Die Funktion von Munc-18 in der Exozytose sekretorischer Granulen

Die Calcium-abhängige Freisetzung von Neurotransmitter aus sekretorischen Organellen wird durch eine Reihe konservierter Proteinfamilien präzise reguliert. Hierzu zählen u.a. SNARE-Proteine (soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor ), Rab-GTPasen und SM-Proteine. SNARE-Proteine sind membranständige Proteine, deren gemeinsames Merkmal das 60-70 Aminosäuren umfassende SNARE-Motiv ist. SNARE-Proteine können sich spontan und irreversibel zu einem Vier-Helix-Bündel („core complex“) zusammenlagern. Die Komplexbildung zwischen SNAREs auf Vesikelmembran und Plasmamembran führt zu einer Verkürzung der Proteine. Hierdurch werden die Membranen einander angenähert, was die Voraussetzung für die Membranfusion darstellt. Die funktionelle Bedeutung von SNARE-Proteinen für die Membranfusion wird durch die Vergiftung von Zellen mit den clostridialen Neurotoxinen Tetanustoxin und Botulinustoxin deutlich, die einzelne SNAREs proteolytisch spalten und die Neurotransmission hemmen. Das synaptische SM-Protein Munc-18-1 bindet hochaffin an das SNARE-Protein Syntaxin1. Hierbei konkurriert die Bindung von Munc-18-1 an Syntaxin1 mit dessen Einbindung in den „core complex“. Dies führte zu einem Modell, demzufolge Munc-18-1 durch seine Bindung an Syntaxin1 eine inhibitorische Rolle in der Exozytose übernimmt. Eine rein inhibitorische Funktion von Munc-18-1 läßt sich jedoch nicht mit seinem essentiellen Charakter für die Membranfusion in allen Spezies vereinbaren. Diese zentrale Rolle des Proteins im Membranfusionsvorgang ist jedoch weitgehend unverstanden. In dieser Arbeit wurde die Bedeutung der Interaktion zwischen Munc-18-1 und Syntaxin1 für die Exozytose von sekretorischen Granulen neuroendokriner Zellen in einem kombinierten Ansatz aus biochemischen und elektrophysiologischen Methoden untersucht. Die Interaktion zwischen Syntaxin1 und Munc-18-1 wurde gestört, indem zum einen Munc-18-1 überexprimiert wurde und zum andern in die Syntaxin1-Bindungsregion des Munc-18-1-Moleküls Punktmutationen eingeführt wurden (D34N und R39C). Nach der biochemischen Dokumentation der Störung der Syntaxin1-Bindung wurden die Mutanten sowie das Wildtyp- (WT-) Protein in der neuroendokrinen Zellinie PC12 überexprimiert und die Folgen der Überexpression mit Hilfe der Kohlefaser-Amperometrie elektrophysiologisch charakterisiert. Die Überexpression des Munc-18-WT-Proteins hatte keinen Effekt, was eine inhibitorische Funktion des Proteins unwahrscheinlich macht. Die R39C-Mutante wies eine gewisse Restbindung an Syntaxin1 auf, wohingegen die Bindung der D34N-Mutante an Syntaxin1 vernachlässigbar war. Trotz dieser gleichsinnig veränderten Syntaxin1-Bindung hatten die Mutanten gegensätzliche Effekte auf die Häufigkeit exozytotischer Ereignisse: die Zahl exozytotischer Ereignisse war durch R39C vermindert und durch D34N erhöht. Dies wies auf die Beteiligung eines weiteren Munc-18-1-Bindungspartners an den beobachteten Effekten hin, woraufhin die Interaktion von Mint1 mit Munc- 18-WT,-D34N und-R39C charakterisiert wurde. Mint1 ist ein synaptisches Multidomänen-Protein mit Phosphotyrosin-Bindungsdomänen sowie PDZ-Domänen, die die Interaktion mit weiteren präsynaptischen Proteinen vermitteln und Mint1 somit in die vielfältigen Protein-Interaktionen der Präsynapse einbetten. Die Beobachtung, daß bei gleichzeitiger Anwesenheit von Syntaxin1 und Mint1 die D34N-Mutante und die R39C-Mutante unterschiedliche Proteininteraktionen bevorzugten (Mint1/Munc-18-Komplex, bzw. Munc-18/Syntaxin1-Komplex), führte zu der Hypothese, daß der stimulatorische Effekt der D34N-Mutante durch die beobachtete gehäuft auftretende Interaktion mit Mint1 verursacht wird. Eine elektrophysiologische Charakterisierung des Mint1-Proteins in PC12-Zellen zeigte, daß Mint1 eine Inhibition der Exozytose verursacht. Die stimulierende Wirkung der D34N-Mutante könnte somit auf eine vermehrte Interaktion mit Mint1 zurückzuführen sein und eine Disinhibition darstellen. Die R39C-Mutante entfaltete ihre inhibitorische Wirkung vermutlich über die abgeschwächte Syntaxin1-Bindung. Zusammengefaßt hat Munc-18-1 eine positive Funktion in der LDCV-Exozytose. Zudem beschränkt sich die Rolle von Munc-18-1 nicht auf seine Interaktion mit Syntaxin1. Munc-18-1 ist über die Interaktion mit Mint1 vermutlich in vielfältige präsynaptische Komplexe involviert und könnte während der Membranfusion ein entscheidendes Bindeglied zwischen der Membranfusionsmaschinerie und strukturgebenden Komponenten der Präsynapse darstellen

