Maria Bykhovskaia - US grants

DESCRIPTION(adapted from applicant's abstract): Neurons communicate with each other through synaptic transmission. Changes in the effectiveness of synapses underlie the ability of neuronal networks to store and retrieve information, the cellular representation of learning and memory. One form of synaptic plasticity is facilitation, a phenomenon by which synapses becomes transiently more effective following repeated use. Facilitation is a ubiquitous phenomenon observed at many synapses and represents a very general process controlling the effectiveness of synapses. This application addresses the question of what mechanisms operate within the synaptic terminal to allow secretion to increase with increased rates of use. The fundamental event in synaptic transmission is the entry of calcium into the synaptic terminal during an action potential, leading to the fusion of a synaptic vesicle with the terminal membrane. The classical interpretation of facilitation was repetitive nerve stimulation increases the Ca++ concentration at presynaptic release sites, which in turn increases the probability of each vesicle to be released. Recently it was demonstrated that the number of synaptic vesicles properly activated to be released (the releasable pool of quanta) is highly dynamic and has a critical role in synaptic plasticity. The goal of the proposed work is to develop and test a quantitative model of neurosecretion, which will clarify the role of the increase in residual calcium, activation of release sites and the increase in the releasable pool of quanta and thereby account for presynaptic facilitation. Facilitation has strictly distinguishable components: short-term facilitation (STF) and long-term facilitation (LTF), which results from different underlying mechanisms. The proposed work will take advantage of the separation of these two components to distinguish between different mechanisms. Experiments will test the hypothesis that STF is determined by an increase of intracellular calcium and vesicle mobilization, while LTF is additionally controlled by activation of previously silent release sites. The approach is to combine computer simulations of presynaptic processes with electrophysiological detection of the number of released vesticles.

DESCRIPTION (provided by applicant): The SNRP-1 Program at UCC is centered around three research topics: norepinephrine modulation of sensitization to cocaine, neuroprotection by nicotine and tobacco cembranoids, and cembranoid mechanism of action on nicotinic receptors. A Natural Products Core provides tobacco and marine cembranoids, both known and new. Each project is performed in collaboration with a collaborator from a research-intensive institution: UCLA, Cornell U. (Ithaca) and the Vollum Institute (OHSU). SNRP-1 was funded since October 1999. Four RO1 proposals were presented to the NIH during the third year, three of which are now in the process of re-submission. Thirty nine manuscripts were published in peer-reviewed journals. Among the scientific discoveries reported, we would like to mention the following: 1) Noradrenergic transmission through alpha-2 receptors mediates some of the inhibitory effects of cocaine in the prefrontal cortex. 2) Both nicotine and tobacco cembranoids protect the hippocampal slice from NMDA-induced excitotoxicity, but the signal transduction pathways are different. 3) Cembranoids are nicotinic antagonists whose inhibitory action can be released by certain cocaine derivatives. We now propose to continue this successful program for a second cycle of five years. Three new scientists will address the issues of oxidative damage in Huntington disease, direct effects of thyroid hormones on the nAChR and on synapse remodelling, and modulation of potassium channels by spermine. Collaborators are from Washington University and UCLA. Program activities include seminars, courses, travel, etc. A transition plan from SNRP-1 to RO1 funding, is proposed for the present investigators. A plan to attract Puerto Rican Ph.Ds to postdoctoral positions with SNRP scientists at UCC is also presented.

