Pharmacology and Molecular Therapeutics Faculty & Staff

Profile

Our laboratory studies the biochemical events that occur following the activation of the mu opioid receptor. It is known that morphine relieves severe pain by activating mu opioid receptors throughout the nervous system. It is also known that repeated exposure of mu opioid receptors to morphine results in an attenuation in the ability of morphine to relieve pain. Our laboratory is also interested in studying the biochemical events that result in the 'desensitization' of the mu receptor signalling system.

When the mu opioid receptor binds an opiate (such as morphine), a series of other proteins associated with the mu receptor signalling pathway become activated. The first protein activated by the morphine-bound mu receptor is a GTP-binding protein (G protein). The G protein requires guanosine triphosphate (GTP) to be active. When the GTP is hydrolyzed to guanosine diphosphate (GDP), the protein becomes inactive. There are a number of different types of G proteins in neurons, and the specific types of G proteins that associate with the mu opioid receptor have recently been identified by our laboratory. We are also investigating how chronic exposure of the mu receptor to morphine results in a diminished responsiveness of neurons to opiates (tolerance). Recently, a new group of proteins has been discovered in the nervous system. These proteins are capable of increasing the rate of hydrolysis of GTP that is bound to G proteins. When GTP is rapidly hydrolyzed, the signalling pathway shuts down. These new proteins that increase the hydrolysis of GTP are called RGS proteins (for Regulators of G protein Signalling). We are interested in determining if chronic morphine administration can increase the synthesis of these RGS proteins and/or cause these RGS proteins to become associated with the G proteins that mediate the effects of the mu receptor.

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Chronic exposure of the mu opioid receptor to morphine results in the interaction of RGS proteins with the G proteins that normally mediate the effects of the mu opioid receptor. The RGS protein inhibits the activity of the G protein by stimulating the hydrolysis of GTP to GDP.

Our laboratory has recently developed antibodies that can selectively immunoprecipitate the mu opioid receptor in an active state in order to co-purify proteins that associate with the mu receptor. These antibodies were raised against fusion proteins that contained 50 to 61 amino acid regions that occur in the mu opioid receptor. These antibodies are currently being used to purify soluble, active mu opioid receptors in association with their interactive G proteins. During this past year, we have identified Gao and Gai1 as the most prominent G proteins that co-precipitate with the rat brain mu opioid receptor. We are also interested in determining if chronic morphine treatment can cause RGS proteins to become associated with the mu receptor/G protein complex. To accomplish this goal, we are also developing antibodies against the newly discovered RGS proteins. Finally, during the coming year, we will be transfecting cells that express the mu opioid receptor with selected RGS proteins to determine if expression of these proteins can diminish mu opioid receptor signalling.

Selected Publications:

Weems, H.B., Chalecka-Franaszek, E., and Cote, T.E. (1996) Solubilization and pharmacological characterization of high affinity, guanine nucleotide sensitive mu-opioid receptors from rat brain. J. Neurochem. 66, 1042-1050.

Chalecka-Franaszek, E., Weems, H.B., Crowder, A.T., Cox, B.M., and Cote, T.E. (2000) Immunoprecipitation of high-affinity, guanine nucleotide-sensitive, solubilized mu opioid receptors from rat brain: co-immunoprecipitation of the G proteins Gao, Gai1 and Gai3. J. Neurochem. 74:1068-78.

Profile

Opiate drugs are indispensable therapeutic agents in the treatment of severe and chronic pain. They act at receptors normally utilized by members of a family of naturally occurring peptides, the endogenous opioid peptides. Several peptides fall into this group, including the enkephalins, ?-endorphin, and dynorphin. A structurally related peptide, nociceptin (also called orphanin FQ; abbreviated N/OFQ) was identified in 1995. Endogenous opioids and N/OFQ act through a set of specific receptors as neural regulators in many parts of the central and peripheral nervous systems. The regulatory role of opioid receptors in pain pathways accounts for the potent analgesic actions of opiate drugs such as morphine. These peptides and their receptors are also important regulators of the central pathways mediating behavioral reinforcement, and thus play a role in the reward mechanisms associated with satisfaction of our needs for fluid replacement, maintained nutrition and reproduction of the species. This makes opioid receptors participants in drug addictions not only for opiate drugs such as heroin but also in the reinforcing actions of other drugs of abuse such as ethanol, amphetamines, cocaine and marijuana. Opioid-regulated neurons show very marked adaptations to chronic opiate drug exposure, resulting in the development of tolerance, physical dependence, and addiction.

