RESUMEN
Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.
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Espectroscopía Dieléctrica/instrumentación , Tauopatías/diagnóstico , Proteínas tau/análisis , Carbazoles/farmacología , Línea Celular , Espectroscopía Dieléctrica/métodos , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Diseño de Equipo , Humanos , Microelectrodos , Tauopatías/tratamiento farmacológicoRESUMEN
Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds.
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Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Mutación , Resonancia Magnética Nuclear Biomolecular , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/química , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/química , Dominios Proteicos , RatasRESUMEN
Mitochondrial defects have been shown to be associated with the pathogenesis of Parkinson's disease (PD). Yet, experience in PD research linking mitochondrial dysfunction, e.g., deregulation of oxidative phosphorylation, with neuronal degeneration and behavioral changes is rather limited. Using the 6-hydroxydopamine (6-OHDA) rat model of PD, we have investigated the potential role of mitochondria in dopaminergic neuronal cell death in the substantia nigra pars compacta by high-resolution respirometry. Mitochondrial function was correlated with the time course of disease-related motor behavior asymmetry and dopaminergic neuronal cell loss, respectively. Unilateral 6-OHDA injections (>2.5 µg/2 µl) into the median forebrain bundle induced an impairment of oxidative phosphorylation due to a decrease in complex I activity. This was indicated by increased flux control coefficient. During the period of days 2-21, a progressive decrease in respiratory control ratio of up to -58 % was observed in the lesioned compared to the non-lesioned substantia nigra of the same animals. This decrease was associated with a marked uncoupling of oxidative phosphorylation. Mitochondrial dysfunction, motor behavior asymmetry, and dopaminergic neuronal cell loss correlated with dosage (1.25-5 µg/2 µl). We conclude that high-resolution respirometry may allow the detection of distinct mitochondrial dysfunction as a suitable surrogate marker for the preclinical assessment of potential neuroprotective strategies in the 6-OHDA model of PD.
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Neuronas Dopaminérgicas/efectos de los fármacos , Haz Prosencefálico Medial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Oxidopamina/toxicidad , Trastornos Parkinsonianos/fisiopatología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Neuronas Dopaminérgicas/patología , Neuronas Dopaminérgicas/fisiología , Relación Dosis-Respuesta a Droga , Lateralidad Funcional , Inmunohistoquímica , Masculino , Haz Prosencefálico Medial/patología , Haz Prosencefálico Medial/fisiopatología , Mitocondrias/fisiología , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fosforilación Oxidativa/efectos de los fármacos , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser](8)CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to >88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser](8)CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage.
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Respiración de la Célula , Ciclosporinas/metabolismo , Maleato de Dizocilpina/metabolismo , Glucosa/metabolismo , Hipocampo/fisiología , Fármacos Neuroprotectores/metabolismo , Oxígeno/metabolismo , Animales , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratas , Ratas WistarRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Técnicas de Cultivo/métodos , Degeneración Nerviosa , Neuronas , Tauopatías , Envejecimiento/metabolismo , Envejecimiento/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Espectroscopía Dieléctrica , Femenino , Humanos , Análisis por Micromatrices , Persona de Mediana Edad , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Neuronas/metabolismo , Neuronas/patología , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/metabolismoRESUMEN
UNLABELLED: 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.
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Lesiones Encefálicas/tratamiento farmacológico , Coma/tratamiento farmacológico , Indanos/uso terapéutico , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Ácidos Sulfónicos/uso terapéutico , Adolescente , Adulto , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/mortalidad , Lesiones Encefálicas/fisiopatología , Coma/etiología , Coma/mortalidad , Interpretación Estadística de Datos , Método Doble Ciego , Femenino , Escala de Coma de Glasgow , Humanos , Indanos/efectos adversos , Indanos/farmacocinética , Estimación de Kaplan-Meier , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Examen Neurológico , Reflejo Pupilar , Ácidos Sulfónicos/efectos adversos , Ácidos Sulfónicos/farmacocinética , Sobrevida , Tomografía Computarizada por Rayos X , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
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Isquemia Encefálica/enzimología , Antígenos CD13/metabolismo , Dipeptidil Peptidasa 4/metabolismo , Regulación Enzimológica de la Expresión Génica/fisiología , Animales , Isquemia Encefálica/complicaciones , Isquemia Encefálica/tratamiento farmacológico , Antígenos CD13/genética , Infarto Cerebral/enzimología , Infarto Cerebral/etiología , Dipeptidil Peptidasa 4/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/uso terapéutico , Lateralidad Funcional , Proteína Ácida Fibrilar de la Glía/metabolismo , Glicoesfingolípidos/uso terapéutico , Masculino , Fosfopiruvato Hidratasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+(cyt) (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+(cyt) (S0.5=60±10 nM). At higher Ca2+(cyt) (>600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-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 Ca2+(cyt), we show that the formation of pyruvate is tightly controlled by Ca2+(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 Ca2+(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, Ca2+(mit) (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+(cyt) is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.
