The Group focusses on pathophysiological mechanisms of different diseases of the central nervous system, especially stroke and Myasthenia gravis.
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Group Leader - Reserach Clinical Neurosciences
Endogenous neuroprotection, Metabolism and Regulation of Cell Death
From single-to multicellular organisms, protective mechanisms have evolved against endogenous and exogenous noxious stimuli. The two most elementary challenges, for living organisms are infection and deprivation of substrate or energy. Our pathophysiological research is focused on mechanisms by which tissue is damaged by noxious stimuli or processes and how to prevent this injury.
Preconditioning paradigms, in which stimulation below the threshold of injury results in subsequent protection of the brain have played an important role in elucidating such endogenous protective mechanisms. Such a stimulus induces a protective state against insults otherwise lethal. Ischemia (“ischemic tolerance”), hypoxia, reactive free oxygen radicals, inflammation, etc. can serve as tolerance inducing stimuli. In general, research on preconditioning aims at understanding endogenous neuroprotection to boost it or to supplement its effectors therapeutically once damage to the brain has occurred, such as after stroke (Dirnagl et al., 2009; Dirnagl et al., 1999; Dirnagl et al., 2003; Mergenthaler et al., 2004).
While the causes of acute (e.g. stroke) and chronic (e.g. Parkinson’s disease) neurological disorders are different, the mechanisms of cell injury – including excitotoxicity, inflammation, and apoptosis overlap. Thus many pathological pathways converge on shared pathways of cell injury, death, and repair and therefore the options for inducing preconditioning and tolerance are not specific to the type of injury. Important for the clinical adaptation of this technique, there are different types of preconditioning stimuli. In summary, these are “cross” preconditioning (stimulus is different from the noxious stimulus), “remote” (preconditioning of one organ leads to protection of a different organ), “immunological” (preconditioning with an inflammatory stimulus, e.g. low dose lipopolysaccharides), “pharmacological” (compounds that trigger preconditioning cascades), “anaesthetic” (preconditionic effect of anaesthetic drugs), “mimetic”, where compounds imitate the main danger signal, and preconditioning with effectors by the administration of downstream mediators of protection such as erythropoietin (Dirnagl et al., 2009).
One of the key regulators of the genomic response after ischemic preconditioning and the adaptation to hypoxia is the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is regulated by an evolutionarily conserved pathway mediated by oxygen dependent post-translational hydroxylation of the HIF-1α subunit. Under hypoxic conditions, HIF-1α is stabilized and, after dimerization with the HIF-1β subunit, HIF-1 changes the expression of genes involved in the regulation of metabolic pathways, immune responses, ion-channel activity, vascular biology, blood coagulation and erythropoiesis (Semenza, 2001; Sharp and Bernaudin, 2004). With the help of diverse methods ranging from models of experimental ischemia and preconditioning in vivo and in vitro to molecular biology and biochemistry, we are working on dissecting the underlying signaling cascades of endogenous tolerant and protective states in the brain. In addition, we are working on the mechanistic description of protein function for endogenous neuroprotection and a. In the context apoptotic regulation of cell death in ischemic damage, the interaction of proteins in large macromolecular complexes is of great importance. We are thus working on the identification and subsequent structural and functional characterization of such multi-protein complexes.
In addition to all standard methods of cell biology, molecular biology and biochemistry our key methods include:
- Cell culture & cell biology: primary neuronal and astro-glial cultures, in vitro models of ischemia & preconditioning, gene transfection and knockdown (RNAi), cell lines, FACS
- Microscopy: live-cell imaging with hypoxia, confocal microscopy, TIRF, FLIM-FRET
- Molecular biology: cloning, gene expression profiling (qRT-PCR)
- Biochemistry: native electrophoresis, mitochondrial preparation and functional assays, immunoprecipitation
- Animal Models: middle cerebral artery occlusion (MCAo), hypoxic and pharmacological preconditioning
- Histology: Immunohistochemistry, immunocytochemistry
Please contact Dr. Philipp Mergenthaler if you are interested in working with us.
Stroke induced Immune Depression (SIDS)
Stroke patients have a much higher risk to develop severe bacterial infections. Pneumonia is the most frequent infection in stroke patients and thus constitutes a major risk factor of mortality.
