Edward L. Stuenkel - US grants

A fundamental problem in the neurosciences is the characterization of the mechanism(s) controlling the release of neurotransmitters and neurohormones at nerve endings. These mechanisms are crucial to nerve cell communication and to a healthy, functioning nervous system. Although the importance of calcium's role in release has been well established, details in its action are poorly understood. This research project is designed to examine, quantitatively, the relationship between calcium currents and intracellular calcium concentrations. Moreover, receptor mediated regulation of the calcium current and its effect on intracellular calcium levels will be directly evaluated. Electrophysiological techniques combined with microspectrofluorometry of calcium sensitive, fluorescent indicators will be used to monitor intracellular calcium levels. Techniques will be developed to allow simultaneous monitoring of neurohormone release from single endings. This research will lead to a better understanding of the properties and actions of molecular components regulating neurohormone release from vertebrate nerve endings.

DESCRIPTION: (Investigator's Abstract): Many key proteins involved in membrane targeting and synaptic vesicle neurotransmitter release have been identified and a fundamental set of interactions defined and placed in a model termed the SNARE hypothesis. However, despite recent rapid progress in identifying molecular components of the membrane targeting and fusion machine, regulatory influences facilitating or inhibiting the protein interactions or the sequence of interactions remain poorly defined. It is the long range goal of the proposed research to identify and understand the regulatory mechanism(s) which govern SNARE protein interactions and, thereby, regulate neurotransmitter and neurohormone release and synaptic plasticity. Preliminary molecular/biochemical studies combined with functional studies monitoring membrane capacitance changes, as a measure of exo-endocytotic activity under whole cell patch clamp, have indicated that proteins of the Sec1 family serve an important regulatory control function. Experiments proposed will test the hypothesis that nSec1 protein (nSec1p) regulates neurosecretion via a specific regulated interaction with syntaxins and that this interaction enhances secretory granule docking. The proposed experiments will utilize nerve endings of the hypothalamo-neurohypophysial system, which possess unique anatomical and electrophysiological advantages allowing resolution of the molecular events of the secretory process at nerve endings to be studied in greater detail than at any other nerve endings. The investigators will utilize a combination of molecular, biochemical, and patch clamp techniques to characterize the regulation of nSec1 protein interactions and to analyze functional effects on Ca2+ currents and on the amplitude and kinetics of secretion with msec resolution at individual nerve endings. The specific aims are: 1) to determine the sites of phosphorylation on nSec1p and examine regulation of protein interactions by specific kinase activity and depolarizing stimuli, 2) to determine the mechanism by which nSec1p regulates secretory granule exocytosis and elucidate the effects of phosphorylation state on secretion, 3) to determine if nSec1p regulation of exocytosis utilizes the molecular mechanisms and machinery proposed by the SNARE hypothesis, and 4) to determine if nSec1p interacts with and activates cdk5, whether this interaction is regulated by nSec1p phosphorylation, and if there are functional consequences on secretion. In summary, an understanding of the mechanisms by which nSec1p regulates SNARE protein interactions may be essential to full understanding of both short and long-term processes of synaptic plasticity and memory.

Information transfer between neurons and the normal functioning of the nervous system is dependent upon the regulated release of neurotransmitter at synapses. A number of psychiatric and neurological conditions are typified by an imbalance of particular neurotransmitters. In addition, many abused and therapeutic drugs that act on the nervous system act at the level of altering synaptic transmission. The long-term objective of the proposed research is to understand molecular mechanisms which regulate neurotransmitter or neurohormone release, as it may ultimately lead to enhanced clinical treatments as well as improved drug design. Small Ras-like GTP binding proteins are key regulators of many cellular processes including morphogenesis, cytoskeletal dynamics, membrane trafficking, transformation, and protein kinase cascades. The general hypothesis to be tested is that the monomeric GTPase Rac1 is essential to secretory responsiveness in that it coordinates events critical for secretory granule availability, priming and SNARE protein interactions. A combination of molecular, biochemical and patch-clamp techniques will be used to determine the sites of Rac1 action within functionally separable stages of the secretory cycle, and to elucidate effector pathways through which it directly exerts effects on secretory responsiveness. The investigations will be performed on bovine chromaffin cells, which present an extensively studied, physiologically relevant, neuroendocrine cell model. The specific aims are: 1) To elucidate the physiological role of Rac1 on regulation of Ca2+- dependent exocytosis and to specifically determine effects on Ca2+ sensitivity, recruitment, and refilling of the readily releasable secretory granule pool, 2) To characterize the properties of Rac1 activation in response to secretory stimuli. We will also determine if IQGAP1, a Rac effector protein that binds Ca2+/calmodulin and F-actin, provides Ca2+-dependent regulation of Rac1, 3) To determine the contribution of the predominant Rac1 effector pathway, i.e. the p21 activated kinases (PAK), to Rac1 effects on secretory responsiveness. In addition, we will establish the role of p35/cyclin dependent kinase 5, an interacting protein kinase that regulates PAK activation, on Rac1 and PAK1 regulation of secretory responsiveness, and 4) To determine the role of Rac1 on phosphatidylinositol 4-phosphate 5- kinase (PIP5K) activity in resting and stimulated chromaffin cells. PIP5K is an essential co-factor for priming of secretory granules in neuroendocrine systems and directly interacts with Rac1. The work proposed attempts to provide a greater understanding of cytosolic and membrane delimited signaling pathways that exert an important regulatory influence on Ca2+- dependent secretory responsiveness.

