Untersuchung von homöostatischen Kalziumströmen in hippokampalen Neuronen mittels “targeted-esterase induced dye loading (TED)”

Calcium ions can activate intracellular signalling cascades that control key functions in all types of neurons. These functions include neuronal excitability and excitation, synaptic plasticity, cell migration, transmitter release, gene transcription, and apoptosis. The major intracellular neuronal store for calcium is the endoplasmic reticulum (ER), a continuous and dynamic, membranous organelle that extends through all parts of neurons, from axons to dendrites. The calcium concentration in the ER is appr. one thousand fold higher than in the cytosol and this calcium gradient is built up by the sarco-/endoplasmic reticulum calcium ATPase (SERCA) pump that pumps calcium from the cytosol into the ER. Despite detailed knowledge about various induced calcium signals within neurons, it was still elusive, how resting neurons maintain their ER calcium content at rest. In order to shed light on the calcium homeostasis at rest, the targeted-esterase induced dye loading (TED) technique was improved. TED allows the direct and non-disruptive visualization of ER calcium in presence of extracellular calcium, thus enabling to visualize the dynamic flow of ER calcium. TED is based on the overexpression of an ER-targeted mouse carboxylesterase. Inside the ER the carboxylesterase cleaves the acetoxymethyl ester calcium dye Fluo5N, AM, thereby converting this dye into a calcium sensitive, low-affinity, cell membrane impermeable calcium indicator that is trapped in the ER. When bound to calcium ions and excited by fluorescent light, its fluorescence intensity increases one hundredfold compared to the calcium-free state. It was observed that calcium withdrawal from resting neurons led to a rapid loss of calcium from both the ER and the cytosol, which recovered upon calcium re-addition. It was concluded that a strong calcium influx and efflux must exist under resting conditions that maintain a constant calcium concentration in neurons at rest. TED calcium imaging could visualize this resting calcium influx event. When the inhibitor of store-operated calcium entry (SOCE), SKF-96365, was acutely added to neurons an immediate decline in ER calcium levels was observed, whereas cytosolic calcium levels remained constant. Based on these findings, a novel calcium homeostasis model is proposed in which a strong SOCE-like calcium influx and a corresponding calcium efflux maintain the ER calcium levels at rest. These fluxes are adapted to disturbances in order to maintain a constant calcium level in resting neurons. This study visualizes for the first time the resting calcium flow into the ER. The calcium enters the neurons via a store-operated calcium entry-like mechanism, a form of calcium influx that was thought to be induced by signalling events.Kalzium kann intrazelluläre Signalkaskaden aktivieren und somit Schlüsselfunktionen in allen Typen von Neuronen kontrollieren. Diese Schlüsselfunktionen umfassen Zellmigration, Freisetzung von Neurotransmittern, synaptische Plastizität, Transkription von Genen und Apoptose. Der wichtigste Speicher für Kalzium in Neuronen ist das endoplasmatische Retikulum (ER); ein kontinuierliches, membranumschlossenes Zellorganell, das sich in alle Teile von Neuronen erstreckt, von Axonen bis zu Dendriten. Im ER ist die Kalziumkonzentration ca. eintausendmal höher als im Zytosol. Dieser Kalziumgradient wird von der sogenannten SERCA, der sarco-/endoplasmic reticulum calcium ATPase, Kalziumpumpe aufrechterhalten, die Kalziumionen aktiv vom Zytosol in das Innere des ER pumpt. Obwohl die einzelnen induzierten Kalziumsignalwege in Neuronen gut untersucht sind, war es lange nicht bekannt wie es Neuronen gelingt die hohe Kalziumkonzentration des ERs in Ruhe aufrechtzuerhalten. Um diese Frage zu klären wurde die sogenannte targeted-esterase induced dye loading (TED) Technik verbessert. TED ermöglicht es ER Kalzium direkt, in intakten Neuronen und in Gegenwart von extrazellulärem Kalzium sichtbar zu machen. Dadurch wird es möglich den Kalziumstrom des ERs direkt zu beobachten. TED beruht darauf, dass eine für das ER bestimmte Carboxylesterase der Maus überexprimiert wird, die sich im ER ansammelt. Im Inneren des ERs wandelt dieses Enzym den synthetischen Kalziumfarbstoff Fluo5N, AM um, der zur Gruppe der Acetoxymethyl-ester gehört. Dadurch wird aus diesem Farbstoff ein kalzium-sensitiver, niedrig-affiner Kalziumindikator, der nicht mehr permeabel für biologische Membranen ist und daher im ER verbleibt. In seiner Kalzium-gebunden Form erhöht sich die Fluoreszenz dieses Indikators einhundertfach im Vergleich zur Kalzium-ungebunden Form, wenn er durch Fluoreszenzlicht angeregt wird. Es wurde beobachtet, dass der Entzug von extrazellulärem Kalzium zu Verlust von Kalzium im ER und Zytosol von Neuronen führt. Bei Rückgabe des Kalziums erholt sich die Kalziumkonzentration wieder. Daraus wurde gefolgert, dass es bei Neuronen in Ruhe einen starken Einstrom und Ausstrom von Kalzium geben muss. Diese Ströme halten den Kalziumgehalt der Neurone konstant. Wenn SKF-96365, ein Inhibitor des sogenannten store-operated calcium entry (SOCE), akut auf die Neurone appliziert wurde, wurde sofort ein Abfall der ER Kalziumkonzentration beobachtet, wohingegen die Kalziumkonzentration des Zytosols gleich blieb. Aufgrund dieser Ergebnisse kann ein neues Kalziumhomöostase-Modell für Neurone vorgeschlagen werden: In ruhenden Neuronen halten ein starker SOCE-ähnlicher Kalziumeinstrom und ein entsprechender Kalziumausstrom den Kalziumgehalt des ERs aufrecht. Diese Ströme werden an Störungen angepasst um eine konstante Kalziumkonzentration zu erreichen. In dieser Studie wird zum ersten Mal der Ruhestrom von Kalzium in das ER sichtbar gemacht. Kalzium gelangt über einen store-operated calcium entry -ähnlichen Mechanismus in die Neurone. Bisher dachte man, dass diese Art von Kalziumeinstrom nur nach Aktivierung von Signalen stattfindet

