…From Single Molecules to Cells…
Fachbereich 14

Professor für Pharmakologie und Klinische Pharmazie

Wissenschaftlicher Werdegang

Akademischer Service und Ehrungen (Auswahl)

Lehre

Forschung

• Über 100 experimentelle Originalpublikation in internationalen Zeitschriften
• ca. 20 weitere  Beiträge (Bücher, Fortbildung).
• Zitationsanalyse (November 2014): 381 Impaktpunkte (ResearchGate), insgesamt ca. 3.200 Zitate, Hirsch-Index: 33 (Google Scholar).

 Forschungsthemen und Expertise

• Neuropharmakologie, insbesondere Pathophysiologie und Pharmakologie der neurodegenerativen Erkrankungen wie z.B. Alzheimer´sche Demenz, Schlaganfall, fötale Alkoholembryopathie.
• Mikrodialyse: Theorie und Praxis. Anwendung bei Neurotransmittern und Metaboliten des Energiestoffwechsels.
• Neurochemie der zentralen cholinergen Systeme. Präsynaptische Kontrolle der Synthese und Freisetzung von Acetylcholin, Cholinesterasen und ihre Inhibitoren.
• Cholin: alle Aspekte, insbesondere Kontrolle der Acetylcholinsynthese durch Präkursoren und Freisetzungswege von Cholin aus cholinhaltigen Phospholipiden.
• Signaltransduktion durch Phospholipasen: Rollen der Lipidbotenstoffe bei Proliferation und Apoptose. Regulation und Funktion der Phospholipase D im Gehirn.
• Suchtstoffe, insbesondere Nikotin, Alkohol (Ethanol) und MDMA.
• Zentral wirksame Pflanzenextrakte und ihre Inhaltsstoffe, insbesondere Ginkgo biloba.

 5 aktuelle Publikationen (aus einer Liste von 104 Publikationen)

• G. Li, J. Klein and M. Zimmermann (2013) Pathophysiological amyloid concentrations induce sustained upregulation of readthrough AChE mediating anti-apoptotic effects. Neuroscience  240: 349-360.
• F. Mohr, M. Zimmermann, J. Klein (2013) Mice heterozygous for AChE are more sensitive to AChE inhibitors but do not respond to BuChE inhibition. Neuropharmacology 67, 37-45.
• T. Horn and J. Klein (2013) Neuroprotective effects of lactate in brain ischemia: dependence on anesthetic drugs. Neurochem. Int. 62, 251-257.
• M. Hillert, I. Imran, M. Zimmermann, H. Lau, S. Weinfurter and J. Klein (2014) Dynamics of hippocampal acetylcholine release during lithium-pilocarpine-induced status epilepticus in rats. J. Neurochem. 131: 42-52.
• U. Burkhardt, B. Wojcik, M. Zimmermann and J. Klein (2014) Phospholipase D is a target for inhibition of astroglial proliferation by ethanol. Neuropharmacology 79: 1-9.

Professor of Pharmacology and Clinical Pharmacy

Scientific career and education

Professional and academic service, honors

• Grant reviewer for various funding organisations (since 1992) including NIH/NCCAM, Alzheimer Association, German Research Foundation etc.
• Ad hoc Reviewer for >100 journal articles from >30 different journals (since 1990)
• More than 60 scientific oral communications at national and international scientific conferences and in departmental seminar series.
• Major advisor for 5 Ph.D. and 7 M.D. students. Member of >20 doctoral committees.
• Mentor and laboratory advisor of 9 predoctoral and 4 postdoctoral associates.
• Member and Chairman, Research Advisory Committee of the TTUHSC School of Pharmacy (2002-2004)
• Member and Chair of several Faculty Search Committees at TTUHSC (2002-2006)
• Member of the Graduate Program Committee and Graduate Council, TTUHSC
• Member in numerous university committees at Univ. of Heidelberg and Univ. of Mainz as representative of students and of scientific staff, including a term (1995-97) as a Senator for the Johannes Gutenberg University of Mainz (elected).
• Member of the Organizing Committee of the Spring Meeting of the German Society for Pharmacology and Toxicology (1996-2002)
• Member of the Local Organizing Committee, and Proceedings Editor, IX. ISCM International Symposium on Cholinergic Mechanisms (Convener: K. Löffelholz)
• State examiner of students of dentistry (1993-2002) and medicine (1997-2002)
• Invited lecturer, Advanced courses in pharmacology and toxicology for postdoctoral fellows pursuing the degree of "Specialist in pharmacology" or "Specialist in toxicology" of the German Society for Pharmacology and Toxicology (1991-2002)
• Boehringer Ingelheim Prize for Medical Research for work on the “Homeostasis of brain choline” (1992)

