TED, a new method to analyze dynamics of intracellular Ca2+ stores in hippocampal networks and to analyze the role of Ca2+ stores in neurotrophin signalling cascades
Molecular and Cellular Neurology and Neuropathology
Final Report Abstract
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.
Publications
- 2012. Role of Na(v)1.9 in activity-dependent axon growth in motoneurons. Human molecular genetics. 21:3655-3667
Subramanian, N., A. Wetzel, B. Dombert, P. Yadav, S. Havlicek, S. Jablonka, M.A. Nassar, R. Blum, and M. Sendtner
(See online at https://doi.org/10.1093/hmg/dds195) - 2013. A de novo gain-of-function mutation in SCN11A causes loss of pain perception. Nature genetics. 45:1399-1404
Leipold, E., L. Liebmann, G.C. Korenke, T. Heinrich, S. Giesselmann, J. Baets, M. Ebbinghaus, R.O. Goral, T. Stodberg, J.C. Hennings, M. Bergmann, J. Altmuller, H. Thiele, A. Wetzel, P. Nurnberg, V. Timmerman, P. De Jonghe, R. Blum, H.G. Schaible, J. Weis, S.H. Heinemann, C.A. Hubner, and I. Kurth
(See online at https://doi.org/10.1038/ng.2767) - 2013. Cell-autonomous axon growth of young motoneurons is triggered by a voltage-gated sodium channel. Channels (Austin). 7:51-56
Wetzel, A., S. Jablonka, and R. Blum
(See online at https://doi.org/10.4161/chan.23153) - 2013. Direct imaging of ER calcium with targeted-esterase induced dye loading (TED). Journal of visualized experiments: JoVE. 75:e50317
Samtleben, S., J. Jaepel, C. Fecher, T. Andreska, M. Rehberg, and R. Blum
(See online at https://doi.org/10.3791/50317) - 2014. High abundance of BDNF within glutamatergic presynapses of cultured hippocampal neurons. Frontiers in cellular neuroscience. 8:107
Andreska, T., S. Aufmkolk, M. Sauer, and R. Blum
(See online at https://doi.org/10.3389/fncel.2014.00107) - 2015. Store-operated calcium entry compensates fast ER calcium loss in resting hippocampal neurons. Cell calcium. 58:147-159
Samtleben, S., B. Wachter, and R. Blum
(See online at https://doi.org/10.1016/j.ceca.2015.04.002) - 2016. Peri-Synaptic Glia Recycles Brain-Derived Neurotrophic Factor for LTP Stabilization and Memory Retention. Neuron. 92:873-887
Vignoli, B., G. Battistini, R. Melani, R. Blum, S. Santi, N. Berardi, and M. Canossa
(See online at https://doi.org/10.1016/j.neuron.2016.09.031) - 2017. Neurobiology of local and intercellular BDNF signaling. Pflugers Archiv : European journal of physiology. 469:593-610
Sasi, M., B. Vignoli, M. Canossa, and R. Blum
(See online at https://doi.org/10.1007/s00424-017-1964-4) - 2018. An open source tool for automatic spatiotemporal assessment of calcium transients and local 'signal-close-to-noise' activity in calcium imaging data. PLoS computational biology. 14:e1006054
Prada, J., M. Sasi, C. Martin, S. Jablonka, T. Dandekar, and R. Blum
(See online at https://doi.org/10.1371/journal.pcbi.1006054) - 2018. NaV1.9 Potentiates Oxidized Phospholipid-Induced TRP Responses Only under Inflammatory Conditions. Frontiers in molecular neuroscience. 11:7
Martin, C., C. Stoffer, M. Mohammadi, J. Hugo, E. Leipold, B. Oehler, H.L. Rittner, and R. Blum
(See online at https://doi.org/10.3389/fnmol.2018.00007) - 2020. Constitutively active TrkB kinase signalling reduces actin filopodia dynamics and cell migration
Gupta, R., M. Bauer, G. Wohlleben, V. Luzak, V. Wegat, D. Segebarth, E. Bady, G. Langlhofer, B. Wachter, S. Havlicek, P. Lüningschrör, C. Villmann, B. Polat, C.M. Monoranu, J. Kuper, and R. Blum
(See online at https://doi.org/10.1101/2020.09.11.292565) - 2020. R-Roscovitine Improves Motoneuron Function in Mouse Models for Spinal Muscular Atrophy. iScience. 23:100826
Tejero, R., S. Balk, J. Franco-Espin, J. Ojeda, L. Hennlein, H. Drexl, B. Dombert, J.D. Clausen, L. Torres-Benito, L. Saal-Bauernschubert, R. Blum, M. Briese, S. Appenzeller, L. Tabares, and S. Jablonka
(See online at https://doi.org/10.1016/j.isci.2020.100826) - 2021. Astrocytic microdomains from mouse cortex gain molecular control over long-term information storage and memory retention. Commun Biol. 4:1152
Vignoli, B., G. Sansevero, M. Sasi, R. Rimondini, R. Blum, V. Bonaldo, E. Biasini, S. Santi, N. Berardi, B. Lu, and M. Canossa
(See online at https://doi.org/10.1038/s42003-021-02678-x) - 2022. Homeostatic calcium fluxes, ER calcium release, SOCE, and calcium oscillations in cultured astrocytes are interlinked by a small calcium toolkit. Cell calcium. 101:102515
Schulte, A., L. Bieniussa, R. Gupta, S. Samtleben, T. Bischler, K. Doering, P. Sodmann, H. Rittner, and R. Blum
(See online at https://doi.org/10.1016/j.ceca.2021.102515) - 2022. Modelling non-local neural information processing in the brain
Balkenhol, J., B. Händel, J. Prada, C.A. Bosman, H. Ehrenreich, J. Grohmann, J.v. Kistowski, S.M. Wojcik, S. Kounev, R. Blum, and T. Dandekar
(See online at https://doi.org/10.1101/2022.01.27.477993) - 2022. Neurodegeneration by alpha-synuclein-specific T cells in AAV-A53T-alpha-synuclein Parkinson's disease mice. Brain Behav Immun. 101:194-210
Karikari, A.A., R.L. McFleder, E. Ribechini, R. Blum, V. Bruttel, S. Knorr, M. Gehmeyr, J. Volkmann, J.M. Brotchie, F. Ahsan, B. Haack, C.M. Monoranu, U. Keber, R. Yeghiazaryan, A. Pagenstecher, T. Heckel, T. Bischler, J. Wischhusen, J.B. Koprich, M.B. Lutz, and C. Wang
(See online at https://doi.org/10.1016/j.bbi.2022.01.007)
