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

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

Die PAC1-Agonisten und PACAP-Rezeptoren in murinen und humanen kleinzelligen Bronchialkarzinom Zelllinien

Zusammenfassung Die pleiotropen Neuropeptide PACAP (pituitary adenylate cyclase activating peptide) und VIP (vasoactive intestinal polypeptide) sind Mitglieder der VIP/PACAP/Glucagon/Secretin-Superfamilie und wirken über G-Proteingekoppelte-Rezeptoren. PACAP wirkt auf den PAC1-Rezeptor und wie VIP auf VPAC1 und VPAC2. Der Nachweis pharmakologischer Wirkungen von Agonisten und Antagonisten der VIP-/PACAP-Familie auf Bronchialkarzinomzelllinien durch Terry Moody lieferte erste Evidenzen, dass Bronchialkarzinomzellen funktionelle VIP-/PACAP-Rezeptoren exprimieren. Allerdings wurde das differentielle PAC1-, VPAC1- und VPAC2-Rezeptorprofil von Bronchialkarzinomzellen bisher noch nicht entschlüsselt. Deshalb setzte sich die vorliegende Arbeit zum Ziel, die mRNAExpressionsprofile der PAC1-, VPAC1- und VPAC2-Rezeptoren inklusive der PAC1-Splicevarianten in der murinen kleinzelligen Lewis-lung-carcinoma-1-(LLC1)-Zelllinie und deren subkutanen und pulmonalen Transplantaten sowie in verschiedenen humanen kleinzelligen Bronchialkarzinomzelllinien und -karzinomen zu differenzieren. Darüber hinaus sollten die Funktionalität des PAC1-Rezeptors in der LLC1-Zelllinie und die Wirkung von PAC1-Agonisten auf die Proliferation bzw. Apoptoserate in der humanen kleinzelligen NCI-H82-BronchiallkarzinomZelllinie getestet werden. Ein weiteres Ziel war, die Expression der tumorrelevanten PAC1-Zielgene Stanniocalcin 1 und Stathmin 1 in der murinen und den humanen Zelllinien und im humanen kleinzelligen Bronchialkarzinom zu bestimmen und den Einfluss PAC1-spezifischer Liganden auf ihre Expression zu erfassen. Folgende wesentliche neue Erkenntnisse wurden erzielt. Es gelang, die RNA-Expressionsprofile von PAC1, VPAC1 und VPAC2 und aller bekannten PAC1-Splicevarianten in der murinen LLC1-Tumorzelllinie und ihren subkutanen und pulmonalen Transplantaten sowie im humanen kleinzelligen Bronchialkarzinom zu differenzieren. Zwei Gruppen humaner Zelllinien kleinzelliger Bronchialkarzinome mit reziproker Abundanz von PAC1 und PACAP mRNA wurden differenziert. In Gruppe 1 war PAC1 hoch exprimiert und PACAP nicht detektierbar, in Gruppe 2 war PAC1 niedrig und PACAP hoch exprimiert. Mithilfe einer FRET-(Förster Resonanz Energie Transfer)-Messung konnte eine funktionell relevante Aktivierung des PAC1-Rezeptors in LLC1-Zellen durch subnanomolare Konzentrationen von PACAP38 nachgewiesen werden, welche die Synthese von cAMP induzierte und zur Freisetzung von Ca 2+ führte. Während die PAC1-Rezeptoraktivierung in der murinen LLC1-Zelllinie zu einer mäßigen Reduktion der Proliferationsrate führte, resultierte eine PAC1-Rezeptoraktivierung in der humanen NCI-H82-Zelllinie nur in einer marginalen Änderung der Proliferationsrate ohne nachweisbare Änderung der durch CisPt induzierten Apoptoserate. Die PAC1-Liganden Maxadilan, PACAP38 und PACAP27 schienen in LLC1-Zellen eine Rückkopplungshemmung der Expression des PAC1-Rezeptors auf mRNA-Niveau zu verursachen. Die murine LLC1-Zelllinie und das murine pulmonale LLC1 -Transplantat wiesen mRNA-Koexpression des PAC1-Rezeptors und seiner putativen Zielgene Stanniocalcin 1 und Stathmin 1 auf. Hingegen war das mRNA Koexpressionsmuster von PAC1 und seinen putativen Zielgenen Stanniocalcin 1 und Stathmin 1 in humanen kleinzelligen Bronchialkarzinomzelllinien und kleinzelligen Bronchialkarzinomen heterogen. Eine eindeutige Regulation der Transkription der genannten Zielgene durch eine PAC1-Rezeptoraktivierung konnte weder für die murine noch für die humanen kleinzelligen Bronchialkarzinomzelllinie(n) nachgewiesen werden. Die Ergebnisse lassen folgende Schlussfolgerungen zu. Da PAC1 und seine putativen Zielgene Stathmin 1 und Stanniocalcin 1 in murinen und humanen Zelllinien kleinzelliger Bronchialkarzinome in der Regel stark exprimiert sind, eignen sie sich sowohl in der präklinischen als auch klinischen Forschung als Bio- bzw. Tumormarker. Zum einen bietet sich an, Maxadilan -PET-Liganden zu entwickeln und für die diagnostische bzw. prognostische Tumorbildgebung einzusetzen. Zum anderen könnten PAC1-Rezeptoren auf kleinzelligen Bronchialkarzinomzellen für „Drug-targeting“ genutzt werden. Angesichts heterogener Ko-Expressionsmuster von PAC1-Rezeptoren und seinen beiden putativen Zielgenen Stanniocalcin 1 und Stathmin 1 in humanen kleinzelligen Bronchialkarzinomzelllinien liegt es nahe, das jeweilige kombinatorische Expressionsprofil von PAC1, PACAP, Stanniocalcin 1 und Stathmin 1 in Tumorbiopsien, Metastasen und Blutproben als Biomarker zur diagnostischen und prognostischen Differenzierung der Malignität von Bronchialkarzinomen im 3 Patienten zu nutzen. Insgesamt offerieren die hier erzielten Ergebnisse einen Beitrag für neue translationale Ansätze in der personalisierten Krebsmedizin. Schlüsselworte: kleinzelliges Bronchialkarzinom, kleinzellige Bronchialkarzinomzelllinien, LLC1-Zelllinie, PACAP, PAC1, VPAC1, VPAC2, PAC1-Splicevarianten, Stanniocalcin 1, Stathmin 1, Genexpression, Maxadila

