Gregory T. Macleod, Ph.D - US grants

DESCRIPTION (provided by applicant): Our overall goal is to determine the mitochondrial mechanisms that influence neurotransmitter release and the impact of these mechanisms across different synapse types. Mitochondria in nerve terminals are well placed to influence neurotransmitter release but their means of influence have resisted clarification. Many facets of mitochondrial function have been directly implicated in synaptic plasticity but due to the interwoven nature of these activities (ATP production; Ca2+ and Na+ handling; extrusion of protons; release of reactive oxygen species) it has been difficult to identify those that make the primary impact. A second area that requires clarification is the role of mitochondria in different forms of short-term synaptic plasticity. Although mitochondria have an established role in the post-tetanic potentiation of synaptic strength, little is known about their impact on other forms of short-term synaptic plasticity. Lastly, while we know that mitochondria influence neurotransmitter release and synaptic plasticity in large nerve terminals very little is known about their influence in small terminals, typical of the mammalian CNS. These are glaring gaps in our knowledge, particularly as synaptic plasticity allows for changes in synaptic strength, a phenomenon underlying learning and memory. More troubling perhaps, is that mitochondrial dysfunction is found at the epicenter of many neurodegenerative conditions for which the pathogenesis and progression are poorly understood. The central hypothesis is that mitochondria influence neurotransmitter release through multiple mechanisms, and the architecture of the nerve terminal and its firing history determines which mechanism is influential. We bring a combined electrophysiological, imaging and genetic approach to address this hypothesis at Drosophila nerve terminals in vivo, and we introduce a novel peripheral synapse with a single release-site as a model for central synapses with the same architecture. We will test the ability of mitochondrial Ca2+ uptake to limit the amplitude of Ca2+ transients and neurotransmitter release during short trains of action potentials - a firing pattern common in central neurons (Aim 1). Emphasis will be placed on single release-site nerve terminals where we observe mitochondria to have a voracious appetite for Ca2+. We will determine if mitochondria in these terminals are more effective at taking up Ca2+ because they are able to take up Ca2+ directly from Ca2+ microdomains (Aim 2). We will determine whether mitochondrial ATP production, rather than Ca2+ uptake, is the principle mechanism that maintains synchronous release during sustained nerve firing (Aim 3). Finally we will test the requirement for mitochondrial Ca2+ release in the post-tetanic potentiation of transmitter release, and examine the transfer of Ca2+ between mitochondria and the endoplasmic reticulum (Aim 4). An understanding of how mitochondrial function influences synaptic transmission under non-pathological conditions will provide the foundation required to understand the role of mitochondria in pathological conditions. PUBLIC HEALTH RELEVANCE: Mitochondria are organelles within all cells of the human body that generate most of our energy. They concentrate within nerve endings where they power communication between nerves, a fundamental activity of the brain. However, little is known about the way in which they contribute to the function of the nervous system and this is troubling, as mitochondrial malfunction is implicated in many diseases of the nervous system. We are currently examining how mitochondria influence the communication between healthy nerves so that we may understand the ways in which they may become involved in neurodegenerative conditions.

DESCRIPTION (provided by applicant): Our overall goal is to elucidate the neuronal mechanisms that control mitochondria to satisfy the energy demands of nerve terminals. Mitochondria accumulate within nerve terminals where they generate most of the ATP required for the release and recycling of neurotransmitters. Neural function, therefore, relies on mitochondria generating sufficient ATP to sustain neurotransmitter release. Similarly, mitochondria power presynaptic Ca2+ homeostasis. A failure in neuronal Ca2+ homeostasis has catastrophic consequences and is a hallmark of many neurodegenerative diseases. Surprisingly, we know very little about the mechanisms that coordinate mitochondrial number and function with presynaptic energy requirements, yet understanding these mechanisms will be critical to understanding the progression of neurodegenerative disease. Our central hypothesis is that neuronal mechanisms control the number and function of mitochondria to accommodate presynaptic energy requirements, and that these mechanisms are synapse specific. We propose to elucidate these mechanisms in the musculoskeletal system of the fruit fly larva, where each motor neuron terminal has a different work rate which we can quantify using electrophysiological and Ca2+-imaging techniques. Diversity in presynaptic energy requirements, genetic tractability and accessibility to neurophysiological techniques, make this an ideal system in which to investigate neuronal mechanisms that control mitochondria to accommodate presynaptic energy requirements. In Aim 1, we will determine whether mitochondria are supplied to motor nerve terminals in numbers that are proportional to their work rate. 3D-EM reconstruction will be used to determine mitochondrial number. We will also probe the relationship between mitochondrial number and function. In Aim 2, we will test the hypothesis that mitochondrial volume is controlled at the level of different terminals on the same axon. Mitochondrial functional parameters will be determined at individual terminals to test whether mitochondrial function may be different between terminals on the same axon. In Aim 3, we will test the hypothesis that, over the course of development, active zone spacing and bouton diameter adjust to the firing rate of the motor neuron to bring presynaptic Ca2+ levels into a range most effective at stimulating mitochondrial energy metabolism during presynaptic activity. In so far as Ca2+ regulation is a heavy consumer of presynaptic ATP, this in situ model of presynaptic bioenergetics will provide an essential context for a better understanding of the early events involving mitochondrial dysfunction and Ca2+ dysregulation in neurodegenerative disease.

