Genexpressionsanalyse an humanen Neuroblastomzellen nach 6-Hydroxydopamin-Behandlung

Morbus Parkinson ist eine chronische neurodegenerative Erkrankung, die durch einen Verlust der dopaminergen (DA) Neurone in der Substantia nigra pars compacta, gekennzeichnet ist. Die Ätiologie ist bisher weitgehend unbekannt. Die Behandlung von Nervenzellen mit 6-Hydroxydopamin (6-OHDA) ist ein etabliertes in vivo und in vitro Modell, um die Auswirkungen des oxidativen Stresses in M. Parkinson Gehirnen nachzuahmen. Um die nachgeschalteten Ereignisse in Zellen nach Exposition mit 6-OHDA zu charakterisieren, haben wir die dopaminerge humane Neuroblastomzell-Linie (SH-SY5Y Zellen) mit 6-OHDA behandelt und anschließend das Profil der Genexpression mittels der reversen arbiträren geprimten Polymerasen-Kettenreaktion (RAP-PCR) ermittelt. Mit der RAP-PCR können die Unterschiede in der Genexpression zwischen zwei oder mehreren Proben ermittelt werden. Reproduzierbare Unterschiede in der Genexpression der mit 6-OHDA behandelten Zellen (die Behandlung erfolgte mit 50μM bzw. 100μM 6-OHDA über 24h) zeigten sich im Vergleich zu unbehandelten Kontrollen. Von diesen wiesen 23 Sequenzen eine hochsignifikante Homologie zu bekannten humanen Kodierungssequenzen auf. Die Ergebnisse wurden mittels RT-PCR, Real-time PCR und Western-Blot Analyse bestätigt. In vier Fällen [Tumoregulin-1 (TMEFF-1), Collapsin response mediator protein 1 (CRMP-1), Neurexin-1 und Phosphoribosylaminoimidazolsynthetase (GART)] wurde eine suppressive Regulation auf mRNA- sowie auf Proteinebene festgestellt. Dies bestätigte, dass die Regulierung, die sich in der RAP-PCR zeigte, nicht nur auf die Transkriptionsebene beschränkt ist, sondern sich auch auf die entsprechende Proteinebene erstreckt. Es bestätigt sich somit, dass die von uns angewandte RAP-PCR als Methode zur Analyse der differentiellen Genexpression von SH-SY5Y im 6-OHDA-Schädigungsmodell in vitro geeignet ist. Diese vier Proteine, die z.T. erstmalig in Bezug in dem Toxizitätsmodell identifiziert wurden, konnten als Proteine charakterisiert werden, die auch physiologisch eine Rolle im Zelluntergang Substantia nigra Neuronen spielen könnten. Weitere Studien für die physiologische Rolle der identifizierten Gene und ihren Einfluss auf die Neurodegeneration im M. Parkinson sind allerdings nötig

Doxorubicin induces caspase-mediated proteolysis of KV7.1

Strigli A, Raab C, Hessler S, et al. Doxorubicin induces caspase-mediated proteolysis of KV7.1. Communications Biology. 2018;1(1): 155.Kv7.1 (KCNQ1) coassembles with KCNE1 to generate the cardiac IKs-channel. Gain- and loss-of-function mutations in KCNQ1 are associated with cardiac arrhthymias, highlighting the importance of modulating IKs activity for cardiac function. Here, we report proteolysis of Kv7.1 as an irreversible posttranslational modification. The identification of two C-terminal fragments of Kv7.1 led us to identify an aspartate critical for the generation of one of the fragments and caspases as responsible for mediating proteolysis. Activating caspases reduces Kv7.1/KCNE1 currents, which is abrogated in cells expressing caspase-resistant channels. Enhanced cleavage of Kv7.1 can be detected for the LQT mutation G460S, which is located adjacent to the cleavage site, whereas a calmodulin-binding-deficient mutation impairs cleavage. Application of apoptotic stimuli or doxorubicin-induced cardiotoxicity provokes caspase-mediated cleavage of endogenous IKs in human cardiomyocytes. In summary, caspases are novel regulatory components of IKs channels that may have important implications for the molecular mechanism of doxorubicin-induced cardiotoxicity