Cellular and molecular basis of TNFa, IL-1ß and LPS mediated signaling in rat dorsal root ganglion

The proinflammatory cytokines TNFa and IL-1ß as well as bacterial lipopolysaccharide (LPS) are known to affect primary afferent functions related to pain and neurogenic inflammation. However, it is not completely understood how these molecules signal to primary sensory neurons of the dorsal root ganglion (DRG). In order to clarify this question RT-PCR, Northern blot, Western blot, RT-PCR in combination with laser capture microdissection (LCM) and in situ hybridization (ISH) with radioactive-labeled probes as well as double ISH were employed. These methods were used to determine the cell-specific expression pattern of TNF, IL-1 and their functional receptors as well as of LPS-related receptors in neuronal and non-neuronal cells of rat DRG as well as in the sensory cell line F11. The following essential new findings and conclusions have been obtained. (1) For the first time, the rat TNFR2 gene was characterized with 10 exons and 9 introns, which are located in chromosome 5q36. Three cDNAs for the rat TNFR2 gene were identified. Their full coding region was found to be identical. Three transcripts of the rat TNFR2 gene were observed in neural tissues (i.e. DRG, spinal cord and brain) and in peripheral tissues (i.e. spleen, lung and kidney). The regulation of TNFR2 transcripts by LPS seemed to occur in a tissue- and cell-specific manner as demonstrated for the spleen and DRG. (2) TNFR1 mRNA was found to be constitutively expressed in all DRG neurons including presumed nociceptive neurons coding for neuropeptides calcitonin gene-related peptide (CGRP), substance P (SP) or vanilloid receptor 1 (VR1) and to be increased after LPS. In contrast to the literature, TNFR2 mRNA was found to be totally absent from DRG neurons of control rats and of rats after LPS challenge. TNFR1 mRNA and TNFR2 mRNA were found to be constitutively expressed in DRG non-neuronal cells and to be increased after systemic LPS. The data provided by this study suggest that TNF may influence DRG sensory functions by directly acting on TNFR1 in neurons or by indirectly acting on both TNFR1 and TNFR2 in non-neuronal cells. (3) Like DRG neurons, the sensory cell line F-11 was found to express TNFR1 but not TNFR2. Therefore, the F11 cell line is uniquely suited to study TNFR1-mediated intracellular signaling and cellular functions independent from that of TNFR2 effects. (4) There was no evidence for but strong evidence against constitutive or LPS-induced expression of TNF and IL-1 mRNAs in DRG neurons. LPS-induced expression of TNF and IL-1 mRNAs in DRG occurred exclusively in DRG non-neuronal cells. Thus, the previously reported concept that TNF and IL-1 are synthesized by DRG neurons should be dismissed. To the contrary, the present data indicate that endogenous TNF and IL-1 in DRG are exclusively synthesized by non-neuronal cells implicating that they may act on DRG neurons in a paracrine manner. (5) In contrast to a previous report indicating that IL-1R1 is expressed in all DRG cells, the present study demonstrated that IL-1R1 mRNA is expressed only in a subpopulation of DRG neurons and in some DRG non-neuronal cells as well. IL-1R1 exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The results of the present study suggest that endogenous and exogenous IL-1 may directly activate DRG neurons via IL-1R1 to preferentially modulate nociceptive functions. In addition, IL-1 may act on DRG non-neuronal cells to cause further release of IL-1. (6) For the first time, the functional LPS receptor-TLR4 was demonstrated to be expressed in DRG neuronal and non-neuronal cells at the mRNA level. The neuronal expression of TLR4 was limited to a subset of DRG neurons where it exhibited substantial coincidence with presumed nociceptive neurons expressing VR1, SP or CGRP. The mRNA coding for the LPS receptor accessory protein CD14 was totally absent from DRG neurons of control rats and of rats after systemic LPS. LPS-induced expression of CD14 occurred in DRG non-neuronal cells. The present data indicate that LPS may directly act on primary sensory neurons via TLR4 or indirectly act on primary sensory neurons via TLR4 and CD14. This implies that primary sensory neurons of DRG may detect an infectious state by directly sensing LPS via TLR4. Taken together, this study provides new insights into the cellular and molecular basis of TNF, IL-1 and LPS mediated primary sensory neurotransmission related to pain and neurogenic inflammation. In addition, the present study provides new evidence that the primary sensory neurons of DRG may have an important role as immunosensors to detect and control microbial infection and inflammation

