Burg bei Magdeburg, Germany
Burg bei Magdeburg, Germany

Time filter

Source Type

Jahnke H.-G.,Center for Biotechnology and Biomedicine | Braesigk A.,Center for Biotechnology and Biomedicine | Mack T.G.A.,German Center for Neurodegenerative Diseases | Mack T.G.A.,KeyNeurotek Pharmaceuticals AG | And 4 more authors.
Biosensors and Bioelectronics | Year: 2012

Alzheimer's disease (AD) and other tauopathies comprise death of cell bodies, synapses and neurites but there is surprising little knowledge of the temporal sequence and the causal relationships among these events. Here, we present a novel biosensoric approach to monitor retrograde neurite degeneration before cell death occurs. We induced tau hyperphosphorylation in organotypic hippocampal slice cultures (OHSC) and applied marker-independent real-time electrical impedance spectroscopy (EIS) for cellular real-time pathology monitoring. Using this approach, we were able to define two distinct phases of neurite degeneration, first a rapid swelling of axonal processes that manifests itself in relative impedance above control levels followed by a slower phase of collapse and subsequent fragmentation indicated by decreased relative impedance below control levels. Initial axon swelling is strictly dose-dependent and swelling intensity correlates with second phase impedance decrease implicating a causative link between both degenerative mechanisms. Moreover, suppressing tau hyperphosphorylation by kinase inhibition nearly prevented both phases of axon degeneration. Our findings demonstrate that the temporal sequence of tau-triggered neurite degeneration can be directly visualized by EIS-based, non-invasive and label-free monitoring. We therefore suggest this approach as a powerful extension of high content applications to study mechanisms of neurite degeneration and to exploit therapeutic options against AD and tau-related disorders. © 2011 Elsevier B.V.


Firsching R.,Otto Von Guericke University of Magdeburg | Piek J.,Chirurgische Universitatsklinik Rostock | Skalej M.,Otto Von Guericke University of Magdeburg | Rohde V.,University of Gottingen | And 2 more authors.
Journal of Neurological Surgery, Part A: Central European Neurosurgery | Year: 2012

Background and Study Object Despite many drug trials, no substance has yet been identified that improves the outcome of severe head injury. The dual cannabinoid CB1/CB2 receptor agonist KN38-7271 mediates potent neuroprotection in animal models. We describe here the first randomized, double-blind, prospective, placebo-controlled clinical phase IIa proof-of-concept trial to investigate the safety, pharmacokinetics, and potential efficacy of a cannabinoid receptor agonist in humans. Patients and Methods Out of the 439, 97 comatose patients at 14 European neurosurgical centers met the inclusion criteria. KN38-7271 was administered within 4.5 hours of the injury, and the patients received 1000, 500 μg, or placebo. The primary analysis was pharmacokinetic; efficacy was measured by survival and by neurological improvement or deterioration 7 and 14 days and 1, 3, and 6 months after the injury. Intracranial pressure (ICP) and cerebral perfusion pressure (CPP) were analyzed from start of treatment to end of day 7. Results Survival rates within 1 month of the injury were significantly better in the treatment groups than in the placebo group (high-dose, Kaplan-Meier difference on day 30 + 0.12 with p = 0.043; low-dose, difference +0.15 with p = 0.011) but this effect was not seen after 6 months. Critical ICP and CPP were less extreme and less frequent in the treatment group. There were no severe and no serious adverse effects that could be attributed to KN38-7271. Conclusions KN38-7271 appeared beneficial in the acute early phase of the comatose patient after a head injury. Its use was safe and well tolerated by patients. These results may provide the basis for further phase II/III trials in larger study populations. © 2012 by Thieme Medical Publishers, Inc.


Gellerich F.N.,KeyNeurotek Pharmaceuticals AG | Gizatullina Z.,Otto Von Guericke University of Magdeburg | Trumbeckaite S.,Kaunas University of Medicine | Nguyen H.P.,University of Tübingen | And 5 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2010

Despite extensive research, the regulation of mitochondrial function is still not understood completely. Ample evidence shows that cytosolic Ca2+ has a strategic task in co-ordinating the cellular work load and the regeneration of ATP by mitochondria. Currently, the paradigmatic view is that Cacyt 2+ taken up by the Ca2+ uniporter activates the matrix enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and isocitrate dehydrogenase. However, we have recently found that Ca2+ regulates the glutamate-dependent state 3 respiration by the supply of glutamate to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier. Since this activation is not affected by ruthenium red, glutamate transport into mitochondria is controlled exclusively by extramitochondrial Ca2+. Therefore, this discovery shows that besides intramitochondrial also extramitochondrial Ca2+ regulates oxidative phosphorylation. This new mechanism acts as a mitochondrial "gas pedal", supplying the OXPHOS with substrate on demand. These results are in line with recent findings of Satrustegui and Palmieri showing that aralar as part of the malate-aspartate shuttle is involved in the Ca2+-dependent transport of reducing hydrogen equivalents (from NADH) into mitochondria. This review summarises results and evidence as well as hypothetical interpretations of data supporting the view that at the surface of mitochondria different regulatory Ca2+-binding sites exist and can contribute to cellular energy homeostasis. Moreover, on the basis of our own data, we propose that these surface Ca2+-binding sites may act as targets for neurotoxic proteins such as mutated huntingtin and others. The binding of these proteins to Ca2+-binding sites can impair the regulation by Ca2+, causing energetic depression and neurodegeneration. © 2010 Elsevier B.V.


