Edwin S. Levitan - US grants

The goal of this proposal is to determine the mechanism and consequence of hormone-dependent regulation of voltage-gated potassium channel expression. The approach taken will be to use biophysical and molecular genetic methods to study steroid hormone induction of potassium channels in clonal pituitary cells. This model system may reveal mechanisms employed by many neurotransmitters, hormones and drugs. The disruption of normal channel expression could induce aberrant electrical activity that provokes seizures in the brain and arrhythmic beating of the heart. Thus, elucidating the mechanisms of regulation of ion channel expression may be essential for understanding and treating epilepsy and heart disease. Dexamethasone, a glucocorticoid hormone agonist, increases both the steady state concentration of mRNA encoding a voltage-gated potassium channel [Kvl] and the voltage-gated potassium current [Ik(i)] in GH3 pituitary tumor cells. The specific aims of this proposal are to test the hypotheses that the dexamethasone-induced increase in Ik(i): 1. is mediated by glucocorticoid receptor-stimulated gene expression. Kvl mRNA will be measured with Northern blots while channel activity will be measured with whole cell and cell-attached patch clamp recording. Inhibitors of transcription and protein synthesis and glucocorticoid receptor agonists and antagonists will be tested for effects on induction of potassium channel expression. 2. is directly caused by the increase in Kvl mRNA Kvl gene expression in GH3 cells or oocytes will be inhibited with antisense oligonucleotides or elevated by overproduction of Kvl mRNA. These conditions will determine if changing Kvl mRNA levels is necessary and sufficient for dexamethasone-mediated induction of voltage-gated potassium current. 3. alters basal and neurotransmitter-evoked cellular electrical activity. The effect of dexamethasone on modulation of potassium, calcium and sodium channels by neuropeptides (e.g. somatostatin, thyrotropin releasing hormone) will be examined with whole cell perforated patch recording. Likewise, the effect of the steroid on spontaneous and transmitter-induced action potentials will be measured. These studies will appraise the impact of potassium channel induction by dexamethasone on cell excitability.

The secretion of prolactin, a hormone that acts on the reproductive and immune systems, is regulated synergistically by dopamine and thyrotropin- releasing hormone (TRH). TRH and dopamine are known to affect tyrosine- and/or serine-threonine kinases, release of intracellular Ca2+, and electrical activity. However, the contribution of each of these actions to the control of prolactin secretion is unclear. Therefore, we have been using membrane capacitance measurements to follow exocytosis from single perforated patch clamped lactotrophs. Our results indicate that secretion is steeply dependent on Ca2+ influx. In addition, we have detected novel effects of TRH and dopamine on action potential activity. Furthermore, we have obtained evidence that protein phosphorylation might affect basal as well as stimulated secretion by maintaining and modulating voltage-gated Ca2+ channel activity. Finally, we have found that TRH acts in three phases to promote secretion under voltage clamp conditions. The multiple temporally distinct effects of TRH may be crucial for producing complex patterns of secretion that can be interactively controlled by dopamine. By combining membrane capacitance recordings with microfluorimetric detection of intracellular Ca2+, we propose to determine the roles of regulating action potential activity, voltage-gated Ca2+ channels, intracellular Ca2+ release and buffering, and the secretory apparatus in TRH-induced secretion. We will then determine if protein kinases and phosphatases are essential for TRH action. These studies will form the basis of further investigations on the mechanisms employed by dopamine to control basal and TRH-induced secretion. The proposed experiments will reveal how multiple modulators that activate distinct signal transduction mechanisms interact to control lactotroph secretory activity. It is likely that similar mechanisms will be utilized for coordinated regulation of peptide secretion by other endocrine cells and neurons.

