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In the first part of this study, we have identified the two steroid hormones progesterone and norgestimate as novel TRPC channel blockers. Both substances blocked TRPC-mediated Ca2+ influx with micromolar activities in fluorometric measurements. TRPC channel inhibition did not seem to be a general steroid effect since another progestin, the norgestimate metabolite levonorgestrel, was not effective. Norgestimate was 4- to 5-fold more active on the TRPC3/6/7 subfamily compared to TRPC4/5, whereas progesterone was similarly potent. This selectivity of norgestimate was confirmed by patch clamp recordings. As norgestimate blocked channels directly gated by DAG with a fast kinetic, we assume the compound acts on the channel protein itself. This view was further substantiated by the lack of effects on IP3R-mediated Ca2+ release from the endoplasmic reticulum, which is activated in parallel with TRPCs by Gq/11-coupled receptor stimulation. Norgestimate did not only block ectopically expressed TRPC channels but also native, TRPC-mediated currents in rat aortic smooth muscle cells with similar activity. The usefulness of norgestimate as a tool compound for the investigation of physiological TRPC functions was tested in isolated vessel rings. Consistent with TRPC6 being an essential component of the alpha-1-adrenoceptor-activated cation channel, we demonstrated a direct vasorelaxant, endothelium-independent effect of norgestimate on rat aortic rings precontracted with phenylephrine. Thus, our results provide further experimental support for a role of TRPC6 in alpha-1-adrenergic vessel constriction. In the second part of this study, we screened a human aorta cDNA-library for novel TRPC4-interacting proteins with a modified yeast two-hybrid (Y2H) system in which the TRPC4-C-terminus was expressed as tetrameric bait protein, thereby mimicking the native channel conformation. Of the eleven interacting proteins found SESTD1 was chosen for further analyses since it contains a phospholipid-binding Sec14p-like domain and thus could be involved in regulation of TRPC channels by phospholipids. After the biochemical validation of the found interaction, the first spectrin domain of SESTD1 was then identified to interact with the CIRB domain of TRPC4 in directed Y2H tests. SESTD1 also co-immunoprecipitated with the closely related TRPC5 protein in which the SESTD1-binding domain is highly conserved. Independent of the CIRB site, co-immunoprecipitation with TRPC6 and the distantly related TRPM8 channel was observed indicating the existence of other sites in these channel proteins that mediate interaction with SESTD1. Analysis of SESTD1 gene expression in human tissues showed that its transcripts are ubiquitously expressed and tissues with significant coexpression with TRPC4 and -5 were identified. We have generated two polyclonal antisera directed against SESTD1 that consistently detected SESTD1 protein in brain, aorta, heart, and in smooth muscle and endothelial cells. The functional consequences of the found interaction were investigated by examination of the TRPC5-mediated Ca2+ influx in a clonal HM1 cell line stably expressing the channel. Since SESTD1 overexpression had no detectable effects on TRPC5-mediated Ca2+ influx, most likely due to expression of endogenous SESTD1, we knocked-down the native protein with specific siRNA. This procedure reduced TRPC5-mediated Ca2+ influx following receptor stimulation by 50%. Parallel biotinylation experiments did not reveal any differences in cell surface expressed TRPC5-protein, suggesting that reduction of TRPC5 activity resulted from a loss of a direct SESTD1 effect on the channel. In addition, in immunofluorescence experiments we observed that reduced SESTD1 protein levels resulted in a redistribution of the multifunctional protein ß-catenin from the plasma membrane to the cytosol. This result may point to an involvement of SESTD1 in formation and maintenance of adherens junctions. SESTD1 contains a phospholipid-binding Sec14p-like domain and we were the first to demonstrate its Ca2+-dependent binding to phosphatidic acid and all physiological phosphatidylinositol mono- and bisphosphates in vitro. The physiological function of this binding activity is not known at present, but it could play a role in regulation of associated TRPC channels. TRPC4 and -5 channels are activated by phospholipid hydrolysis and also bind phospholipids directly. The identification of SESTD1 as novel TRPC-interacting protein could thus be an important step forward in the investigation and better comprehension of the complex molecular mechanisms of TRP channel regulation by lipids.
