Ege T. Kavalali, Ph.D. - US grants

DESCRIPTION (provided by applicant): Synaptobrevin-2/VAMP-2 (syb2) is an abundant synaptic vesicle protein essential for normal synaptic transmission in the brain. Syb2's interaction with the plasma membrane proteins, syntaxin 1 and SNAP-25, is critical for synaptic vesicle fusion and neurotransmitter release. These proteins are collectively called SNAREs (acronym for soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and belong to a family of proteins that mediate vesicle trafficking and fusion in the secretory pathway in eukaryotes. Despite extensive progress in the characterization of molecular interactions among SNAREs and their role in fusion, their precise role in synaptic vesicle trafficking events after fusion remains elusive. Rapid coupling of vesicle fusion and retrieval during neurotransmission have led us to hypothesize that SNARE proteins that drive rapid Ca2+ dependent fusion may also be responsible for ensuring rapid synaptic vesicle retrieval. Indeed, our studies in the previous grant period have revealed an essential role for syb2 in rapid synaptic vesicle endocytosis. Moreover, our initial results suggest that this function of syb2 may not be shared by SNAP-25. This observation suggests a specific role for syb2 in ensuring faithful coupling between exocytosis and endocytosis. In the next award period, we aim to investigate the role of syb2 and related v-SNAREs in exo-endocytic coupling, synaptic vesicle trafficking after endocytosis as well as fusion pore regulation using a powerful combination of fluorescence imaging, electrophysiology and electron microscopy. For this purpose, we propose three aims. In the first aim, we will define the role of v-SNAREs in coupling exocytosis and endocytosis via monitoring trafficking of fluorescently-tagged v-SNAREs synaptic vesicle proteins. In the second aim, we will determine the function of v-SNAREs in postendocytic trafficking of synaptic vesicles by detecting uptake and release fluorescent probes, monitoring neurotransmitter release and electron microscopy. Lastly, we will determine the impact of v-SNAREs on unitary neurotransmission and glutamate release kinetics using optical and electrophysiological measures. Collectively, these experiments will elucidate the degree of overlap between the fusion machinery and endocytic machinery in central synapses and the role of SNAREs in directing synaptic vesicle trajectories during retrieval, vesicle reuse as well as neurotransmitter release. Information attained from these studies will provide new insight to the synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia. PUBLIC HEALTH RELEVANCE: The experiments proposed for this project present a systematic and comprehensive effort to address the role of key SNARE molecules in the regulation of synaptic vesicle fusion, retrieval and recycling. Currently, a thorough analysis of the role of SNAREs in synaptic vesicle trafficking beyond vesicle fusion is lacking. In this project, we aim to establish the basic principles of SNARE-dependent regulation of synaptic vesicle trafficking in mammalian central synapses. Information attained from these studies will provide new insight to the molecular synaptic substrates that may be affected by a number of in neuropsychiatric and neurological disorders including mental retardation, autism and schizophrenia.

DESCRIPTION (provided by applicant): In response to high frequency action potential firing synapses exhibit extensive depression. This common form of synaptic plasticity brings computational advantage to synaptic circuits by increasing the sensitivity of neurons to subtle temporal changes in synaptic inputs. This depression is thought to be a direct outcome of the dynamics of vesicle recycling and vesicle depletion in presynaptic terminals. However, a clear mechanistic link between the dynamics of synaptic vesicle cycle and phases of synaptic depression is still lacking. The main goal of this project is to bridge this gap using a powerful combination of electrophysiology, optical imaging, electron microscopy and molecular biology in hippocampal synapses. Our studies have recently shown that synaptotagmin7 forms a molecular switch controlling the rate of vesicle recycling in a bi-directional manner through its alternative splice variants. This observation sets the stage for studies aimed at understanding the exact relationship between synaptic vesicle recycling and synaptic output. To fulfill this goal three specific aims are proposed. In the first specific aim, the regulation of vesicle recycling and synaptic depression by activity and second messengers, such as calcium and diacylglycerol, will be studied using fluorescent measurement of vesicle recycling and electrophysiology. In the second specific aim, the fast and slow recycling will be molecularly dissected through overexpression of synaptotagmin7 splice variants to examine their respective roles in regulation of presynaptic dynamics and neurotransmitter release. These functional experiments will be complemented by morphological analysis of synapse structure using electron microscopy. In the third specific aim, structural elements within synaptotagmin7 that control synaptic vesicle recycling will be identified through structure-function analysis in transfected synapses. These concerted investigations will enable us to understand with increasing precision the mechanistic link between recycling of synaptic vesicles and short-term synaptic plasticity. This information will also be critical for a better understanding of the pathologies underlying several neurological and psychiatric illnesses ranging from epilepsy to schizophrenia.

