Stephen D. Meriney - US grants

DESCRIPTION: (Adapted from applicant's abstract) Communication between cells in the nervous system is dependent on the precise control of neurotransmitter release. The rapid onset and termination of evoked release indicate that the specific proteins that participate in the regulation of transmitter release, it is apparent that the selective targeting of presynaptic proteins to release sites occurs rapidly following specific contact between a nerve cell and its target. Ion channels (especially Ca++ and calcium-activated K+) are known to be of critical importance in the regulation of transmitter release, and are among the proteins known to be selectively targeted to presynaptic active zones. Little is known, however, about the identity, properties, and development of nerve terminal ionic currents. The applicant proposes to study the induction of presynaptic specialization (Ca++ and calcium-activated K+ channels) expressed at transmitter-releasing varicosities that form along neurites in cultures of Xenopus spinal cord neurons and myocytes. These are newly formed synapses where there are no surrounding glial cells to interfere with access. Using patch clamp techniques, the applicant proposes to (1) characterize directly the types of currents present at newly formed presynaptic structures, (2) determine their role in transmitter release regulation, (3) identify cell-cell interactions that regulate the expression of these specializations, and (4) identify basal lamina components that can induce the expression of presynaptic specialization. From these studies should come a more thorough understanding of motor nerve terminal Ca++ and calcium activated K+ channels, their role in transmitter release and the induction of presynaptic specialization.

Cells in the nervous system communicate at specialized regions called synapses. When a nerve fires, calcium ions enter synapses from the surrounding extracellular fluid and cause neurotransmitter molecules to be released into the synaptic cleft between the communicating cells. However, the sequence of molecular events leading from calcium entry to transmitter release remains unknown. To better understand this important process, this research will combine laboratory experiments and large-scale computer simulations to build detailed 3-D models of calcium-triggered neurotransmitter release at the synapse between a nerve and muscle cell in the frog. In many respects, the frog synapse is quite similar to many mammalian synapses (including human), and yet it presents unique opportunities for combined laboratory and computational studies. This makes it an ideal choice for creation of highly realistic and accurate models. Using these models, hypotheses will be developed and tested to explain how synapses use calcium to trigger neurotransmitter release, and how repeated nerve firing changes release, as occurs at all synapses as they respond to changing behavioral conditions. Development of realistic 3-D computer models also presents unique opportunities for transformational training and education. Undergraduate, graduate, and postdoctoral trainees will participate directly in these studies, and high quality animations of the models will be adapted for use in the classroom. The project co-directors (S. Meriney, Univ. of Pittsburgh, and J. Stiles, Carnegie Mellon Univ.) and faculty colleagues will use the new teaching material at the University of Pittsburgh, the Pittsburgh Supercomputing Center, and Carnegie Mellon University, in undergraduate and graduate Neuroscience, Biology, and Computational Biology courses, a multi-institutional summer institute (www.ccbb.pitt.edu/bbsi), workshops, and web-based tutorials (www.mcell.psc.edu/tutorials/tlist.htm). New teaching material will also be featured in the CMIST program, an undergraduate and K-12 outreach program at the National Resource for Biomedical Supercomputing, directed by Dr. Stiles (www.nrbsc.org/cmist).

Decades of work comparing neuromuscular synapses in both crayfish and lizards have led to a general acceptance of the dogma that the structure of a synapse has little impact on its function. As a result, the study of structure-function relationships at synapses has been stymied, and, instead, investigators have focused mechanistic studies on the identification of differences in molecules and events associated with transmitter release and calcium signaling. In this project, a comparative study of neuromuscular junctions from frog and mouse skeletal muscle will be carried out using new techniques and a predictive computational model developed in the project collaborators' laboratories. These animal models offer several important advantages. The neuromuscular junctions of frogs and mice serve similar roles (as opposed to previously studied synapses that served different roles in the crayfish and lizard animal models) and yet, have distinct differences in their physiological properties and pre-synaptic structure and organization. The driving hypothesis is that key physiological features of neuromuscular synapses are determined by the structural arrangement of basic building blocks of synaptic structure. This work will offer opportunities for training undergraduate, graduate, and post-graduate students, and high school teachers. Because this is a collaborative project, all trainees will have the unique opportunity to learn both experimental and computational approaches. Importantly, it is expected that the MCell computational model will provide an example of unprecedented scale and realism for the illustration of nerve terminal structure and function with the potential to literally transform a student's grasp of difficult biochemical concepts in space and time. This model will also illustrate and teach the use of Monte Carlo simulations, sophisticated visualization and animation software, and a variety of statistical analysis methods.

