R35NS127219

Fusion Pores in Endocrine and Synaptic Exocytosis - Project Summary/Abstract

Neurons and endocrine cells release signaling molecules through Ca2+-triggered exocytosis. Ca2+ enters a nerve terminal or endocrine cell, binds to a Ca2+ sensor protein, and triggers the fusion of vesicle and plasma membranes to expel neurotransmitters and hormones. To investigate the mechanisms of exocytosis, our research focuses on fusion pores and Ca2+. Ca2+ triggers the opening and evolution of the fusion pore; the fusion pore is an aqueous passage between the vesicle interior and cell exterior. All secreted molecules pass through a fusion pore, which is strategically situated to exert finely tuned control over secretion.

We use biophysical techniques to probe fusion pores at the single-pore level, track their transitions, and monitor their responses to biological signals. Studies of the fusion pore have given us valuable insights into the roles of specific proteins in the control of exocytosis. We showed that SNARE protein transmembrane domains alter flux through initial fusion pores in both endocrine and synaptic exocytosis. We have made important advances in understanding the nascent fusion pores of endocrine exocytosis, but progress has been slow in understanding endocrine fusion pore expansion and how fusion pores impact synaptic transmission. Innovations from this laboratory have created opportunities to take on these new challenges.

Project 1: Late-Stage Endocrine Fusion Pores
We have developed a new method for analyzing amperometric recordings to probe the dynamics of late-stage endocrine fusion pores. This method tracks fusion pore permeability as vesicles lose catecholamine and led to the novel findings that a fusion pore sequentially expands, contracts, and settles into a metastable state. We will use measurements of late-stage fusion pores to address long-standing questions about the biological control of secretion. We will probe late-stage fusion pores for control by lipid bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin.

Project 2: Synaptic Fusion Pores
To study synaptic fusion pores, we developed a co-culture system with neurons and HEK293 cells expressing four postsynaptic proteins: neuroligin 1, GLUA2, stargazin, and PSD95. These HEK cells serve as sensors of synaptic release, yielding miniature synaptic current data of exceptional quality in which fusion pore contributions are more clearly resolved. In parallel with Project 1, we will use HEK cell-neuron co-cultures to determine how synaptic fusion pores are controlled by bilayer elasticity, Ca2+, synaptotagmins, and synaptophysin/dynamin. The results on endocrine and synaptic fusion pores will be synthesized into a comprehensive framework for regulated secretion. We will then adapt this co-culture system to the study of synaptic kiss-and-run and presynaptic contributions to synaptic plasticity.

Project 3: Human Stem Cell-Derived Neurons
We will adapt HEK cell synaptic sensors to the study of synaptic release from neurons derived from human stem cells. Collaborators have been recruited to provide neurons, which we will use to evaluate synaptic release and fusion pores in Down syndrome, fragile X mental retardation, aging, Parkinson's disease, and tuberous sclerosis complex. This work will provide insight into the molecular mechanisms of exocytosis, illuminate its molecular control, and show us how synaptic release goes awry in diseases.