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In recent studies, we have found that the expression of enkephalins, N/OFQ, their precursor peptides and their mRNAs are significantly increased in neurons and/or glial cells following neuronal activation or exposure to cytokines, to oxidative stress, or ischemia and re-perfusion. In neurons, expression of these peptides is increased by neuronal depolarization or elevation of intracellular calcium. It is not surprising, therefore, that N/OFQ levels are increased in brain by mechanical injury, seizures or neurotoxic agents including those inducing symptoms closely reminiscent in Parkinson's Disease. We are now studying the expression and functions of N/OFQ and other opioids in regulating central dopamine neurons in experimental models of Parkinson's Disease, or after seizure generation.

Profile

• A.B., Biochemistry, Smith College
• Ph.D, Biochemistry, Tufts University Sackler School of Biomedical Sciences
• Postdoctoral, Laboratory of Cellular and Molecular Biology, NCI/NIH
• Postdoctoral, Division of Pulmonary, Critical Care and Sleep Medicine, Tufts University/New England Medical Center

Cell biology and signal transduction of tissue repair

Cell biology and signal transduction of normal tissue repair and fibrotic remodeling in the lung; adult stem cells that participate in lung repair; anoikis; apoptosis; cytoskeletal rearrangement; cell proliferation; focal adhesion and src kinases; rhoA and Rac1; reactive oxygen species in signal transduction and antioxidant cellular defenses.

Recent findings in fibrotic remodeling diseases of a number of organs including lung, heart, kidney and liver have shown that hepatocyte growth factor (HGF) can mitgate the development of fibrosis, and stimulate normal tissue repair. Our laboratory is interested in identifying HGF signal transduction pathways which lead to normal repair, thus inhibiting fibrotic remodeling by transforming growth factor- beta1 (TGF b1) and angiotensin II (Ang II). Using two animal models for lung fibrosis, the bleomycin model and the thoracic irradiation model, we are exploring changes in protein expression, signal transduction as well as cellular composition of the lung. Our laboratory makes extensive use of primary lung cell culture to study signal transduction by HGF, and mechanisms by which TGF 1 and Ang II signaling for anoikis and apoptosis can be inhibited.

Collaborative Projects:

Protection from Acute and Delayed Radiation Toxicity:

Gamma radiation exposure in humans is highly toxic. The injuries induced by radiation have been divided into two categories: acute effects, which often result in lethality within ~30 days, (including the hematopoietic syndrome and the gastrointestinal syndrome); and delayed effects, which may require months to develop (including lung injuries, pneumonitis and pulmonary fibrosis). Our laboratory in collaboration with Dr. Michael R. Landauer at the Armed Forces Radiobiology Research Institute, is investigating the activity and mechanisms of agents to prevent both types of injuries.

Program Affiliation:

Cellular and Molecular Biology

Selected publications (of 50)

Stacey, D.W., Roudebush, M., Day, R., Mosse, S.C., Gibbs, J.B., Feig, L.A. (1991) Dominant inhibitory Ras mutants demonstrate the requirement for Ras activity in the action of tyrosine kinase oncogenes. Oncogene 6: 2297-2304.

Day, R.M., Cioce, V., Breckenridge, D., Castagnino, P. Bottaro, D.P. (1999) MAP kinase and PI 3-kinase activation are not sufficient for mitogenic signaling by the hepatocyte growth factor and its truncated isoform NK2. Oncogene 18: 3399-340.

Thannickal, V.J., Bastien, M., Day, R.M., Klinz, S.G., Fanburg,B.L. (2000) Ras-dependent and independent regulation of reactive oxygen species by mitogenic growth factors and TGFb. FASEB J. 14: 1741-1748.