Research expertise

• In vivo-microdialysis to evaluate central cholinergic systems (rats and mice).
• Middle cerebral artery occlusion in mice (stroke model).
• Basic animal experimentation: anesthesia, injections, blood and CSF withdrawal, arterio-venous differences.
• Measurements of cholinergic parameters: HACU/LACU, AChE etc.
• Basic behavioural analysis of rats and mice (neurological and cognitive testing).
• Brain slices as in vitro-models of ischemia; synaptosomes and synaptoneurosomes,e.g. for ion flux assays.
• Primary cell cultures of neurons and astrocytes, e.g. for fluorescence microscopy (fura-2) and neurotoxicity assays.
• High-performance liquid chromatography for the determination of acetylcholine and choline in the fmol range.
• Analysis of phospholipids and choline metabolites.
• Measurements of phospholipase activation, e.g. phospholipase D by transphosphatidylation assay.
• Basic techniques in protein chemistry, cell and molecular biology (e.g. protein purification, electrophoresis, PCR, cell permeabilization with bacterial toxins).

Research Accomplishments

• Identification of dihydrodiol dehydrogenase as a detoxifying pathway in the metabolism of carcinogenic aromatic hydrocarbons.
• Description of dynamics of choline entry into and release from the brain, and the parameters determining precursor control of acetylcholine synthesis. Development of the “homeostasis of brain choline” hypothesis.
• Development of a novel choline release assay to follow NMDA receptor activation during hypoxia, and to monitor membrane breakdown in vivo.
• Demonstration of neuroprotective properties and anti-edema effects of bilobalide (from Ginkgo) and hyperforin (from St. John´s wort) using brain slices and middle cerebral artery occlusion in vivo. Contributions to bilobalide´s mechanism of action.
• Investigations of acetylcholine release in animal models such as rats with neuronal grafts, amyloid-bearing mice, transgenic mice overexpressing acetylcholinesterase (AChE), and AChE knockout animals.
• Suggestion of a novel hypothesis to explain fetal alcohol syndrome by the ethanolspecific disruption of the phospholipase D pathway.

Funding

My laboratory was or is being funded by the NIH (National Institute of Aging, National Center for Complementary and Alternative Medicine), the Alzheimer Association, the German Research Association, the Stiftung VERUM, the Alfried Krupp von Bohlen und Halbach Stiftung/German Scholars Organisation, by Novartis Company and by Schwabe Pharmaceutical Co.

Membership in professional societies

• American Society for Pharmacology and Experimental Therapeutics (ASPET)
• American Association of Pharmaceutical Scientists (AAPS)
• Society for Neuroscience (SfN)
• German Society for Pharmacology and Toxicology (DGPT)
• German Pharmaceutical Society (DPhG)
• German Society for Neuroscience (NWG)

Current Research interests

1. Central cholinergic systems: synthesis of acetylcholine
   In my postdoctoral work with Konrad Löffelholz, we developed the hypothesis of the "homeostasis of brain choline" which claims that the extracellular concentration of choline in the brain is regulated within narrow limits by homeostatic mechanisms. Our data explained why the administration of choline (or lecithin) was not successful as a treatment for patients with central cholinergic dysfunction (e.g. Alzheimer´s disease). Using in vivo-approaches such as microdialysis, we later found that the synthesis of acetylcholine can be facilitated in a synergistic manner by the combined application of presursors (choline, glucose) when the turnover of acetylcholine is increased, e.g. pharmacologically or during exploratory behaviour. The combined administration of glucose plus choline has not been clinically tested.