Vital capacity and valvular dysfunction could serve as non-invasive predictors to screen for exercise pulmonary hypertension in the elderly based on a new diagnostic score

Introduction: Exercise pulmonary hypertension (exPH) has been defined as total pulmonary resistance (TPR) >3 mm Hg/L/min and mean pulmonary artery pressure (mPAP) >30 mm Hg, albeit with a considerable risk of false positives in elderly patients with lower cardiac output during exercise. Methods:We retrospectively analysed patients with unclear dyspnea receiving right heart catheterisation at rest and exercise (n=244) between January 2015 and January 2020. Lung function testing, blood gas analysis, and echocardiography were performed. We elaborated a combinatorial score to advance the current definition of exPH in an elderly population (mean age 67.0 years±11.9). A stepwise regression model was calculated to non-invasively predict exPH. Results: Analysis of variables across the achieved peak power allowed the creation of a model for defining exPH, where three out of four criteria needed to be fulfilled: Peak power ≤100 Watt, pulmonary capillary wedge pressure ≥18 mm Hg, pulmonary vascular resistance >3 Wood Units, and mPAP ≥35 mm Hg. The new scoring model resulted in a lower number of exPH diagnoses than the current suggestion (63.1% vs. 78.3%). We present a combinatorial model with vital capacity (VCmax) and valvular dysfunction to predict exPH (sensitivity 93.2%; specificity 44.2%, area under the curve 0.73) based on our suggested criteria. The odds of the presence of exPH were 2.1 for a 1 l loss in VCmax and 3.6 for having valvular dysfunction. Conclusion: We advance a revised definition of exPH in elderly patients in order to overcome current limitations. We establish a new non-invasive approach to predict exPH by assessing VCmax and valvular dysfunction for early risk stratification in elderly patients