Pituitary adenylate cyclase-activating polypeptide mediates differential signaling through PAC1 receptor splice variants and activates non-canonical cAMP dependent gene induction in the nervous system - Implications for homeostatic stress-responding

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. PAC1 occurs in different splice variants of the third intracellular loop, designated PAC1null, hop or hip, affecting second messenger generation as shown in non-neural cells. At the splanchnico-adrenomedullary synapse, PACAP is required for prolonged catecholamine secretion from chromaffin cells to restore homeostasis during prolonged periods of stress. In the central nervous system, PACAP is neuroprotective in neurodegenerative conditions associated with e.g., stroke. In the present study, PAC1 splice variant-specific second messenger production and activation of homeostatic responses were investigated in neuroendocrine and neural cells. Heterologous expression of the major PAC1 splice variant of adrenomedullary chromaffin cells, PAC1hop, in PC12-G cells reconstituted a PACAP-mediated Ca2+ and prolonged secretory response similar to the one observed in primary chromaffin cells. The Ca2+ response mediated by PAC1null was somewhat smaller and PAC1hip failed to couple to Ca2+. Neither variant conferred prolonged catecholamine release, suggesting that expression of the hop cassette in the third intracellular loop of the receptor is required for sustained catecholamine release from neuroendocrine cells. In NG108-15 cells, heterologous expression of the PAC1hop, null and hip receptor conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca2+]i occurred efficiently in PAC1hop- and to a lesser extent in PAC1null-expressing cells. Expression of PAC1hip did not confer an intracellular Ca2+ response, indicating that PAC1hop is the receptor variant most efficiently coupled to combinatorial signaling through cAMP and Ca2+. PAC1hop-mediated signaling activated the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases 1 and 2 (ERK1/2). Signaling to ERK proceeded through cAMP independently of the cAMP dependent protein kinase (PKA). PACAP induced transcription of the gene encoding the putative neuroprotectant stanniocalcin 1 (STC1), which has previously been implicated in neuronal resistance to hypoxic/ ischemic insult; gene induction proceeded through ERK but not PKA. Cyclic AMP generation by forskolin did not activate ERK in NG108-15 cells, but rather induced STC1 mRNA elevation through the canonical PKA dependent pathway. This suggests that activation of non-canonical cAMP signaling, mediating ERK-dependent gene induction, requires additional signaling through Ca2+ via PAC1hop in these cells. Primary rat cortical neurons expressed predominantly the PAC1hop and null variants. Exposure of cortical neurons to PACAP resulted in elevation of the two second messengers cAMP and Ca2+, activation of ERK1/2, and induction of STC1 gene transcription. PACAP-mediated ERK activation proceeded through cAMP but not PKA, and STC1 was induced via ERK but not PKA. Pharmacological stimulation of adenylate cyclases by forskolin also resulted in increased ERK phosphorylation and STC1 mRNA elevation independently of PKA. These results indicate that cAMP production alone is sufficient to activate ERK in differentiated cortical neurons, unlike in the less differentiated NG108-15 cell line. Induction of another PACAP target gene, brain-derived neurotrophic factor (BDNF), occurred through the canonical cAMP/PKA pathway. PACAP has been shown by our laboratory and others to be neuroprotective against ischemia in rodent stroke models. To begin to define the mechanism of this neuroprotection, we employed two cell culture stroke models. Rat cortical neurons subjected to either oxygen-glucose-deprivation or glutamate-induced excitotoxicity underwent cell death as expected. However, treatment with PACAP did not increase neuronal survival in either of the two models, and STC1 over-expression also failed to increase resistance to neuronal cell death during glutamate-induced excitotoxicity. These data suggest that the protective effects of the neurotrophic peptide PACAP and the putative neuroprotectant STC1 during neurodegenerative conditions in vivo are mediated through cells absent in cultures of cortical neurons, such as glial cells. In conclusion, the present study has demonstrated that expression of different PAC1 splice variants determines the degree of activation of two different second messenger pathways that may mediate different functional outcomes during stress-responding. PACAP mediates ERK activation and STC1 induction via non-canonical cAMP signaling. The selective pharmacological activation of this potentially neuroprotective pathway, which is different from the cAMP/PKA pathway critical for learning and memory, could have therapeutic implications for neuroprotection in vivo