   Representative publications
   • J. Klein, O. Weichel, J. Ruhr, C. Dvorak and K. Löffelholz (2002) A homeostatic mechanism counteracting K+-evoked choline release in adult brain. J. Neurochem. 80, 843-849.
   • S.R. Kopf, M.L. Buchholzer, M. Hilgert, K. Löffelholz and J. Klein (2001) Glucose and choline improve passive avoidance behaviour and increase hippocampal acetylcholine release in mice. Neuroscience 103, 365-371.
   • J. Klein, A. Köppen and K. Löffelholz (1998) Regulation of free choline in rat brain: Dietary and pharmacological manipulations. Neurochem. Int. 32, 479-485.

2. Central cholinergic systems: release of acetylcholine
   In recent years, we perfected the microdialysis technique in mice to investigate acetylcholine levels in brains of transgenic animals. We use this technique to characterize a variety of animal models including models of Alzheimer´s disease; grafted animals; and transgenic mice which overexpress or are deficient for cholinergic proteins such as cholinesterases.

   Representative publications
   • J. Hartmann, C. Kiewert, E.G. Duysen, O. Lockridge, N.H. Greig and J. Klein (2007) Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429.
   • J. Hartmann, C. Erb, U. Ebert, K.H. Baumann, A. Popp, G. König and J. Klein (2004) Central cholinergic functions in human amyloid precursor protein knock-in/ presenilin-1 transgenic mice. Neuroscience 125, 1009-1017.
   • M.L. Buchholzer and J. Klein (2002) NMDA-induced acetylcholine release in mouse striatum: role of NO synthase isoforms. J. Neurochem. 82, 1558-1560.
   • C. Erb, J. Troost, S. Kopf, U. Schmitt, K. Löffelholz, H. Soreq and J. Klein (2001) Compensatory mechanisms facilitate hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase. J. Neurochem. 77, 638-646.

3. Centrally acting plant constituents
   We have an ongoing interest in centrally acting plant extracts and have focused our studies on two major constituents, bilobalide from Ginkgo biloba and hyperforin from St. John´s wort. Bilobalide was found to be a potent antagonist of the NMDA receptor-induced breakdown of phosphatidylcholine which is mediated via calcium influx and PLA2 activation. Our studies on hyperforin showed that hyperforin, at low doses, induces the release of acetylcholine by a depolarisation-type effect while high doses inhibit high-affinity choline uptake and ACh synthesis. Current work focuses on characterizing potential neuroprotective properties of Ginkgo and St. John´s wort extracts in models of neuronal cell death and edema formation (e.g., middle cerebral artery occlusion in mice).

   Representative publications

   • A. Mdzinarishvili, C. Kiewert, V. Kumar, M. Hillert and J. Klein (2007) Bilobalide prevents ischemia-induced edema formation in vitro and in vivo. Neuroscience 144, 217-222.
   • C. Kiewert, V. Kumar, O. Hildmann, M. Rueda, J. Hartmann, R.S. Naik and J. Klein (2007) Role of GABAergic antagonism in the neuroprotective effects of bilobalide. Brain Res. 1128, 70-78.
   • C. Kiewert, M.L. Buchholzer, J. Hartmann, S.S. Chatterjee and J. Klein (2004) Stimulation of hippocampal acetylcholine release by hyperforin, a constituent of St. John’s wort. Neurosci. Lett. 364, 195-198.
   • M. Buchholzer, C. Dvorak, S.S. Chatterjee and J. Klein (2002) Dual modulation of striatal acetylcholine release by hyperforin, a constituent of St. John´s wort. J. Pharmacol. Exp. Ther. 301, 714-719.

4. Ischemia and phosphatidylcholine breakdown
   Release of choline from phosphatidylcholine can be catalyzed by phospholipase A2 or by phospholipase D. We find that PLA2 is mostly responsible for choline release under pathological conditions such as hypoxia and ischemia. Choline release can serve as an indicator of membrane breakdown in vitro and in vivo, both in animal and in human studies. We have characterized metabolic changes in human skeletal muscle during surgery and have observed release of choline during ischemia. Choline release was also observed in the context of intestinal ischemia in pigs. We now monitor choline release from the brain by microdialysis during stroke in mice. Choline release serves as a tissue marker of ischemia and may be useful to monitor the effects of neuroprotective drugs.