A physically based material model for the simulation of friction stir welding

A physically based material model, taking into account the interdependence of material microstructure and yield strength, is presented for an Al 5182 series aluminum alloy for the simulation of friction stir welding using continuum mechanics approaches. A microstructure evolution equation considering dislocation density and grain size is used in conjunction with a description of yield stress. In order to fit experimental stress-strain curves, obtained from compression tests at various strain rates and temperatures, phenomenological relationships are developed for some of the model parameters. The material model is implemented in smoothed particle hydrodynamic research code as well as in the commercial finite element code Abaqus. Simulations for various strain rates and temperatures were performed and compared with experimental results as well as between the two discretization methods in order to verify the material model and the implementation. Simulations provide not only an accurate approximation of stress based on temperature, strain rate, and strain but also an improved insight into the microstructural evolution of the material

CAPS1 Regulates Catecholamine Loading of Large Dense-Core Vesicles

SummaryCAPS1 is thought to play an essential role in mediating exocytosis from large dense-core vesicles (LDCVs). We generated CAPS1-deficient (KO) mice and studied exocytosis in a model system for Ca2+-dependent LDCV secretion, the adrenal chromaffin cell. Adult heterozygous CAPS1 KO cells display a gene dosage-dependent decrease of CAPS1 expression and a concomitant reduction in the number of docked vesicles and secretion. Embryonic homozygous CAPS1 KO cells show a strong reduction in the frequency of amperometrically detectable release events of transmitter-filled vesicles, while the total number of fusing vesicles, as judged by capacitance recordings or total internal reflection microscopy, remains unchanged. We conclude that CAPS1 is required for an essential step in the uptake or storage of catecholamines in LDCVs

Protein Expression of the Microglial Marker Tmem119 Decreases in Association With Morphological Changes and Location in a Mouse Model of Traumatic Brain Injury

The activation of microglia and the infiltration of macrophages are hallmarks of neuroinflammation after acute brain injuries, including traumatic brain injury (TBI). The two myeloid populations share many features in the post-injury inflammatory response, thus, being antigenically indistinguishable. Recently Tmem119, a type I transmembrane protein specifically expressed by microglia under physiological conditions, was proposed as a tool to differentiate resident microglia from blood-borne macrophages, not expressing it. However, the validity of Tmem119 as a specific marker of resident microglia in the context of acute brain injury, where microglia are activated and macrophages are recruited, needs validation. Our purpose was to investigate Tmem119 expression and distribution in relation to the morphology of brain myeloid cells present in the injured area after TBI. Mice underwent sham surgery or TBI by controlled cortical impact (CCI). Brains from sham-operated, or TBI mice, were analyzed by in situ hybridization to identify the cells expressing Tmem119, and by Western blot and quantitative immunofluorescence to measure Tmem119 protein levels in the entire brain regions and single cells. The morphology of Iba1+ myeloid cells was analyzed at different times (4 and 7 days after TBI) and several distances from the contused edge in order to associate Tmem119 expression with morphological evolution of active microglia. In situ hybridization indicated an increased Tmem119 RNA along with increased microglial complement C1q activation in the contused area and surrounding regions. On the contrary, the biochemical evaluation showed a drop in Tmem119 protein levels in the same areas. The Tmem119 immunoreactivity decreased in Iba1+ myeloid cells found in the contused cortex at both time points, with the cells showing the hypertrophic ameboid morphology having no Tmem119 expression. The Tmem119 was present on ramifications of resident microglia and its presence was decreased as a consequence of microglial activation in cortical areas close to contusion. Based on the data, we conclude that the decrease of Tmem119 in reactive microglia may depend on the process of microglial activation, which involves the retracting of their branchings to acquire an ameboid shape. The Tmem119 immunoreactivity decreases in reactive microglia to similar levels than the blood-borne macrophages, thus, failing to discriminate the two myeloid populations after TBI.This work was supported by the ERA-NET NEURON, JTC 2016: LEAP, NEURON9-FP-044 from the following national funding institutions: Italian Ministry of Health (Ministero della Salute), Italy; Ministerio de Economía, Industria y Competitividad (PCIN-2017-035) Spain; 01EW1703, Bundesministerium für Bildung und Forschung (BMBF), Germany.Peer reviewe