Neurological diseases associated with neuronal death are also accompanied by axonal denervation of connected brain regions. In these areas, denervation leads to a decrease in afferent drive, which may in turn trigger active central nervous system (CNS) circuitry rearrangement. This rewiring process is important therapeutically, since it can partially recover functions and can be further enhanced using modern rehabilitation strategies. Nevertheless, the cellular mechanisms of brain rewiring are not fully understood. We recently reported a mechanism by which neurons remodel their local connectivity under conditions of network-perturbance: hippocampal pyramidal cells can extend spine head protrusions (SHPs), which reach out toward neighboring terminals and form new synapses. Since this form of activity-dependent rewiring is observed only on some spines, we investigated the required conditions. We speculated, that the actin-associated protein synaptopodin, which is involved in several synaptic plasticity mechanisms, could play a role in the formation and/or stabilization of SHPs. Using hippocampal slice cultures, we found that ~70 % of spines with protrusions in CA1 pyramidal neurons contained synaptopodin. Analysis of synaptopodin-deficient neurons revealed that synaptopodin is required for the stability but not the formation of SHPs. The effects of synaptopodin could be linked to its role in Ca(2+) homeostasis, since spines with protrusions often contained ryanodine receptors and synaptopodin. Furthermore, disrupting Ca(2+) signaling shortened protrusion lifetime. By transgenically reintroducing synaptopodin on a synaptopodin-deficient background, SHP stability could be rescued. Overall, we show that synaptopodin increases the stability of SHPs, and could potentially modulate the rewiring of microcircuitries by making synaptic reorganization more efficient.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited neurodegenerative disorder with preferential affection of Purkinje neurons, which are known as integrators of calcium currents. The expansion of a polyglutamine (polyQ) domain in the RNA-binding protein ataxin-2 (ATXN2) is responsible for this disease, but the causal roles of deficient ATXN2 functions versus aggregation toxicity are still under debate. Here, we studied mouse mutants with Atxn2 knockout (KO) regarding their cerebellar global transcriptome by microarray and RT-qPCR, in comparison with data from Atxn2-CAG42-knock-in (KIN) mouse cerebellum. Global expression downregulations involved lipid and growth signaling pathways in good agreement with previous data. As a novel effect, downregulations of key factors in calcium homeostasis pathways (the transcription factor Rora, transporters Itpr1 and Atp2a2, as well as regulator Inpp5a) were observed in the KO cerebellum, and some of them also occurred subtly early in KIN cerebellum. The ITPR1 protein levels were depleted from soluble fractions of cerebellum in both mutants, but accumulated in its membrane-associated form only in the SCA2 model. Coimmunoprecipitation demonstrated no association of ITPR1 with Q42-expanded or with wild-type ATXN2. These findings provide evidence that the physiological functions and protein interactions of ATXN2 are relevant for calcium-mediated excitation of Purkinje cells as well as for ATXN2-triggered neurotoxicity. These insights may help to understand pathogenesis and tissue specificity in SCA2 and other polyQ ataxias like SCA1, where inositol regulation of calcium flux and RORalpha play a role.
Dieser Arbeit war zum Ziel gesetzt, Methoden zur Simulation von neuronalen Prozessen zu entwickeln, zu implementieren, einzusetzen und zu vergleichen. Ein besonderes Augenmerk lag dabei auf der Frage, wo eine volle räumliche Auflösung der Modelle benötigt wird und wo darauf zugunsten von vereinfachenden niederdimensionalen Modellen, die wesentlich weniger Ressourcen und mathematischen Sachverstand erfordern, verzichtet werden kann. Außerdem wurde speziell bei der Beschreibung der verschiedenen Modelle für die Elektrik der Nervenzellen das Anliegen verfolgt, deren Zusammenhänge und die Natur vereinfachender Annahmen herauszuarbeiten, um deutlich zu machen, an welchen Stellen Probleme bei der Benutzung der weniger komplexen Modelle auftreten können.
