Timothy A. Ryan - US grants

DESCRIPTION: One of the major forms of communication between neurons in the brain is that which occurs at chemical synapses. The control of synaptic communication is a crucial means by which the nervous system directs information flow in the brain. Understanding the detailed physiology of synaptic terminals in the central nervous system (CNS) will thus be critical to determining the nature of this control in both normal and diseased states of brain function. The long term objectives are to determine the mechanisms of control of synaptic transmission in the CNS. Traditional electrophysiological characterization gives a complex picture of a mixture of presynaptic and postsynaptic properties from an indeterminate number of synapses. Individual synaptic terminal properties remain poorly characterized. The optical tracer dye FM 1-43 labels synaptic vesicles specifically during membrane recycling; this allows one to cleanly separate presynaptic contributions to synaptic function and to obtain detailed information of presynaptic physiological parameters at the single synapse level. Three specific aims are proposed: Characterize the control of synaptic vesicle exocytosis by extracellular Ca2+ in individual synaptic terminals during action potential stimulation and determine the role of specific Ca2+ channels in determining this control. Determine the maximal amount of exocytosis per action potential at individual synapses and how it is controlled. Examine the role of synaptotagmin-1 in the control of Ca2+-mediated exocytosis by measuring unitary presynaptic properties in neurons derived from synaptotagmin deficient mice. This work should lead to a much better understanding of the functioning of synaptic machinery, the major target of most therapies of CNS disorders such as epilepsy, depression, and schizophrenia.

[unreadable] DESCRIPTION (provided by applicant): The Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional Training Program in Chemical Biology comprises combined faculty from Cornell University (CU) in Ithaca , NY and the Weill Medical College of Cornell University (WCMC), The Rockefeller University (RU), and the Sloan-Kettering Institute (SKI), the research arm of the Memorial Sloan-Kettering Cancer Center (MSKCC) in New York City (NYC)- the latter group of three biomedical research and educational institutions are in close geographic proximity. [unreadable] [unreadable] The main theme of our proposed project is to create a new training environment that will be specifically tailored to train a future generation or scientists with expertise at the interface of chemistry and the biomedical sciences. This new training environment, referred to as the Training Program in Chemical Biology (TPCB) attempts to address the challenges of melding two disciplines with diverse scientific cultures and approaches, i.e., that of chemistry with that of biomedical research. In order to maintain and insure the rigorous training typical of graduate programs in chemistry, students that participate in this program must first be admitted to the graduate program in Chemistry at Cornell in Ithaca and complete an expanded and accelerated schedule of graduate classes in the first year. During the summer preceding their first year of classes as well as during the winter-break period of the first year, students carry out short research internships in biomedical research laboratories at the New York City participating institutions. The second year of training takes place on the campuses of the New York City participating institutions and gives the students training in cutting edge aspects of biomedical research. In addition to course and lab work in each location, students are also exposed to a series of research seminars to give them maximal exposure to the research environments at the 4 institutions. [unreadable] [unreadable] The program has recruited its third class of entering students bringing the current enrollment to 25 students. The goal of the program is to train chemists in a biomedical science environment, not merely to have students switch from chemistry to biology. The need to examine biomedical problems with the mindset of a chemist is lever more pressing in the post-genomics era. The goal of this project is to help meet the future needs defined in terms of these challenging research problems by training students simultaneously in these two different research disciplines. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This application is for support of collaborative research conducted by Dr. Timothy Ryan (U.S. Principal Investigator) and Dr. Felipe Barros (Foreign Collaborator; Chile). The U.S. P.I. has a long standing interest on the regulation of vesicle traffic in synaptic terminals, and how this traffic impacts synaptic function. The current proposal extends the breadth of his investigations by considering how synaptic activity is matched by glucose transport and how the metabolic load is shared between neurons and their surrounding astrocytes. The Foreign Collaborator, Dr. Felipe Barros, has experience on the mechanisms of glucose transport activation by metabolic stress and has recently developed methods based on confocal microscopy for realtime measurement of hexose transport in single astrocytes and neurons. The proposal, based on fluorescence imaging, will combine the know how of the US laboratory on synaptic transmission with the expertise of the Chilean laboratory on glucose transport. It is expected that this research will shed new light on the molecular mechanisms that underlie functional imaging of the brain in vivo. [unreadable] [unreadable]

Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function[unreadable] and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal[unreadable] projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic[unreadable] terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales[unreadable] than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may[unreadable] well be different than for those more typical "fast" synapses. Here we propose to examine details of the[unreadable] presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to[unreadable] characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic[unreadable] veshicles. We will make use of technologies previously developed in the lab to examine many aspects of[unreadable] the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures.[unreadable] These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family[unreadable] members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative[unreadable] information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures[unreadable] of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this[unreadable] initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic[unreadable] vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates,[unreadable] replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well[unreadable] as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion[unreadable] directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter[unreadable] release.

Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may well be different than for those more typical "fast" synapses. Here awe propose to examine details of the presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic veshicles. We will make use of technologies previously developed in the lab to examine many aspects of the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures. These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates, replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter release.

Our long-term objectives are to determine the sub-cellular processes, and their molecular substrates, which control the efficiency of synaptic transmission in the central nervous system (CMS). Our approach is to use methods that allow us to isolate and study distinct steps in the cycling of synaptic vesicles in the presynaptic terminal. Our previous work demonstrates the usefulness and applicability of a number of different optical probes, including the fluorescent tracer FM 1-43 and the more recently-developed pH-based sensor of synaptic activity, svnapto-DHIuorin. for studying synaptic vesicle recycling in single presynaptic terminals in dissociated neuronal cell cultures. Here we propose to examine several aspects of the coupling of exocytosis and endocytosis and how it is controlled using biophysical, molecular and genetic means. Our working hypothesis is that the proper functioning of the complete synaptic vesicle cycle is critical to coordinating information flow in the brain. Because of its cyclic nature, modulation at any point in the synaptic vesicle cycle could prove important in controlling synaptic efficacy. Similarly, dysfunction of any of the steps in the vesicle cycle, such as might arise in specific mutations in diseased states, could lead to impairment of synaptic transmission. One of the critical steps in the synaptic vesicle cycle is the endocytic retrieval of synaptic vesicle membrane and proteins for future reuse. We propose 4 specific aims to examine how endocvtosis is controlled and how different molecules participate in endocvtosis for different types of physiological stimuli. 1) Examine the role of dynamin-1 in synaptic vesicle endocytosis at CMS nerve terminals 2) Examine the role of synaptojanin-1 in synaptic vesicle endocytosis at CMS nerve terminals 3) Examine the role of clathrin in in synaptic vesicle endocytosis at CMSnerve terminals 4) Examine the role of different endocytic adaptors as well as the importance of sorting motifs and interactions with synaptophysin in controlling synaptic vesicle membrane protein recapture and endocytosis.

DESCRIPTION (provided by applicant): Synapses represent key transduction machines that convert incoming electrical information in the form of action potentials into a secreted chemical message which in turn is converted back into a postsynaptic electrical response. The orchestration of these events is thought to underlie critical mechanisms of learning and memory, and dysfunction of synaptic communication is suspected to be central in a number of diseased states of brain function. Synapses however are located at significant distances from cell bodies, and the highly-localized cell biological machinery must rely on local sources of ATP for function. In addition to the ion pumps that are present on both pre and postsynaptic membranes that work to restore ionic balance following activity both compartments contain high concentrations of proteins that must consume ATP in their role carrying out signal transduction, as well as key membrane trafficking events delivering and retrieving proteins and lipids to and from the plasma membrane. Because of these high energy needs, a large fraction of nerve terminals and postsynaptic dendrites are endowed with local mitochondria. Little is known however about intracellular ATP concentrations at these sites, how these concentrations are impacted by synaptic activity, how metabolic needs are coupled to activity, how the presence of local mitochondria impacts local ATP levels, or the extent to which local synaptic ATP generation relies on glycolysis versus oxidative phosphorylation. A local direct reporter of ATP levels is required to access this information. Given that mitochondrial dysfunction has been implicated in a number of neurodegenerative diseases, the ability to directly measure ATP concentration dynamics at individual nerve terminals will be valuable for examining ATP metabolism in these diseased states as well. Here we propose to develop imaging methodology to help fill this information gap. Our approach will be to develop a combined chemo-luminescence and fluorescence approach in the form of a genetically encoded and synaptically targeted ATP indicator that will provide calibrated, dynamic, intracellular synaptic ATP concentration profiles in living nerve terminals. PUBLIC HEALTH RELEVANCE: Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. The functioning of the human brain relies on the careful orchestration of delivering neurotransmitter-laden vesicles to sites at nerve terminals where they can be used to deliver this chemical message on demand. Many known genetic mutations in diseases such as Parkinson's disease, migraine headache and schizophrenia are linked to proteins that control synapse function. Our work is aimed at understanding the machinery at a molecular level to better ensure the success of future therapies for these types of neuronal diseases. Here we are proposing to develop technologies that will allow us to examine how synapses regulate their energy supply, which is thought to be a critical area of malfunction in certain neurological diseases.