Gellerich F.N.,Leibniz Institute for Neurobiology | Gellerich F.N.,Otto Von Guericke University of Magdeburg | Gellerich F.N.,KeyNeurotek Pharmaceuticals AG | Gizatullina Z.,Leibniz Institute for Neurobiology | And 12 more authors.
Biochemical Journal | Year: 2012

The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca 2+ cyt (cytosolic Ca 2+ ) (S 0.5 =225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca 2+ cyt (S 0.5 =60±10 nM). At higher Ca 2+ cyt (>600 nM), BM accumulate Ca 2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca 2+ - induced increments of state 3 respiration decrease with substrate in the order glutamate>α- oxoglutarate>isocitrate>α- glycerophosphate>pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca 2+ cyt, we show that the formation of pyruvate is tightly controlled by Ca 2+ cyt. Through its common substrate couple NADH/NAD + , the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate-aspartate shuttle) with aralar as a central component. A rise in Ca 2+ cyt in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a 'gas pedal'. In contrast, Ca 2+ mit (intramitochondrial Ca 2+ ) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca 2+ cyt is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons. ©The Authors Journal compilation © 2012 Biochemical Society.


Krebiehl G.,University of Tübingen | Ruckerbauer S.,University of Tübingen | Burbulla L.F.,University of Tübingen | Kieper N.,University of Tübingen | And 11 more authors.
PLoS ONE | Year: 2010

Background: Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson's disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined. Methodology/Principal Findings: Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2. Conclusions/Significance: We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson's disease. © 2010 Krebiehl et al.


Gizatullina Z.Z.,Leibniz Institute for Neurobiology | Gizatullina Z.Z.,KeyNeurotek Pharmaceuticals AG | Gaynutdinov T.M.,Academy of Science of the Republic Tatarstan | Svoboda H.,KeyNeurotek Pharmaceuticals AG | And 12 more authors.
Mitochondrion | Year: 2011

We studied the functional properties of isolated brain mitochondria (BM) prepared from total rat brain (BMtotal) or from cerebral subregions under basal and Ca2+ overload conditions in order to evaluate the effects of cyclosporine A (CsA) in a regiospecific manner. CsA-induced effects were compared with those of two derivatives-the none-immunosuppressive [O-(NH2(CH2)5NHC(O)CH2)-D-Ser]8-CsA (Cs9) and its congener, the immunosuppressive [D-Ser]8-CsA. The glutamate/malate-dependent state 3 respiration of mitochondria (state 3glu/mal) differed in region-specific manner (cortex > striatum = cerebellum > substantia nigra > hippocampus), but was significantly increased by 1μM CsA (+21±5%) in all regions. Ca2+ overload induced by addition of 20μM Ca2+ caused a significant decrease of state 3glu/mal (-45 to -55%) which was almost completely prevented in the presence of 1μM CsA, 1μM Cs9 or 1μM [D-Ser]8-CsA. Mitochondrial Ca2+ accumulation thresholds linked to permeability transition (PT) as well as the rate and completeness of mitochondrial Ca2+ accumulation differed between different brain regions. For the first time, we provide a detailed, regiospecific analysis of Ca2+-dependent properties of brain mitochondria. Regardless of their immunosuppressive impact, CsA and its analogues improved mitochondrial functional properties under control conditions. They also preserved brain mitochondria against Ca2+ overload-mediated PT and functional impairments. Since Cs9 does not mediate immunosuppression, it might be used as a more specific PT inhibitor than CsA. © 2010 Elsevier B.V. and Mitochondria Research Society.


Krinke D.,University of Leipzig | Jahnke H.-G.,University of Leipzig | Mack T.G.A.,KeyNeurotek Pharmaceuticals AG | Hirche A.,University of Leipzig | And 2 more authors.
Biosensors and Bioelectronics | Year: 2010

Herewith we developed a novel 3D in vitro Alzheimer's disease (AD) model, based on the human neuroblastoma cell line SH-SY5Y, which is well differentiated without the application of any agents. Furthermore AD-like pathological neurodegeneration can be induced by okadaic acid (OA) mediated hyperphosphorylation of the microtubule associated protein tau. Moreover, we established stable " rapid tauopathy cell lines" expressing additional EGFP-fused (enhanced green fluorescent protein) wildtype or a pathology-promoting mutant tau variant (P301L) by lentiviral transduction. For the sensitive and feasible quantitative detection of pathological effects on neuronal 3D-cultures by electrochemical impedance spectroscopy (EIS) we optimized and redesigned a microcavity array (MCA). The cellular contribution to impedance could be increased by the factor of 2.5 and the variance decreased by 40%. Using our optimized MCA and impedance measurement setup we were able to detect quantitatively an OA concentration- and time-dependent decrease of the impedance in 3D SH-SY5Y cultures. Moreover, we were able to detect and quantify distinct, AD-related effects triggered by tau-mutant (P301L) expression and hyperphosphorylation in our organotypic 3D-cultures with the help of impedance spectroscopy. © 2010 Elsevier B.V.