It is obvious that factors such as immobilization, reduced bulbar reflexes, drowsiness and, subsequently, a higher risk of aspiration promote pulmonary infections. Although these are risk factors for bacterial infection, they cannot fully explain the observed high risk of infection. A large meta-analysis showed that other factors had to be equally important. In particular, a reduced function of the immune system was postulated. Very recently, we were able to prove that immunodepression following stroke causes severe bacterial infections. In a mouse model we demonstrated that, three days after stroke, severe bacterial infections (mostly pneumonia and sepsis) develop spontaneously. Stroke induces an overactivation of the sympathetic nervous system (SNS) and hypothalamo-pituitary-adrenal axis (HPA) leads to a rapid, severe, and sustained lymphopenia as well as to a disturbed function of lymphocytes and monocytes (see figure). Interestingly, there is also evidence for an overactivation of the sympathetic nervous system within the first two days after stroke in humans. Furthermore, we were able to show that a disturbed secretion of interferon-g from T cells and NK cells is central to these severe infections. Both a cellular reconstitution of the immune system and the application of interferon-g prevented infections. Our studies provide for the first time a mechanistic explanation for the clinical phenomenon of increased susceptibility to infections after stroke.
Approaches to prevent infection after stroke seem to be desirable. Preventive anti-infective therapy is expected to reduce a number of negative prognostic factors such as fever, infection induced arterial hypotension, and the systemic release of pro-inflammatory cytokines. In addition, with the prevention of severe infections, an earlier mobilization and rehabilitation becomes feasible. In an experimental stroke model, we were able to demonstrate that preventive anti-infective therapy with an antibiotic after focal cerebral ischemia not only reduces mortality and the infarct sizes, but also decreases the functional deficit. These experimental results lead to the initiation of a Phase IIb trial for preventive antibacterial short-term therapy in patients with acute ischemic media-infarction (Preventive ANti-infective THERapy In Stroke; PANTHERIS, Berlin).
- Neuro-immune mechanisms of post-ischemic infection
- impact of the autonomic nerve system on post-stroke pneumonia
- immunemodulation of the intestinal and respiratory tract and its influence on postischemic sepsis and pneumonia
- - Studying mechanisms of stroke-induced immune depression and functional relevance for immune tolerance
- Bench to bedside project: immune depression and pneumonia in stroke patients:
- effect on outcome (survival and neurological deficit) of Preventativ ANtiifective THerapy in acute Ischemic Stroke (PANTHERIS-Trial)
Epigenetics in ischemic preconditioning and neuroprotection against brain ischemia
Epigenetics is conventionally defined as all mitotically and meiotically heritable changes in gene expression patterns which are not coded in DNA sequence.
Considering the dynamic nature of chromatin modifications that are not necessarily heritable but still result in changes in gene expression, epigenetics could be also described as the structural adaptation of chromatin regions to register, signal or perpetuate altered activity states (Bird et al., 2007). Epigenetic mechanisms define a cell’s identity by regulating its characteristic pattern of gene expression and mainly include DNA methylation, RNA associated post-transcriptional gene silencing and covalent histone modifications such as acetylation and methylation of histone tails. Epigenetic modifications singly and/or in combination regulate the accessibility of cognate regulatory DNA elements for transcription machinery, thereby lead to a wide range of different biological readouts.
Recent years have witnessed a major increase in the number of reports supporting integral roles for epigenetic mechanisms in neuronal gene expression. Ischemic preconditioning (IPC), a phenomenon described as a brief sub-lethal ischemia which renders organs/cells resistant to subsequent severe ischemic injury, is accompanied by a dramatic change in gene expression programs suggesting that IPC induces a fundamental genomic reprogramming of cells that confer cytoprotection and survival. Certainly, the switching “on–off” of gene expression is the province of transcription factors and HIF-1α, CREB and NF-κB are known to be the main transcription factors driving neuroprotective gene expression in brain after an IPC stimulus. Nevertheless, as transcription is not occurring on naked DNA, but rather in the context of chromatin, it is today widely appreciated that regulation of such transcriptional activity requires the orchestrated effort of not only transcription factors, but also the protein complexes that modify chromatin structure.
