Clinical Neurosciences

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.

Methods
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 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).

Ongoing projects:

1. 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
2. - Studying mechanisms of stroke-induced immune depression and functional relevance for immune tolerance
3. 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)

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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.

Ongoing project: 

• Role of genetic (DNA damage and repair) and epigenetic modifications (Histon-Modification, DNA-Methylation) in endogenous neuroprotection

Approaches

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).

Perspectives

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.

Techniques 

• 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

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