Impact of the first COVID lockdown on accident- and injury-related pediatric intensive care admissions in Germany - a multicenter study

Children’s and adolescents’ lives drastically changed during COVID lockdowns worldwide. To compare accident- and injury-related admissions to pediatric intensive care units (PICU) during the first German COVID lockdown with previous years, we conducted a retrospective multicenter study among 37 PICUs (21.5% of German PICU capacities). A total of 1444 admissions after accidents or injuries during the first lockdown period and matched periods of 2017–2019 were reported and standardized morbidity ratios (SMR) were calculated. Total PICU admissions due to accidents/injuries declined from an average of 366 to 346 (SMR 0.95 (CI 0.85–1.05)). Admissions with trauma increased from 196 to 212 (1.07 (0.93–1.23). Traffic accidents and school/kindergarten accidents decreased (0.77 (0.57–1.02 and 0.26 (0.05–0.75)), whereas household and leisure accidents increased (1.33 (1.06–1.66) and 1.34 (1.06–1.67)). Less neurosurgeries and more visceral surgeries were performed (0.69 (0.38–1.16) and 2.09 (1.19–3.39)). Non-accidental non-suicidal injuries declined (0.73 (0.42–1.17)). Suicide attempts increased in adolescent boys (1.38 (0.51–3.02)), but decreased in adolescent girls (0.56 (0.32–0.79)). In summary, changed trauma mechanisms entailed different surgeries compared to previous years. We found no evidence for an increase in child abuse cases requiring intensive care. The increase in suicide attempts among boys demands investigation

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) is involved in lysosomal cholesterol export

The intracellular transport of cholesterol is subject to tight regulation. The structure of the lysosomal integral membrane protein type 2 (LIMP-2, also known as SCARB2) reveals a large cavity that traverses the molecule and resembles the cavity in SR-B1 that mediates lipid transfer. The detection of cholesterol within the LIMP-2 structure and the formation of cholesterol - like inclusions in LIMP-2 knockout mice suggested the possibility that LIMP2 transports cholesterol in lysosomes. We present results of molecular modeling, crosslinking studies, microscale thermophoresis and cell-based assays that support a role of LIMP-2 in cholesterol transport. We show that the cavity in the luminal domain of LIMP-2 can bind and deliver exogenous cholesterol to the lysosomal membrane and later to lipid droplets. Depletion of LIMP-2 alters SREBP-2-mediated cholesterol regulation, as well as LDL-receptor levels. Our data indicate that LIMP-2 operates in parallel with Niemann Pick (NPC)-proteins, mediating a slower mode of lysosomal cholesterol export.Peer reviewe

Year in review in Intensive Care Medicine 2009: II. Neurology, cardiovascular, experimental, pharmacology and sedation, communication and teaching

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Combined Fluorescence-Guided Resection and Intracavitary Thermotherapy with Superparamagnetic Iron-Oxide Nanoparticles for Recurrent High-Grade Glioma: Case Series with Emphasis on Complication Management

Background: Concepts improving local tumor control in high-grade glioma (HGG) are desperately needed. The aim of this study is to report an extended series of cases treated with a combination of 5-ALA-fluorescence-guided resection (FGR) and intracavitary thermotherapy with superparamagnetic iron oxide nanoparticles (SPION). Methods: We conducted a single-center retrospective review of all recurrent HGG treated with FGR and intracavitary thermotherapy (n = 18). Patients underwent six hyperthermia sessions in an alternating magnetic field and received additional adjuvant therapies on a case-by-case basis. Results: Nine patients were treated for first tumor recurrence; all other patients had suffered at least two recurrences. Nine patients received combined radiotherapy and thermotherapy. The median progression-free survival was 5.5 (95% CI: 4.67–6.13) months and median overall survival was 9.5 (95% CI: 7.12–11.79) months. No major side effects were observed during active treatment. Thirteen patients (72%) developed cerebral edema and more clinical symptoms during follow-up and were initially treated with dexamethasone. Six (33%) of these patients underwent surgical removal of nanoparticles due to refractory edema. Conclusions: The combination of FGR and intracavitary thermotherapy with SPION provides a new treatment option for improving local tumor control in recurrent HGG. The development of cerebral edema is a major issue requiring further refinements of the treatment protocol