University Of Wisconsin System

(1) TO SUPPORT EXTRAMURAL RESEARCH FUNDED BY THE NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE (NINDS) INCLUDING: BASIC RESEARCH THAT EXPLORES THE FUNDAMENTAL STRUCTURE AND FUNCTION OF THE BRAIN AND THE NERVOUS SYSTEM, RESEARCH TO UNDERSTAND THE CAUSES AND ORIGINS OF PATHOLOGICAL CONDITIONS OF THE NERVOUS SYSTEM WITH THE GOAL OF PREVENTING THESE DISORDERS, RESEARCH ON THE NATURAL COURSE OF NEUROLOGICAL DISORDERS, IMPROVED METHODS OF DISEASE PREVENTION, NEW METHODS OF DIAGNOSIS AND TREATMENT, DRUG DEVELOPMENT, DEVELOPMENT OF NEURAL DEVICES, CLINICAL TRIALS, AND RESEARCH TRAINING IN BASIC, TRANSLATIONAL AND CLINICAL NEUROSCIENCE. THE INSTITUTE IS THE LARGEST FUNDER OF BASIC NEUROSCIENCE IN THE US AND SUPPORTS RESEARCH ON TOPICS INCLUDING BUT NOT LIMITED TO: DEVELOPMENT OF THE NERVOUS SYSTEM, INCLUDING NEUROGENESIS AND PROGENITOR CELL BIOLOGY, SIGNAL TRANSDUCTION IN DEVELOPMENT AND PLASTICITY, AND PROGRAMMED CELL DEATH, SYNAPSE FORMATION, FUNCTION, AND PLASTICITY, LEARNING AND MEMORY, CHANNELS, TRANSPORTERS, AND PUMPS, CIRCUIT FORMATION AND MODULATION, BEHAVIORAL AND COGNITIVE NEUROSCIENCE, SENSORIMOTOR LEARNING, INTEGRATION AND EXECUTIVE FUNCTION, NEUROENDOCRINE SYSTEMS, SLEEP AND CIRCADIAN RHYTHMS, AND SENSORY AND MOTOR SYSTEMS. IN ADDITION, THE INSTITUTE SUPPORTS BASIC, TRANSLATIONAL AND CLINICAL STUDIES ON A NUMBER OF DISORDERS OF THE NERVOUS SYSTEM INCLUDING (BUT NOT LIMITED TO): STROKE, TRAUMATIC INJURY TO THE BRAIN, SPINAL CORD AND PERIPHERAL NERVOUS SYSTEM, NEURODEGENERATIVE DISORDERS, MOVEMENT DISORDERS, BRAIN TUMORS, CONVULSIVE DISORDERS, INFECTIOUS DISORDERS OF THE BRAIN AND NERVOUS SYSTEM, IMMUNE DISORDERS OF THE BRAIN AND NERVOUS SYSTEM, INCLUDING MULTIPLE SCLEROSIS, DISORDERS RELATED TO SLEEP, AND PAIN. PROGRAMMATIC AREAS, WHICH ARE PRIMARILY SUPPORTED BY THE DIVISION OF NEUROSCIENCE, ARE ALSO SUPPORTED BY THE DIVISION OF EXTRAMURAL ACTIVITIES, THE DIVISION OF TRANSLATIONAL RESEARCH, THE DIVISION OF CLINICAL RESEARCH, THE OFFICE OF TRAINING AND WORKFORCE DEVELOPMENT, THE OFFICE OF PROGRAMS TO ENHANCE NEUROSCIENCE WORKFORCE DEVELOPMENT, AND THE OFFICE OF INTERNATIONAL ACTIVITIES. (2) TO EXPAND AND IMPROVE THE SMALL BUSINESS INNOVATION RESEARCH (SBIR) PROGRAM, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, TO INCREASE SMALL BUSINESS PARTICIPATION IN FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION. TO UTILIZE THE SMALL BUSINESS TECHNOLOGY TRANSFER (STTR) PROGRAM, TO STIMULATE AND FOSTER SCIENTIFIC AND TECHNOLOGICAL INNOVATION THROUGH COOPERATIVE RESEARCH AND DEVELOPMENT CARRIED OUT BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO FOSTER TECHNOLOGY TRANSFER BETWEEN SMALL BUSINESS CONCERNS AND RESEARCH INSTITUTIONS, TO INCREASE PRIVATE SECTOR COMMERCIALIZATION OF INNOVATIONS DERIVED FROM FEDERAL RESEARCH AND DEVELOPMENT, AND TO FOSTER AND ENCOURAGE PARTICIPATION OF SOCIALLY AND ECONOMICALLY DISADVANTAGED SMALL BUSINESS CONCERNS AND WOMEN-OWNED SMALL BUSINESS CONCERNS IN TECHNOLOGICAL INNOVATION.

93.853 - Extramural Research Programs in the Neurosciences and Neurological Disorders

National Institute of Neurological Disorders and Stroke (NNDS) [HHS - NIH]

Madison, Wisconsin 53715 United States

Single Zip Code

Research Program Award (R35 Clinical Trial Optional) (RFA-NS-21-020)

Amendment Since initial award the total obligations have increased 444% from $671,036 to $3,648,126.