Investigation of Homeostatic Calcium Fluxes in Hippocampal Neurons by Means of Targeted-Esterase Induced Dye Loading (TED)

Calcium ions can activate intracellular signalling cascades that control key functions in all types of neurons. These functions include neuronal excitability and excitation, synaptic plasticity, cell migration, transmitter release, gene transcription, and apoptosis. The major intracellular neuronal store for calcium is the endoplasmic reticulum (ER), a continuous and dynamic, membranous organelle that extends through all parts of neurons, from axons to dendrites. The calcium concentration in the ER is appr. one thousand fold higher than in the cytosol and this calcium gradient is built up by the sarco-/endoplasmic reticulum calcium ATPase (SERCA) pump that pumps calcium from the cytosol into the ER. Despite detailed knowledge about various induced calcium signals within neurons, it was still elusive, how resting neurons maintain their ER calcium content at rest. In order to shed light on the calcium homeostasis at rest, the targeted-esterase induced dye loading (TED) technique was improved. TED allows the direct and non-disruptive visualization of ER calcium in presence of extracellular calcium, thus enabling to visualize the dynamic flow of ER calcium. TED is based on the overexpression of an ER-targeted mouse carboxylesterase. Inside the ER the carboxylesterase cleaves the acetoxymethyl ester calcium dye Fluo5N, AM, thereby converting this dye into a calcium sensitive, low-affinity, cell membrane impermeable calcium indicator that is trapped in the ER. When bound to calcium ions and excited by fluorescent light, its fluorescence intensity increases one hundredfold compared to the calcium-free state. It was observed that calcium withdrawal from resting neurons led to a rapid loss of calcium from both the ER and the cytosol, which recovered upon calcium re-addition. It was concluded that a strong calcium influx and efflux must exist under resting conditions that maintain a constant calcium concentration in neurons at rest. TED calcium imaging could visualize this resting calcium influx event. When the inhibitor of store-operated calcium entry (SOCE), SKF-96365, was acutely added to neurons an immediate decline in ER calcium levels was observed, whereas cytosolic calcium levels remained constant. Based on these findings, a novel calcium homeostasis model is proposed in which a strong SOCE-like calcium influx and a corresponding calcium efflux maintain the ER calcium levels at rest. These fluxes are adapted to disturbances in order to maintain a constant calcium level in resting neurons. This study visualizes for the first time the resting calcium flow into the ER. The calcium enters the neurons via a store-operated calcium entry-like mechanism, a form of calcium influx that was thought to be induced by signalling events

