David Cool, PhD
- Cellular mechanisms involved in neurodegeneration and neuroinflammation in response to diseases and chemical toxicity.
- Proteomic and genomic changes in response to endocrine diseases.
- Developing protocols and facilities for proteome, lipid and carbohydrate analysis.
The focus of my laboratory has evolved over the past decade to investigate hormones of the hypothalamic, pituitary, adrenal, pancreatic-axis (HPAP-Axis) under normal, disease or chemically challenged conditions. My research involves studying the expression, sorting, processing and secretion of HPAP peptide hormones such as vasopressin, oxytocin, insulin and ACTH. I have been funded to study these processes in diseases such as familial neurohypophyseal diabetes insipidus, autism and in response to nerve agents such as sarin. I am also involved in research on obstetrical/gynecological related diseases, such as pre-eclampsia, preterm birth, interstitial cystitis, and endometriosis. My long-term goals are to identify and investigate the effects that these diseases have on the regulation and dysfunction of the neuroendocrine and endocrine system.
Research Line 1:
Protection against sarin-induced neurotoxicity via an in vivo caspase inhibitor
The latest avenue of research in my laboratory has been to determine if neurodegeneration and neuroinflammation in the brain in response to the nerve agent sarin can be attenuated or affected by an inhibitor of apoptosis, i.e., Q-VD-OPh. The experiments involved exposure to sarin followed at one hour by the administration of Q-VD-OPh with or without diazepam. Neurodegeneration is analyzed by staining for TUNEL, GFAP, and other stains designed to test for cell death. Neuroinflammation was determined by using the Bio-Rad BioPlex 200 bead based instrument to analyze up to 23 different cytokines in amygdala, hippocampus and piriform cortex of the brain. We also have studied apoptosis using a new Bioplex kit that can measure four different apoptotic markers. This project was funded by a Countermeasures Against Chemical Threats (CounterACT) Exploratory/Developmental Projects in Translational Research (R21) NIH-R21NS072103 that ran from 2010 to 2013. We are currently analyzing the data from experiments with plans to complete and publish in 2014.
Research Line 2:
Proteomic changes in the hypothalamic-pituitary axis in response to chemical nerve agents, prophylactic treatments and insecticides
In this research line we have been investigating the effects that exposure to chemical nerve agents, i.e., sarin; prophylactics, e.g., physostigmine or pyridostigmine; or insecticides, e.g., chlorpyrifos, have on endocrine disruption of the HP-axis and endocrine peptides and proteins in mice. This has become an exciting new area of research for my laboratory. The data indicate that specific proteins in the HP-axis and pancreas are affected by the acetylcholinesterase inhibitors. This is significant because it suggests mechanisms for toxicity as well as treatment in cases of nerve agent exposure.
- This research has developed along two connected pathways. In the first, we studied the effects of pyridostigmine (PB), physostigmine (PYR) and sarin (GB) to affect hypothalamic acetylcholinesterase activity and protein. Several papers were conflicted on whether pyridostigmine could cross the blood brain barrier. We show that there are acute (< less than 15 min) effects of PB on the AChE activity in the hypothalamic region of the brain, but not the cortex, similar to the effects PHY has on these areas. We also show a similar effect with sarin.
- In the second, we have investigated the effect of these chemical agents, i.e., PB, PYR and GB, on the proteins and peptide hormones of the HP-axis. We have found significant changes with subacute treatment of the tissues. We used SELDI-TOF mass spectrometry and now MALDI-TOF/TOF MS for these projects.
Research Line 3:
Miami Valley Hospital — Translational Research Training
Maternal-Fetal Medicine Fellowship Program, 2008–Present
I am working with maternal-fetal medicine fellows in the Boonshoft School of Medicine Department of Obstetrics and Gynecology at MVH to develop research projects based on proteomic analysis of patient tissue/fluid samples. This research provides a framework for the fellows future as clinical research scientists in translational medicine. Current projects involve analyzing cervico-vaginal secretions, amniocentesis fluid, and serum for biomarkers of preterm labor or pre-eclampsia. Other projects are to analyze lung cells for surfactant secretion following treatment with various drugs or other therapeutics. I have also been working to develop clinically oriented research projects with ob/gyn residents. Through focus groups, we have identified several topics of initial interest to develop projects around, e.g., interstitial cystitis, endometriosis, and pre-eclampsia. These projects are in the early stages, but will pair master’s level students in my lab at WSU with a specific project to help provide the hands necessary to analyze samples supplied by the residents. Our goal is to develop proteomic profiles at the mass spec and cytokine levels to establish biomarkers for the diseases that can then be developed into a better diagnostic tool. Furthermore, we will be investigating the lipid components of the tissues and fluids to determine whether there are variations in the lipids that may impact the disease. Eventual results could result in improved diagnosis or therapeutic care.