Rubin, J.S., Day, R.M., Breckenridge, D., Atabey, N., Taylor, W.G., Stahl, S.J., Wingfield, P.T., Kaufman, J.D., Schwall, R., Bottaro, D.P. (2001) Dissociation of heparan sulfate and receptor binding domains of hepatocyte growth factor reveals that heparan sulfate-c-Met interaction facilitates signaling. J. Biol. Chem. 276: 32977-83.

Day, R.M., Yang, Y.Z., Suzuki, Y.J., Stevens, J., Perlmutter, A., Pathi, R., Fanburg, B.L., Lanzillo, J.J. (2001) Bleomycin upregulates gene expression of angiotensin converting enzyme via MAP kinase and Egr-1 transcription factor. Am. J. Resp. Cell. Mol. Biol. 25:613-619.

Day, R.M., Bridges, B., Patel, B.K.R., Corey, S.J., Bottaro, D.P. (2002) Mitogenic complementarity between HGF-mediated PI 3-kinase activation and IL-4-mediated Stat6 activation. Oncogene 21:2201-11.

Day, R.M., Suzuki, Y.J., Lum, J.L., White, A.C., Fanburg, B.L. (2002) Bleomycin upregulates gene expression of g-glutamylcysteine synthetase in pulmonary artery endothelial cells. Am. J. Physiol. Lung Cell. Biol. 282:L1349-1357.

Thannickal, V.J., Lee, D.Y., White, E.S., Cui, Z., Larios, J.M., Horowitz, J.C., Day, R.M., Thomas, P.E. (2003) Myofibroblast differentiation by TGF-b1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. J. Biol. Chem. 278:12384-12389.

Day, R.M., Thiel, G., Lum, J.M., Chevere, R.D., Yang, Y., Stevens, J., Sibert, L., Fanburg, B.L. (2004) Hepatocyte growth factor regulates angiotensin converting enzyme gene expression. J. Biol. Chem. 279:8792-8801.

Lee YH, Mungunsukh O, Tutino RL, Marquez AP, and Day RM. (2010) Angiotensin II-induced apoptosis requires SHP-2 regulation of nucleolin and Bcl-xL in primary lung endothelial cells. J Cell Sci, 123: 1634-43.

Davis TA, Landauer MR, Mog SR, Barshishat-Kupper M, Zins S, Amare MF, Day RM. (2010) Timing of captopril administration determines radiation protection or radiation sensitization in a murine model of total body irradiation. Exp Hematol, 38: 270-81.

Mungunsukh O, Lee YH, Marquez AP, Cecchi F, Bottaro DP, Day RM. (2010) A tandem repeat of a fragment of Listeria monocytogenes Internalin B protein induces cell survival and proliferation. Am J Physiol Lung Cell Biol, 299:L905-14.

Lee YH, Marquez AP, Mungunsukh O, Day RM. (2010) Hepatocyte growth factor inhibits apoptosis by the profibrotic factor angiotensin II via ERK1/2 in lung endothelial cells and lung explants. Mol Biol Cell, 21: 4240-50.

Barshishat-Kupper M, Mungunsukh O, Tipton AJ, McCart EA, Panganiban RAM, Davis TA, Landauer MR, Day RM. (2011) Captopril modulates HIF and erythropoietin responses in a murine model of total body irradiation. Exp Hematol, 39: 293-304.

Day RM, Lee YH, Han L, Kim Y-C , Feng Y-H. (2011) Angiotensin II activates AMPK for energy-dependent and -independent apoptotic signaling. Am J Physiol Lung Cell Biol, (In revision).

Panganiban, R.A., Day R.M. (2011) Hepatocyte Growth Factor in Lung Repair and Pulmonary Fibrosis (Invited review). Acta Pharmacol Sin 32: 12-20.

Day, R.M., Lee, Y.H., Han, L., Kim, Y-C., Feng, Y-H. (2011) Angiotensin II activates AMPK for energy-dependent and independent apoptotic signaling. Am J Physiol Lung Cell Biol, 301:L772-81.