DESCRIPTION (provided by applicant): Intellectual merit: At the cellular level, learning and memory are governed by changes in the efficacy of synaptic transmission and in particular, by the dynamic regulation of neuronal transmitter release. Neurotransmitters are packaged into synaptic vesicles that dock at the synaptic membrane, undergo a series of preparatory steps, open a pore, and fuse with the synaptic membrane, resulting in neurotransmitter release into the synaptic cleft. This process is very dynamic, plastic, and highly regulated. Although molecular components of docking and fusion have been identified, it is not yet understood how they interact to regulate the dynamics of docking, pore opening, and fusion. In particular, little is known about the detailed mechanics of protein interactions that regulate synaptic vesicle fusion. The present application will focus on this critical question by combining modeling and experimentation to investigate the molecular machinery that regulates synaptic vesicle docking and fusion. Vesicles tightly dock at the plasma membrane via a specialized protein complex (SNARE), which is thought to provide the necessary force to overcome inter-membrane repulsion and thus mediate vesicle fusion. Stimulus evoked fusion is triggered by an influx of Ca2+ ions that interact with a vesicle protein, synaptotagmin (Syt), which is tightly coupled with the SNARE complex. Fusion pore opening is thought to be controlled by the interaction of Syt and a small protein complexin (Cpx) with the SNARE complex. Although molecular interactions of these proteins have been studied with biochemical and molecular biology tools, there is still a lack of understanding of how these proteins interact dynamically and how the forces of the protein fusion machinery counterbalance forces generated within the synaptic and vesicle membranes. To elucidate these mechanisms, we propose to build a molecular model of the fusion machinery and to perform computer simulations of the dynamics of the fusion complex. To understand the interactions between the vesicle, synaptic membrane and the protein fusion machinery, we will develop a coarse grain model of membrane/vesicle dynamics and integrate it with the atomic model of the fusion protein complex. To validate the model, we will simulate the effect of single point mutations in the fusion complex on the release kinetics and test our predictions experimentally. The experiments will be performed at Drosophila neuromuscular junctions (NMJ), a model system ideally amendable to genetic manipulations. To test the predictions of the model, we will combine electrophysiology and optical fluorescent microscopy to assess release kinetics in NMJs where the fusion machinery is modified by point mutations with computationally predicted effects on membrane fusion. This research will be performed by a multidisciplinary team that includes experts in molecular modeling (Dr. Jagota), membrane mechanics and dynamics (Dr. Hui), synaptic physiology (Dr. Bykhovskaia) and Drosophila neurobiology (Dr. Littleton). An attack on this problem by a collaborative team with balanced representation of all its aspects will lead to new, detailed and quantitative, understanding of the regulated synaptic vesicle fusion process. Broader impact: Universidad Central del Caribe (UCC) is a Hispanic serving institution in Puerto Rico (U.S. Commonwealth). The proposed project will allow the UCC to develop tight links with highly regarded mainland institutions and will thus create training and employment opportunities for students with diverse backgrounds. The PI, Dr. Bykhovskaia, directs the Specialized Neuroscience Research Program (SNRP) at UCC (funded by NIH), that has a goal of raising research standards in institutions with a predominant enrollment of underrepresented minorities. Thus, the proposed project will involve underrepresented B.S., M.S., Ph.D., and M.D. students in biomedical research. Furthermore, Dr. Jagota directs the undergraduate and graduate Bioengineering programs at Lehigh University, an institution with a balanced emphasis on research, teaching and training. The proposed research will be performed by graduate students, and will actively involve undergraduate students through research for credit and summer opportunities. This research will be incorporated into the undergraduate bioengineering curriculum through a course on Biomolecular and Cellular Mechanics, developed by Dr. Jagota at Lehigh University. It will be tightly integrated with other activities, including student exchange and transdisciplinary seminars, and thus will promote integration of research and training across diverse intellectual and ethnic backgrounds.

Numerous neurological disorders result from disruptions in the communication between nerve cells. Neurons communicate by releasing neuronal transmitters from nerve endings. Impaired transmitter release may produce certain forms of epilepsy. Transmitter molecules are packed into synaptic vesicles, and the preparation of vesicles for release is regulated by numerous proteins. Synapsin is the most abundant synaptic vesicle protein, and its deficiency causes epileptic seizures. Past work shows elimination of another vesicle protein, Rab3a, rescues epilepsy caused by synapsin deletion. This project will investigate how these two proteins interact within nerve endings. To do this, genetically engineered mice that lack synapsin, Rab3a, or both proteins, will be studied. High resolution microscopy to observe vesicle dynamics, and electrical recording will be performed to monitor neuronal activity. In addition, a computational model of the nerve ending will be developed to test whether hypotheses of the protein interaction correctly predict how synapsin and Rab3a affect the release of transmitters. The results of our study will elucidate the mechanisms of synapsin-dependent epilepsy and will suggest new strategies for its treatment.

The Universidad Central del Caribe (UCC) is a Hispanic serving institution which strives to provide Puerto Rican students with first-rate education and scientific training. The present project creates a foundation for an excellent training program. The experiments will be performed by graduate, undergraduate, and medical students, and thus the project will provide a support for the students' careers. The project involves collaboration between the UCC and the Wayne State University, and this creates advantageous opportunities for Puerto Rico students to have a practice in a research intense institution. Thus, this project will integrate research and training, advance careers of Puerto Rico students, and help to promote workforce diversity.