DESCRIPTION (provided by applicant): Neuropeptides influence mood, sensation, learning and memory, and the function of peripheral organs. Their release typically begins slowly only after bursts of electrical activity and is limited so that distal terminals are not easily emptied. Our goal is to understand how these unique properties are generated. Initially, we used live cell imaging of calcium and a GFP-tagged neuropeptide/hormone along with patch clamping to study peptide release by cultured cells. These experiments demonstrated great diversity in the handling of peptidergic dense core vesicles (DCVs). We then generated transgenic animals to study DCV dynamics in synaptic boutons. In vivo experiments with Drosophila synapses suggest that calcium influx, triggered by depolarization or electrical activity, increases DCV motion within boutons to facilitate recruitment for neuropeptide release. Furthermore, we generated a new fluorescent protein construct to measure the delay in release following fusion pore formation, and have produced transgenic flies that express this protein. Also, we generated animals for inducible expression of fluorescent DCVs. Finally, we began to study the basis of regulated DCV motion. We are now poised to study processes that could govern the time and activity dependence of neuropeptide release. Aim 1 will determine the activity and calcium dependence of DCV motion within synaptic boutons and neuropeptide release. Aim 2 examines the mechanistic basis for regulated DCV movement within boutons. Aim 3 will determine whether the releasable DCVs in synapses are replenished first by refractory DCVs that were already present in boutons, or by new DCVs transported into the bouton from the axon. Aim 4 will detect the initial fusion with the plasma membrane to learn about the location and speed of neuropeptide exocytosis. Understanding DCV dynamics is essential for determining how neuropeptide release is uniquely controlled. This could provide a basis for controlling secretion of neuropeptides that are involved in pain, mood, hunger, and sleep