G protein

[unreadable] DESCRIPTION (provided by applicant): The assembly of SNARE proteins into a ternary core complex is essential for neurotransmitter release. Precise regulation of SNARE complex assembly ultimately determines the site and dynamics of the exocytotic event. Our objective is to understand the mechanisms that regulate temporal and spatial assembly of these SNARE complexes. Tomosyn is a protein that is critical in setting the level effusion- competent SNARE complexes. Its regulatory action has been proposed to be primarily mediated by its interaction with the Q-SNARE syntaxinl A, which results in the formation of non-fusogenic SNARE complexes. The goal of the research proposed is to provide an understanding of the molecular mechanisms and signal transduction pathways governing the assembly/disassembly of tomosyn-SNARE complexes in the regulated release of neurotransmitter. Our general hypothesis is that tomosyn-SNARE complex formation is promoted by Rho-GTPase signaling pathways and antagonized by protein kinase A-dependent pathways, with the balance of activation of these pathways fine-tuning the level of fusion-competent SNARE complexes. Aim 1 will test the hypothesis that the formation of tomosyn-SNARE complexes is under dynamic control by secretory agonists to regulate secretion. In addition, we will identify N-terminal tomosyn domains important for this regulation, and quantity effects of endogenous tomosyn on granule priming and exocytosis. Aim 2 will test the hypothesis that the level of tomosyn-SNARE complexes and their functional effects on the exocytotic pathway are finely regulated by a balance between the activation state of Rho, to promote tomosyn-SNARE complex assembly, and PKA, to antagonize tomosyn-SNARE complex assembly. Aim 3 will define the spatial properties of tomosyn-SNARE complexes on the plasma membrane and determine if RhoA and PKA mediate spatially delimited effects on the assembly and disassembly of these complexes. Experiments will be performed using a combination of biochemical, optical and electrophysiological approaches on adrenal chromaffin cells, a highly characterized cell model for neurotransmitter release. Understanding the regulation of neurotransmitter release is essential to understanding the function of the nervous system and fundamental to development of therapeutic treatments in the many psychiatric and neurological conditions typified by an imbalance of particular neurotransmitters. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application seeks continuation of support for nine predoctoral positions on our training grant, 'Early Stage Training in the Neurosciences'. This training grant serves as the centerpiece in an integrated plan of support for pre-candidate students in the Interdepartmental Neuroscience Graduate Program at the University of Michigan. The first two years of this training program ensure that all students graduate as broadly trained neuroscientists. In the first year, students take an intensive lab course, an interdisciplinary course in the principles of neuroscience, human neuroanatomy, statistics, and a course in research responsibility and ethics, in addition to performing two laboratory research rotations. During their second year, students take elective courses, participate in a seminar course in which they give an oral presentation, complete their lab rotations and begin the initial work on the doctoral thesis. Once students complete pre-candidate training, they perform full-time doctoral research in the laboratory of one of 98 different training faculty members. Approximately half of these faculty members hold appointments in basic science departments, and the other half hold appointments in clinical departments. In both the classroom and laboratory, our students are exposed a broad range of research topics including Cellular and Molecular Neurobiology, Developmental Neurobiology and Regeneration, Clinical and Translational Neuroscience, Sensory and Computational Neuroscience, Behavioral, Affective, and Integrative Neuroscience, and Cognitive Neuroscience and Neuropsychiatry. Upon completion of their training, our graduates are poised to tackle a host of public health issues from the molecular basis of neurodegenerative disorders to brain circuit abnormalities in psychiatric disease. The University of Michigan is proud of its history of recruiting and training underrepresented minority students. The Neuroscience Graduate Program strives to increase the diversity of its trainees, and we describe our accomplishments and a detailed plan for recruiting and retaining underrepresented minorities. We also present detailed plans for the evaluation of the program and for instruction in the responsible conduct of research. This training grant is critical to the success of the Neuroscience Program's training mission. It has provided support for roughly 40% of the students admitted to the program in the last 4 years, and has supported roughly half of the current students in the program at one point in their training. The training grant supports a variety of trainee-related activities including classroom laboratory training, student travel to scientific conferences, and speakers at neuroscience symposia and colloquia. This training grant is key to our recruitment of outstanding students and their educational success. PUBLIC HEALTH RELEVANCE: This is an application to support Early Stage Neuroscience training for students in the University of Michigan Neuroscience Graduate Program. One of the oldest of its kind, this interdisciplinary training program is essential to for the development of the next generation of laboratory scientists capable of using state-of-the-art methodologies to understand the normal and disordered function of the brain and nervous system. Trainees in this program are poised to tackle a host of public health issues from the molecular basis of neurodegenerative disorders to the brain circuit abnormalities in psychiatric disease.