   Representative publications

   • A. Walter, U. Korth, M. Hilgert, J. Hartmann, O. Weichel, M. Hilgert, K. Fassbender, A. Schmitt and J. Klein (2004) Glycerophosphocholine is elevated in cerebrospinal fluid of Alzheimer patients. Neurobiol. Aging 25, 1299-1303.
   • U. Korth, H. Krieter, C. Denz, C. Janke, K. Ellinger, T. Bertsch, C. Henn and J. Klein (2003) Intestinal ischaemia during cardiac arrest and resuscitation: comparative analysis of extracellular metabolites by microdialysis. Resuscitation 58, 209-217.
   • U. Korth, G. Merkel, F. Fernandez, O. Jandewerth, T. Koch, K. van Ackern, O. Weichel and J. Klein (2000) Tourniquet-induced changes of energy metabolism in human skeletal muscle monitored by microdialysis. Anesthesiology 93, 1407-1412.
   • J. Klein (2000) Membrane breakdown in acute and chronic neurodegeneration: focus on choline-containing phospholipids. J. Neural Transm. 107, 1027-1063.

5. Regulation and function of phospholipase D in the brain
   In our earlier work, we characterized the activation of hippocampal phospholipase D (PLD) by glutamate which occurs via metabotropic glutamate receptors. We have also described modulations of synaptosomal PLD activity under depolarising conditions which suggest a role of the enzyme in neurotransmitter release. Recent work, however, focussed on the role of phospholipase D in the proliferation of astrocytes. The PLD pathway is activated by mitogenic agents but is disrupted in the presence of ethanol, a well-known teratogen causing reduced brain and body growth (alcoholic embryopathy). In the presence of ethanol, PLD forms phosphatidylethanol instead of phosphatidic acid, and thereby phosphatidic acid formation is suppressed. We presented evidence that the disruption of the PLD pathway is the cause for the ethanol-induced impairment of astroglial proliferation. Furthermore, we found that ethanol increases ceramide levels and induces apoptosis in cultured astrocytes. The experiments with ethanol indicated that ceramide formation is secondary to inhibition of the PLD pathway, and we hypotehsized that the phosphatidic acid/ceramide ratio determines cell fate in proliferating astrocytes.

   Representative publications

   • J. Klein (2005) Functions and pathophysiological roles of phospholipase D in the nervous system. J. Neurochem. 94, 1473-1487.
   • B. Schatter, S. Jin, K. Löffelholz and J. Klein (2005) Ethanol-induced apoptosis in astrocytes: cross-talk between phosphatidic acid and ceramide. BMC Pharmacology 5:3 (11 pages).
   • B. Schatter, I. Walev und J. Klein (2003) Mitogenic effects of phospholipase D and phosphatidic acid in transiently permeabilized astrocytes: effects of ethanol. J. Neurochem. 87, 95-100.
   • K. Kötter, S. Jin, C. von Eichel-Streiber, J.B. Park, S.H. Ryu and J. Klein (2000) Activation of astroglial phospholipase D by phorbol ester involves ARF and Rho proteins. Biochim. Biophys. Acta 1485, 153-162.
   • K. Kötter and J. Klein (1999) Ethanol inhibits astroglial cell proliferation by disruption of phospholipase D-mediated signalling. J. Neurochem. 73, 2517-2523.

Five chosen publications (of 91 publications)

• B. Schatter, S. Jin, K. Löffelholz and J. Klein (2005) Ethanol-induced apoptosis in astrocytes: cross-talk between phosphatidic acid and ceramide. BMC Pharmacology 5:3 (11 pages).
• A. Mdzinarishvili, C. Kiewert, V. Kumar, M. Hillert and J. Klein (2006) Bilobalide prevents ischemia-induced edema formation in vitro and in vivo. Neuroscience 144, 217-222.
• J. Hartmann, C. Kiewert, E.G. Duysen, O. Lockridge, N.H. Greig and J. Klein (2007) Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429.
• J. Klein (2005) Functions and pathophysiological roles of phospholipase D in the nervous system. J. Neurochem. 94, 1473-1487. (Übersichtsarbeit)
• K. Löffelholz and J. Klein (2006) Chapter 6: Precursors: choline and glucose. In The Brain Cholinergic System (E. Giacobini and G. Pepeu, eds.). informa/Taylor & Francis, London, pp. 75-83. (Buchbeitrag)