DESCRIPTION (provided by applicant): The Cellular Biophysics ofthe Neuron Training Program (CBNTP) at the University ofTexas Southwestern Medical Center at Dallas (UTSW) will prepare trainees to become exceptional biophysical scientists addressing complex neuroscience questions using quantitative methods and advanced analytical tools. Transformative discoveries in this field will come from scientists who combine a deep appreciation of biophysical principles and thorough understanding of optical and electrophysiological tools with an intimate knowledge of problems in neuroscience research. The program's central goal is to train scientists who can cultivate synergistic interactions between cellular biophysicts and scientists focused on complex neuroscience problems including cellular/molecular basis of neurological, neurodevelopmental and neuropsychiatric disorders. CBNTP faculty members employ a wide range of biophysical methodologies in their research, emphasizing relevance to human health, physiology and disease. Scientific opportunities in the CBNTP ranging from mechanistic analyses of neuronal function in diverse model systems to detailed biophysical studies of neuron-relevant processes in non- neuronal cellular systems will offer uniquely exceptional prospects for training. Trainees will gain state-of-the- art expertise in experimental biophysical principals and benefit from intimate exposure to how these principles are applied in advanced neuroscience research. Students will receive rigorous formal training in membrane biophysics, quantitative analyses and cellular neurophysiology. Additionally, they will have guided interactions with established, world class role models and benefit from enrichment activities inside and outside of the institution. Students will join the CBNTP in the fall of their third year and remain as trainees for two years. Trainee selection is competitive. The program steering committee will select trainees based on prior credentials, current performance and commitment ofthe student and mentor to pursue training consistent with research goals of the program. We are requesting 2 positions in the first year, a total of 4 positions in the second and for each subsequent year of this 5-year period. RELEVANCE: Explosive growth of advanced biophysical methods now impacts almost every dimension of neuroscience research. By leveraging interactions between quantitative molecular & cellular biophysicts with neuroscientists focused on neurological, neurodevelopmental and neuropsychiatric diseases and those working on model systems, this unique training program equips students with the sophisticated skill sets needed among scientific leaders in neuroscience research.

DESCRIPTION (provided by applicant): In this application, we propose to employ bacteriorhodopsin, a light-activated proton pump from Halobacterium salinarium, to manipulate the pH gradient in synaptic vesicles. Synaptic vesicle filling with neurotransmitters is highly sensitive to intravesicular pH, which is regulated by an intrinsic vesicular proton pump, vacuolar ATPase (v-ATPase). Recent studies, including work from our group, suggests that inhibition of v-ATPase by small molecule inhibitors (e.g. bafilomycin) results in fast use-dependent rundown of synaptic responses and blockade of neurotransmitter release. In this project, we will exploit this strict pH-dependence of the synaptic vesicle refilling process by using bacteriorhodopsin targeted to synaptic vesicles in neurons to emulate the effect of v-ATPase inhibitors in a light-induced and rapidly reversible fashion without global changes in the membrane excitability. In addition, targeting of bacteriorhodopsin to other secretory organelles such as lysosomes that critically depend on the intravesicular pH for their proper operation can be a powerful tool to investigate their role(s) in neuronal function. We propose to develop this project in three stages: First, we aim to selectively target bacteriorhodopsin in a functional conformation to synaptic vesicles. Second, we will optimize light-induced proton pump activity of the vesicular bacteriorhodopsin in cultured hippocampal neurons. Finally, we will express optimized bacteriorhodopsin constructs in vivo using specific neuronal promoters in Drosophila for light-induced manipulation of Drosophila behavior. Taken together the research proposed here has significant potential in bridging synaptic functional studies in vitro and information processing in the intact brain. This approach will enable acute manipulation of synaptic inputs into a particular area of the brain to test how they may influence function as well as behavioral output.