In etlichen Beispielen wurde daraufhin untersucht, inwieweit die Vereinfachung auf ein eindimensionales Kabelmodell sowie der Verzicht auf die Betrachtung einzelner Ionensorten die realistische Darstellung der zellulären Elektrik beeinträchtigen können. Dabei stellte sich heraus, dass alle betrachteten Modelle für das rein elektrische Verhalten der Neuronen im Wesentlichen dieselben Ergebnisse liefern, weshalb zu dessen Simulation in den allermeisten Fällen ein 1D-Kabelmodell völlig ausreichend und angezeigt sein dürfte.
Nur wenn Größen von Interesse sind, die in diesem Modell nicht erfasst werden, etwa das Außenraumpotential oder die Ionenkonzentrationen, muss auf genauere Modelle zurückgegriffen werden. Außerdem ist in einer Konvergenzstudie exemplarisch vorgeführt worden, dass bereits eine recht grobe Darstellung der zugrundeliegenden Rechengitter genügt, um korrekte Ergebnisse bei der Simulation der rein elektrischen Signale sicherzustellen.
In scharfem Kontrast steht hierzu die Simulation von einzelnen Ionen-Dynamiken. Bereits in der Untersuchung des Poisson-Nernst-Planck-Modells für das Membranpotential erwies sich, dass für eine korrekte Simulation der diffusiven Anteile der Ionenbewegung wesentlich feinere Gitter benötigt werden.
Noch viel deutlicher wurde dies in Simulationen von Calcium-Wellen in Dendriten, wo -- neben anderen Einsichten -- aufgezeigt werden konnte, dass nicht nur eine feine axiale
(und Zeit-) Auflösung der Dendritengeometrie zur Sicherstellung exakter Ergebnisse notwendig ist, sondern auch die räumliche Auflösung in die übrigen Dimensionen wichtig ist, weswegen eine eindimensionale Kabeldarstellung der Calcium-Dynamik erheblich fehlerbehaftet und
(jedenfalls im Zusammenhang mit Ryanodin-Rezeptorkanälen) von deren Nutzung dringend abzuraten ist. Auch die Darstellung von Kanälen als eine kontinuierliche Dichte in der Membran kann, wie darüber hinaus vorgeführt wurde, problematisch sein.
Ihre exaktere Modellierung, etwa durch Einbettung auch probabilistischer Einzelkanaldarstellungen in das räumliche Modell sollte in zukünftigen Arbeiten noch mehr thematisiert werden.
Mit Blick auf die Wiederverwendbarkeit bereits implementierter Funktionalität innerhalb dieser Arbeiten wurden spezielle Teile dieser Funktionalität hier in einem gesonderten
Kapitel genauer beschrieben. Als komplexes Beispiel für das, was simulationstechnisch bereits im Bereich des Machbaren
liegt, und gleichsam für eine Anwendung, die zeigt, wie möglichst viele der im Rahmen dieser Arbeit entwickelten Methoden miteinander kombiniert werden können, wurde die
Calcium-Dynamik eines kompletten Dendriten innerhalb eines großen aktiven neuronalen Netzwerks simuliert.
Dual-energy CT (DECT) has emerged into clinical routine as an imaging technique with unique postprocessing utilities that improve the evaluation of different body areas. The virtual non-calcium (VNCa) reconstruction algorithm has shown beneficial effects on the depiction of bone marrow pathologies such as bone marrow edema. Its main advantage is the ability to substantially increase the image contrast of structures that are usually covered with calcium mineral, such as calcified vessels or bone marrow, and to depict a large number of traumatic, inflammatory, infiltrative, and degenerative disorders affecting either the spine or the appendicular skeleton. Therefore, VNCa imaging represents another step forward for DECT to image conditions and disorders that usually require the use of more expensive and time-consuming techniques such as magnetic resonance imaging, positron emission tomography/CT, or bone scintigraphy. The aim of this review article is to explain the technical background of VNCa imaging, showcase its applicability in the different body regions, and provide an updated outlook on the clinical impact of this technique, which goes beyond the sole improvement in image quality.
