Yuriy M. Usachev, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): In addition to their bioenergetic function, mitochondria are critical regulators of Ca signaling in neurons. Mitochondria efficiently buffer Ca2+ influx during excitation and limit the amplitude of the cytosolic Ca2+ concentration ([Ca2+]i) increase. Rapid Ca2+ uptake is followed by a slower Ca release from mitochondria, completing stimulus-induced mitochondrial Ca2+ cycle. By shaping [Ca2+]i response, mitochondria can modulate numerous Ca2+-dependent neuronal functions. At the same time, impairment of mitochondrial Ca2+ transport is the key factor leading to neuronal damage in stroke and in a number of neurodegenerative disorders. Despite significant progress, many questions remain about the spatiotemporal organization, function and modulation of mitochondrial Ca2+ cycling in neurons and, specifically, about the mechanisms regulating the transition from physiology to pathophysiology. Our overall hypothesis is that mitochondrial Ca2+ cycling controls diverse neuronal functions and that the decision between physiological and pathological outcomes is influenced by reversible phosphorylation of mitochondrial proteins. We will initially focus on two physiological processes, transmitter release (Aim 1) and activation of transcription (Aim 2), by studying spatiotemporal organization and the role of mitochondrial Ca2+ transport in two morphologically and functionally distinct cellular compartments, presynaptic boutons and the cell soma, respectively. We will then investigate how protein kinase A and protein phosphatase 2A modulate mitochondrial Ca2+ signaling and Ca2+-dependent processes, such as neurotransmission, transcription activation and excitotoxicity (Aim 3). Both enzymes are targeted to the outer mitochondrial membrane (OMM), but exhibit opposite effects on cell survival, and are predicted to differentially influence mitochondria-dependent functions. The proposed studies will advance our understanding of how mitochondrial Ca2+ transporters interplay with protein kinases and phosphatases in neurons to trigger a specific physiological or pathological response. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The immune system plays a critical role in the pathogenesis of pain triggered by injury or illness. A precise understanding of the mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for the development of more efficacious treatment strategies. The complement system is a principal component of innate immunity. This proposal focuses on two highly active split products of the complement system, C3a and C5a. Increased production of C3a and C5a has been reported in various pathological states associated with pain, including arthritis, pancreatitis, inflammatory bowel disease, burns and surgical trauma. Blocking the synthesis of C3a and C5a or antagonizing their receptors produces analgesic effects in animal models of inflammatory, neuropathic and postoperative pain. Moreover, direct administration of the complement fragments increases nociceptive sensitivity to heat and mechanical stimuli in animal models. In spite of this strong evidence implicating the complement system in the development of pain hypersensitivity, the underlying mechanisms are not understood. The goal of this project is to investigate mechanisms that are responsible for the enhanced pain sensitivity produced by the generation of C3a and C5a in the affected tissues. We hypothesize that C3a and C5a receptors are expressed and functionally coupled with TRPV1 in a subset of primary nociceptors; activation of these receptors sensitize nociceptors via protein kinase C-dependent modulation of the TRPV1 channel, which is known to function as a molecular integrator of pain producing stimuli. We will use a multidisciplinary approach involving immunohistochemistry, single-cell RT-PCR, [Ca2+]i imaging, patch-clamp analysis, single-fiber recordings and measurement of nociceptive behavior to test our central hypothesis. In Aim 1, we will characterize the expression and subcellular distribution of C3a and C5a receptors (C3aR and C5aR, respectively) and TRPV1 in various classes of sensory neurons identified by specific molecular markers. We will also use Ca2+ imaging and patch-clamp recordings to test functional coupling of TRPV1 with C3aR and C5aR. In Aim 2, we will investigate intracellular signaling cascades that link the activation of C3aR and C5aR with TRPV1 sensitization. In Aim 3, we will use single-fiber recordings and behavioral studies to examine the role of PKC- dependent modulation of TRPV1 downstream of C3aR and C5aR activation in regulating nociceptor excitability as well as heat and chemical sensitization of nociceptors. The proposed studies will help to characterize the novel roles of C3a and C5a receptors in the regulation of nociceptor function, and may lead to the development of new therapeutic strategies targeting the complement system to alleviate pain.

