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TED, eine neue Methode für die Analyze intrazellulärer Ca2+ Speicher in hippocampalen Netzwerken und zur Analyse der Funktion von Ca2+ Speichern in Neurotrophin Signalkaskaden

Fachliche Zuordnung Molekulare Biologie und Physiologie von Nerven- und Gliazellen
Molekulare und zelluläre Neurologie und Neuropathologie
Förderung Förderung von 2011 bis 2021
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 194101929
 
Erstellungsjahr 2022

Zusammenfassung der Projektergebnisse

Changes in free calcium concentration are involved in many different cellular processes. While cytosolic calcium signals are well investigated, homeostatic calcium fluxes between the extracellular space and the endoplasmic reticulum (ER) are barley described. In the year 2008, we described TED – targeted esterase-induced dye loading, an approach allowing direct ER calcium imaging. The method depends on overexpression of a Carboxylesterase 2 (CES2) in the ER lumen. Then cells are loaded with a lowaffinity calcium indicator (e.g. Fluo5N-AM). This synthetic calcium dye is enzymatically released and trapped in the ER lumen. The overarching hypothesis of this project has been that TED is a method suited to clarify how ER calcium dynamics shapes calcium signals. In the first funding phase, we first created new vectors for TED and validated them in different cell types (Samtleben et al., 2013). TED works very well in cultured astrocytes and some cell lines. In neurons, it was useful to investigate ‘slow’ homeostatic calcium fluxes (time range: seconds to minutes). However, TED suffers from disadvantageous properties of Fluo5N. Excitation light can rapidly destroy the calcium-sensitive dye properties (‘flashing’). While investigating resting homeostatic calcium fluxes with TED, we found that ER calcium levels in neurons are maintained by a continuous, resting store-operated calcium entry (SOCE). Acute blocking of this resting SOCE causes a rapid drop in ER calcium levels. We further asked whether resting SOCE is reflected in calcium imaging data. Indeed, we found a way (wavelet ridgewalking on 1D calcium imaging raw data) to objectively quantify ‘signal-close-to-noise’ calcium activity by resting SOCE. In 2017, a new tool for ER calcium analysis, the genetic calcium indicator ER-GCamp6-150, was published. We compared TED with ER-GCamp6-150 in cultured astrocytes as a cell model. TED was faster for measuring signal onsets, while ER-GCamp6-150 was very bleach resistant. Then we developed dual color imaging (ER-cytosol) and transcriptome analysis to link candidates of the calcium toolkit of astrocytes with homeostatic calcium signals. The data show a rather strong contribution of homeostatic calcium fluxes in shaping IP3-induced and calcium-induced calcium release as well as spatiotemporal components of intracellular calcium oscillations. To improve ER calcium analysis in neural circuits, we developed a mouse model for TED and expressed an advanced TED vector (RedCES2) under control of the Thy1-promoter. However, ER calcium dynamics could not be analyzed when we combined TED and Fluo5N in hippocampal neurons, acute slices, or organotypic slice cultures from RedCES2 transgenic mice. We will not give up and will try to use the mice for 3D-ER structure tracing in neurons. One aim of our project was to establish TED for analyzing fast, induced calcium signals by the neurotrophin BDNF, via its receptor TrkB, in hippocampal neurons. However, these experiments were not conclusive and disappointing. It has been suggested in my work that NaV1.9, a voltage-gated sodium channel, would be a mediator of fast, instructive BDNF-induced neuronal excitation. I followed this controversial concept by looking at it from different sides and in different cell models. These studies contributed new and clinically relevant information about neuronal excitability caused by Nav1.9, for instance in motoneurons and DRG neurons. I am now sure that NaV1.9 is not responsible for fast BDNF- induced calcium effects in hippocampal neurons. Anyhow, we learned more about BDNF signaling aspects that could help us in the future. For instance, with super-resolution microscopy, we could confirm, with 20 nm resolution, high abundance of BDNF in small vesicles within the presynapse of glutamatergic neurons. While we tried to establish TrkB vectors for analysis of ER calcium signals downstream of BDNF/TrkB, we made a surprising additional finding. We observed that the intracellular TrkB kinase domain as well as oncogenic intracellular NTRK2-gene fusions (de facto protumorigenic TrkB-fusion proteins) activate an intracellular self-activation signaling cascade that is different from classical BDNF/TrkB signaling. The study is clinically very relevant, as we also found evidence for immature, phosphoactive Trk in Nestin-positive glioblastoma tissue. We suggest to use immature, phospho-active Trk protein as biomarker to find out which glioblastoma patients would best profit from an anti-tumorigenic Trk inhibitor therapy.

Projektbezogene Publikationen (Auswahl)

 
 

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