Normal sulfation levels regulate spinal cord neural precursor cell proliferation and differentiation

Abstract Background Sulfated glycosaminoglycan chains are known for their regulatory functions during neural development and regeneration. However, it is still unknown whether the sulfate residues alone influence, for example, neural precursor cell behavior or whether they act in concert with the sugar backbone. Here, we provide evidence that the unique 473HD-epitope, a representative chondroitin sulfate, is expressed by spinal cord neural precursor cells in vivo and in vitro, suggesting a potential function of sulfated glycosaminoglycans for spinal cord development. Results Thus, we applied the widely used sulfation inhibitor sodium chlorate to analyze the importance of normal sulfation levels for spinal cord neural precursor cell biology in vitro. Addition of sodium chlorate to spinal cord neural precursor cell cultures affected cell cycle progression accompanied by changed extracellular signal-regulated kinase 1 or 2 activation levels. This resulted in a higher percentage of neurons already under proliferative conditions. In contrast, the relative number of glial cells was largely unaffected. Strikingly, both morphological and electrophysiological characterization of neural precursor cell-derived neurons demonstrated an attenuated neuronal maturation in the presence of sodium chlorate, including a disturbed neuronal polarization. Conclusions In summary, our data suggest that sulfation is an important regulator of both neural precursor cell proliferation and maturation of the neural precursor cell progeny in the developing mouse spinal cord.</p

ER stress-mediated BK dysfunction in the DRG underlies pain in a model of multiple sclerosis

ABSTRACTNeuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went to assess disease-mediated changes in the functional properties of Ca2+-sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype.</jats:p

Endoplasmic reticulum stress in the dorsal root ganglia regulates large-conductance potassium channels and contributes to pain in a model of multiple sclerosis

Neuropathic pain is a common symptom of multiple sclerosis (MS) and current treatment options are ineffective. In this study, we investigated whether endoplasmic reticulum (ER) stress in dorsal root ganglia (DRG) contributes to pain hypersensitivity in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Inflammatory cells and increased levels of ER stress markers are evident in post-mortem DRGs from MS patients. Similarly, we observed ER stress in the DRG of mice with EAE and relieving ER stress with a chemical chaperone, 4-phenylbutyric acid (4-PBA), reduced pain hypersensitivity. In vitro, 4-PBA and the selective PERK inhibitor, AMG44, normalize cytosolic Ca2+ transients in putative DRG nociceptors. We went on to assess disease-mediated changes in the functional properties of Ca2+-sensitive BK-type K+ channels in DRG neurons. We found that the conductance-voltage (GV) relationship of BK channels was shifted to a more positive voltage, together with a more depolarized resting membrane potential in EAE cells. Our results suggest that ER stress in sensory neurons of MS patients and mice with EAE is a source of pain and that ER stress modulators can effectively counteract this phenotype