DESCRIPTION (provided by applicant): Mitochondria play a central role in cell bioenergetics and control multiple aspects of neuronal life and death, including regulation of Ca2+ signaling. By buffering Ca2+ during excitation, and subsequently releasing Ca2+ back to the cytosol, mitochondria shape [Ca2+]i signals and regulate numerous Ca2+-dependent functions in neurons, such as excitability, synaptic plasticity and gene expression. However, excessive load of mitochondria with Ca2+ trigger neurotoxic processes in stroke and in Alzheimer's, Parkinson's and Huntington's diseases. In spite of the importance of neuronal mitochondrial Ca2+ transport, the molecules mediating mitochondrial Ca2+ uptake and release in neurons are not known. This knowledge gap presents a major obstacle in our progress toward understanding and therapeutically exploiting mitochondrial functions. Thus, the first objective of this proposal is to identify the molecular components of mitochondrial Ca2+ uptake in neurons. Mitochondria are highly dynamic organelles that rapidly undergo fission and fusion (MFF), which affects transport of mitochondria, synaptic plasticity and neuronal survival. Notably, mitochondrial fission is an early event in stroke, and fragmented mitochondria are prevalent in Alzheimer's and Huntington's disease. Given the central role of mitochondrial Ca2+ transport in neuronal life and death, it is possible that the effects of MFF on neuronal survival are mediated in part through changes in mitochondrial Ca2+ handling, although this idea has not been tested. Thus, our second objective is to determine how MFF status affects mitochondrial Ca2+ transport and Ca2+ homeostasis in neurons. Based on our preliminary data and published literature, we hypothesize that CCDC109A, CCDC109B and MICU1-3 are essential molecular components of mitochondrial Ca2+ uptake in neurons, and that the MFF process provides important control of CCDC109A and/or CCDC109B activities, mitochondrial Ca2+ transport and Ca2+ homeostasis in neurons exposed to neurotoxic conditions. We will use innovative approaches, including genetically encoded mitochondrial Ca2+ sensors, electron probe X-ray microanalysis and novel genetic mouse strains, to test our hypothesis in three specific aims. Aim 1 will identify the roles of novel proteins CCDC109A, CCDC109B and MICU1, 2 and 3 in mitochondrial Ca2+ uptake in neurons. Aim 2 will determine how mitochondrial restructuring regulates mitochondrial Ca2+ transport in neurons and examine specific roles of CCDC109A and CCDC109B phosphorylation in this process. Aim 3 will examine the function of MFF in maintaining neuronal Ca2+ homeostasis under neurotoxic conditions, such as excessive exposure to glutamate and ischemia. This project will provide insight into the molecular organization of mitochondrial Ca2+ transport in neurons and will establish mechanistic links between mitochondrial dynamics, Ca2+ signaling and neuronal Ca2+ homeostasis. We anticipate that these studies will be transformative because they will identify new molecular and genetic tools for exploring many functions of mitochondrial Ca2+ uptake in neurons and may lead to new therapeutics targeting mitochondrial Ca2+ transport and MFF for treating stroke and neurodegeneration.

? DESCRIPTION (provided by applicant): Activity-dependent changes in gene expression in nociceptors play a crucial role in the pathogenesis of pain. These changes are triggered by increased electrical activity following tissue or nerve injury, or inflammation. Dozens of genes associated with the development of persistent pain have been identified (e.g., ion channels, receptors and neuromodulators). Pharmacological targeting of the expression of groups of genes that depend on common transcription factors may allow preventing the development or maintenance of chronic pain states. Yet, the specific mechanisms and transcription factors responsible for activity-dependent gene regulation in nociceptors are largely unknown. Ca2+ and Ca2+-dependent transcription factors play key roles in excitation- transcription coupling in neurons. Here, we focus on the Ca2+-dependent transcription factor NFAT as an attractive candidate for regulating gene expression in nociceptors for the following reasons. First, four NFAT isoforms (NFATc1-c4; NFATc3 being the predominant) are expressed in DRG neurons and regulated by action potentials and pain producing compounds such as capsaicin, bradykinin and NGF. Second, NFAT is highly sensitive to [Ca2+]i elevations in DRG neurons (activation trheshold~300 nM) mediated by Ca2+ entry via voltage-gated Ca2+ channels and TRPV1 receptors. Third, NFAT regulates the expression of a number of genes implicated in pain, such as COX-2, BDNF, GluA2, IL-6, chemokine receptor CCR2, and based on our pilot data, CGRP and voltage-gated Na+ channel Nav1.7. Fourth, our preliminary studies using NFATc3 KO mice indicate that NFATc3 contributes to inflammation-induced pain sensitization. Collectively, these observations suggest that NFAT plays an important role in pain control. However the roles of specific NFAT isoforms in this process and the underlying mechanisms are not known. We hypothesize that NFATc3 plays a critical role in activity-dependent gene regulation in DRG neurons, which contributes to inflammation- and injury-induced nociceptor sensitization and to the pathogenesis of inflammatory and neuropathic pain. This hypothesis will be tested in two specific aims. In Aim 1, we will examine the functional significance of NFATc3 in pain hypersensitivity following inflammation, tissue and nerve injury by comparing thermal and mechanical sensitization in WT and NFATc3 KO (complete and sensory-neuron-specific KO) mice using models of persistent pain produced by inflammation (intraplantar complete Freund's adjuvant/CFA), tissue injury (postincisional pain) and nerve injury (spared nerve injury/SNI), respectively. In Aim 2, we will establish the role of NFATc3 in regulating the expression of two important molecules implicated in pain, CGRP and Nav1.7, by testing the effects of depolarization on the expression of CGRP and Nav1.7 in DRG neurons from WT and NFATc3 KO mice. These studies will advance our understanding of the mechanisms that control activity- dependent gene regulation in nociceptors and pain sensitization, and are expected to lead to the development of new strategies for alleviating pain by targeting specific NFAT isoforms and their regulatory mechanisms. RELEVANCE: Pain management remains one of the most serious public health problems. The proposed studies will help to better understand how the activity-dependent gene regulation in primary nociceptors, and specifically, the Ca2+ -dependent transcription factor NFAT, contribute to the pathogenesis of pain caused by surgical trauma, inflammation or nerve injury, and may lead to the development of new therapeutic strategies targeting NFAT and the associated signaling mechanisms to alleviate pain.