Research Line 4:
Establish protocols and facilities for proteomic analysis
My laboratory has been using protein procedures to study changes in cells and tissues for years. However, now with the advent of “proteomics” we have begun to push forward into new areas of research using advanced proteomic tools, i.e., IonTrap mass spectrometry, MALDI-TOF mass spectrometry, Free-Flow Electrophoresis for protein separation, 2D-IEF gel electrophoresis, protein and peptide in-gel digests and extraction, and peptide and protein sequencing.
- The Kettering Foundation provided funding to purchase a Bruker HTCUltra High Capacity nanoESI IonTrap Mass Spectrometer and Free flow electrophoresis system. These two pieces of equipment will be used to establish a proteomic center.
- Through the Boonshoft Innovation Fund we purchased and are using a Bruker Autoflex III MALDI-TOF/TOF mass spectrometer for high end mass spectrometry. We have used this equipment to study peptide hormones in the HPA/P axis in response to many different types of treatment.
- We have also been using it for peptide identification for a number of projects in collaboration with numerous other laboratories.
- We have been collaborating with Dr. Nadja Grobe in our department to develop use of the MALDI-TOF MS for MALDI-Imaging. In this technique, tissue cryosections are applied to a conductive slide and imaged in the MS by rastering the laser over the cryosection. In this way, proteomic and lipidomic profiles can be established and analyzed in the tissue sections.
- Likewise, we have been working to develop TLC-MALDI-Imaging to analyze lipids separated by organic solvents on conductive TLC plates. This technology is very exciting and allows us to develop a lipidomic profile in addition to the proteomic profile. It also allows us to more accurately assess the types of lipids and modifications to lipids expressed in cells or released from cells.
- We have developed protocols for separating proteins on FPLC using the Bio-Rad DuoFlow and Bio-Rad NGC FPLC systems and 2D-IEF gels (BioRad-Criterion System).
- We have purchased a Bio-Rad BioPlex 200 bead based system for analyzing proteins such as cytokines, metabolites, e.g., insulin, adiponectin, and other markers of disease in humans, mice and rats. This instrument allows us to take protein analysis beyond the limitations of ELISA by multiplexing upwards of 40 different analytes per 50 microliter aliquot of tissue lysate or body fluid. Analyses have been completed on: serum, cervicovaginal secretions, amniotic fluid, urine, and CSF.
Research Line 5:
Fractionation and analysis of snake venom proteins on neuronal, lung, muscle, spleen and keloid fibroblasts.
The goal of this project was to determine if snake venom proteins could be separated using FPLC followed by desalting and analyzing them by mass spectrometry, cell cultures, and in vitro assays. We have tested a number of cell lines with the snake venom and determined that some fractions are benign while others contain proteins that are inhibitory to cell growth as well as some that seem to cause cell proliferation.
Rutherford, C.M.. ,Grunwald, Jr., W. C., Garrett, C.M., Cool, D.R., Cutaneous effect of chlorpyrifos on acetylcholinesterase and endocrine tissues in rats. J. Env. Imm. Toxicol. 2(2) 63-71. 10.7178/jeit.27 2014
Joshi, K., Rapp, C.R., Garrett, T.L., Schlager, J.T., Davidson, M.B., Cool, D.R., Lucot. J.B.,The effects of 8-OH-DPAT on neurodegeneration after sarin exposure. Toxicology.2013 Aug 9;310:22-8. doi: 10.1016/j.tox.2013.05.005. Epub May 18 2013
Ventolini, G., Gigax, S.E. Adelson, M.E., Cool, D.R., Vulvodynia and Fungal Association: A Preliminary Report, Medical Hypotheses 81 (2013) 228–230
Grobe, N., Elased, K., Cool, D.R., Morris, M., Mass Spectrometry for the molecular imaging of angiotensin metabolism in kidney. Am J Physiol Endocrinol Metab.2012 Apr;302(8):E1016-24. doi: 10.1152/ajpendo.00515.2011. [Abstract]
Yan, Z, Hoffmann, A., Kelly-Kaiser, E., Gruwald, Jr.,W.C., and Cool, D.R., Misfolding of mutated vasopressin causes ER-retention and activation of ER-stress markers in Neuro-2a cells. TONEUROJ 4: 136-146 2011DOI: 10.2174/1876528901104010136.