DESCRIPTION (provided by applicant): Information flow in the brain is mediated by transduction of electrical information into chemical information and back again at chemical synapses. Synapses are made up of crucial cellular machineries that orchestrate a balance of membrane traffic to and from the plasma membrane. Our goal is to develop detailed quantitative understanding of the synapse both in terms of physiological responses to action potential stimuli as well as the molecular underpinnings of its function. One of the most important functional elements is the voltage-gated calcium channel as it converts the electrical signal into a flux of calcium that drives neurotransmitter release in a highly non-linear fashion. We recently developed sensitive approaches that allow us to characterize key properties of these channels and the electrical signal that controls them in their native environment, the nerve terminal. The goal of this project is determine the molecular basis of key mechanisms that determine VGCC function. The first aim will examine the mechanisms of a novel form of adaptive plasticity whereby changes in VGCC number at nerve terminals in turn changes the shape of the electrical signal (the action potential). These experiments make use of an emerging technology of genetically-encoded fluorescent voltage indicators that allow one to quantitatively measure the action potential waveform in the nerve terminal and how it is controlled. Our second Aim will use another new technology allowing us to determine how long VGCCs typically stay resident in the active zone, whether they can recycle through an intracellular presynaptic compartment and how these dynamics are controlled by activity, calcium influx and a number of presynaptic proteins. The third Aim will examine how different active zone proteins, in particular Munc13 and Rim1, control different functional and cell biological aspects of VGCC function at nerve terminals.

DESCRIPTION (provided by applicant): Synapses represent key transduction machines that convert action potential-based signals into secreted chemical messages which in turn are converted back into postsynaptic electrical responses. Modulation of these processes is thought to underlie critical mechanisms of learning and memory, and dysfunction of synaptic communication is suspected to be central in a number of diseased states of brain function. It has long been known that the critical trigger for neurotransmitter release is the opening of voltage-gated calcium channels within the presynaptic terminal which in turn leads to the influx of calcium. The highly-non-linear relationship between calcium entry and exocytosis efficiency places the control of calcium channel function and abundance as a potent potential leverage point in sculpting synaptic strength. We recently demonstrated that expression of a calcium channel subunit, alpha2delta, is rate-limiting in determining how many calcium channels are present at nerve terminals in hippocampal neurons. Our work showed that it acts at 2 distinct molecular steps: it acts in a forward trafficking step to allow calcium channels to traffic to synapses and it acts locally at nerve terminals to allow channels to function at the presynaptic membrane. This second step requires the integrity of a predicted domain within alpha2delta that encodes a metal-ion-dependent adhesion site. In many other proteins this domain confers binding to an extracellular partner. We predict that proper alpha2delta function requires interaction with an as-yet-discovered binding partner on the synaptic surface. The goal of this proposal is to use biochemical approaches to identify this(ese) binding partner(s).

Abstract Age-related neurodegenerative diseases place a substantial and increasing socioeconomic burden on society. Age-related dementias including Alzheimer?s disease represent some of the greatest unmet medical challenges facing the aging population in the US. To date clinical interventions for these diseases have had very modest impact despite major efforts to develop new therapeutics. This landscape suggests that we are still missing fundamental information regarding the root cause of these diseases and the specific cellular vulnerabilities that lead to disease progression. We propose that a critical element of theses disease may relate to local synaptic metabolism. The brain is highly vulnerable from a metabolic point of view: severe hypoglycemia results in overt and severe neurological problems including delirium and coma. Furthermore, as we age (and aging is the strongest correlate of all these afflictions) the efficiency with which we can deliver fuel to tissues (including the brain) and convert this fuel into the useful biochemical currency, the high-energy intermediate adenosine tri-phosphate (ATP), both degrade. Although these neurodegenerative disorders ultimately lead to neuronal death it is thought that much earlier symptomatic problems arise from synaptic dysfunction. My laboratory recently discovered that nerve terminals represent one of the likely loci of the brain?s metabolic vulnerability: they consume large amounts of ATP but store little rapidly usable high-energy molecules and must therefore locally synthesize ATP to maintain function. We also discovered that synapses relay on several mechanisms to upregulate ATP that are essential for synapse function. Additionally, we discovered resting nerve terminals consume large amounts of ATP to maintain the synaptic vesicles proton gradient but that this energy burden likely varies across neurotransmitter type. We propose to test the hypothesis that neurodegenerative diseases have a strong local metabolic component by examining how genetic drivers of neurodegenerative disease specifically impact the local metabolic balance and do to determine if this might be a driver of disease-driven synapse impairment. Although certain neurodegenerative diseases disease initially present with other overt symptoms (for example movement disorders) over time they most frequently convert to dementias in the majority of patients. Here using quantitative approaches we will determine how nerve terminals in a metabolically vulnerable neuron population rely upon glycolysis versus oxidative phosphorylation to support function, examine if maintaining the vesicle proton gradient places a large energetic burden on the nerve terminals (Aim1), determine if the disease mutations associated with mitochondrial integrity specifically impact the balance of ATP (AIM2) and determine if a number of other known disease associated mutations increase metabolic vulnerability by altering the local balance of ATP production versus consumption in this critical neuron population (Aim3). The lessons and insights learned from these studies should then prove valuable in informing the pathology of a larger class of dementias.