[unreadable] DESCRIPTION (provided by applicant): Midbrain dopamine neurons express inhibitory D2 dopamine autoreceptors. Therefore, D2 receptor antagonists such as the antipsychotic drug haloperidol act acutely to excite these cells. However, chronic haloperidol acts after a delay to decrease dopamine release and dopamine dependent behavior. The long-term regulation of dopamine neuron activity, which might contribute to the therapeutic action of antipsychotic drugs, has been a source of controversy because of the confounding effects of general anesthetics present during in vivo recording. We have used an experimental approach that bypasses the need for anesthetics to demonstrate that chronic haloperidol dampens the intrinsic excitability of young rat midbrain dopamine neurons. This is caused by upregulation of Kv4.3 A-type K+ channels. Furthermore, we find that this effect can be recapitulated in cell culture with chronic exposure to the D2 receptor antagonist sulpiride. In this proposal, we will determine: (i) whether Kv4.3 auxiliary subunit expression is also regulated by the antipsychotic drug, (ii) the ionic basis for irregular pacemaker activity induced by chronic haloperidol, (iii) whether an atypical antipsychotic drug acts similarly to remodel dopamine neuron excitability, (iv) the role of D2 receptors and second messengers in the long-term effect in vitro, and (v) how remodeling of dopamine neuron excitability depends on age and duration of antipsychotic drug treatment. These experiments will determine how D2 receptors and clinically used antipsychotic drugs remodel dopamine neuron intrinsic pacemaker activity. This long-term regulation may operate during normal development and in response to changes in D2 receptor activity induced by addictive and antipsychotic drugs. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Potassium channels control the function of excitable cells such as neurons, smooth muscle cells and cardiac myocytes. Potassium channel regulation is also important in programmed cell death in a variety of cell types. Although a great deal is understood about the function and acute modulation of potassium channels, little is known about long-term control of potassium channel function. Yet, manipulating potassium channel expression in vascular smooth muscle cells, cardiac myocytes and neurons could be a valuable therapeutic approach for controlling high blood pressure and reducing the incidence of cardiac arrhythmias and epileptic seizures. Here we pursue three aims focused on our ongoing studies of potassium channel expression and activity. Aim 1 will determine how Angiotensin II (Ang II) acts on cardiac myocytes to downregulate Kv4.3 channel expression. Experiments will test the hypothesis Ang II acts via NADPH oxidase- generated reactive oxygen species (ROS) to destabilize the 3' untranslated region of the channel messenger RNA. Aim 2 will determine how a protein and a chemical identified by high throughput screening stimulate Kir2.1 activity. Since total channel expression is unaffected, experiments will focus on whether these two activators affect channel trafficking and function. Aim 3 will determine how voltage-gated potassium (Kv) channel activity is slowly increased as a critical step in apoptosis. We will determine whether phosphorylation triggers insertion of new homomeric Kv2.1 channels in the cell surface. Furthermore, we will test whether native channels found in vascular smooth muscle and the heart are subject to similar regulation. This proposal will reveal fundamental insights into novel physiological, pharmacological and pathological mechanisms that produce long-term regulation of potassium channel activity in the heart, blood vessels and the brain. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Serotonin neurons of the raphe nuclei control mood and are sensitive to drugs of abuse such as the amphetamine MDMA (3,4-Methylenedioxymethamphetamine, Ecstasy) and cocaine. Because serotonin release by axon-derived varicosities, cell bodies and dendrites cannot be assayed and compared directly in intact tissue, the contribution of each cellular compartment to release evoked by electrical activity and drugs has not been quantified. Likewise, the properties of release sites near a dorsal raphe nucleus serotonin neuron that arise from that neuron, from neighboring neurons in the same nucleus and projections from other raphe nuclei have not been determined with current methods. Therefore, we have been developing a new optical technique for studying serotonin neurons in brain slices. Multiphoton monoamine imaging (MMI) utilizes a fluorescent serotonin analog that is excited by 725 nm pulsed infrared light from Ti:sapphire laser. We have demonstrated that MMI detects uptake and depolarization-evoked release from serotonin neuron cell bodies and varicosities in dorsal raphe nucleus brain slices. Here, experiments are presented to establish the utility of MMI for studying release in brain slices induced by relevant stimuli (action potentials and MDMA) at identified compartments (axonal boutons, dendrites and cell bodies) of serotonin neurons. This further development will ensure that MMI will be able to address fundamental questions concerning the effects of abused drugs on serotonin neurons. PUBLIC HEALTH RELEVANCE: Drug abuse continues to be a major public health problem. It is known that the neurotransmitter serotonin affects mood (e.g. depression, anxiety) and dopaminergic reward systems involved in addiction. This CEBRA application will further develop a new optical method called Multiphoton Monoamine Imaging for studying transmitter release by serotonin neurons. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Heart failure is associated with remodeling of the electrical and mechanical function of the heart. A common feature of electrical remodeling seen under a wide variety of pathologic states in many mammals is decreased expression of transient outward current (Ito) channels, which alters heart function and may contribute to arrhythmias that cause sudden death. Experiments in the current funding period showed that Kv4.3 messenger RNA (mRNA), which limits Ito channel expression in humans, is destabilized in cultured cardiac myocytes by stretch and Angiotensin II (AII), a hormone implicated in hypertension and congestive heart failure. Destabilization is induced by Nadph oxidase (Nox)-generated superoxide and activation of ASK1 and p38 kinase, resulting in induced expression of AUF1, a protein upregulated in human heart failure that can directly bind to a non-canonical sequence in the channel mRNA. Our recent studies show that AII acts via endosomes and CamKII (calmodulin dependent protein kinase II) to induce biphasic activation of p38 kinase. Furthermore, the AUF1 promoter is activated, implicating transcriptional regulation. Finally, preliminary experiments suggest that AUF1 knockout mice are compromised in their response to transverse aortic constriction (TAC), showing that AUF1 is important for the in vivo cardiac response to pressure overload. Here we study the signaling responsible for downregulating Kv4.3 gene expression, because (a) this channel is an evolutionarily conserved target of cardiac electrical remodeling, (b) AUF1 may regulate expression of many genes in the pathologic heart, (c) Nox, CamKII and p38 kinase have been implicated in heart failure and cardiac myocyte apoptosis, and (d) delayed endosome-induced p38 kinase signaling may be a therapeutic target for maintaining cardiac function without arrhythmia during heart failure. Aim 1 will determine the temporal organization of endosome-superoxide signaling in cardiac myocytes. Aim 2 will determine the mechanisms responsible for enhanced expression and function of AUF1. Aim 3 will use knockout mice to elucidate in vivo how AUF1 affects the healthy and pathologic heart. New molecular and cellular mechanisms for controlling cardiac myocyte gene expression will be revealed by these studies.

