurrent neural activity. Further, we will identify the molecular mechanisms by which this is achieved. In Aim 1, We will ask how the brain states affect synaptic architecture across the animal's nervous system by testing different types of connections throughout the animals for their response to wakefulness and sleep. We will then ask whether the number or size of connections is affected. We will also identify the molecular regulators of the brain state-dependent synaptic changes, and visualize synaptic components in each brain state to determine how and when synapses are altered. These studies would provide the first evidence of broad sleep-dependent synaptic remodeling in C. elegans. In Aim 2, We will characterize the activity calcium transients (GCaMP6/7), cGMP fluxes (WincG) and neuropeptide-driven GPCR ligand binding (D-lite adapted reporters) of the entire brain of C. elegans as it sleeps and compare that to the wakeful animal. Using this information we will assess the pattern of these changes and relate them to the structure of synaptic components within the units that change most. We will attempt to understand if the synaptic changes are dispersed brain-wide and whether these connections are responsible for stabilizing sleep dependent changes in behavior. We will then alter the brain activity using optical manipulation of ChR, Arch, our cGMP-sponge WincD and cell and timing specific regulation of neuropeptide processing during sleep to test the requirement for patterned activity to direct changes in connections that we observe. In this way, we will begin to understand how brain state effects structural changes that affect behavior. These studies would provide the first brain wide understanding of the interplay between the activity and structure of connections in any animal. This understanding will provide insights into novel approaches to therapies aimed at mitigating activity-driven changes in human brain activity that promote maladaptive responses to activity such as addiction and post traumatic stress disorder.