David R. Cool, Ph.D.
Director, Proteome Analysis Laboratory
Campus Address: 240 Health Sciences Building
Phone: (937) 775-2457
Fax: (937) 775-7221
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
- Cellular mechanisms involved in neurodegeneration of magnocellular neurons in familial neurohypophyseal diabetes insipidus.
- Proteomic changes in the hypothalamic-pituitary axis in response to chemical nerve agents and insecticides.
- Proteomic changes in response to endocrine diseases such as diabetes.
- Establish protocols and facilities for proteomic analysis.
The focus of my laboratory is to investigate the proteome and genome 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 am currently funded to study these processes in diseases such as familial neurohypophyseal diabetes insipidus, type 1 diabetes, autism and in response to nerve agents such as sarin. My long term goals are to identify and investigate the effects that these disease have on the regulation and dysfunction of the neuroendocrine system.
Research Line 1:
Cellular mechanisms involved in neurodegeneration of magnocellular neurons in familial neurohypophyseal diabetes insipidus
Familial neurohypophyseal diabetes insipidus (FNDI) is an autosomally dominant inherited disorder that typically presents prior to one to six years of age. The genetic component for FNDI is localized to the arginine vasopressin-neurophysin II (AVP-NPII) prohormone precursor gene, locus 20p13. It contains three exons and two introns. Mutations that cause FNDI have been found to occur primarily within the signal peptide of the prepro-AVP-neurophysin II precursor and the neurophysin II region. Only one mutation has been identified in the AVP peptide hormone region.
- My laboratory has investigated the structural requirements for sorting pro-vasopressin into the regulated secretory pathway (RSP). To do this we have generated a number of deletion mutations of the pro-vasopressin cDNA coupled to EGFP in a plasmid.
- We have also been studying the effect that FNDI mutations have on sorting pro-vasropressin into the RSP. For this we have created 10 mutations to pro-vasopressin in the GFP plasmid. These have been expressed in Neuro-2a cells to determine whether they are sorted correctly. To further explore this relationship, we have co-expressed the wild-type pro-vasopressin with the FNDI-mutated pro-vasopressin in the Neuro-2a cells. The wild-type pro-vasopressin has been inserted into a plasmid containing the dsRed gene and is identified as a red fluorescence on the fluorescence microscope.
- We are currently investigating the effects of FNDI-mutants on the protein synthesis process in the ER and Golgi compartments of the cell. We are also investigating the effects of the FNDI-mutants on cell death via apoptosis.
- Finally, we have been characterizing the different angiotensin receptors in the Neuro-2a cell line. This has led us to discover that the AT1a, AT1b and AT2 receptors are all present in the cell line. By using specific inhibitors for the AT1 and AT2 receptors we have begun to determine the effects that each has on the function of the cells.
Research Line 2:
Proteomic changes in the hypothalamic-pituitary axis in response to chemical nerve agents, prophylactic treatments and insecticides
In this research, 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 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 have used SELDI-TOF mass spectrometry for these projects.
Research Line 3:
Prohormone Processing Changes in Knockout Mice
Prohormone processing involves the concerted actions of multiple enzymes and compartment of the cell. In this new line of research, we have studied prohormone processing in processing enzyme null mice and in prohormone knockout mice. In the preliminary study, we have used SELDI-TOF mass spectrometry to determine that some prohormones are differentially processed in the pituitaries of PC2-null mice while others are processed the same (Hardiman et al., 2005).
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, SELDI- and 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.
- Recently the Kettering Foundation provided funding to purchase a High Capacity nanoESI IonTrap Mass Spectrometer and Free flow electrophoresis system. These two pieces of equipment will be used to establish a proteomic center.
- As part of the DOD project, we purchased a Ciphergen SELDI-TOF mass spectrometer. 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.
- We have developed protocols for separating proteins on 2D-IEF gels (BioRad-Criterion System). To evaluate these gels, we have purchased the BioRad PDQuest software package.
Research Line 5:
Role of the Peptide Hormone Oxytocin in Autism
Autism is a neurodevelopmental disorder in which afflicted individuals exhibit characteristic impairments in social interaction, communication, cognition, imagination, and behavior. Though the root cause of autism is unknown, a defect in the oxytocin peptide hormone has been proposed to play a possible role in the development of autism. In this study we sequenced the oxytocin gene of two autistic children and eight non-autistic control individuals. There was a lack of sequence polymorphism within the three exons of the oxytocin gene from patient samples and the control group. SELDI-TOF mass spectrometry analysis of the oxytocin peptide in the plasma revealed the presence of mature oxytocin. We conclude that in these patients, defects within the oxytocin peptide do not play a role in autism.
Kwiatek, A.M., Minshall, R.D., Cool, D.R., Skidgel, R, Malik, A.B. and Tiruppathi, C., Caveolin-1 Regulates Store-Operated Ca2+ Influx by Binding of its Scaffolding Domain to TRPC1 in Endothelial Cells. Mol. Pharmacol. 70(4): 1174-1183 (2006). [Abstract]
Polito III, A., 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). [Abstract]
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). [Abstract]
Elased K, Cool D.R., Morris M. Novel Mass Spectrometric Methods for Evaluation of Plasma ACE1 and Renin. Hypertension, 46: 953-959 (2005). [Abstract]
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). [Abstract]
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).
Hardiman, A., Friedman, T.C., Grunwald, W.C., Furuta, W., Steiner, D.F. and 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.
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).
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). [Abstract]
Key, M., Wirick, B., Cool, D.R. and Morris, M., Quantitative in situ hybridization for peptide mRNAs in mouse brain. Brain Res. Protocols 8: 9-15 (2001). (Journal Cover)