ABSTRACT Synaptic vesicle cycling is highly dynamic and plastic, and subtle disruptions in its regulation may contribute to severe neurological disorders, including schizophrenia, epilepsy, Huntington's and Parkinson's Disease. Although a number of molecular determinants of vesicle cycling at synaptic terminals have been identified, our understanding of how vesicle cycling contributes to plasticity remains sparse. Using the Drosophila neuromuscular junction (NMJ), the lab of Dr. Bykhovskaia has discovered a pathway of presynaptic enhancement that may enable a nerve terminal to sustain and enhance its activity upon intense stimulation. Specifically, it was found that vesicle abundance in synaptic boutons is regulated by activity, and that intense stimulation increases vesicle number in nerve terminals. Importantly, these results suggest a functional implication for this form of plasticity: enhanced activity upon a subsequent intense stimulation. This application will employ a combination of molecular biology and genetics, live confocal imaging, and electron microscopy to unravel the mechanisms by which the terminal regulates its vesicle numbers in response to activity. This project will be performed in collaboration with the lab of Dr. Littleton at the Massachusetts Institute of Technology. Dr. Littleton is a leader in the field of Drosophila genetics and neurobiology, and thus this project will enable the Bykhovskaia lab to encompass Drosophila genetics approaches and to take the full advantage of this fruitful experimental preparation. Further, the project will test whether the phenomenon observed at the Drosophila glutamatergic synapse could be also detected in the mammalian central or peripheral synapses. Thus, the project combines multidisciplinary approaches at two genetic model organisms, mice and Drosophila, and involves a collaboration with the Massachusetts Institute of Technology, and as such it represents an excellent training vehicle for the graduate students from underrepresented groups. Dr. Bykhovskaia has a solid mentoring record, and the project will support two graduate research assistants, and thus it will contribute to the integration of research and training components.

DESCRIPTION (provided by applicant): Our major goal is to advance diversity by creating a strong neuroscience center in Puerto Rico that will develop Hispanic American neuroscientists through a program that integrates research, graduate education, undergraduate engagement, and career guidance. Neuroscience research at Universidad Central del Caribe (UCC) has grown significantly over the last decade, and we are now are positioned to bring neuroscience at UCC to the next level, one defined by continued excellence in basic research, growth in translational research, collaborations with top mainland universities, multidisciplinary training o students from underrepresented minority groups, and fostering professional development in these individuals. At the core of this effort will be a state-of-the-art Institute for Neuroscience that integrates high performance in basic and translational research with educational programs and ongoing mentoring of students and young faculty. The specific objectives of our program are to: 1) increase diversity and develop excellence in neuroscience research in Puerto Rico, and 2) provide first-rate training in neuroscience research and career development guidance for faculty, graduate students, medical students, and undergraduates that are Hispanic Americans and often from disadvantaged backgrounds. Our research program is focused on the mechanisms of neuronal plasticity and neuroprotection against stroke, thus creating a bridge between basic and translational research on neuronal mechanisms. The proposed research program creates a multi-university cross-disciplinary network of neuroscientists that will facilitae mentoring relationships, amplify scientific synergism, and at the same time broaden the research focus of every member of the group as well as bridge basic and translational research. Building on a foundation grounded in an interdisciplinary, collaborative research network and research program addressing health-related problems through basic and translational neuroscience, we will develop a comprehensive training and mentoring program that serves both faculty and students. By creating a strong and efficient training and mentoring center in neuroscience on the Island of Puerto Rico that serves Hispanic Americans, the proposed program has the potential to significantly promote workforce diversity in the U.S.

Development; Neurosciences Research; programs; Puerto Rico; Research; Research Training;

The objectives of the Program Assessment component are to conduct both formative and summative evaluations to assess (1) program implementation and progress (formative), (2) impact of the program on the participating faculty, graduate students, undergraduates, the University, and the field (formative and summative), and (3) the long-term impact of the program on i) training models to enhance diversity and research in neuroscience with an emphasis on NINDS mission-related activities and ii) careers, placements, and accomplishments of the participants. These longer-term outcomes will require tracking information beyond the formal end date for the proposed SNRP program. We will use a mixed-methods approach to assessment that incorporates both quantitative and qualitative data (Westat, 2010). We have designed a cost-effective, comprehensive, and not overly burdensome approach to assessment. The SNRP has engaged Dr. Richard McGee as a consultant to head the assessment of the program. A portion of the effort requested for a SNRP staff person in the Administrative Core will be allocated to assist and support Dr. McGee and be responsible for collection of objective data from students, faculty and program leadership. Dr. McGee will work with the SNRP leadership, SNRP staff, the UCC administration, and the PAC on the design of assessment instruments, implementation of the assessment protocols, data collection, and data management He will independently conduct confidential interviews and/or focus groups with students and faculty, analyze the assessment data, and prepare annual reports for the SNRP leadership that will be made part of annual Progress Reports to the PAC and NINDS. Dr. McGee will present his findings annually at the PAC meetings. He will meet at least twice a year with SNRP leadership during years 1-3 of the program (one meeting in conjunction with the PAC meeting) and, if needed, more frequently by teleconference. Assuming the program continues for an additional two years, he will continue heading the assessment component.