Seidel D.,University of Leipzig | Krinke D.,University of Leipzig | Jahnke H.-G.,University of Leipzig | Hirche A.,University of Leipzig | And 6 more authors.
PLoS ONE | Year: 2012

Tauopathies including Alzheimer's disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer's disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner. Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer's disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy. Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates. © 2012 Seidel et al.


PubMed | KeyNeurotek Pharmaceuticals AG
Type: | Journal: Journal of neuroinflammation | Year: 2012

Cerebral inflammation is a hallmark of neuronal degeneration. Dipeptidyl peptidase IV, aminopeptidase N as well as the dipeptidyl peptidases II, 8 and 9 and cytosolic alanyl-aminopeptidase are involved in the regulation of autoimmunity and inflammation. We studied the expression, localisation and activity patterns of these proteases after endothelin-induced occlusion of the middle cerebral artery in rats, a model of transient and unilateral cerebral ischemia.Male Sprague-Dawley rats were used. RT-PCR, immunohistochemistry and protease activity assays were performed at different time points, lasting from 2 h to 7 days after cerebral ischemia. The effect of protease inhibitors on ischemia-dependent infarct volumes was quantified 7 days post middle cerebral artery occlusion. Statistical analysis was conducted using the t-test.Qualitative RT-PCR revealed these proteases in ipsilateral and contralateral cortices. Dipeptidyl peptidase II and aminopeptidase N were up-regulated ipsilaterally from 6 h to 7 days post ischemia, whereas dipeptidyl peptidase 9 and cytosolic alanyl-aminopeptidase were transiently down-regulated at day 3. Dipeptidyl peptidase 8 and aminopeptidase N immunoreactivities were detected in cortical neurons of the contralateral hemisphere. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were identified in activated microglia and macrophages in the ipsilateral cortex. Seven days post artery occlusion, dipeptidyl peptidase IV immunoreactivity was found in the perikarya of surviving cortical neurons of the ipsilateral hemisphere, whereas their nuclei were dipeptidyl peptidase 8- and amino peptidase N-positive. At the same time point, dipeptidyl peptidase IV, 8 and aminopeptidase N were targeted in astroglial cells. Total dipeptidyl peptidase IV, 8 and 9 activities remained constant in both hemispheres until day 3 post experimental ischemia, but were increased (+165%) in the ipsilateral cortex at day 7. In parallel, aminopeptidase N and cytosolic alanyl-aminopeptidase activities remained unchanged.Distinct expression, localization and activity patterns of proline- and alanine-specific proteases indicate their involvement in ischemia-triggered inflammation and neurodegeneration. Consistently, IPC1755, a non-selective protease inhibitor, revealed a significant reduction of cortical lesions after transient cerebral ischemia and may suggest dipeptidyl peptidase IV, aminopeptidase N and proteases with similar substrate specificity as potentially therapy-relevant targets.


The endocannabinoid system is crucially involved in the regulation of brain activity and inflammation. We have investigated the localization of cannabinoid CB1 and CB2 receptors in adult rat brains before and after focal cerebral ischemia due to endothelin-induced transient occlusion of the middle cerebral artery (eMCAO). Using immunohistochemistry, both receptor subtypes were identified in cortical neurons. After eMCAO, neuronal cell death was accompanied by reduced neuronal CB1 and CB2 receptor-linked immunofluorescence. In parallel, CB1 receptor was found in activated microglia/macrophages 3 days post eMCAO and in astroglia cells at days 3 and 7. CB2 receptor labeling was identified in activated microglia/macrophages or astroglia 3 and 7d ays post ischemia, respectively. In addition, immune competent CD45-positive cells were characterized by pronounced CB2 receptor staining 3 and 7 days post eMCAO. KN38-72717, a potent and selective CB1 and CB2 receptor agonist, revealed a significant, dose-dependent and long-lasting reduction of cortical lesion sizes due to eMCAO, when applied consecutively before, during and after eMCAO. In addition, severe motor deficits of animals suffering from eMCAO were significantly improved by KN38-7271. KN38-7271 remained effective, even if its application was delayed up to 6h post eMCAO. Finally, we show that the endocannabinoid system assembles a comprehensive machinery to defend the brain against the devastating consequences of cerebral ischemia. In summary, this study underlines the therapeutic potential of CB1 and/or CB2 receptor agonists against neurodegenerative diseases or injuries involving acute or chronic imbalances of cerebral blood flow and energy consumption.

Loading KeyNeurotek Pharmaceuticals AG collaborators
Loading KeyNeurotek Pharmaceuticals AG collaborators