Diseases such as cardiac arrhythmias, CPVT and other issues of the human heart still remain largely unexplored. To contribute to this field of research, it is necessary to create tools to control the spatial and temporal release and reuptake of Ca2+ from the sarcoplasmic/endoplasmic reticulum (SR/ER). Ca2+ release and uptake by the ryanodine receptor (RyR) and Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), respectively, are essential for the function of excitable cells. In this process, the rapid Ca2+ release from the SR/ER and the associated contraction in muscle cells is modulated by RyR. However, diseases due to calcium leakage, such as cardiac arrhythmias, seizures and contractile dysfunction, are also caused by RyR. The resting Ca2+ concentration in the cytosol, which is important for the cell, is kept in balance by Ca2+ release and reuptake into the SR/ER. This reuptake is controlled quite considerably by SERCA. SERCA is important for development and muscle function in both nematodes such as C. elegans and mammals, though there is also a great need for tools that can help study precise function.
To advance towards the goal of developing tools for optogenetic stimulation of intracellular Ca2+ release from the SR/ER, the model organism C. elegans was chosen. Its advantages are the fully sequenced genome and the neural network connectome. In addition, the ease of maintenance, self-fertilisation, transparency and rapid generation cycles, as well as the fact that it is a eutelic animal, are advantages for the application of the optogenetic approach.
So far, tools for light-induced Ca2+ release (LICR) have already been developed, involving the creation of ChR2 versions with higher Ca2+ conductivity based on the "CatCh" variant and further improving their conductivity through several established mutations. In addition, the pharynx of C. elegans was modified to produce an optogenetically stimulated muscle pump that resembles mammalian cardiac muscle cells. In this work, both optoUNC-68 (optically excitable RyR) and SERCA/LOV2 were generated in different variants by CRISPR/Cas9 and plasmid-based genome editing to achieve light-driven manipulation of calcium homeostasis in C. elegans. Here, LICR was triggered by LOV2 domains in an opto-mechanical manipulation of RyR as well as SERCA. This approach was made possible by recently published high-resolution cryoEM structural images. In addition, alternative approaches using Ca2+ conductance-optimised channelrhodopsin variants were tested in C. elegans body wall muscle cells.
By inserting ChR-XXM into C. elegans and subsequent fluorescence microscopy of the co-introduced GFP, an expression in body wall muscle cells could be detected. Furthermore, in contraction assays, ChR-XXM was demonstrated to induce contractions of the animals of up to 16% compared to the original body length in both medium (0.8mW/mm²) and high (1.4mW/mm²) stimulation at 470nm. ChR-XXM was thus identified as an excellent candidate for the development of an optogenetic tool, as it exhibits significantly increased Ca2+ conductivity compared to other ChR2 variants.
The use of CRISPR/Cas9 to insert AsLOV2 domains (L404-L546) into different insertion sites of RyR allowed the generation of a transgenic strain of C. elegans that could be stimulated to elongate during 0.3mW/mm² photostimulation. This demonstrated that RyR can be manipulated by photostimulation, spatiotemporally through conformational changes in the LOV2 domain and the resulting disruption of the pore region.
The CRISPR/Cas9 method was also used to insert LOV2 domains into SERCA. Here it could be demonstrated that a conformational change of the LOV2 domains induced by photostimulation leads to a stop or impairment of Ca2+ ion translocation by SERCA from the cytosol into the SR/ER. In contrast to LOV2 in RyR, this resulted in a contraction of C. elegans body length.
The data presented here indicate that the intracellular Ca2+ cycle involving the SR/ER and cytosol can be successfully manipulated by the introduction of optogenetic tools. It turned out that the manipulation/impairment of individual components of this system, such as RyR or SERCA, is usually insufficient to achieve a clear response. Therefore, simultaneous manipulation of the two main actors RyR and SERCA is arguably the best way to take another step towards creating optogenetic tools for light-stimulated manipulation of Ca2+ release and reuptake from the SR/ER.