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Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Malatos/metabolismo , Mitocondrias/metabolismo , Ácido Pirúvico/metabolismo , Animales , Cinética , Ratones , Fosforilación OxidativaRESUMEN
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.
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Técnicas Biosensibles/instrumentación , Espectroscopía Dieléctrica/instrumentación , Hipocampo/citología , Neuritas/patología , Tauopatías/patología , Animales , Carbazoles/farmacología , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Diseño de Equipo , Neuritas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tauopatías/tratamiento farmacológico , Proteínas tau/antagonistas & inhibidoresRESUMEN
Reactive oxygen species (ROS) are key players in ischemia-induced neurodegeneration. We investigated whether hippocampal neurons may lack sufficient redox-buffering capacity to protect against ROS attacks. Using organotypic hippocampal slice cultures (OHSCs) transiently exposed to oxygen and glucose deprivation (OGD) and gerbils suffering from a two-vessel occlusion (2VO) as complementary ex vivo and in vivo models, we have elucidated whether the intrinsic redox systems interfere with ischemia-induced neurodegeneration. Cell- type-specific immunohistological staining of hippocampal slice cultures revealed that pyramidal neurons, in contrast to astrocytes and microglia, express free thiols only weakly. In addition, free thiol levels were extensively decreased throughout the hippocampal formation immediately after OGD, but recovered within 24 hr after reperfusion. In parallel, progressive glia activation and proliferation were observed. Increased neuronal exposure to ROS was monitored by dihydroethidium oxidation in hippocampal pyramidal cell layers immediately after OGD. Coadministration of reduction equivalents (α-lipoic acid) and thiol-stimulating agents (enalapril, ambroxol) decreased ischemia-induced neuronal damage in OGD-treated OHSCs and in gerbils exposed to 2VO, whereas single drug applications remained ineffective. In summary, limited redox buffering capacities of pyramidal neurons may underlie their exceptional vulnerability to cerebral ischemia. Consistently, multidrug treatments supporting endogenous redox systems may offer a strategy to promote valid neuroprotection.
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Isquemia Encefálica/patología , Neuronas/patología , Oxidación-Reducción , Daño por Reperfusión/patología , Animales , Muerte Celular , Modelos Animales de Enfermedad , Etidio/análogos & derivados , Etidio/metabolismo , Fluoresceínas/metabolismo , Gerbillinae , Proteína Ácida Fibrilar de la Glía/metabolismo , Glucosa/deficiencia , Glicoproteínas/metabolismo , Hipocampo/citología , Hipoxia , Lectinas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Técnicas de Cultivo de Órganos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno , Rodaminas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Ácido Tióctico/farmacología , VersicanosRESUMEN
We studied the functional properties of isolated brain mitochondria (BM) prepared from total rat brain (BM(total)) or from cerebral subregions under basal and Ca(2+) 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-(NH(2)(CH2)(5)NHC(O)CH(2))-D-Ser](8)-CsA (Cs9) and its congener, the immunosuppressive [D-Ser](8)-CsA. The glutamate/malate-dependent state 3 respiration of mitochondria (state 3(glu/mal)) differed in region-specific manner (cortex > striatum = cerebellum > substantia nigra > hippocampus), but was significantly increased by 1µM CsA (+21±5%) in all regions. Ca(2+) overload induced by addition of 20µM Ca(2+) caused a significant decrease of state 3(glu/mal) (-45 to -55%) which was almost completely prevented in the presence of 1µM CsA, 1µM Cs9 or 1µM [D-Ser](8)-CsA. Mitochondrial Ca(2+) accumulation thresholds linked to permeability transition (PT) as well as the rate and completeness of mitochondrial Ca(2+) accumulation differed between different brain regions. For the first time, we provide a detailed, regiospecific analysis of Ca(2+)-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 Ca(2+) overload-mediated PT and functional impairments. Since Cs9 does not mediate immunosuppression, it might be used as a more specific PT inhibitor than CsA.