DESCRIPTION (provided by applicant): Intellectual merit: At the cellular level, learning and memory are governed by changes in the efficacy of synaptic transmission and in particular, by the dynamic regulation of neuronal transmitter release. Neurotransmitters are packaged into synaptic vesicles that dock at the synaptic membrane, undergo a series of preparatory steps, open a pore, and fuse with the synaptic membrane, resulting in neurotransmitter release into the synaptic cleft. This process is very dynamic, plastic, and highly regulated. Although molecular components of docking and fusion have been identified, it is not yet understood how they interact to regulate the dynamics of docking, pore opening, and fusion. In particular, little is known about the detailed mechanics of protein interactions that regulate synaptic vesicle fusion. The present application will focus on this critical question by combining modeling and experimentation to investigate the molecular machinery that regulates synaptic vesicle docking and fusion. Vesicles tightly dock at the plasma membrane via a specialized protein complex (SNARE), which is thought to provide the necessary force to overcome inter-membrane repulsion and thus mediate vesicle fusion. Stimulus evoked fusion is triggered by an influx of Ca2+ ions that interact with a vesicle protein, synaptotagmin (Syt), which is tightly coupled with the SNARE complex. Fusion pore opening is thought to be controlled by the interaction of Syt and a small protein complexin (Cpx) with the SNARE complex. Although molecular interactions of these proteins have been studied with biochemical and molecular biology tools, there is still a lack of understanding of how these proteins interact dynamically and how the forces of the protein fusion machinery counterbalance forces generated within the synaptic and vesicle membranes. To elucidate these mechanisms, we propose to build a molecular model of the fusion machinery and to perform computer simulations of the dynamics of the fusion complex. To understand the interactions between the vesicle, synaptic membrane and the protein fusion machinery, we will develop a coarse grain model of membrane/vesicle dynamics and integrate it with the atomic model of the fusion protein complex. To validate the model, we will simulate the effect of single point mutations in the fusion complex on the release kinetics and test our predictions experimentally. The experiments will be performed at Drosophila neuromuscular junctions (NMJ), a model system ideally amendable to genetic manipulations. To test the predictions of the model, we will combine electrophysiology and optical fluorescent microscopy to assess release kinetics in NMJs where the fusion machinery is modified by point mutations with computationally predicted effects on membrane fusion. This research will be performed by a multidisciplinary team that includes experts in molecular modeling (Dr. Jagota), membrane mechanics and dynamics (Dr. Hui), synaptic physiology (Dr. Bykhovskaia) and Drosophila neurobiology (Dr. Littleton). An attack on this problem by a collaborative team with balanced representation of all its aspects will lead to new, detailed and quantitative, understanding of the regulated synaptic vesicle fusion process. Broader impact: Universidad Central del Caribe (UCC) is a Hispanic serving institution in Puerto Rico (U.S. Commonwealth). The proposed project will allow the UCC to develop tight links with highly regarded mainland institutions and will thus create training and employment opportunities for students with diverse backgrounds. The PI, Dr. Bykhovskaia, directs the Specialized Neuroscience Research Program (SNRP) at UCC (funded by NIH), that has a goal of raising research standards in institutions with a predominant enrollment of underrepresented minorities. Thus, the proposed project will involve underrepresented B.S., M.S., Ph.D., and M.D. students in biomedical research. Furthermore, Dr. Jagota directs the undergraduate and graduate Bioengineering programs at Lehigh University, an institution with a balanced emphasis on research, teaching and training. The proposed research will be performed by graduate students, and will actively involve undergraduate students through research for credit and summer opportunities. This research will be incorporated into the undergraduate bioengineering curriculum through a course on Biomolecular and Cellular Mechanics, developed by Dr. Jagota at Lehigh University. It will be tightly integrated with other activities, including student exchange and transdisciplinary seminars, and thus will promote integration of research and training across diverse intellectual and ethnic backgrounds.