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Abstract
Spontaneous Ca2+ transients and actin dynamics in primary motoneurons correspond to cellular differentiation such as axon elongation and growth cone formation. Brain-derived neurotrophic factor (BDNF) and its receptor trkB support both motoneuron survival and synaptic differentiation. However, in motoneurons effects of BDNF/trkB signaling on spontaneous Ca2+ influx and actin dynamics at axonal growth cones are not fully unraveled. In our study we addressed the question how neurotrophic factor signaling corresponds to cell autonomous excitability and growth cone formation. Primary motoneurons from mouse embryos were cultured on the synapse specific, β2-chain containing laminin isoform (221) regulating axon elongation through spontaneous Ca2+ transients that are in turn induced by enhanced clustering of N-type specific voltage-gated Ca2+ channels (Cav2.2) in axonal growth cones. TrkB-deficient (trkBTK−/−) mouse motoneurons which express no full-length trkB receptor and wildtype motoneurons cultured without BDNF exhibited reduced spontaneous Ca2+ transients that corresponded to altered axon elongation and defects in growth cone morphology which was accompanied by changes in the local actin cytoskeleton. Vice versa, the acute application of BDNF resulted in the induction of spontaneous Ca2+ transients and Cav2.2 clustering in motor growth cones, as well as the activation of trkB downstream signaling cascades which promoted the stabilization of β-actin via the LIM kinase pathway and phosphorylation of profilin at Tyr129. Finally, we identified a mutual regulation of neuronal excitability and actin dynamics in axonal growth cones of embryonic motoneurons cultured on laminin-221/211. Impaired excitability resulted in dysregulated axon extension and local actin cytoskeleton, whereas upon β-actin knockdown Cav2.2 clustering was affected. We conclude from our data that in embryonic motoneurons BDNF/trkB signaling contributes to axon elongation and growth cone formation through changes in the local actin cytoskeleton accompanied by increased Cav2.2 clustering and local calcium transients. These findings may help to explore cellular mechanisms which might be dysregulated during maturation of embryonic motoneurons leading to motoneuron disease.
Highlights
It is widely recognized that neurotrophic factor signaling contributes to motoneuron survival (Arakawa et al, 1990; Sendtner et al, 1991, 1992; Hughes et al, 1993; Henderson et al, 1994; Pennica et al, 1995, 1996)
In order to investigate whether impaired BDNF/trkB signaling affects axon elongation and growth cone formation we started to culture embryonic trkBTK−/− motoneurons on synapse specific laminin-221/211 for 5 days (Figure 1)
In axonal growth cones of wild type motoneurons trkB receptors and Cav2.2 calcium channels clustered in close proximity at axon tips (Figure 1A, Figure S1B)
Summary
It is widely recognized that neurotrophic factor signaling contributes to motoneuron survival (Arakawa et al, 1990; Sendtner et al, 1991, 1992; Hughes et al, 1993; Henderson et al, 1994; Pennica et al, 1995, 1996). Beta 2-chain laminins are reported to associate with the pore-forming (Cav) subunit of N- and P/Q type calcium channels (Nishimune et al, 2004) In embryonic motoneurons this interaction leads to Cav2.2 accumulation which corresponds to increased spontaneous Ca2+ influx at axonal growth cones modulating axonal extension which is not observed on β1-chain laminins such as laminin111 (Jablonka et al, 2007). Local translation is induced through Src-dependent phosphorylation of the β-actin mRNA zipcode binding protein 1 (ZBP1) leading to growth cone turning in correspondence to cellular Ca2+ availability (Zhang et al, 1999, 2001; Hüttelmaier et al, 2005; Yao et al, 2006; Sasaki et al, 2010)
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