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Encéfalo/efectos de los fármacos , Ciclosporina/metabolismo , Inhibidores Enzimáticos/metabolismo , Mitocondrias/efectos de los fármacos , Animales , Calcio/metabolismo , Respiración de la Célula/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Masculino , RatasRESUMEN
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.
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Bioensayo/instrumentación , Técnicas Biosensibles/instrumentación , Espectroscopía Dieléctrica/instrumentación , Análisis por Micromatrices/instrumentación , Tauopatías/metabolismo , Tauopatías/patología , Proteínas tau/metabolismo , Línea Celular Tumoral , Sistemas de Computación , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Miniaturización , Coloración y EtiquetadoRESUMEN
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 Cacyt2+ taken up by the Ca2+ uniporter activates the matrix enzymes pyruvate dehydrogenase, alpha-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.
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Calcio/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa , Animales , Antiportadores/metabolismo , Canales de Calcio/metabolismo , Modelos Animales de Enfermedad , Complejo IV de Transporte de Electrones/metabolismo , Ácido Glutámico/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Ratones , Ratones Transgénicos , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Proteínas Mitocondriales/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Modelos Biológicos , Oxidorreductasas/metabolismo , Consumo de Oxígeno , Canales Aniónicos Dependientes del Voltaje/metabolismoRESUMEN
We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca(2+) in physiological concentration ranges (S(0.5) = 360 nM Ca(2+)). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca(2+) uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca(2+)-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca(2+). At micromolar concentrations, Ca(2+) can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca(2+) regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca(2+).
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Calcio/farmacología , Ácido Glutámico/metabolismo , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Adenosina Difosfato/farmacología , Animales , Respiración de la Célula/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Ratas , Ratas Wistar , Rojo de Rutenio/metabolismoRESUMEN
Mitochondrial dysfunction is a hallmark of almost all diseases. Acquired or inherited mutations of the mitochondrial genome DNA may give rise to mitochondrial diseases. Another class of disorders, in which mitochondrial impairments are initiated by extramitochondrial factors, includes neurodegenerative diseases and syndromes resulting from typical pathological processes, such as hypoxia/ischemia, inflammation, intoxications, and carcinogenesis. Both classes of diseases lead to cellular energetic depression (CED), which is characterized by decreased cytosolic phosphorylation potential that suppresses the cell's ability to do work and control the intracellular Ca(2+) homeostasis and its redox state. If progressing, CED leads to cell death, whose type is linked to the functional status of the mitochondria. In the case of limited deterioration, when some amounts of ATP can still be generated due to oxidative phosphorylation (OXPHOS), mitochondria launch the apoptotic cell death program by release of cytochrome c. Following pronounced CED, cytoplasmic ATP levels fall below the thresholds required for processing the ATP-dependent apoptotic cascade and the cell dies from necrosis. Both types of death can be grouped together as a mitochondrial cell death (MCD). However, there exist multiple adaptive reactions aimed at protecting cells against CED. In this context, a metabolic shift characterized by suppression of OXPHOS combined with activation of aerobic glycolysis as the main pathway for ATP synthesis (Warburg effect) is of central importance. Whereas this type of adaptation is sufficiently effective to avoid CED and to control the cellular redox state, thereby ensuring the cell survival, it also favors the avoidance of apoptotic cell death. This scenario may underlie uncontrolled cellular proliferation and growth, eventually resulting in carcinogenesis.