Structural Determinants of M-Type KCNQ (Kv7) K+ Channel Assembly

Schwake M. Structural Determinants of M-Type KCNQ (Kv7) K+ Channel Assembly. Journal of Neuroscience. 2006;26(14):3757-3766.The ability of KCNQ (Kv7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise ∼30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2. Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane

Protein trafficking of members of the CLC and KCNQ ion channels

Titel Inhaltsverzeichnis 1 1 Einleitung 3 2 Material 29 3 Methoden 34 4 Ergebnisse 57 5 Diskussion 93 6 Abkürzungen 109 7 Literaturverzeichnis 111 8 Anhang 123Mutationen in den Genen für die Kaliumkanäle KCNQ2 und KCNQ3 führen zu einem vererbbaren epileptischen Syndrom mit der englischen Bezeichnung benign familial neonatal convulsions (BFNC). Diese beiden spannungsabhängigen Kaliumkanäle kommen fast ausschließlich im ZNS vor und ihre Expressionsmuster stimmen im Gehirn sehr stark überein. KCNQ2 und KCNQ3 bilden Heteromere mit neuen biophysikalischen Eigenschaften und vermitteln wahrscheinlich den physiologisch sehr wichtigen M-Strom, der die neuronale Erregbarkeit moduliert. Im Rahmen dieser Arbeit wurden die Ursachen für den Anstieg des Stroms untersucht, den man bei Koexpression von KCNQ2 und KCNQ3 in Xenopus Oozyten im Verglich zu den jeweiligen homomeren Kanälen beobachten kann. Dazu wurden die Einzelkanalleitfähigkeiten und Offenwahrscheinlichkeiten (popen) von homomeren KCNQ2 und KCNQ3 und heteromeren KCNQ2/KCNQ3 Kanälen bestimmt. Die Einzelkanalleitfähigkeiten und popen von homomeren KCNQ2 (i» 18pS, popen» 0,61) und heteromeren KCNQ2/KCNQ3 (i» 16pS, popen» 0,72) sind annähernd gleich. Die Einzelkanalleitfähigkeit von KCNQ3 (i» 7,3) ist im Vergleich ungefähr nur halb so groß und die Offenwahrscheinlichkeit (popen» 0,42) etwas kleiner. Die Zunahme des Stroms bei Koexpression der beiden KCNQ-Kanäle konnte somit nicht mit diesen Parametern erklärt werden. Koexpression von KCNQ3 mit KCNQ2 erhöhte die Oberflächenexpression von KCNQ2 um einen Faktor von ungefähr fünf. Umgekehrt wurde die Oberflächenexpression von KCNQ3 durch KCNQ2 um einen Faktor von ungefähr zehn stimuliert. Eine KCNQ2 Mutante, die den C-Terminus des Kanals verkürzt, erreichte nicht die Zelloberfläche und konnte auch nicht die Oberflächenexpression von KCNQ3 erhöhen. Hingegen verhielten sich Missense-Mutationen in der Porenregion und in der Transmembrandomäne S6 von KCNQ2 und KCNQ3 in ihrer Oberflächenexpression annähernd wie die jeweiligen Wildtyp Kanäle. Zusammenfassend kann also festgehalten werden, daß der Anstieg des Stroms bei Koexpression von KCNQ2 und KCNQ3 vorwiegend durch eine erhöhte Anzahl aktiver Kanäle in der Plasmamembran verursacht wird. Die Interaktion und damit die Zielsteuerung der homo- und heteromeren Kanäle ist abhängig von einem intakten C-Terminus. ClC-5 kommt hauptsächlich in den endozytotischen Vesikeln des proximalen Tubulus der Niere vor, für deren effiziente Ansäuerung er maßgeblich verantwortlich ist. ClC-5 defiziente Mäuse weisen eine stark verminderte Endozytose (sowohl rezeptorvermittelt als auch aus der Flüssigphase) in der apikalen Region des proximalen Tubulus auf. Mutationen innerhalb des CLCN5 Gens sind die Ursache der Dent`schen Erkrankung, deren Hauptsymptome das Ausscheiden von Proteinen niederen Molekulargewichtes mit dem Urin und die Bildung von Nierensteinen sind. In der vorliegenden Arbeit wurde nach Endozytosemotiven innerhalb der Aminosäuresequenz von ClC-5 gesucht, um deren Einfluß auf die Zielsteuerung des Proteins zu bestimmen. Dabei wurden zwei Internalisierungsmotive identifiziert, ein PY Motiv und ein YF Motiv. Das PY Motiv konnte mit verschiedenen Methoden charakterisiert werden, für das YF Motiv steht eine derartige Analyse noch aus. Mutationen innerhalb des PY Motivs erhöhten den ClC-5 vermittelten Strom und die Oberflächenexpression um einen Faktor von ungefähr zwei. Die Analyse der Halbwertszeit des Wildtyps und des Kanals ohne funktionelles PY Motiv in der Plasmamembran zeigte, daß die Halbwertszeit der Mutante gegenüber dem Wildtyp verlängert ist. Dieses Ergebnis deutete darauf hin, daß es sich bei dem PY Motiv um ein Signal handelt, daß die Degradation des Kanalproteins