Kim, Y-C., Day, R.M. (2012) Angiotensin II regulates activation of Bim via Rb/E2F1 during 1/2 and Cdk4.bapoptosis: Involvement of interaction between AMPK Am J Physiol Lung Cell Biol, in press.

Panganiban, R.A.M., Mungunsukh, O., Day, R.M. (2012) X Irradiation Induces ER Stress, Apoptosis, and Senescence in Primary Pulmonary Artery Endothelial Cells. Int J Radiat Biol, in press.

Profile

• B.S. The Cooper Union for the Advancement of Science and Art
• Ph.D. University of Rochester School of Medicine and Dentistry

Regulation of Sphingolipid Biosynthesis

Sphingolipids are involved in maintaining the structural integrity of cell membranes, serve as intra and intercellular signaling molecules and are highly enriched in lipid rafts implicated in protein trafficking. The rate limiting step in sphingolipid biosynthesis is catalyzed by the heteromeric protein serine palmitoyltransferase (SPT) found in the endoplasmic reticulum (ER), and mutations in the gene encoding the subunit common to both catalytic isoforms are responsible for the late-onset neurologic disorder Hereditary Sensory Neuropathy Type I (HSAN1), a disease selectively affecting peripheral sensory neurons. We study the organization and regulation of this ubiquitously expressed enzyme, as well as downstream components of the sphingolipid biosynthetic pathway. Our current focus is elucidation of the roles, intracellular localization and membrane topology of the two known SPT isoforms, and identification of mammalian orthologues (SPTLCx) of the yeast protein Tsc3p that modulates the activity of yeast SPT. We employ a combination of classical protein purification, proteomics, and molecular biology to address these questions, as well as the use of novel covalent subunit chimera.

• B.S., Chemistry and Mathematics, University of Nebraska-Lincoln
• Ph.D., Pathology and Microbiology, University of Nebraska Medical Center
• M.D., University of Nebraska Medical Center
• Postdoctoral Training, National Institutes of Health

Targeting RTK/Ras signaling in cancer

Oncogenic mutations in RTK/Ras signaling pathways account for 30-50% of tumors. For cancers driven by mutated or hyperactivated RTKs, targeted therapeutics have shown enormous potential, however, resistant cells often emerge that continue to rely on RTK signaling to drive their oncogenic phenotype. Similarly, for cancers driven by the Ras effector B-Raf, resistance to kinase inhibit ors often involves hyperactivation of RTK signaling.  It is therefore incumbent to find alternative therapeutic targets within the RTK/Ras pathway that when inactivated will limit oncogenesis.

Simultaneous targeting of multiple signaling proteins within the same pathway has the potential to overwhelm a cancer cell’s ability to mutate and become resistant to therapy. A major goal of our laboratory is to elucidate those signaling proteins downstream of RTKs that can be targeted either alone or in combination with current therapeutics to treat cancer.

Examining novel Sos-dependent functions in RTK/Ras signaling

The RasGEFs (Guanine Exchange Factors) Sos1 and Sos2 (Son of Sevenless 1 and 2) are central to signal transduction from receptor tyrosine kinases (RTKs) to the small G protein Ras, and recruitment of the RasGEF Sos1 by the adaptor Grb2 to receptor signaling complexes is an essential early step in normal Ras activation. In T cells, Sos1 and Grb2 show multipoint binding that nucleate higher-order protein complexes at the cell surface necessary to drive cell fate decisions (Kortum, Sci. Signaling, 2013).  We are currently examining whether similar mechanisms are important to both normal and oncogenic RTK/Ras signaling

Profile

Ph.D., Neurophysiology, Tarbiat Modarres University, Tehran, Iran, 2003
Postdoctoral, Neuroscience, Brown University