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Metabolismo Energético/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Adenosina Trifosfato/metabolismo , Apoptosis/fisiología , Proliferación Celular , Supervivencia Celular/fisiología , Glucólisis/fisiología , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/genética , Enfermedades Neurodegenerativas/genética , Fosforilación OxidativaRESUMEN
Tauopathies such as Alzheimer's disease (AD) belong to the group of neurodegenerative diseases that are characterised by hyperphosphorylation of the protein tau. Hyperphosphorylation of tau is one of the salient events leading to neuronal cytotoxicity and cognitive impairments. In this context, inhibition of tau hyperphosphorylation by specific tau kinase inhibitors can provide an excellent drug target for the treatment of AD and other tau-related neurodegenerative diseases. To improve the identification, optimisation and validation during the high-cost hit-to-lead cycle of AD drugs, we established a fast and sensitive label-free technique for testing the efficacy of tau kinase inhibitors in vitro. Here, we report for the first time that microelectrode-based impedance spectroscopy can be used to detect the pathological risk potential of hyperphosphorylated tau in the human neuroblastoma cell line SH-SY5Y. Our findings provide a novel real-time recording technique for testing the efficiency of tau kinase inhibitors or other lead structures directed to tau hyperphosphorylation on differentiated SH-SY5Y cells.
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Glucógeno Sintasa Quinasa 3 , Procedimientos Analíticos en Microchip/métodos , Proteínas tau/metabolismo , Análisis de Varianza , Carbazoles , Línea Celular Tumoral , Impedancia Eléctrica , Inhibidores Enzimáticos , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Laminina , Microelectrodos , Neuroblastoma , Ácido Ocadaico , Fosforilación , EstaurosporinaRESUMEN
Huntington disease (HD) is characterized by polyglutamine expansions of huntingtin (htt), but the underlying pathomechanisms have remained unclear. We studied brain mitochondria of transgenic HD rats with 51 glutamine repeats (htt(51Q)), modeling the adult form of HD. Ca(free)(2+) up to 2 mum activated state 3 respiration of wild type mitochondria with glutamate/malate or pyruvate/malate as substrates. Ca(free)(2+) above 2 mum inhibited respiration via cyclosporin A-dependent permeability transition (PT). Ruthenium red, an inhibitor of the mitochondrial Ca(2+) uniporter, did not affect the Ca(2+)-dependent activation of respiration but reduced Ca(2+)-induced inhibition. Thus, Ca(2+) activation was mediated exclusively by extramitochondrial Ca(2+), whereas inhibition was promoted also by intramitochondrial Ca(2+). In contrast, htt(51Q) mitochondria showed a deficient state 3 respiration, a lower sensitivity to Ca(2+) activation, and a higher susceptibility to Ca(2+)-dependent inhibition. Furthermore htt(51Q) mitochondria exhibited a diminished membrane potential stability in response to Ca(2+), lower capacities and rates of Ca(2+) accumulation, and a decreased Ca(2+) threshold for PT in a substrate-independent but cyclosporin A-sensitive manner. Compared with wild type, Ca(2+)-induced inhibition of respiration of htt(51Q) mitochondria was less sensitive to ruthenium red, indicating the involvement of extramitochondrial Ca(2+). In conclusion, we demonstrate a novel mechanism of mitochondrial regulation by extramitochondrial Ca(2+). We suggest that specific regulatory Ca(2+) binding sites on the mitochondrial surface, e.g. the glutamate/aspartate carrier (aralar), mediate this regulation. Interactions between htt(51Q) and distinct targets such as aralar and/or the PT pore may underlie mitochondrial dysregulation leading to energetic depression, cell death, and tissue atrophy in HD.
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Encéfalo/metabolismo , Calcio/metabolismo , Enfermedad de Huntington/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Encéfalo/patología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Colorantes/farmacología , Ciclosporina/farmacología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Inhibidores Enzimáticos/farmacología , Humanos , Proteína Huntingtina , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/genética , Mitocondrias/genética , Mitocondrias/patología , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Consumo de Oxígeno/efectos de los fármacos , Consumo de Oxígeno/genética , Ratas , Ratas Transgénicas , Rojo de Rutenio/farmacologíaRESUMEN
A better understanding of the cellular and molecular pathomechanisms of Alzheimer's disease (AD) is a prerequisite for the development of efficient treatments. We have used a novel assay system based on virus-transduced organotypic hippocampal slice cultures that mimics important aspects of tau-driven AD pathology in a short time frame. Human tau P301L, when expressed in pyramidal neurons of hippocampal slice cultures, was increasingly phosphorylated at several disease-relevant epitopes, leading to progressive neuronal dystrophy and formation of RIPA-insoluble tau. AD-like tau hyperphosphorylation was reduced by the tau kinase inhibitors lithium and SRN-003-556, but RIPA-insoluble tau remained unaffected after treatment with any of these substances. Only SRN-003-556 was able to protect hippocampal neurons from synaptic damage that was presumably caused by a toxic soluble tau fraction. These data provide first mechanistic insights towards the functional benefits of SRN-003-556 that have been observed in vivo.