Lee, A.G, Cool, D.R., Grunwald, Jr., W.C, Neal, D.E., Buckmaster, C.L. Cheng, M.Y., Hyde, S., Lyons, D.M., and Parker, K.J., A novel oxytocin peptide in New World monkeys. Biology Letters, 7(4):584-587 2011 doi: 10.1098/rsbl.2011.0107
Southerland, B. Kulkarni-Datar, K, Grunwald, W, Ketcha, D. M., Cool, D.R., and Brown, T.L, Q-VE-OPh, a negative control for O-phenoxy-conjugated (-OPh) caspase inhibitors, Q-VD-OPh. J. Cell Death, 3: 33-40, 2010.
Grigsby, C., Rizki, M, Tamburino, L, Pitsch, R., Shiyanov, P, Cool, D.R., Metabolite Differentiation and Discovery Lab (MeDDL): A New Tool for Biomarker Discovery and Mass Spectral Visualization, Analytical Chemistry 82: 4386–4395,2010.
Cool, D.R., Jackson, S. & Waddell, K.S. Structural Requirements for Sorting Pro-Vasopressin to the Regulated Secretory Pathway in a Neuronal Cell Line. The Open Neuroendocrinology Journal 1: 1-8 (2008)
Ropp, S. A., W.C. Grunwald, Jr., Morris, M. and Cool, D.R., Pyridostigmine modulation of hypothalamic acetylcholinesterase in mice: evidence for a central effect. J. Med Chem Bio Rad Def. 6 (2008).
Polito III, A., Goldstein, D., Sanchez, L., Cool, D.R., and Morris, M, Urinary Oxytocin as a Non-Invasive Biomarker for Neurohypophyseal Hormone Secretion. Peptides 27(11): 2877-2884 (2006).
Chen, Y., Hoffmann, A., Cool, D.R. and Morris, M, Adenovirus Mediated Small Interference RNA for In Vivo Silencing of Angiotensin AT1a Receptors in Mouse Brain. Hypertension 47:1-8 (2006).
Hoffmann, A. and Cool, D.R., Characterization of two polyclonal peptide antibodies that recognize the carboxy-terminus of angiotensin II type 1A and 1B receptors.Clin.Exp. Pharmacol. Physiol. 32 (11): 936-943 (2005).
Elased K, Cool D.R., Morris M. Novel Mass Spectrometric Methods for Evaluation of Plasma ACE1 and Renin. Hypertension, 46: 953-959 (2005).
Hardiman, A., Friedman, T.C., Grunwald, W.C., Furuta, W., Steiner, D.F. and Cool, D.R., Endocrinomic Analysis of Vasopressin and Oxytocin processing in PC1/3 and PC2 Processing Enzyme Knockout Mice by SELDI-TOF Mass Spectrometry J. Mol. Endocrinol. 34: 739-751 (2005).
Chen, Y., Liu-Stratton, Y., Hassanain, H., Cool, D.R. and Morris, M., Dietary Sodium Regulates Angiotensin AT1a and AT1b mRNA Expression in Mouse Brain Analyzed by Quantitative Real-time PCR. Experimental Neurology, 188: 238-245 (2004).
† Cool, D.R. and Hardiman, A. C-terminal sequencing of peptide hormones using carboxypeptidase Y and SELDI-TOF mass spectrometry. Biotechniques 36: 32-34 (2004)
† Recommended Read by Faculty of 1000 WebSite, March 2004—Factor 3.0
Cool, D.R. and DeBrosse, D., Extraction of oxytocin and arginine-vasopressin from serum and plasma for radioimmunoassay and SELDI-TOF mass spectrometry. J. Chromatography B, 792: 375-380 (2003).
Hoffmann, A. and Cool, D.R. Angiotensin II receptor types 1A, 1B and 2 expression in Neuro-2a cells. J. Receptors and Signal Transduction, 23: 111-121 (2003).
Biology, B.S. (1985), Wright State University, Dayton, OH
Biology, M.S. (1988), Wright State University, Dayton, OH
Biochemistry, Ph.D. (1991), The Medical College of Georgia, Augusta, GA
Postdoctoral Research Fellow (1991-1996), National Institutes of Health, Bethesda, MD