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,
Objectives: To determine
the mechanisms of brain-derived
neurotrophic factor (BDNF)-mediated
control of gene expression in the
nervous system. The specific aims are:
-
Characterization of the cytosolic
and nuclear calcium signals, evoked
through full length TrkB (TrkBFL)-mediated
activation in neurons,
-
Investigation of the role of the
truncated isoforms TrkB T1 and T2 in
glial gene expression,
-
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: |
|
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. |