This project entitled "International Program for the Advancement of Neurotechnology (IPAN)" is about understanding the complexity and mysteries of the brain. It is cited by many as the biggest scientific challenge of this century. In this International Program for the Advancement of Neurotechnology (IPAN), the researchers are creating a holistic system for studying brain activity by closely integrating hardware from leading neurotechnologists with novel software from leading neuroscientists. Enabling this large-scale collaboration should accelerate the rate of discovery in neuroscience. This in turn will pave the way to improved treatments for neurological disorders and to breakthroughs in artificial intelligence in the next decade. The PIRE team will also provide advanced educational opportunities for undergraduates with the express purpose of recruiting future U.S. STEM (science, technology, engineering and mathematics) researchers. Graduate students and postdocs will also be enrolled in a unique cross-training program between neuroscience and neurotechnology laboratories. The resulting experience will prepare a new generation of globally-connected multi-disciplinary engineers and scientists while driving critical advances in neurotechnology.

IPAN is an explicit partnership of leading neuroscientists and technologists to develop and deliver a hardware and software system that fundamentally simplifies the ability of a neuroscientist to (i) identify recorded neuron types, (ii) reconstruct local neural circuits, and (iii) deliver biomimetic or synthetic inputs in a cell-specific targeted manner. This project teams the University of Michigan, New York University, Howard Hughes Medical Institute, and the University of Puerto Rico with the University of Freiburg, the University of Hamburg-Eppendorf, the Korea Institute of Science and Technology, Singapore?s Institute for Microelectronics, and University College London. Complementary strengths, world-class infrastructures, and strong student exchange programs are an important part of this IPAN team, with major thrusts in Technology, Neuroscience, and Education. The enabling technology to meet these three system goals (i-iii) will be next-generation neural probes equipped with novel optoelectronics, high-density recording interfaces, and low-noise multiplexed digital outputs. The neuroscience thrust will help define the technology from the onset and are developing novel software tools to accelerate the analysis of large neurophysiological data sets. The team includes leading system neuroscientists with unique capabilities specializing in memory, sensory, fear, and development, and will work with technologists to validate both the technology and the software tools in distinctive neuroscience applications.