DESCRIPTION (provided by applicant): Communication between cells in the nervous system underlies all complex behaviors, and occurs at specialized regions of the nerve cell called synapses. Synapses work by releasing chemical transmitter from a region called the active zone, which activates a neighboring cell. We propose to characterize the relationship between active zone function and structural organization within frog and mouse neuromuscular synapses. We hypothesize that neuromuscular active zones are assembled from a basic transmitter release building block: the unreliable single-vesicle release site consisting of a docked synaptic vesicle and its associated Ca2+ channels. We further hypothesize that major aspects of synaptic function and presynaptic homeostatic plasticity can be explained by changes in the number and organization of these single-vesicle release sites within active zones. Our approach is characterized by a seamless collaboration between three labs with expertise in computer simulations of cellular physiology (Dittrich lab), synaptic anatomy, physiology, and Ca2+ imaging (Meriney lab), and super-resolution imaging of the number and spatial distribution of synaptic proteins (Blanpied lab). Importantly, as part of this proposal, trainees from all three laboratories will receive crosstraining in each lab. We will use this collaborative approach to develop a comprehensive MCell computer model of the presynaptic transmitter release site that will significantly increase our understanding of the relationship between active zone organization and synaptic function. This insight will not only lead to a better understanding of presynaptic mechanisms of homeostatic plasticity but also aid in our understanding of synaptic diseases, which are known to underlie a large number of neurological disorders. Intellectual Merit: A significant number of neurological diseases are known to affect the synapse by targeting synaptic organization and function. While most research on this important topic has to date focused on postsynaptic adaptations, it has become increasingly clear that presynaptic homeostatic changes are likely to be just as important. Thus, a better understanding of the role of presynaptic structure and organization in synaptic function under both control and disease conditions is needed. Broader Impacts: The MCell model that we will develop will enhance our teaching mission in many ways. It will provide an example of unprecedented scale and realism for the illustration of nerve terminal structure and function. This material will be used in courses and programs at the University of Pittsburgh, the University of Maryland, and Carnegie Mellon University. These include undergraduate and graduate Neuroscience courses, a Computational Biology PhD program that spans PITT and Carnegie Mellon University, summer workshops, and web-based tutorials (www.mcell.org). These simulations will expand previous models that already have been converted into instructive 3D movies, which are routinely shown to a broad range of audiences during open houses, student visits or classroom teaching. This work will also provide source material for teaching examples tailored to high school outreach programs at the Pittsburgh Supercomputing Center, particularly the CMIST program (Computational Modules in Science Teaching, www.cmist.org) of the National Resource for Biomedical Supercomputing (NRBSC) directed by Dr. Dittrich. Our proposed work will have a broad impact on K-12 education, undergraduate teaching and training, graduate and post-graduate training, community outreach, STEM teaching, training at underrepresented minority institutions, and knowledge of synaptic function in the field. Dr. Meriney is a member of the Neuroscience outreach committee at the University of Pittsburgh (PITT), which organizes a variety of community events. Dr. Meriney's laboratory is in the Arts and Sciences College, so the proposed research would contribute to undergraduate teaching via undergraduate research participation in the proposed work, and changes to content for undergraduate courses based on new research insights. Dr. Dittrich will also train undergraduate students in his laboratory as participants in the proposed work. He is training faculty in the NSF funded TECBio REU program at the PITT and typically mentors 1-2 students in computational projects as part of the program. In addition, Dr. Dittrich is a training faculty in the PA Governors School for the Sciences, an intense summer program for talented high school students in Pennsylvania. Drs. Dittrich, Meriney, and Blanpied will bring graduate researchers and postdoctoral fellows into their labs who will directly participate in the proposed experiments, receive cross training in all three laboratories, and receive career training. Lastly, Dr. Ulises Ricoy (an under-represented minority faculty member) from Northern New Mexico College will visit during each summer to learn new research, teaching, and training tools to bring back to underrepresented minority undergraduates at Northern New Mexico College. This will expose these underrepresented minority students to an intense academic research environment and aid in their training and career planning.

