Mathew C. Tantama - US grants

? DESCRIPTION (provided by applicant): Extracellular ATP mediates purinergic signaling throughout the nervous system, and purinergic signaling mechanisms have been associated with multiple brain pathologies including stroke, aging-related neurodegenerative diseases, and epilepsy. Therefore, methods to accurately and precisely detect extracellular ATP are essential to the study of its physiological role. To this end, our broad goal is to develop a set of quantitative optical tools to image and study the signaling dynamics of extracellular ATP in real-time at central synapses. In particular, evidence in recent decades suggests that astrocytes modulate synaptic transmission and plasticity through activity-dependent release of gliotransmitters that include extracellular ATP. Spillover of neurotransmitters during synaptic activity can activate G-protein coupled receptors on perisynaptic astrocytes. It is proposed that this activation of astrocyte receptors elicits a calcium response that can lead to release of ATP as a gliotransmitter. Subsequently, the released ATP or its metabolite adenosine can bind to and activate neuronal purinergic receptors, causing either homosynaptic or heterosynaptic neuromodulation. Thus, extracellular ATP released from astrocytes might act as a feedback signal to modulate synaptic efficacy and network behavior. However, there is an unmet need for new analytical tools to measure extracellular ATP, and the limitations of current detection methods have impeded the resolution of important questions regarding the mechanisms and physiological relevance of ATP as a gliotransmitter. To meet this need, the central hypothesis of this proposal is that genetically-encoded fluorescent biosensors can be engineered to sense extracellular ATP with sensor properties suitable for studying physiologically relevant purinergic signaling. We will test our hypothesis in two working aims: Aim 1 is to engineer cell surface-tethered biosensors to quantitatively image ATP release and clearance; Aim 2 is to use these new biosensors to measure activity- dependent ATP release and clearance in primary cultures of astrocytes and neurons as well as in brain slices. Upon completion of these aims, we will be able to provide both new optical tools for measuring extracellular ATP and imaging protocols that are of broad use to the purinergic signaling community.

? DESCRIPTION (provided by applicant): Neuropeptides are neuromodulators that regulate the physiology of cells, synapses, and neural circuits in the brain. For example, opioid neuropeptides are involved in pain and analgesia, and altered opioid neuropeptide levels have been observed in addiction, depression and anxiety, as well as a number of other neurological disorders including Parkinson's disease. Clearly, neuropeptides play a significant role in modulating behavior and cognition, but many aspects of neuropeptide signaling are not well understood. Unlike classical fast synaptic transmitters such as glutamate and GABA, neuropeptides can be released both at the synapse and outside the synapse. Neuropeptides can also diffuse significant distances from where they were released. At distant sites, neuropeptides can continue to function as signals at low concentrations by activating high affinity G-protein coupled receptors. Thus, neuropeptides engage in signaling over much broader spatial and temporal scales than synaptic transmission, and these spatiotemporal characteristics have made it difficult to precisely study how neuropeptide signals propagate throughout the brain. This experimental barrier has left important gaps in our knowledge of how the neural circuits that are responsible for behavior and cognition are modulated. Therefore, in order to overcome this critical barrier and enable neuropeptides to be directly studied in healthy and diseased brain tissue, it is the goal of this proposal is to develop new genetically-encoded optical tools to (1) record and (2) modulate neuropeptide signaling.

PROJECT SUMMARY/ABSTRACT Leucine-Rich Repeat Kinase 2 (LRRK2) is a potential therapeutic target for Parkinson?s disease (PD) intervention. Mutations in LRRK2 are currently the most common genetic causes of PD, and several of these mutations directly or indirectly increase LRRK2 kinase activity. Furthermore, LRRK2 kinase activity has been implicated in its toxicity to neurons, and LRRK2 mutations may be pathogenic in other cell types such as microglia and those in peripheral tissues, causing symptoms associated with inflammation and gastrointestinal distress. The recent identification of endogenous LRRK2 kinase substrates as well as biochemical and genetic studies suggest that LRRK2 mutations cause dysfunction in a host of processes including autophagy, cytoskeletal dynamics, mitochondrial function, and synaptic transmission; however, the precise mechanisms by which LRRK2 mutants cause pathology are not understood. Elevation of oxidative stress is a major cellular mechanism of pathogenesis that has been associated with LRRK2, and recent evidence suggests that PD-associated mutations in LRRK2 increase levels of reactive oxygen species originating from damaged mitochondria. However, there are currently no tools to directly measure LRRK2 kinase activity in real-time in live cells in correlation with oxidative stress. Thus, in this project we will develop genetically-encoded fluorescent protein based sensors to overcome this technological barrier. Furthermore, these sensors will provide a key technology to directly analyze LRRK2 kinase activity in live cells, tissues, and animals, and they will be particularly well- suited for longitudinal experiments with respect to aging or drug treatments. The primary outcome of this project will be sensor technologies that are optimized for use by the research communities studying Parkinson?s disease, LRRK2, and oxidative stress.