vermittelt. Unterstützung findet diese Schlußfolgerung durch Koexpressionsexperimente mit WWP2 und nicht-funktionellen Konstrukten dieses Proteins. Dominant-negative Mutanten des WWP2 Proteins erhöhen den ClC-5 vermittelten Strom zweifach, allerdings nur, solange das PY Motiv intakt ist. Da es sich bei dem WWP2 Protein um eine Ubiquitin-Protein Ligase handelt, ist es wahrscheinlich, daß entweder ClC-5 oder ein drittes, noch unbekanntes Protein ubiquitiniert wird. Zusätzlich konnte im Rahmen dieser Arbeit gezeigt werden, daß ClC-5 vermittelte Ströme abnehmen, wenn die Endozytose durch rab5 oder rab5 Q79L in Xenopus Oozyten stimuliert wird. Umgekehrt nahmen diese Ströme bei Inhibition der Endozytose durch rab5 S34N zu. Die Zerstörung des PY Motivs hatte zur Folge, daß die Ströme nicht mehr von der Endozytoserate beeinflußt werden konnten. Zusammenfassend kann also festgehalten werden, daß sich ein Endozytosemotiv im C-Terminus von ClC-5 befindet, das die Degradation des Proteins vermittelt bzw. beeinflußt. Die vorliegenden Studien führen also zu einer Erweiterung unserer Kenntnisse über Zielsteuerung und Protein-Protein- Interaktionen von physiologisch sehr wichtigen Mitgliedern der CLC- und der KCNQ-Ionenkanalfamilie.Mutations in either KCNQ2 or KCNQ3 cause benign familial neonatal convulsions (BFNC), an inherited epilepsy of newborns. Both proteins are co-expressed in the brain and associate to form heteromeric potassium channels. They are thought to give rise to the so called M-currents, which are important regulators of neuronal excitability. In this work the basis for the current increase seen upon co-expression of both KCNQ subunits in Xenopus oocytes was investigated. Therefore the single-channel conductances and open-probabilities of homo- and heteromeric KCNQ2 and KCNQ3 channels were determined ((KCNQ2: i» 18pS, popen» 0,61) (KCNQ2/KCNQ3: i» 16pS, popen» 0,72) (KCNQ3: i » 7,3, popen» 0,42)). The mean single channel conductance and popen of heteromeric channels were not significantly different from those of homomeric KCNQ2 channels, whereas the conductance of KCNQ3 was about half that value. This indicates that the increase of currents observed upon co-expressing both subunits in Xenopus oocytes cannot be explained by an increase in these parameters. Co- expression with KCNQ3 increased the surface expression of KCNQ2 by a factor of 5, and co-expression of KCNQ3 with KCNQ2 led to a >10fold increase of KCNQ3 surface expression. A KCNQ2 mutant associated with BFNC that truncates the cytoplasmic carboxyterminus neither reached the surface nor stimulated KCNQ3 surface expression. By contrast, BFNC missense mutations in the transmembrane regions of KCNQ2 or KCNQ3 left surface expression unchanged. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyterminus. In the kidney ClC-5 chloride channel resides mainly in vesicles of the endocytotic pathway and contributes to their acidification. Its disruption in mice entails a broad defect in renal endocytosis and causes secondary changes in calciotropic hormone levels. Inactivating mutations in Dent?s disease lead to proteinuria and kidney stones. Possibly by recycling, a small fraction of ClC-5 also reaches the plasma membrane. We identified a carboxy-terminal internalization motif in ClC-5. It resembles the so called PY motif which is crucial for the endocytosis and degradation of epithelial Na+-channels (ENaC). Disrupting this motif increases surface expression and currents about 2-fold. This is likely due to interactions with WW-domains as dominant negative mutants of the ubiquitin-protein ligase WWP2 increased surface expression and currents of ClC-5 only when its PY motif was intact. Stimulating endocytosis by expressing rab5 or its GTPase-deficient Q79L mutant decreased WT ClC-5 currents, but did not affect channels with mutated motifs. Similarly, decreasing endocytosis by expressing the inactive S34N mutant of rab5 increased ClC-5 currents only if its PY motif was intact. Thus, the endocytosis of ClC-5, which itself is crucial for the endocytosis of other proteins, depends on the interaction of a carboxy-terminal internalization signal with ubiquitin-protein ligases containing WW-domains