Mitochondria play a central role in cell metabolism and control multiple aspects of neuronal signaling. By efficiently buffering Ca2+ influx during neuronal excitation and slowly releasing Ca2+ back into the cytosol, mitochondria shape [Ca2+]i transients and regulate Ca2+-dependent neuronal functions, such as excitability, synaptic transmission and gene expression. Ca2+ rise in the mitochondrial matrix stimulates Ca2+-dependent dehydrogenases and boosts ATP production to meet the increase in energy demand during excitation. However, mitochondrial overload with Ca2+ can kill neurons, and mitochondrial Ca2+ dysregulation is implicated in neuronal damage during stroke and in neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. Despite the importance of mitochondrial Ca2+ transport to neuronal life and death, the molecules that mediate mitochondrial Ca2+ uptake and release in neurons are not known. This knowledge gap presents a major obstacle in our progress toward understanding and therapeutically correcting mitochondrial functions in neurons. The main objectives of this proposal are to identify molecules that mediate mitochondrial Ca2+ uptake in peripheral and central neurons, and to establish their roles in neuronal Ca2+ signaling, ATP synthesis, synaptic transmission and excitotoxicity. Our preliminary studies indicate that two novel molecules, MCU (CCDC109A) and MCUb (CCDC109B), are broadly expressed in the peripheral and central nervous systems, and that MCU is required for mitochondrial Ca2+ uptake in neurons whereas MCUb inhibits this Ca2+ transport mechanism. Moreover, our pilot data using MCU KO mice showed that MCU loss dramatically, but not completely, reduced mitochondrial Ca2+ uptake, altered Ca2+ signaling and mitochondrial function and provided remarkable protection against glutamate-induced toxicity. Our central hypothesis is that MCU and MCUb play important but opposite roles in the regulation of mitochondrial Ca2+ uptake in neurons, bioenergetics, Ca2+ signaling and synaptic transmission, and that knockout of MCU, but not of MCUb, protects neurons from excitotoxicity and reduces neuronal damage in ischemic stroke. We will employ a multidisciplinary approach involving genetic Ca2+ and ATP sensors, patch-clamp recording, knockout mice and a mouse model of ischemic stroke to test this hypothesis in three specific aims. Aim 1 will establish the roles of MCU and MCUb in mitochondrial Ca2+ transport and Ca2+ signaling in central and peripheral neurons. Aim 2 will examine the impact of MCU and MCUb on presynaptic Ca2+ signaling and synaptic transmission. Aim 3 will establish the roles of MCU and MCUb in excitotoxicity and ischemic stroke. We anticipate that this work will be transformative because it will establish the molecular basis for genetic and pharmacological manipulation of mitochondrial Ca2+ transport in neurons, and may lead to the development of new therapeutics that target mitochondrial Ca2+ uniporters for treating stroke and other neurological disorders associated with excitotoxicity.