The Neuropeptide PACAP Mediates Stimulus-Transcription Coupling in Hypothalamic-Pituitary-Adrenocortical Axis and Sympathetic Nervous System - Implications for Acute and Chronic Stress Responses

Stress is a vital response of all organisms to the demands of life. By adjusting to stimuli from the outside world, and stimuli arising from its internal organs, the body is continually at work to ensure its proper function under a widely variable range of conditions. Thus, acute and adequate responses to such stimuli (stressors) are essential. However, when these responses are either insufficient or excessive, the well-being of the organism is at risk. Furthermore, if stress becomes chronic and the cost of continual adjustment rises, a plethora of illnesses can result. This phenomenon has grown into epidemic proportions, particularly in Western societies, with the physical and mental health of millions severely affected. Despite much research, the mechanisms underlying responses to stressors are still incompletely understood. Our experiments show that the neuropeptide PACAP is required for normal responses to acute stressor exposure. Expanding previous results from our laboratory, evidence is provided for PACAP-dependent regulation of the catecholaminergic system in the adrenal medulla during responses of the ANS. By inducing the expression of enzymes required for epinephrine biosynthesis (tyrosine hydroxylase, phenylethanolamine N-methyltransferase), as well as neuropeptides involved in modulation of adrenal secretory activity (galanin, Tac1, VIP), PACAP appears to provide a mechanism for plasticity during periods of high demand. Our data suggest that this PACAP-dependent stimulus-transcription coupling may proceed via stressor-specific mechanisms, as the induction of a number of transcription factors which are putatively responsible for the regulation of enzymes and neuropeptides (e.g. Egr1, Fos, Nur77) is PACAP-dependent in response to restraint, but not hypoglycemia. Furthermore, PACAP controls upregulation of transcripts encoding potential cytoprotectants (Ier3, Stc1) in the adrenal glands, in response to hypoglycemia and restraint. Most importantly, our present work is the first to show that the endogenous PACAPergic system is required for activation of the HPA axis in response to stressor exposure. This appears to be mediated at the central level, via PACAP-dependent stimulation of hypophysiotropic neurons, as restraint-induced upregulation of CRH mRNA in the PVN is completely abolished in PACAP-deficient mice. Consequently, restraint-induced secretion of ACTH and corticosterone is blunted, particularly when stressor exposure is prolonged, while serum concentrations of both hormones in untreated mice are equivalent to those in wild-types. These PACAP-dependent effects seem to involve PACAP-dependent stimulus-transcription coupling throughout the HPA axis, and possibly rely on inducible transcription factors from the Nr4a family of orphan nuclear receptors. Thus, rapid regulation of Nur77 (Nr4a1), Nurr1 (Nr4a2) and Nor1 (Nr4a3) in the PVN, pituitary gland and adrenal cortex occurs in a PACAP-dependent pattern. The fact that stressor-induced upregulation of transcripts encoding steroidogenic acute regulatory protein (StAR) and steroidogenic factor 1 (SF-1) is significantly attenuated in adrenal glands from PACAP-deficient mice provides a link between PACAP-dependent central control of the HPA axis and peripheral corticosterone production. Beyond the acute regulation of responses to stressor exposure, our results have implications for the understanding, and potentially the treatment, of disease states associated with chronic stress. In this regard, a crucial finding from the present work concerns stressor-induced corticosterone secretion. The initial phase is largely intact in PACAP-deficient animals, while more sustained secretion during prolonged stressor exposure becomes increasingly blunted. This suggests that chronic hypersecretion of glucocorticoids, such as during certain psychiatric illnesses, could be targeted by blockade of the PACAPergic system, without compromising acute HPA responses that are necessary for survival and health. As mentioned, our experiments suggest that PACAP is a central regulator of the HPA axis, controlling activation in response to stressors at the level of the hypothalamic PVN. Future work will address the exact signaling mechanisms employed during PACAP-dependent stress responses, in order to reveal potential avenues for therapeutic intervention. To further clarify the involvement of this neuropeptide in chronic stress-related diseases, the PACAP-deficient mouse model will continue to be used as a valuable tool in experiments concerning the behavioral, physiological, cellular and molecular mechanisms of stress