? DESCRIPTION (provided by applicant): The objective of this proposal is to elucidate the molecular mechanisms that cause denervation of aged neuromuscular junctions (NMJs) and how exercise ameliorates NMJ denervation in aging. In the elderly, progressive denervation of NMJs decreases neuromuscular function, decreases quality of life from frailty, and increases the risk of falling and fractures. However, there are knowledge gaps about the molecular mechanisms that underlie NMJ denervation in aging and how exercise ameliorates this condition. Our long-term goal is to elucidate these mechanisms and to identify new intervention strategies to improve neuromuscular function in the elderly. Here, we hypothesize that an age-related loss of synaptic vesicle release sites - active zones - causes NMJ denervation, and that exercise ameliorates this denervation by restoring active zones at the aged NMJs. This hypothesis has been formulated on the basis of our published and preliminary data that strongly suggest that age-related loss of active zone organizer laminin ?2 causes active zone depletion and NMJ denervation, and that exercise restores active zones and NMJ innervations by increasing laminin ?2 protein expression. Furthermore, these data are firmly supported by our published studies showing the molecular mechanism that organizes NMJ active zones involves interactions between laminin ?2, a specific receptor for laminin ?2 (presynaptic P/Q-type voltage-dependent calcium channels, VDCC), and the active zone protein Bassoon. The specific aims for testing the hypothesis are as follows: (1) Elucidate molecular defects that cause denervation of aged NMJs and how exercise ameliorates the defects. Aged and exercised aged rodents will be analyzed using confocal + super resolution microscopy; (2) Test the hypothesis that exercise maintains aged NMJs by increasing laminin ?2 expression level using transgenic mice; and (3) Evaluate systemic effects of a novel calcium channel agonist on neuromuscular function of aged mice using electrophysiology, electromyography, and behavior tests. This project is innovative because it is based on a novel, untested concept, namely the causality of active zone integrity in NMJ denervation. It is thus distinct from current aging research approaches. Another innovation is the testing of a novel calcium channel agonist specific for the P/Q-type VDCC that was invented by the Co-PI. The significance of this project is that it will yield: (i) the molecular mechanisms of NMJ denervation in aging, (ii) the exercise activated mechanism that ameliorates NMJ denervation in aging, and (iii) a VDCC agonist that may function as an intervention for age-related neuromuscular dysfunction and potentially as an exercise mimetic. Cross-disciplinary investigators from developmental and aging neurobiology, electrophysiology, gerontology, imaging, nephrology, and physiology fields have been assembled for this project.

Nerve cells communicate with each other using travelling electrical pulses called action potentials. These pulses arrive at the end of nerve cells (at structures specialized for chemical communication with neighboring nerve cells called synapses or terminals), where they can trigger electrical pulses in neighboring nerve cells. Despite the fact that these communication events are crucial to everything that the nervous system does, and can be compromised by neural diseases, we know surprisingly little about what shapes the effectiveness of these electrical pulses at synapses, and how diseases change this process. This project uses nerves that cause muscles to contract as a model, and combines physiology and pharmacology measurements in nerve terminals with microscopy to determine the density and distribution of functionally-important proteins. These details are used to development a new computer modeling approach that uses structural and functional information to produce detailed models of electrical pulse generation. The new data and models that project produces will advance basic scientific knowledge about synapse function, and enhance our understanding of the mechanisms that underlie neural disease. The proposed work will also have a broad impact on K-12 education, undergraduate teaching and training, graduate and post-graduate training, community outreach, and science training at under-represented minority institutions.

The presynaptic events that control transmitter release at synapses are incompletely understood, particularly with respect to the role of various ion channels positioned with transmitter release sites (active zones). We hypothesize that the structure-function relationships between active zone ion channels regulates the presynaptic action potential waveform within healthy synapses, and that this relationship is disrupted in disease states. We will approach these issues using a collaborative team of investigators from four universities using an approach broken into four aims: (1) voltage imaging to characterize the shape of the presynaptic action potential, including the effects in disease model synapses, (2) patch clamp measurements of the effects of action potential waveforms on ionic currents, (3) characterization of the density and distribution of presynaptic ion channels in motor nerve terminals using super-resolution imaging, and (4) using a combination of data from prior studies with those collected here, we will develop a novel modeling approach that combines modeling ion channel activation and ion flux in a realistic nerve terminal environment with a voltage simulator that predicts the effects of these ion fluxes on the shape of presynaptic action potentials. The proposed studies will advance basic science issues related to presynaptic function and also enhance understanding of the mechanisms that underlie neuromuscular diseases. Our proposed work will also have a broad impact on K-12 education, undergraduate teaching and training, graduate and postgraduate training, community outreach, training at under-represented minority institutions, and fundamental knowledge about synaptic function.

This grant was cofunded by the Cellular Dynamics and Function Cluster in the Division of Molecular and Cellular Biosciences, and the Division of Emerging Frontiers in the Directorate for Biological Science.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.