Abstract: A severe health burden imposed by many neuropsychiatric and neurological diseases can be linked to limitations in, or disruption of, molecular pathways which guide development, maintenance or plasticity of synaptic connections. Importantly, as many neural circuits are persistently active the individual synaptic connections must undergo continuous and coordinated homeostatic changes to counteract continual synaptic strengthening/weakening and development of network instability. Thus, developing a functional map of sites of activity- dependent dynamic coordination of the molecules and signaling pathways that define the process is central to enabling an understanding of many CNS diseases. The work proposed is uniquely important as it will elucidate novel and yet potent molecular signaling pathways that mediate accurate and reproducible activity-dependent adaptations in synaptic efficacy. Specifically, investigations focus on understanding how post-synaptic sensing of activity via the mTORC1/BDNF signaling pathway mediates increased presynaptic neurotransmitter release during reduced excitatory input to the post-synaptic element. Investigations will test the hypothesis that tomosyn-1 is a central presynaptic target of mTORC1/BDNF/TrkB receptor signaling and that trans-synaptic adjustments in presynaptic neurotransmitter release via this signaling pathway occur via UPS regulation of tomosyn-1 protein levels. We will also define specific sites within the secretory pathway by which tomosyn exerts control over presynaptic neurotransmitter release. Commonality of this trans-synaptic mechanism will also involve comprehensive evaluation at Mossy fiber/CA3 and at CA3/CA1 synapses of hippocampal brain slices, as these circuits are known to exhibit strikingly divergent synaptic plasticity. In addition, we will characterize the E3 ligase responsible for UPS regulation of tomosyn, determine if the E3 ligase activity is sensitive to mTORC1/BDNF/TrkB signaling and if ubiqutination of tomosyn by the E3 ligase regulates tomosyn protein levels and neurotransmitter release. The investigations employ state of the art optical imaging of vesicle cycling, genetic models targeting protein/signaling function, optogenetic control of neuronal excitability and analysis of synaptic function in cultures of hippocampal neurons and hippocampal slices. Biochemical assays will quantify and establish activity dependent control on tomosyn protein via the UPS. This information will significantly advance understanding on mechanisms by which activity- dependent changes in post-synaptic mTORC1 activity coordinate spatial and temporal adjustments in presynaptic neurotransmitter release.

Abstract Our goal is to meet the nation's research needs by enhancing diversity in the neuroscience workforce. The focus will be to increase the representation of under-represented minorities (URMs) and achieve this by targeting specific challenges in transitions along the Neuroscience education and career continuum. Our four aims propose activities that focus on the four transition points relevant to our University of Michigan (UM) Neuroscience Graduate Program (NGP). Aim 1 focuses on transition from Undergraduate Institutions (UGI) to a Graduate Institution. It will build relationships with UGIs with high numbers of URMs through visits by UM NGP faculty and students to these UGI and visits to UM by UGI Program Directors with selected students. UM NGP faculty and students will attend SACNAS and ABRCMs meetings to provide information on NGPs in general and the UM-NGP in particular. There will also be a ?Preview Program? with visits to UM-NGP by URM students. A key component of Aim 1 will be a Summer Program for Undergraduate Research in Neuroscience, for rising Sophomores, Juniors, and Seniors with research in NGP faculty labs, professional development workshops as well as mentoring and networking. Aim 2 focuses on the transition into Graduate School. It will have peer mentoring workshops on topics such as communicating with mentors and imposter syndrome and workshops on time management, study skills, critical thinking, scientific writing and communication. Aim 2 will also have activities and courses on Mentorship directed to the NGP Faculty. Finally there will be workshops for NGP faculty, fellows and students on diverse cultures and their needs. Aim 3 focuses on the transition through Graduate Studies towards Ph.D.. Aim 3 will bring in speakers/mentors with diverse backgrounds from outside UM for research presentations, mentoring and networking. It will also send URM students to small focused research meetings (such as Gordon Conferences). It will send students and faculty to SACNAS and ABRCMs meetings (overlap with Aim 1) and have professional development courses/activities, workshops on rigor and ethics, mentor-student relationship as well as workshops on scientific communication. Aim 4 focuses on the transition to post-doctoral studies and/or neuroscience related careers. Activities will include: workshops and speakers representing a wide range of traditional and non-academic career opportunities; Alumni Days which focus on interactions with UM-NGP alumni; Opportunities for internships in neuroscience related companies; Certificate Programs such as in teaching; and sending students to small meetings (overlap with Aim 3). The proposed programs have all been successfully piloted by the UM NGP and have resulted in a 40% rate of diversity in our 2016 class of 18 students, increasing to 58% in our incoming 2017 class of 14 students. We believe our success in pilot programs and the proposed Aim 1-4 activities can make us a model program for addressing URM challenges in navigating the NGP training continuum to a successful conclusion. Evaluation will be accomplished through use of an independent agency and will generate publications. 1