- Role of genetic (DNA damage and repair) and epigenetic modifications (Histon-Modification, DNA-Methylation) in endogenous neuroprotection
Our colleagues have previously demonstrated that aberrant DNA methylation is associated with augmented brain injury after mild middle cerebral artery occlusion (MCAo) in mice (Endres et al., 2000). Suppression of DNA methylation, by genetic as well as by pharmacological means, conferred resistance to ischemia. Transcriptional dysfunction associated with impaired histone acetylation and/or elevated histone methylation patterns were recently linked to manifestation of numerous neurodegenerative conditions such as Hungtington’s disease, Alzheimer’s disease and amyotrophic lateral sclerosis. We have observed that levels of histone acetylation, a master epigenetic switch for activation of gene expression processes, were drastically decreased in neurons after ischemic injury. In contrast, ischemic preconditioning (IPC) enhanced acetylation levels of histones and conferred robust neuroprotection against cerebral ischemia. These epigenetic changes appear to facilitate global changes in transcription and support the concept of genomic reprogramming by IPC. In line, enhancement of histone acetylation by the potent HDAC inhibitor Trichostatin A (TSA) proved significant neuroprotection in experimental stroke models. We characterized gelsolin as a major mediator of TSA’s neuroprotective properties (Meisel et al. 2006, Yildirim et al. 2008).
We investigate histone modification patterns and the members of epigenetic machinery that are essentially involved in neuronal ischemic preconditioning and in neuroprotection. Subsequently, by utilizing a genome-wide approach we would like to characterize gene targets of such epigenetic proteins. Ultimately, mimicry and further acceleration of these epigenetic mechanisms by pharmacological agents, such as more specific HDAC inhibitors and/or HAT activators, may provide yet another avenue of therapeutic neuroprotection and thereby improve the treatment of stroke tremendously.
- Chromatin immunoprecipitation (ChIP)
- Real time qRT-PCR
- Cloning, conventional PCR, agarose gel electrophoresis
- SDS-PAGE, Western immunoblotting
- HAT/HDAC ELISA-based enzyme activity assays
- Immuno -cyto/-histochemistry
- Primary cortical neuron cultures of mice and rat
- Cell death/survival assays such as LDH measurement, MTT test, PI staining etc
- Oxygen-glucose deprivation
- As in vitro ischemic injury model - As in vitro ischemic preconditioning paradigm
- Middle cerebral artery occlusion (MCAo) of mice
- As in vivo ischemic injury model
- As in vivo ischemic preconditioning paradigm
- - Hematoxylin/Eosin staining of cryostat-sectioned brain slices
- - Computer assisted brain lesion assessment
Myasthenia gravis: Pathogenesis and new treatments
Myasthenia gravis (MG) is an autoimmune disease which is characterized by ocular and / or generalized muscle weakness.
Auto-antibodies against essential structures of the neuromuscular synapse, e.g. the acetlycholinreceptor (AChR) and the muscle-specific tyrosine kinase (MuSK) can be detected in about 90% of patients and have been shown to cause the clinical symptoms. The underlying cause for the development of these auto-antibodies in MG patients is unknown; however the high prevalence of thymus pathology, i.e. thymoma or thymic hyperplasia, suggests a role for thymic abnormalities in MG pathogenesis. Consequently, general immunosuppression and, though its exact mode of action is unknown, surgical removal of the thymus gland are well accepted and frequently utilized treatment options. However serious treatment side effects and a substantial number of non-responders exemplify the need for a more detailed understanding of disease pathogenesis and improved treatment strategies.
A clinical study has already been started in order to determine relevant immunoparameters as well as AChR-specific B and T cell immunity in MG patients and controls. Furthermore thymic samples and blood of patients before and at different time points after thymectomy will be analysed. For the analysis of immune mechanisms and the effects of new experimental treatment strategies in vivo, an experimental mouse model for MG (EAMG) is currently being established in the lab.
We aim at identifying mechanisms that explain the strong association between thymus pathology and MG. A longitudinal investigation of thymectomy and its impact on (auto-) immunity in MG patients should provide new insights into its mode of action and help to discriminate responders and non-responders in advance. The availability of an in vivo mouse model of MG will help to further investigate pathomechanisms in MG and give the opportunity to test new experimental treatment strategies like a transfer of AChR-specific T suppressor cells or the depletion of AChR-producing plasma cells.