Lysosomal Membrane Proteins

Schwake M, Schröder B, Saftig P. Lysosomal Membrane Proteins and Their Central Role in Physiology. Traffic. 2013;14(7):739-748.The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non-mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways

Surface Expression and Single Channel Properties of KCNQ2/KCNQ3, M-type K+ Channels Involved in Epilepsy

Schwake M, Pusch M, Kharkovets T, Jentsch TJ. Surface Expression and Single Channel Properties of KCNQ2/KCNQ3, M-type K+ Channels Involved in Epilepsy. Journal of Biological Chemistry. 2000;275(18):13343-13348.Mutations in either KCNQ2 or KCNQ3 underlie benign familial neonatal convulsions (BFNC), an inherited epilepsy. The corresponding proteins are co-expressed in broad regions of the brain and associate to heteromeric K+ channels. These channels mediate M-type currents that regulate neuronal excitability. We investigated the basis for the increase in currents seen after co-expressing these subunits in Xenopus oocytes. Noise analysis and single channel recordings revealed a conductance of ≈ 18 pS for KCNQ2 and ≈7 pS for KCNQ3. Different conductance levels (ranging from 8 to 22 pS) were seen upon co-expression. Their weighted average is close to that obtained by noise analysis (16 pS). The open probability of heteromeric channels was not increased significantly. Co-expression of both subunits increased the surface expression of KCNQ2 and KCNQ3 by factors of 5 and >10, respectively. A KCNQ2 mutant associated with BFNC that has a truncated cytoplasmic carboxyl terminus did not reach the surface and failed to stimulate KCNQ3 surface expression. By contrast, several BFNC-associated missense mutations in KCNQ2 or KCNQ3 did not alter their surface expression. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyl terminus