DESCRIPTION (provided by applicant): Dopamine is important in Parkinson's disease and the action of abused drugs (e.g. amphetamines) and clinical psychiatric drugs (e.g. antipsychotics). Because dopamine cannot be seen directly in living neurons with the light microscope, it is difficult to localize and quantify Ca2+-dependent vesicular and amphetamine- induced nonvesicular dopamine release from the soma, dendrites and terminals in living brain tissue. Likewise, the hypothesis that drugs accumulate in and are released from monoamine vesicles has not been tested. Such corelease of antipsychotic drugs with their target transmitters would result in concentrated drug delivery when and where drug action is needed (i.e., at active dopamine and serotonin synapses) resulting in greater efficacy and specificity. To be able to visualize dopamine and drug dynamics in living neurons, we have been developing new experimental approaches based on multiphoton microscopy in the rodent brain slice. First, we found that the clinically used anxiolytic antipsychotic drug cyamemazine produces visible fluorescence upon multiphoton excitation. Multiphoton imaging in midbrain slices showed that cyamemazine is subject to acidic trapping and Ca2+-dependent release. Second, multiphoton microscopy detected autofluorescence in substantia nigra dopamine neurons. Amphetamine induced dopamine transporter- mediated depletion of this signal. Likewise, depolarization induced Ca2+-dependent depletion. These results support the hypothesis that intrinsic multiphoton autofluorescence is derived from dopamine. This proposal builds on these preliminary results to first determine whether multiphoton microscopy in the brain slice can image a dopamine-derived signal that reveals content and release. Then the hypothesis that there is colocalized release of an antipsychotic drug with its target monoamine transmitters (i.e., dopamine and serotonin) is tested. These experiments will explore new optical approaches for studying dopamine and drugs in living brain tissue. Furthermore, determining whether there is corelease of an anxiolytic antipsychotic drug with serotonin and dopamine would be important for establishing a new paradigm for psychiatric drug action.

? DESCRIPTION (provided by applicant): Antipsychotic drugs are D2 dopamine receptor competitive antagonists. It has long been speculated that these drugs, which are weak base amines that cross the blood brain barrier, accumulate by acidic trapping in acidic organelles including synaptic vesicles. Previous experiments addressing this hypothesis relied on acidophilic dyes that are not psychiatric drugs and cell cultures that do not recapitulate D2 receptor-dependent neurotransmission. Therefore, we identified a clinically used antipsychotic drug that can be directly imaged in the brain slice by two-photon microscopy. Our experiments established that this antipsychotic is subject to acidic trapping, including in midbrain dopamine neuron synaptic vesicles, which contain the native transmitter. Furthermore, we showed that the antipsychotic drug can be released from vesicles by action potentials, which requires Ca2+, or an amphetamine, which requires the dopamine transporter and the vesicular monoamine transporter. Vesicular accumulation and release were seen at a therapeutic concentration in vitro and with systemic administration in animals. Thus, these results demonstrated for the first time that an antipsychotic drug is subject to vesicular release with dopamine at synapses. This finding is intriguing because it implies that local antipsychotic drug concentration will scale wit dopamine exactly where and when dopamine synapses are active. Given that synaptic vesicular release of an antipsychotic drug has now been established, this proposal determines the functional impact of vesicular antipsychotic drug release on dopaminergic transmission mediated by D2 receptors in the substantia nigra and the striatum. Our working hypothesis is that efficacy of the vesicular drug will increase with repetitive firing, which is relevant for behavior. As acidic trapping is passive and cannot be disrupted without inhibiting vesicular storage of dopamine, experiments make use of the striking difference in kinetics of vesicular trapping (i.e., it occurs over hours and then is long lasting) and acute application of the drug vi the bath (i.e., in minutes) that was revealed by two-photon microscopy. Striatum experiments are focused on electrochemically measured dopamine overflow, which is subject to frequency dependent inhibition by D2 autoreceptors. Substantia nigra experiments utilize patch clamping of dopamine neurons to assay spontaneous miniature and evoked inhibitory currents that are induced by somatodendritic D2 receptors. Together, these complementary approaches will ascertain the impact of activity-dependent vesicular release of antipsychotic drug on synaptic D2 receptor function.

Project Description Neuropeptides and neurotrophins are packaged in dense-core vesicles (DCVs) in the soma and released at synaptic terminals to control development, mood and numerous behaviors. Despite the importance of neuronal DCVs, little was known about how they are delivered to terminals and release their contents at synapses. Our GFP-based imaging studies combined with Drosophila genetics have revealed that control of synaptic peptide stores and exocytotic release do not operate as advertised in text books. First, instead of a one-way trip mediated by anterograde axonal transport, DCVs circulate in and out of synapses with capture occurring during both anterograde and retrograde transport. Moreover, contrary to long-held assumptions, capture limits the size of presynaptic neuropeptide pool. Furthermore, we have recently found that capture efficiency is directionally controlled by activity, neuron subtype and disease-related genes. Second, preliminary studies suggest that DCV transport appears to be adjusted for extensive innervation with many boutons (e.g. ~1000) and injury in the terminal. The latter effect is associated with intraterminal Ca2+ release, which may potentiate synaptic function to compensate for loss of boutons induced by injury. Third, a monoamine neuromodulator and intracellular cAMP evoke robust synaptic peptide release, with the latter displaying mechanistic differences from release evoked by action potentials. These results suggest that, in addition to conventional transmission evoked by electrical activity, synaptic peptide release is evoked by intracellular Ca2+ release and cAMP signaling. Here fluorescent protein imaging in Drosophila neurons addresses three questions posed by our prior work on this project: 1. What molecular mechanisms selectively control presynaptic DCV capture and neuropeptide release? 2. How is vesicle circulation regulated to support extensive, diverse and injured terminals? 3. What is the release mechanism and physiological impact of intracellular Ca2+ and/or cAMP signaling evoked by injury and neuromodulators? These studies will yield fundamental insights into the maintenance of terminals and the regulation of synaptic release of neuropeptides and neurotrophins. Furthermore, the proposal will show how these processes are affected by acute injury and disease-related genes.