- Stroke models of mice and rat (middle cerebral artery occlusion; MCAO), Infarct-volumetry
- Pneumonia models of mice (Streptococcus pneumoniae)
- Broncho-alveolar lavage in mice
- Models of Ischemic Preconditioning:
- hypoxic preconditioning
- pharmacological preconditioning (desferrioxamine)
- primary cortical neurons and astrocytes of mice and rat
- Cell culture model of stroke: oxygen glucose deprivation (OGD)
- hypoxic preconditioning -pharmacological preconditioning (desferrioxamine)
- Transfection of primary cortical neurons (Amaxa technology)
Mergenthaler P, Kahl A, Kamitz A, van Laak V, Stohlmann K, Thomsen S, Klawitter H, Przesdzing I, Neeb L, Freyer D, Priller J, Collins TJ, Megow D, Dirnagl U, Andrews DW, Meisel A. (2012). Mitochondrial hexokinase II (HKII) and phosphoprotein enriched in astrocytes (PEA15) form a molecular switch governing cellular fate depending on the metabolic state. Proc Natl Acad Sci U S A. 109(5):1518-23. PubMed PMID: 22233811
Isaev, N.K., Stelmashook, E.V., Lukin, S.V., Freyer, D., Mergenthaler, P.,and Zorov, D.B. (2010). Acidosis-induced zinc-dependent death of culturedcerebellar granule neurons. Cell Mol Neurobiol 30, 877-883.
Stelmashook, E.V., Lozier, E.R., Goryacheva, E.S., Mergenthaler, P., Novikova, S.V., Zorov, D.B., and Isaev, N.K. (2010). Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity.
Neurosci Lett 482, 151-155.
Royl G, Balkaya M, Lehmann S, Lehnardt S, Stohlmann S, Lindauer U, Endres M, DirnaglU, Meisel A (2009). Effects of the PDE5-inhibitor vardenafil in a mouse stroke model. Brain Res. 1265:148-571
van Zwam M, Huizinga R, Melief MJ, Wierenga-Wolf AF, van Meurs M, Voerman JS, Biber KP, Boddeke HW, Höpken UE, Meisel C, Meisel A, Bechmann I, Hintzen RQ, 't Hart BA, Amor S, Laman JD, Boven LA 2008. Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE. J Mol Med. 2008 Dec 3. [Epub ahead of print]
Klehmet J, Harms H, Richter M, Prass K, Volk HD, Dirnagl U, Meisel A*, Meisel C* (2009). Stroke-induced immunodepression and post-stroke infections: Lessons from the Preventive Antibacterial Therapy in Stroke trial. Neuroscience. 158:1184-93.
Rückert JC, Ismail M, Swierzy M, Sobel H, Rogalla P, Meisel A, Wernecke KD, Rückert RI, Müller JM (2008). Thoracoscopic thymectomy with the da Vinci robotic system for myasthenia gravis. Ann N Y Acad Sci. 1132:329-335.
Harms H, Prass K, Meisel C, Klehmet J, Rogge W, Drenckhahn C, Göhler J, Bereswill S, Goebel U, Wernecke K, Wolf T, Arnold G, Halle E, Volk HD, Dirnagl U, Meisel A (2008). Preventive antibacterial therapy in acute ischemic stroke: a randomized controlled trial. PloSOne, 3(5):e2158.
Yildirim F, Gertz K, Kronenberg G, Harms C, Fink KB, Meisel A, Endres M 2008. Inhibition of histone deacetylation protects wildtype but not gelsolin-deficient mice from ischemic brain injury. Exp Neurol. 210: 531-542
Braun JS, Prass K, Dirnagl U, Meisel A, Meisel C 2007. Protection from brain damage and bacterial infection in murine stroke by the novel caspase-inhibitor Q-VD-OPH. Exp Neurol. 206:183-191.
Lehnardt S, Lehmann S, Kaul D, Tschimmel K, Hoffmann O, Cho S, Krueger C, Nitsch R, Meisel A*, Weber JR* 2007. Toll-like receptor 2 mediates CNS injury in focal cerebral ischemia. J Neuroimmunol. 190: 28-33
Prass K, Braun JS, Dirnagl U, Meisel C, Meisel A 2006. Stroke Propagates Bacterial Aspiration to Pneumonia in a Model of Cerebral Ischemia. Stroke 37: 2607-2612.
Meisel A Harms C, Yildirim F, Bosel J, Kronenberg G, Harms U, Fink KB, Endres M 2006. Inhibition of histone deacetylation protects wild-type but not gelsolin-deficient neurons from oxygen/glucose deprivation. J Neurochem. 98:1019-31.
Kohler S, Wagner U, Pierer M, Kimmig S, Oppmann B, Möwes B, Jülke K, Romagnani C, Thiel A (2005). Post-thymic in vivo proliferation of naive CD4+ T cells constrains the TCR repertoire in healthy human adults. Eur J Immunol. 35:1987-94.
Meisel C, K Prass, J Braun, I Victorov, T Wolf, D Megow, E Halle, HD Volk, U Dirnagl, A Meisel 2004. Preventive antibacterial treatment improves the general medical and neurological outcome in a mouse model of stroke. Stroke. 35:2-6.