Synaptic plasticity of the Reward Pathway

Since the discovery of synaptic plasticity as the cellular correlate of learning and memory, strong overlaps between neural and cellular substrates of learning, drug addiction and stress-related disorders have been recognized. Yet it remains a major challenge to identify the neural circuits and synaptic mechanisms contributing to abnormalities in dopamine signaling induced by addictive drugs and adverse early life experiences. The major focus of my laboratory is the elucidation of synaptic mechanisms underlying reward learning, drug addiction and neuropsychiatric disorders such as depression, with particular emphasis on the midbrain dopamine system originating from the ventral tegmental area (VTA) and its control by the lateral habenula (LHb). Research in our laboratory also explores effects of severe early life stress on synaptic transmission and plasticity of distinct VTA dopamine circuits to identify the neural circuits and molecular mechanisms contributing to abnormalities in dopamine signaling induced by adverse early life experiences. To achieve these goals, we use a combination of immunohistochemical, biochemical, epigenetic, electrophysiological and behavioral techniques. We expect that identifying novel epigenetic mechanisms in the regulation of synaptic plasticity and memory formation in midbrain dopamine circuits will point to new directions in pharmacotherapy for drug addiction and stress-related disorders.

Selected Publications

Authement M.E., Kodangattil J.N.,Gouty,S., Rusnak, M., Symes A.J., Cox B.M., and Nugent F.S., Histone deacetylase inhibition rescues maternal deprivation-induced GABAergic metaplasticity through restoration of AKAP signaling, Neuron, 86: 1240–1252 (2015)

Kodangattil J.N., Dacher M.A., Authement, M.E. and Nugent F.S., Spike timing-dependent plasticity at GABAergic synapses in the Ventral tegmental area. Journal of Physiology, 591.19: 4699-710 (2013)

Dacher, M. A., Gouty, S., Dash, S., Cox, B.M., and Nugent, F.S., A-kinase anchoring protein-calcineurin signaling in long-term depression of GABAergic synapses, Journal of Neuroscience, 33:2650-60 (2013)

Nugent, F.S., Penick, E.C., Kauer, J.A., Opiates block long-term potentiation of GABAergic synapses. Nature, 466: 1086-1095 (2007)

Postdoctoral Fellows: Dr. Haifa Kassis and Dr. Ludovic Langlois

Graduate Students: Michael Authement and Ryan Shepard

Lab Alumni: Dr. Matthieu Dacher (Université Pierre et Marie Curie, France), Dr. Jayaraj N. Kodangattil and Steven Dash

Profile

• B.S., Biochemistry, Auburn University
• Ph.D., Biochemistry, Tulane University
• Post-Doctoral Training, National Institutes of Health

Amyloid is a highly-ordered filamentous protein aggregate that is notorious for its association with terrible human disorders such as Parkinson's and Alzheimer's diseases. The formation of amyloid is generally regarded as a misfolding event in which proteins that are normally soluble accumulate into fibrous structures. The determinants of amyloid formation and toxicity are largely unknown, and determining the atomic-level structure of amyloid is challenging because it is not amenable to the conventional structural techniques of x-ray crystallography and solution NMR. Much of the focus of our laboratory is on fundamental issues concerning amyloid formation, structure and toxicity.

Prions

Prions (infectious proteins) of the yeast Saccharomyces cerevisiae represent a special case of amyloid. While most amyloids are not considered infectious, many yeast prions are based on a self-propagating amyloid that is passed between cells during mating or from mother to daughter cells during replication. Because yeast prions are not infectious to people, they offer an easy and safe way to study the fundamentals of amyloid propagation and transmission. Also, the yeast prions are an excellent example of an epigenetic phenomenon; in this case, heritable information is structurally encoded in cytoplasmic proteins instead of within the sequence of DNA. Our laboratory is pursuing issues of prion structure, initiation and propagation.

Electron micrographs of various amyloids:

Profile

• B.S., Biology, Duke University
• Ph.D., Immunology, Stanford University
• Postdoctoral Training, National Institutes of Health