Intravenous Inoculation of a Bat-Associated Rabies Virus Causes Lethal Encephalopathy in Mice through Invasion of the Brain via Neurosecretory Hypothalamic Fibers

The majority of rabies virus (RV) infections are caused by bites or scratches from rabid carnivores or bats. Usually, RV utilizes the retrograde transport within the neuronal network to spread from the infection site to the central nervous system (CNS) where it replicates in neuronal somata and infects other neurons via trans-synaptic spread. We speculate that in addition to the neuronal transport of the virus, hematogenous spread from the site of infection directly to the brain after accidental spill over into the vascular system might represent an alternative way for RV to invade the CNS. So far, it is unknown whether hematogenous spread has any relevance in RV pathogenesis. To determine whether certain RV variants might have the capacity to invade the CNS from the periphery via hematogenous spread, we infected mice either intramuscularly (i.m.) or intravenously (i.v.) with the dog-associated RV DOG4 or the silver-haired bat-associated RV SB. In addition to monitoring the progression of clinical signs of rabies we used immunohistochemistry and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to follow the spread of the virus from the infection site to the brain. In contrast to i.m. infection where both variants caused a lethal encephalopathy, only i.v. infection with SB resulted in the development of a lethal infection. While qRT-PCR did not reveal major differences in virus loads in spinal cord or brain at different times after i.m. or i.v. infection of SB, immunohistochemical analysis showed that only i.v. administered SB directly infected the forebrain. The earliest affected regions were those hypothalamic nuclei, which are connected by neurosecretory fibers to the circumventricular organs neurohypophysis and median eminence. Our data suggest that hematogenous spread of SB can lead to a fatal encephalopathy through direct retrograde invasion of the CNS at the neurovascular interface of the hypothalamus-hypophysis system. This alternative mode of virus spread has implications for the post exposure prophylaxis of rabies, particularly with silver-haired bat-associated RV

Charakterisierung humaner hippocampaler Astrozytenvorläuferzellen in Langzeitkultur zur Anwendung in der ex-vivo Gentherapie

Astrozytäre Vorläuferzellen wurden erstmals aus postnatalem humanen Hippocampus isoliert und als Primärkultur in vitro für mehr als 6 Monate expandiert. Die Zellen wiesen die typische Morphologie protoplasmatischer Astrozyten auf und zeigten sich in der immunzytochemischen Analyse positiv für GFAP, Nestin, Vimentin und S-100. Außerdem konnte eine Positivität für die neuronalen Marker -III-Tubulin und NeuN nachgewiesen werden, was für Astrozytenvorläufer bislang nicht beschrieben wurde. Proliferierende Zellen konnten mit dem Ki-67 Antikörper identifiziert werden. Dabei betrug die initiale Generationszeit zwei bis drei Tage und verlangsamte sich nach der achten Passage in Zellkultur. Die Zellproliferation wurde in Anwesenheit von 10% FCS oder anderen Wachstumsfaktoren bestimmt. Durch Giemsa Färbung konnte ein diploider Chromosomensatz bestimmt werden und eine Transplantation sowohl in Nacktmäuse, als auch in Nacktratten ergab keinen Anhalt für ein tumorigenes Potential der Zellen. Die humanen hippocampalen Astrozytenvorläufer sezernieren NGF und BDNF in Konzentrationen von 36,2 bzw. 16,7 pg / 105 Zellen pro Tag. Die Zellen konnten effizient durch einen EGFP-exprimierenden adenoviralen Vektor transduziert werden. Transplantierte EGFP-positive Zellen überlebten im Empfängergehirn und zeigten eine typisch astrozytäre Morphologie. Humane astrozytäre Vorläuferzellen stellen somit eine vielversprechende Quelle für eine ex vivo Gentherapie bei neurodegenerativen Erkrankungen dar