Endres M, D Biniszkiewicz, RW Sobol, C Harms, M Ahmadi, A Lipski, J Katchanov, P Mergenthaler, U Dirnagl, SH Wilson, A Meisel, R Jaenisch 2004. Increased postischemic brain injury in mice deficient in uracil-DNA glycosylase. J Clin Invest 113: 1711-21.
Prass K, C Meisel, C Höflich, J Braun, E Halle, T Wolf, K Ruscher, IV Victorov, J Priller, U Dirnagl, HD Volk, A Meisel 2003. Stroke-induced immunodeficiency promotes spontaneous bacterial infections and is mediated by sympathetic activation - reversal by post-stroke Th1-like immunostimulation. J. Exp. Med. 198: 725-736.
Prass K, A Scharff, K Ruscher, D Löwl, C Muselmann, I Victorov, K Kapinya, U Dirnagl, A Meisel 2003. Hypoxia induced stroke tolerance in the mouse is mediated by erythropoietin. Stroke 34:1981-6.
Ruscher K, D Freyer, M Karsch, N Isaev, D Megow, B Sawitzki, J Priller, U Dirnagl, A Meisel 2002. Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: Evidence from an in vitro model. J Neuroscience 22, 10291-301.
Prass K, K Ruscher, M Karsch, N Isaev, D Megow, J Priller, A Scharff, U Dirnagl, A Meisel 2002. Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro. J Cereb Blood Flow Metab 22, 520-525.
Trendelenburg G, K Prass, J Priller, K Kapinya, A Polley, C Muselmann, K Ruscher, U Kannbley, AO Schmitt, S Castell, F Wiegand, A Meisel, A Rosenthal, U Dirnagl 2002. Serial analysis of gene expression identifies metallothionein II as major neuroprotective gene in mouse focal cerebral ischemia. J Neuroscience 22, 5879-5888.
Endres M*, A Meisel*, D Biniszkiewicz, S Namura, K Prass, K Ruscher, A Lipski, R Jaenisch, MA Moskowitz, U Dirnagl 2000. DNA methyltransferase contributes to delayed ischemic brain injury. J Neuroscience 20: 3175-3181
Meisel C, Meisel A. (2011). Suppressing immunosuppression after stroke. N Engl J Med. 365(22):2134-6. PubMed PMID: 22129259
Mergenthaler P, Dirnagl U. (2011). Protective conditioning of the brain: expressway or roadblock? J Physiol. 589(Pt 17):4147-55. PubMed PMID: 21708907
Kunz, A., Dirnagl, U., and Mergenthaler, P. (2010). Acute pathophysiological processes after ischaemic and traumatic brain injury. Best Pract Res Clin Anaesthesiol. 24, 495-590.
Dirnagl U, Becker K, Meisel A (2009). Preconditioning and tolerance against cerebral ischaemia: from experimental strategies to clinical use. Lancet Neurology 8: 398-412.
Capela JP, Carmo H, Remião F, Bastos ML, Meisel A, Carvalho F 2009.Molecular and Cellular Mechanisms of Ecstasy-Induced Neurotoxicity: An Overview. Mol Neurobiol. Apr 17. [Epub ahead of print]
Kohler S, Thiel A (2009). Life after the thymus: CD31+ and CD31- human naive CD4+ T-cell subsets. Blood 113:769-74.
Meisel A, Meisel C (2008). Stroke-induced immunodepression: consequences, mechanisms and therapeutic implications. Future Neurology 3, 551-563.
Dirnagl U, Meisel A. (2008). Endogenous neuroprotection: Mitochondria as gateways to cerebral preconditioning? Neuropharmacology. 334-344.
Dirnagl U, Klehmet J, Braun JS, Harms H, Meisel C, Ziemssen T, Prass K, Meisel A (2007). Stroke-induced immunodepression: experimental evidence and clinical relevance. Stroke 38:770-773.
Meisel C, Schwab JM, Prass K, Meisel A, U Dirnagl (2005). Central nervous system injury-induced immune deficiency syndrome. Nature Reviews Neuroscience 6: 775-786.
Mergenthaler P, U Dirnagl, A Meisel 2004. Pathophysiology of stroke: lessons from animal models. Metab Brain Dis 19: 151-167
Harms H, K Prass, U Dirnagl, A Meisel 2004. Therapierelevante Pathophysiologie des akuten ischämischen Schlaganfalls: Was ist gesichert? Akt Neurologie. 31, 113-121.