?DESCRIPTION (provided by applicant): Synapses are key elements of communication and signal processing in the healthy brain. Furthermore, synapses are severely perturbed in several neurological and psychiatric diseases. This proposal requests support for a scientific meeting on Synaptic Transmission as part of the Gordon Research Conference (GRC) series to be held at the Waterville Valley Conference Center, New Hampshire, during the week of August 14- 19, 2016. GRC will be preceded by a Gordon Research Seminar (GRS) targeted towards graduate students and post-doctoral fellows on August 13-14 at the same location. We plan to bring together a group of scientists who all are highly interested in synaptic function, but examine synapses at different levels (molecular, cellular, and systems level), look at them from different perspectives (bottom-up, top-down), and use different approaches (molecular biology, super resolution imaging, cryo-electron microscopy, optogenetics, subcellular electrophysiology, modeling, and many others). The specific aims of the 2016 conference are (1) to include a new focus on subcellular nano-scale analysis of synapse structure and function, synapse development and synapse evolution, (2) to enhance the contribution of students, postdocs, and young faculty members, and (3) to achieve the highest possible scientific quality, while further optimizing the balance of gender, age, and nationality at all levels (speakers, discussion leaders, and short talk presenters). We envisage having ~32 speakers representing critical areas of synaptic transmission research with a total of ~150 participants for an intense five day conference in a retreat like setting. The program will have two Keynote Lectures entitled Synaptic transmission: From nanostructures to microcircuits in health and disease and eight sessions that address different aspects of synapse structure, synaptic signaling, including mechanisms of exocytosis, presynaptic terminals, synaptic spines, pre- and postsynaptic plasticity, synapse development, synapse evolution, synaptic diseases, and function of synapses in microcircuits and neuronal networks. Short talks sessions and evening poster sessions on all four days will permit all participants to contribute to these topics. The health an disease relevance of this application is substantial. Although many presentations will focus on basic science, the presented data have far-reaching implications for a wide range of devastating brain disorders, particularly neurodegenerative diseases such as Parkinson's and Alzheimer's disease, mood disorders, schizophrenia, autism spectrum disorders, mental retardation, drug addiction, and loss of peripheral sensory function, which are collectively emerging as synaptopathies. Thus, we are convinced that the results presented at this meeting will help clinical researchers to develop new therapeutic strategies for major brain diseases. We anticipate that the GRC Synaptic Transmission will have a major shaping influence on both basic and disease-related neuroscience in the next decade.

The broad, long-term objective of this multi-PI grant application is to understand the importance of calcium (Ca2+) signaling for synaptic spine dysfunction and loss in Alzheimer's disease (AD). Understanding the molecular mechanisms that lead to initial synaptic dysfunction and synapse loss in AD is essential for early disease detection as well as for the development of effective therapeutic interventions. However, primary steps that result in synaptic dysfunction and loss in AD remain poorly understood. There is an increasing consensus that alterations in neuronal Ca2+ signaling is a key contributor to the pathogenesis of AD, however, the impact of these Ca2+ signaling alterations on long term synapse homeostasis and control of synapse stability is unclear. In our studies we will focus on two major neuronal Ca2+ signaling pathways - neuronal store-operated Ca2+ entry (SOC) pathway and Ca2+ influx via NMDAR driven by spontaneous vesicle release (SVR) from the presynaptic terminals. Both of these pathways are active chronically and maintain baseline synaptic Ca2+ signals. Working independently, our two groups have identified key Ca2+ signaling pathways that are active chronically and maintain synapse stability We will investigate roles played by these pathways in synaptic maintenance and analyze dysregulation of these pathways in neurons from AD mouse models. Specifically, we will (1) investigate the role of spontaneous glutamate release-mediated NMDAR signaling in AD-related alterations in hippocampal synaptic Ca2+ homeostasis. Spontaneous glutamate release-mediated Ca2+ signaling measurements will be performed with primary neuronal cultures from presenilin 1 M146V knock-in (PS1KI) and APP knock-in (APPKI) mouse models; (2) investigate the changes in synaptic Ca2+ homeostatic mechanisms in AD neurons. We will test the hypothesis that a balance in activity of CaMKII and Calcineurin (CaN) is shifted in synaptic spines of AD neurons, leading to synaptic loss; (3) evaluate synaptic Ca2+ homeostatic mechanisms as potential target for AD treatment. Pharmacological and genetic experiments will be performed in this aim. Results obtained in our studies provide essential mechanistic information about causes of synaptic loss in AD and offer new potential therapeutic targets for treatment of AD.