Expression von TNFa und seinen Rezeptoren p55TNFR und p75TNFR im Gehirn der Maus nach SEB- und LPS-Stimulation

Zwischen Gehirn und peripherem Immunsystem besteht eine bidirektionale Kommunikation, die u.a. durch lösliche Faktoren wie Zytokine mediiert wird. Eines der dabei beteiligten Proteine ist der Tumor-Nekrose-Faktor-alpha (TNFa), ein proinflammatorisch und apoptotisch wirkendes Zytokin, das im ZNS von residenten Mikrogliazellen und Astrozyten synthetisiert werden kann. Die Voraussetzung einer Rezeptor-mediierten lokalen Wirkung des TNFa im Gehirn ist durch die nachgewiesene Expression der TNFa Rezeptoren p55TNFR und p75TNFR erfüllt. Allerdings sind die Zellentypen ebenso wie die Mechanismen, die zur Synthese der TNFa Rezeptoren führen bislang unklar. Die vorliegende Arbeit charakterisiert die zelluläre Verteilung von TNFa, p55TNFR und p75TNFR im Gehirn der Maus unter physiologischen Bedingungen und zu verschiedenen Zeitpunkten (1, 4, 8, 12 und 24h) nach intraperitonealer Injektion von Lipopolysaccharid (LPS) oder Staphylokokken Enterotoxin B (SEB). Beide Mitogene erhöhen die Serumspiegel von TNFa. Die unterschiedlichen Aktivierungsmechanismen der beiden Mitogene erlauben aber zu unterscheiden, ob Serum-TNFa alleine oder nur in Kombination mit der lokalen Wirkung eines der Mitogene direkt im ZNS zu einer Induktion der TNFa-Rezeptoren im Gehirn führt. Im unstimulierten Gehirn fand sich eine panneuronale Expression der p55TNFR mRNA, die in einzelnen Hirnnervenkernen besonders stark ausgeprägt war. Kleine, non-neuronalen Zellen im gesamten Gehirn exprimierten die p75TNFR mRNA. LPS als auch SEB führten zu einer neuronalen Aktivierung im ZNS, die sich in der Induktion von c-fos mRNA im Nucleus Paraventricularis (PVN) nachweisen ließ. Keines der beiden Mitogene beeinflusste die konstitutive Expression der p55TNFR mRNA. Dagegen führte nur die Gabe von LPS, nicht aber von SEB zu einer Erhöhung der TNFa mRNA und zu einer Induktion der p75TNFR mRNA im Bereich der Zirkumventrikulären Organe (circumventricular organs, CVOs). Aus dem Vergleich der zerebralen Reaktionen auf die periphere Gabe von SEB oder LPS lässt sich folgern, dass der afferente Schenkel der neuroimmunen Kommunikation des TNFa-Systems Mitogen-abhängig ist. Im Serum erhöhte Zytokinspiegel alleine können innerhalb der „immune-to-brain communication“ zwar Einfluss auf die neuronale Aktivität wie im PVN ausüben, sie haben aber keine modulierende Wirkung auf das TNFa-System im Gehirn