Rabies virus replication outside the central nervous system - Implications for disease transmission

Rabies is a fatal disease in mammals which is transmitted by the neurotropic Rabies virus (RV). Most often, classical RV infections originate from muscle tissue after a bite through an infected canine and ascend to the central nervous system (CNS) via peripheral nerves. In contrast, transfer of non-classical RV by bat bites or scratches, the most common cause for human rabies in North America and also an emerging disease in Europe, most likely introduces RV in rather small amounts superficially into a new host. In both cases, classical and non-classical RV can have access to lymph and/or blood. The impact and effects of the hematogenously and lymphatically distributed share of the viral inoculum is unclear. Taking this into account combined with recent RV infections through unrecognized RV infected organ transplantations the questions arose whether RV is able to infect peripheral organs primarily via a vascular route or only by centrifugal spread via neuronal pathways from the CNS and if this postulated route is strain dependent. Subsequently it was thought to be elucidated, whether RV is able to replicate in organs and if its target cells for direct invasion of organs are different from those it reaches after centrifugal spread from the CNS. With regard to the transmission of RV by tissue transplants it was also investigated whether RV originating from organs is more likely to ascend into the CNS by neuronal pathways or on alternative routes. In order to answer these questions, mice were infected either with a dog-derived classical RV (DOG4) or a bat-derived non-classical RV (rSB) as representatives for the two RV strains with the largest impact in naturally occurring human rabies, and monitored for weight loss and disease symptoms. To maximize the hematogenous dissemination of the inoculum, mice were infected intravenously (i.v.) and compared to mice inoculated intramuscularly (i.m.). A TaqMan® probe based quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify strain-specifically negativestranded as well as positive-stranded viral RNA in various tissues. For confirmation of replicating RV, virus was isolated from tissues and the nature of virus-positive cells in the periphery determined by immunohistochemistry. A kinetic study was undertaken to trace the pathways of RV into and within the CNS after i.v. and i.m. inoculation. I.m. inoculation with either DOG4 or rSB led to hind limb paralysis and death within twelve days. Viral RNA was detected in the CNS and all analyzed organs (lungs, heart, liver, kidneys) from morbid animals. rSB killed mice in a dose-dependent way also when injected i.v., however without causing typical symptoms of rabies. Surprisingly, i.v. inoculation of DOG4 rendered the infection completely harmless. The mice recovered from a short period of mild weight loss and survived for longer than eight months, showing no signs of viral replication in organs, but low virus load in blood cells and CNS. This and persistent high virus neutralizing antibody (VNA) titers suggest an ongoing immune-controlled latent RV infection after DOG4 i.v. inoculation. After rSB i.m. inoculation, the spread of RV to the periphery was only detected after viral progression throughout the CNS. Importantly, viral RNA was detected at early time points in organs after i.v. inoculation and infectious RV was isolated from the heart before it was isolated from the brain. After i.m. as well as after i.v. inoculation with rSB only neuronal cells were found to be positive for viral antigen. This data reveal for the first time the possibility of a primary infection of peripheral ganglionic cells in organs by rSB via a nonneuronal route. Immunohistochemical kinetic studies of CNS tissue after rSB i.m. inoculation confirmed the motor pathway from the muscle to the brain as the main route for viral invasion whereby the sensory system was affected only secondarily through its connections to the motor system. In contrast, the forebrains of i.v. inoculated mice were infected independently from the presence of viral antigen in spinal cord and brain stem. Our immunohistochemical findings suggest for the first time a direct invasion of the CNS by rSB from the vascular system, most preferentially through hypothalamic neurosecretory axons in the neurohypophysis and the median eminence, whereas retrograde neuronal transport of RV from peripheral organs to the CNS proved to be unlikely