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Hipocampo/citología , Inhibidores de Proteínas Quinasas/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Proteínas tau/metabolismo , Animales , Animales Recién Nacidos , Ensayo de Inmunoadsorción Enzimática/métodos , Fluoresceínas , Proteínas Fluorescentes Verdes/metabolismo , Degeneración Nerviosa/genética , Proteínas de Neurofilamentos/metabolismo , Técnicas de Cultivo de Órganos , Compuestos Orgánicos/metabolismo , Ratas , Ratas Wistar , Serina/metabolismo , Estadísticas no Paramétricas , Factores de Tiempo , Transducción Genética/métodosRESUMEN
FK506 and FK506-derived inhibitors of the FK506-binding protein (FKBP)-type peptidylprolyl cis/trans-isomerases (PPIase) display potent neuroprotective and neuroregenerative properties in various neurodegeneration models, showing the importance of neuroimmunophilins as targets for the treatment of acute and chronic neurodegenerative diseases. However, the PPIase activity targeted by active site-directed ligands remains unknown so far. Here we show that neurotrophic FKBP ligands, such as GPI1046 and N-[methyl(ethoxycarbonyl)]cycloheximide, inhibit the calmodulin/Ca(2+) (CaM/Ca(2+))-regulated FKBP38 with up to 80-fold higher affinity than FKBP12. In contrast, the non-neurotrophic rapamycin inhibits FKBP38.CaM/Ca(2+) 500-fold less affine than other neuroimmunophillins. In the context of the high expression of FKBP38 in neuroblastoma cells, these data suggest that FKBP38.CaM/Ca(2+) inhibition can mediate neurotrophic properties of FKBP ligands. The FKBP38-specific cycloheximide derivative, N-(N',N'-dimethylcarboxamidomethyl)cycloheximide (DM-CHX) was synthesized and used in a rat model of transient focal cerebral ischemia. Accordingly, DM-CHX caused neuronal protection as well as neural stem cell proliferation and neuronal differentiation at a dosage of 27.2 mug/kg. These effects were still dominant, if DM-CHX was applied 2-6 h post-insult. In parallel, sustained motor behavior deficits of diseased animals were improved by drug administration, revealing a potential therapeutic relevance. Thus, our results demonstrate that FKBP38 inhibition by DM-CHX regulates neuronal cell death and proliferation, providing a promising strategy for the treatment of acute and/or chronic neurodegenerative diseases.
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Isquemia Encefálica/patología , Encéfalo/patología , Cicloheximida/análogos & derivados , Cicloheximida/farmacología , Factores de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/farmacología , Proteínas de Unión a Tacrolimus/antagonistas & inhibidores , Calcio/metabolismo , Línea Celular Tumoral , Cicloheximida/química , Humanos , Cinética , Ligandos , Modelos Químicos , Enfermedades Neurodegenerativas , Neuronas/metabolismoRESUMEN
OBJECTIVE: The aim of the present work was the detection of Mitochondrial dysfunction of Huntington's disease (HD). METHODS: We investigated muscle and muscle mitochondria of 14- to 16-week-old R6/2 mice in comparison with wild-type mice. RESULTS: Atrophic fibers, increased fuchsinophilic aggregates, and reduced cytochrome c oxidase (15%) were found in HD muscle. With swelling measurements and Ca2+ accumulation experiments, a decreased stability of HD mitochondria against Ca2+-induced permeability transition was detected. Complex I-dependent respiration of HD mitochondria was more sensitive to inhibition by adding 10 microm Ca2+ than wild-type mitochondria. INTERPRETATION: Data suggest that the decreased stability of HD mitochondria against Ca2+ contributes to energetic depression and cell atrophy.