Project Summary / Abstract All forms of life rely on biochemical processes and these processes are either accelerated or inhibited according to the concentration of protons (pH) in their immediate vicinity. In the nervous system, pH buffering mechanisms provide a stable pH environment for biochemical reactions. Volume-averaged estimates of pH reveal only modest fluctuations in cytosolic and interstitial pH. Yet changes in pH, much like changes in Ca2+, are likely to be spatially non- uniform, and pH microdomains of substantial magnitude may develop close to the membranes across which acid equivalents flow. As many membrane-associated receptors, transporters, ion channels and enzymes are pH sensitive, pH-microdomains could have a significant impact on the fundamental neuronal properties underpinning normal operations of the nervous system. Our long range goal is to understand the influence of pH-microdomains on neuronal processes such as membrane excitability, neurotransmission and short term synaptic plasticity, and the extent to which near-membrane pH can influence the recovery of neural function after ischemic events. Our central hypothesis is that, as Ca2+ is ejected across the plasma-membrane, substantially acidic pH-microdomains develop at the cytosolic face of plasma-membrane Ca2+- ATPases (PMCAs) as a result of H+ exchange for Ca2+. The synaptic cleft will also alkalinize as a result of PMCA activity. Technological limitations have prevented investigations into the magnitude of pH microdomains, and their temporal and spatial characteristics. In an investigation of pH microdomains at the synapse, we will overcome current limitations by targeting pH Indicators to the plasma-membrane of pre- and post-synaptic compartments of the Drosophila neuromuscular junction (NMJ), and to the synaptic cleft. This approach requires the creation of a number of transgenic flies with ratiometric Genetically Encoded pH Indicators (GEpHIs) fused to proteins with well characterized distributions at the NMJ. We also introduce a technique to trap chemical pH indicators in the synaptic cleft through the introduction of a tetracysteine motif to an extracellular loop of the endogenous presynaptic voltage-gated Ca2+- channel. High speed fluorescence imaging techniques will be used to measure changes in fluorescence during the action potentials which initiate neurotransmission. Changes in fluorescence will be calibrated to quantify the underlying changes in pH.

Synaptic strength is subject to activity-dependent changes over periods of milliseconds to minutes, a phenomenon referred to as short-term synaptic plasticity (STSP). STSP has a direct influence on computations performed by neural circuits and must be understood to fully understand brain function. The synaptic environment is subject to significant activity-dependent pH fluctuations but their impact on the pH-sensitive mechanisms underlying neurotransmission is rarely considered despite their likely influence of multiple mechanisms underlying STSP. We have developed fluorescent genetically-encoded pH indicators allowing single action potential resolution of pH dynamics in the synaptic cleft of the Drosophila NMJ. Our preliminary data reveal the surprising extent to which the cleft alkalinizes (see preliminary data) and it is highly likely that this also happens at vertebrate synapses that employ the Ca2+/H+ exchanging plasmamembrane Ca2+-ATPase (PMCA). Furthermore, our preliminary data point to cleft alkalinization potentiating both quantal size and Ca2+ entry during burst firing. Our long-term goal is to elucidate the means by which pH fluctuations are incorporated into STSP mechanisms. Within this proposal we will examine the hypothesis that activity-dependent cleft alkalinization has been incorporated into gain mechanisms that sustain neurotransmission during burst firing. Using molecular genetic techniques, electrophysiology and fluorescence imaging we will test our working hypotheses that presynaptic voltage-gated Ca2+ channels (VGCCs) and postsynaptic ionotrophic glutamate receptors (iGluRs) are potentiated by alkalinization at their extracellular faces in the cleft. Our Research Strategy is broken down into three separate aims: Aim 1: Elucidate the influence of synaptic cleft alkalinization on presynaptic Ca2+ entry during bursts. Aim 2: Elucidate the mechanisms by which synaptic cleft alkalinization affects quantal size during bursts. Aim 3: Investigate the impact of neurotransmitter release on cleft pH change at individual active zones. Here we develop a test bed for investigating the contribution of activity-dependent pH fluctuations to mechanisms underlying STSP. Beyond their immediate employment in addressing the aims above, the reagents we develop will be useful for subsequent investigations into the contribution of pH-sensitive STSP mechanisms to circuit function and behavior in Drosophila, potentially providing insight into neurological disorders with an acid-base imbalance component such as seizure disorders and certain intellectual disabilities.

ABSTRACT Our overall goal is to elucidate the different mechanisms available to a neuron to control mitochondria at a subcellular level, and the genetic bases of these capabilities. Mitochondria accumulate within nerve terminals where they generate most of the ATP required to package and recycle neurotransmitters and to maintain transmembrane ion-balances. Neural function is reliant on mitochondrial function to sustain neurotransmitter release, and mitochondrial dysfunction is a hallmark of many neurodegenerative diseases. It is therefore imperative to gain a better understanding of the mechanisms that neurons use to control mitochondria at the sub-cellular level of nerve terminals, and how this might differ between neuron types. Here we present the hypothesis that sites at which mitochondria interact with the plasma membrane (PM) represent a form of mitochondrial utilization that confers advantages in those parts of a neuron with high power demands, such as nerve terminals. We propose to elucidate the functional significance of such interactions, and their genetic underpinnings. To do this we are adopting a structure-function approach, exploiting the small size and genetic tools of Drosophila. In Aim 1 we will use serial block face scanning electron microscopy to determine the neuron types, and subcellular regions served by mitochondrial-PM interactions. In Aim 2 we will use a novel form of super-resolution to investigate the formation and disassembly of these interactions, and the functional consequences for presynaptic physiology and neurotransmission. In Aim 3 we will investigate the role of a select group of genes identified as candidates for a role in mitochondrial-PM interactions. The significance of this proposal lies in its potential to uncover novel neuronal and sub-cellular specific mitochondrial functions, and the genetic bases of these functions, which may throw light on the selective neuronal vulnerability observed in different neurodegenerative diseases and neurological conditions.