Michael Xi Zhu - US grants

? DESCRIPTION (provided by applicant): Pertussis toxin (PTX) sensitive Gi/o proteins are traditionally thought to only exert inhibitory effects on neurons through inhibition of adenylyl cylases and voltage-gated Ca2+ channels, as well as activation of G protein-gated inwardly rectifying K+ channels. These actions cause either decreased neurotransmitter release or reduced membrane excitability. However, emerging evidence suggest that the Gi/o proteins have other effectors and among them, two members of the Transient Receptor Potential Canonical (TRPC) family, TRPC4 and TRPC5, are particularly noteworthy because they respond to the activation of Gi/o signaling with membrane depolarization and intracellular Ca2+ concentration rise, owning to their function as non-selective cation channels. However, the activation of TRPC4/C5 by Gi/o proteins depends on other factors, with coincident activation of phospholipase C (PLC) being the most effective. Thus, these channels act by integrating signals from Gi/o and Gq/11-PLC? (or tyrosine kinase- PLC?) pathways. Consistent with the high expression levels in the nervous systems, TRPC4/C5 channels have been implicated in neurotransmission, neurite outgrowth and neurodegeneration. However, to what extent the Gi/o signaling pathway is involved and how it integrates with PLC signaling in these processes are poorly understood. This project aims to elucidate how Gi/o signaling triggered by metabotropic neurotransmitter receptors act in concert with the PLC pathway to regulate growth and function of brain neurons. Using TRPC knockout mice, we have found that TRPC4 is critical for integrating multiple neurotransmitter inputs, acting through both Gi/o and Gq/11 pathways, to alter excitation of lateral septal neurons. We also show that TRPC4 plays a key role in dendritic branching of hippocampal neurons through integration of signals from Gi/o proteins and neurotrophin receptors. Building upon these findings, we will first investigate how TRPC4 integrates Gi/o and neurotrophin signals to regulate dendritic arborization using cultured hippocampal neurons (Specific aim I). We found this regulation to be dependent on TRPC4 expression and Gi/o protein activation via metabotropic glutamate receptors and neurotrophin receptors. We will then examine the mechanisms and implications of coincident stimulation of Gi/o and Gq/11 pathways on postsynaptic response using mouse lateral septal neurons in brain slices as examples to elucidate Gi/o-TRPC4 coupling in neurotransmission (Specific aim II). We found that a number of G protein-coupled receptors activate native TRPC4 channels in this brain region through co-stimulation of Gq/11 and Gi/o proteins and the resulting channel activity strongly impacts excitability of lateral septal neurons. We will combines molecular and cellular biology, pharmacology, genetic mouse models and electrophysiological approaches for these studies. Accomplishing the proposed work will strongly enhance our understanding on PTX-sensitive G proteins in neurotransmission and neuronal development and help devise new strategies to treat neurological disease.

PROJECT SUMMARY Tissue acidosis is a major contributing factor to neuronal cell death associated with neurological diseases, such as stroke, traumatic brain and spinal cord injuries, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS), as well as Alzheimer's, Huntington, and Parkinson?s diseases. It has been well established that acid-sensing ion channels subtype 1a (ASIC1a) is critically involved in acidosis-induced neuronal cell death in both in vitro and in vivo models. The protective effects of ASIC1a knockout and pharmacological inhibition of ASIC1a function shown in the mouse models of ischemic stroke, MS, HD, and ALS testify the potential of targeting ASIC1a to mitigate neuronal damages in multiple types of neurological disorders. However, the mechanism(s) by which ASIC1a activation causes neuronal death remains mysterious despite extensive investigations. Conventionally, ASIC1a is believed to form cell surface cation channels activated by extracellular protons to mediate Na+ and Ca2+ entry into the cell. The ion conducting function, especially Ca2+ influx, is thought to cause Ca2+ overload that eventually leads to acid-induced cytotoxicity. However, our recent results suggest that the cell killing effect of ASIC1a is dependent not on its channel conductance, but on the recruitment and phosphorylation of serine/threonine kinase receptor interaction protein 1 (RIP1) to the C-terminus of ASIC1a protein. RIP1 is a key mediator of death receptor-induced necroptotic pathway. In rodent model of ischemic stroke, middle cerebral artery occlusion (MCAO), inhibiting RIP1, just like inhibiting ASIC1a, was shown to be neuroprotective even when the drug was administered several hours after the onset of brain ischemia. Therefore, acidosis neuronal death most likely occurs through ASIC1a-RIP1 physical coupling and the consequent activation of RIP1-dependent necroptosis. The goal of the proposed project is to elucidate this novel mechanism of acid-induced, ASIC1a/RIP1-mediated necroptotic cell demise in neurons. Aim I will define the death pathway mediated by ASIC1a-RIP1 interaction in response to acidosis through systematic evaluation of key factors involved in ASIC1a-mediated cell demise in cultured neurons and in the mouse MCAO model. Aim II will examine a novel mechanism by which a chaperone protein facilitates RIP1 activation through disruption of an intramolecular interaction between the cytoplasmic N- and C-termini of ASIC1a. Aim III will elucidate how the C-terminal RIP1 interaction domain of ASIC1a triggers and mediates acidosis-induced necroptosis and test whether disrupting such interaction can mitigate neuronal damage caused by acidosis and brain ischemia. Successful completion of this project will provide a better understanding of the molecular mechanism of neuronal acidotoxicity, which will shed lights on new treatment strategies for several major types of neurological disorders.