Project Summary / Abstract Presenting with chronic pain or loss of sensation, peripheral diabetic neuropathy (PDN) is a debilitating comorbidity of diabetes that affects at least half the diabetic patient population. Since only palliative treatments are available, there is an urgent need for therapies that prevent or reverse the ?dying back? degeneration of peripheral axons in PDN. Recent evidence suggests that diabetes compromises mitochondrial structure and function in sensory neurons. However, the underlying mechanisms are unknown. Mitochondrial shape is controlled by opposing fission and fusion events. Mutations in mitochondrial fusion enzymes cause neurological disorders that present similarly to neurological complications in diabetic patients. Specifically, mitofusin-2 mutations result in Charcot-Marie-Tooth disease type 2A, a peripheral neuropathy characterized by primary axon degeneration, while mutations in Opa1 cause dominant optic atrophy, the most common form of hereditary blindness. The mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is activated by dephosphorylation of a highly conserved inhibitory PKA phosphorylation site. Two phosphatases target this site to promote mitochondrial fission, the Ca2+-dependent phosphatase calcineurin and a neuron-specific and mitochondria- localized isoform of protein phosphatase 2A containing the B?2 regulatory subunit (PP2A/B?2). We generated a mouse knock-out (KO) of B?2 and found elongated mitochondria in neurons, consistent with deletion of a Drp1 activator. B?2 KO results in a striking reduction in infarct volume following ischemic stroke, indicating that mitochondrial elongation is neuroprotective. Conversely, knocking out A Kinase Anchoring Protein 1 (AKAP1), the protein that recruits PKA to the outer mitochondrial membrane to maintain Drp1 in a phosphorylated and inhibited state, causes mitochondrial fragmentation and exacerbates stroke injury. Supported by preliminary evidence that B?2 KO mice are resistant to peripheral neuropathy in both type-1 and type-2 diabetes models, the present proposal seeks proof-of-concept evidence for B?2 (and other, as yet undiscovered, neuron-specific Drp1 activators) as a drug target for the treatment of PDN. We further propose to investigate how diabetes causes mitochondrial fragmentation in sensory neurons and how inhibiting mitochondrial fragmentation protects peripheral axons in diabetes. Using new mouse models and innovative in vivo imaging approaches, we will test the overarching hypothesis that dysregulation of the mitochondrial fission/fusion equilibrium contributes to the pathogenesis of diabetic neuropathy, and that inhibition of Drp1- dependent mitochondrial fission provides neuroprotection via improvement of mitochondrial metabolism, reduction of ROS, modulation of mitochondrial Ca2+ transport and enhanced regeneration of sensory axons. We anticipate that these studies will shed light on PDN etiology, suggest new therapeutic strategies, and thus help improve quality of life for a rapidly growing diabetic population.

PROJECT SUMMARY / ABSTRACT Persistent pain affects 100 million Americans and 15 million Ukrainians. The immune system critically contributes to pathogenesis of inflammatory and neuropathic pain, and precise understanding of mechanisms through which particular immune mediators contribute to sensitization of nociceptive neuronal pathways will be essential for developing more efficacious treatment strategies. The complement system is a principal component of innate immunity that contributes to host defenses via diverse mechanisms. In spite of growing evidence implicating the complement system in various chronic pain states, the underlying mechanisms are not well understood. Our main objectives for this collaborative proposal between the US and Ukrainian groups are to elucidate complement-dependent spinal mechanisms that contribute to the development of neuropathic pain, and at a broader level, to promote building and strengthening sustainable research capacity in Ukraine. Mechanical hypersensitivity and spontaneous pain are common features of neuropathic pain. The main nociceptive output pathway from the spinal cord to the brain underlying this abnormal pain processing is lamina I projection neurons (PNs) of dorsal horn (DH). Our patch-clamp recordings from these neurons using an innovative intact spinal cord preparation demonstrate abnormal regulation of spinal cord output following spared nerve injury (SNI), a common model of neuropathic pain that well reproduces many features of clinical neuropathic pain. Recent studies suggest that neuropathic pain is associated with a robust upregulation of complement effectors in the spinal cord, which ultimately leads to production of a highly active complement product, C5a. Intrathecal administration of C5a produces allodynia, whereas C5 knockout (KO) and C5a receptor (C5aR1) antagonists produce analgesic effects in animal models of neuropathic pain. Our preliminary data show that C5aR1 KO prevents mechanical hypersensitivity following SNI. C5aR1 in the DH is found primarily on microglia that is known to be activated in the DH after SNI. Moreover C5aR1 expression is increased after SNI. We will use a multidisciplinary approach including patch-clamp recordings, optogenetic stimulation and multi-photon Ca2+ imaging in innovative intact spinal cord preparation combined with behavioral pharmacology to test our central hypothesis that C5a/C5aR1 signaling plays important roles in neuropathic pain processing by impacting central sensitization via microglia-dependent signaling that enhances the output of lamina I PNs of the DH to the supraspinal structures. This proposal will provide mechanistic insight into the function of the complement system in the CNS pain processing, and may lead to the development of new analgesic drugs that target complement system. In its broader impact, this project will promote establishment of Center for Excellence in brain disorder research in Ukraine, and help attracting young Ukrainian scientists to this field, providing their training and advancing chronic pain research in this country.