Antigen Receptor Signaling & Lymphocyte Homeostasis

The overall number and associated functions of white blood cells known as T and B lymphocytes are carefully regulated by biochemical signals that can activate or kill a given cell during different phases of an immune response. Many of these signals are initiated by engagement of the antigen receptor on the cell surface. For example, repeated engagement of the T cell receptor (TCR) on an activated T cell triggers a self-regulatory apoptosis program known as restimulation-induced cell death (RICD). However, the intracellular biochemical processes that determine the ultimate outcome of antigen receptor signaling in a "na?ve" versus a "previously activated" lymphocyte, including survival, proliferation, or programmed cell death, remain poorly defined. The major focus of our laboratory is to identify and characterize those genes, proteins, and their associated modifications that distinguish between survival and death signaled through antigen receptor stimulation in both T and B cells. We are also interested in elucidating how the loss or mutation of key genes (e.g. SLAM-associated protein (SAP)) alters apoptosis sensitivity and contributes to dysregulation of immune homeostasis. Our goal in studying these molecular pathways is to advance our knowledge of how the immune system maintains balanced responses, and illuminate new genes and biomarkers that explain aberrant lymphocyte regulation in diseases involving excessive lymphocyte proliferation and/or leukemia and lymphoma.

Profile

• Ph.D. Biochemistry, University College London, UK
• Postdoctoral Fellowship: Neurology, Massachusetts General Hospital/ Harvard Medical School

Research Interests:

Our lab aims to understand the mechanisms through which the brain responds to traumatic injury and more specifically to delineate the pathways through which detrimental neuroinflammatory cascades can be altered to enhance recovery after injury. The initial lesion can produce significant tissue damage and breakdown of the blood brain barrier. These immediate effects lead to activation of many secondary cascades involving interplay between endogenous surviving neurons and glia, and infiltrating cells and molecules from the bloodstream. Injury therefore leads to acute neuroinflammatory cascades, and also to more chronic inflammatory effects.  Indeed, activated microglia have been documented in postmortem brains many years after the initial insult. Our lab focuses on understanding the molecular signaling cascades that occur after injury in addition to testing potential therapeutics to enhance recovery from injury. We currently utilize several different rodent models of traumatic brain injury (TBI), with assays ranging from behavioral through molecular to understand the complex interactions that occur after injury, and to determine how manipulations may impact on functional recovery. We also utilize primary cell cultures to tease apart the molecular signaling pathways that contribute to inflammation after injury.

TGF-β signaling after injury

TGF-β is an injury induced cytokine that has complex roles in the central nervous system. After brain injury TGF-β can promote astrogliosis and enhance the deposition of molecules inhibitory to regeneration in the glial scar. Yet TGF-β is also neuroprotective indicating the sometimes conflicting roles of this cytokine. We have shown that after a penetrating brain injury mice that lack expression of the TGF-β signaling molecule Smad3, form a glial scar more quickly, with a smaller scar, than wild type mice. However, we also found that Smad3 null mice have more pronounced neuronal loss after injury. Thus, global interference with TGF-β signaling is not a desirable therapeutic option. Further, TGF-β is anti-inflammatory in some contexts – so chronic repression of TGF-β signaling pathways may enhance inflammation. In primary microglial cultures we have shown that inflammatory pathways downregulate TGF-β receptor expression, allowing for prolonged enhancement of the inflammatory activated state.

Angiotensin receptor blockers as potential therapeutics for TBI

Angiotensin receptor blockers (ARBs) are widely used FDA-approved anti-hypertension drugs with a favorable side effect profile that have been used as a mechanism for reducing the amount of TGF-β signaling in several different organ systems. In the CNS, ARBs have also been shown to be neuroprotective, anti-inflammatory and protective of the cerebral blood flow (CBF). The brain possesses its own renin angiotensin system, with many different components of the system expressed in the different cell types in the brain. However, in addition to acting as antagonists at the Angiotensin II receptor 1 (AT1R) some ARBs also possess potent PPARγ agonist activity that enhances their anti-inflammatory action. We have demonstrated that the ARB candesartan, lessens inflammation, reduces lesion volume, limits glial reactivity, increases neuronal survival and improves motor and cognitive recovery up to 28 days after injury. These beneficial effects are seen when low-dose candesartan is administered up to 6 hours after injury, a clinically acceptable therapeutic window. Our data suggest that both AT1 receptor antagonism and PPARγ agonism contribute to the efficacy of ARB treatment after TBI and it is this multimodal action by a single drug that makes it a strong candidate for TBI clinical trials. We are pursuing translational studies for candesartan for brain injury in addition to investigating the consequences of TBI on the endogenous renin-angiotensin system in the brain.