Project Summary The long-term objective of this project is to discover novel, highly targeted approaches for treating ongoing pain by defining critical mechanisms of ongoing activity (OA) in primary nociceptors that drive this pain. Recent discoveries revealed that the OA generated spontaneously in probable nociceptors and linked to ongoing pain after spinal cord injury (SCI) is associated with all three electrophysiological alterations that, in principle, can promote OA. These are depolarization of resting membrane potential (RMP), reduced voltage threshold for action potentials (APs), and increased frequency of large, transient, depolarizing spontaneous fluctuations (DSFs). Two extrinsic mediators related to inflammation, serotonin (5-HT) and capsaicin (mimicking endogenous TRPV1 activators), also promote OA, in large part by enhancing DSFs. Virtually nothing is known about mechanisms underlying large DSFs. Three specific aims will test hypotheses about DSF generation and potentiation, employing whole cell patch recording, stimulation by Ca2+ uncaging, pharmacological and transgenic approaches, in vivo recording, and behavioral tests. Aim 1 will define ion conductance and cell signaling (Ca2+ and cAMP) contributions to the acute generation of large DSFs, taking advantage of the ability of 5-HT, forskolin, and capsaicin to rapidly stimulate large DSFs, using naïve rats and transgenic mice. The focus will include HCN channels, T-type Ca2+ channels, and Nav1.8 channels. Special attention will be paid to TRPC4/5 channels, which are important for OA and have unusual properties that account for unique features of large DSFs. Aim 2 will define ion conductances and cell signals that promote large DSF generation in chronic SCI and in a subacute peripheral inflammation model (hindpaw injection of complete Freund's adjuvant - CFA). The channels found in Aim 1 to be important for large DSFs will be tested for altered contributions and expression in each model. Alterations promoting OA are predicted to be shared in these models (and thus to potentially drive many forms of ongoing pain). Aim 3 will test the prediction that combined interventions selectively blocking large DSFs and elevating AP threshold will reduce ongoing pain. A novel analgesic strategy will be tested, which combines a drug that prevents large DSF generation (a TRPC4/5 blocker) with a drug that selectively elevates AP threshold in nociceptors (a Nav1.8 blocker). The combination should efficiently suppress nociceptor OA and consequent ongoing pain at doses lower than required to observe any effect on ongoing pain from either drug alone. This prediction will be tested in vivo both on C-fiber OA recorded from dorsal roots of anesthetized rats and on ongoing pain in SCI rats and in rat and mouse CFA models. This targeted approach could lay the foundation for new treatments for severe ongoing pain that have relatively few side effects and provide an alternative to opioids, with their attendant risks.

PROJECT SUMMARY Chronic pain is debilitating medical problem that affects millions of people. However, current clinical therapy relying on opioids and non-steroidal anti-inflammatory drugs has limited efficacy because of severe adverse effects and abuse potential. To overcome these limitations, more in-depth illustration of the mechanism that underlies the development and maintenance of chronic pain will be extremely helpful. Pain perception consists of both peripheral and central components. While the peripheral mechanisms of pain have been well studied, our current understanding of the central mechanism of pain perception, especially with respect to chronic pain, remains rather limited. The current project focuses on the mechanism by which anterior cingulate cortex (ACC) of the brain participates in pain perception. It has been well-established that synaptic plasticity in ACC represents one of the most critical mechanisms underlying the transition of pain from acute to chronic. Using mouse models of chronic pain induced by peripheral inflammatory and spared nerve injury, the research team has obtained strong evidence that acid-sensing ion channel isoform 1a (ASIC1a) plays a pivotal role in both the development and maintenance of chronic pain. Not only did ACC neuron specific ablation of ASIC1a gene mitigated inflammatory hyperalgesia and mechanical allodynia, but in situ pharmacological inhibition of ASIC1a at ACC also quickly reversed the pre-established pain hypersensitivity. More intriguingly, in situ focal application of an ASIC1a activator at ACC enhanced sensitivity to peripheral thermal and mechanical stimulation within 10 minutes in the absence of peripheral inflammation or injury, indicating a crucial role of ACC ASIC1a activity in pain processing. The current project aims to elucidate the mechanism by which ACC ASIC1a regulates central pain processing at molecular, cellular and functional levels. The central hypothesis is that in ACC excitatory neurons that receive persistent nociceptive inputs, ASIC1a, in an ion conduction-independent manner, facilitates cingulate long-term potentiation through promoting forward trafficking of AMPA receptors. The enhanced synaptic efficacy in turn leads to altered sensitivity and reactivity of the pain pathways. The two specific aims are to define molecular underpinnings of ASIC1a regulation of AMPAR trafficking during the course of LTP induction and expression in ACC excitatory neurons (AIM 1) and illustrate functional relevance of molecular interactions that control AMPAR trafficking in cingulate LTP and chronic pain (AIM 2). The collaborative project will combine the unique strengths of the two laboratories in biochemical and cell biological analysis (US lab) and electrophysiological and behavioral study of plasticity and pain (China lab) to accomplish the goals. The project will greatly enhance our understanding on mechanism of ASIC1a regulation of synaptic plasticity, especially as it relates to pain hypersensitivity through enhancing synaptic efficacy at supraspinal levels, and shed new lights on more effective ways to treat chronic pain with minimal side effects.