Alternative pre-mRNA splicing
of ion channels


WP5: Control of calcium-dependent gene expression through receptor tyrosine kinase TrkB isoforms

WP5: Control of calcium-dependent gene expression through receptor tyrosine kinase TrkB isoforms

Scientific team: Robert Blum, Olga Garaschuk, Christine Rose, Tanja Ziegler, Martin Sumser, Alex Lepier, Arthur Konnerth

Objectives: To determine the mechanisms of brain-derived neurotrophic factor (BDNF)-mediated control of gene expression in the nervous system. The specific aims are:

  1. Characterization of the cytosolic and nuclear calcium signals, evoked through full length TrkB (TrkBFL)-mediated activation in neurons,

  2. Investigation of the role of the truncated isoforms TrkB T1 and T2 in glial gene expression,

  3. Identification of the signal transduction pathway underlying BDNF-mediated gene expression in hippocampal neurons and their influence on trkB splicing.

Description of the work: BDNF is secreted from highly active neurons in the CNS, presumably locally at synapses. It functions in the low nanomolar range, indicating that it is the most potent excitatory substance in the central nervous system. Secretion and biosynthesis of BDNF is enhanced by electrical or synaptic stimulation. BDNF binds to two structurally unrelated plasma membrane receptor types, the p75 neurotrophin receptors and the receptor tyrosine kinase TrkB, the activation of which triggers intracellular signalling cascades and can result in changes in cellular gene expression. Both TrkB and p75 receptors can act independently but also interact with each other. TrkB receptors form multiple splice variants generating at least three proteins. The large intron 16 contains a human specific ChAB4 repeat element (STOILOV ET AL. 2002). Regulated splicing of this intron results in full-length and truncated receptors. Full-length receptors (TrkBFL) possess an intracellular tyrosine kinase domain and are considered as those TrkB receptors that mediate the critical effects of BDNF or NT-4/5. Truncated receptors (TrkB-T1; TrkB-T2), in contrast, lack tyrosine kinase activity. Surprisingly, TrkB-T1 receptors mediate rapid BDNF-induced activation of PLC and IP3-induced calcium release from intracellular stores in glial cells, which will be further analysed in aim 1. We showed that besides modulating or activating ion channels through intracellular signalling cascades within seconds to minutes, BDNF-induced activation of TrkBFL directly gates ion channels within milliseconds . BDNF effects on regulation of splicing factors will be tested in RNA preparations from BDNF-stimulated cultures with the regulator chip of WP6. In addition to the TTX-insensitive Na+ channel Nav1.9, shown to be BDNF-dependent, we will identify other channels by sequence comparison and electrophysiological methods or by imaging ion-activated fluorogenic dyes. In aim 2, we will elucidate the signaling pathway from TrkB-T1 to Ins(1,4,5)P3-dependent glial calcium stores, using Ca++-release inhibitors and imaging techniques. Astrocytes predominately express TrkB-T1 and respond to brief BDNF application with rapid calcium release signals from intracellular stores . Points of this signaling cascade will be compared for cross-talk with the splicing machinery determined in WP3. In aim 3 the dependency of trkB alternative splicing on neuronal activity will be studied by stimulating cells with BDNF followed by RT-PCR, as well as in animal models with manipulated cholinergic balance (WP6). Together with WP3, an intron containing the ChAB4 element will be inserted into a trkB expression construct, allowing us to study its potential influence on splicing regulation.


Previous work related to the project:
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Rose, C.R., et al., Truncated TrkB-T1 mediates neurotrophin-evoked calcium signalling in glia cells. Nature, 2003. 426(6962): p. 74-8.

Kovalchuk, Y., et al., Postsynaptic Induction of BDNF-Mediated Long-Term Potentiation. Science, 2002. 295(5560): p. 1729-34.

Blum, R., K.W. Kafitz, and A. Konnerth, Neurotrophin-evoked depolarization requires the sodium channel Na(V)1.9. Nature, 2002. 419(6908): p. 687-93.

Rose, C.R. and A. Konnerth, Stores not just for storage. intracellular calcium release and synaptic plasticity. Neuron, 2001. 31(4): p. 519-22.

Kafitz, K.W., et al., Neurotrophin-evoked rapid excitation through TrkB receptors. Nature, 1999. 401(6756): p. 918-21.