Summary/Abstract for R21 Pyridine nucleotides are redox coenzymes that are essential for bioenergetics, metabolism and DNA damage responses. For decades, autofluorescence from reduced pyridine nucleotides (i.e., NADH and NADPH or NAD(P)H) was used to study redox in the nucleus, cytoplasm and mitochondria, while the endoplasmic reticulum (ER) NAD(P)H pool was assumed to be insignificant. This assumption has now been overturned by our recent brain slice two-photon NAD(P)H imaging experiments that demonstrated that much of somatic NAD(P)H in dopamine neurons is in the ER and coupled to mitochondrial function. We also discovered that an amphetamine acts via dopamine vesicles to rapidly reduce NAD(P)H, thus potentially reducing compensation for redox stress. Therefore, our studies have revealed new NAD(P)H organelle distribution and redox coupling, as well as NAD(P)H regulation by an important abused drug. Here we use fluorescence lifetime microscopy (FLIM) and newly generated ER- targeted indicators for individual pyridine nucleotides and their redox ratios (e.g. NAD+/NADH) to determine the identity and location of pyridine nucleotides involved in the amphetamine response and ER-mitochondria NAD(P)H coupling. Then the roles of shuttles and ER-mitochondria proximity in interorganelle redox coupling will be determined. Finally, the ER NAD(P)H pool will be perturbed to test the novel hypothesis that the ER buffers pyridine nucleotide redox in other organelles. This study will yield fundamental insights into the native and pharmacological control of pyridine nucleotides in organelles and guide future development of therapies for reducing dopamine neuron redox stress associated with Parkinson?s disease and amphetamine abuse.

Neuropeptides modulate synapses and circuits to control mood and a wide variety of behaviors including appetite, pain perception and circadian rhythms. Despite the importance of neuropeptides, it is not possible to detect synaptic neuropeptide release in the intact living brain with current methods. However, we recently developed a new optical approach for imaging exocytosis of neuropeptide-containing dense-core vesicles (DCVs) at intact living Drosophila synapses. This approach is based on inserting a fluorogen activating protein (FAP), which confers fluorescence on the normally nonfluorescent dye malachite green (MG), into a proneuropeptide, thus targeting the FAP to the DCV lumen. Following extracellular application of membrane impermeant MG derivatives that are small enough to pass through fusion pores, activity-evoked fusions of individual DCVs can readily be resolved at the Drosophila neuromuscular junction. Furthermore, we detected a novel mode of DCV spontaneous exocytosis that is distinguished by its sensitivity to perturbations of the secretory apparatus (e.g. resistance to tetanus toxin). Finally, preliminary studies show that the neuropeptide-FAP approach is applicable to studying circadian peptidergic neurons in the intact adult Drosophila brain. Therefore, to demonstrate the utility of FAP imaging, this approach will be used to answer fundamental questions regarding the function of the circadian circuit in the Drosophila. For example, we will determine the timing of neuropeptide release by multiple neurons and address whether a newly discovered mode of spontaneous release accounts for tetanus toxin resistant behavioral effects of a fly neuropeptide. Furthermore, we will use new dyes, a second spectrally distinct FAP variant and genomic engineering to enable simultaneous imaging of synaptic release of two neuropeptides under native transcriptional control. In addition to testing specific hypotheses about peptidergic transmission in the circadian circuit of the adult fly brain, these studies will serve as proof of principle examples for applying real time neuropeptide-FAP imaging to other systems including the mammalian central nervous system.