No abstract

The capacity of neurons to develop a long axon and multiple dendrites defines neuron connectivity in the CNS. The highly conserved microRNA-9 (miR-9) is expressed in both neuronal precursors and some post-mitotic neurons, and we detected miR-9 expression in the axons of primary cortical neurons. We found that miR-9 controlled axonal extension and branching by regulating the levels of Map1b, an important protein for microtubule stability. Following microfluidic separation of the axon and the soma, we found that miR-9 repressed Map1b translation and was a functional target for the BDNF-dependent control of axon extension and branching. We propose that miR-9 links regulatory signaling processes with dynamic translation mechanisms, controlling Map1b protein levels and axon development.

Spectraplakins are large actin-microtubule linker molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. Expression data for the mammalian spectraplakin ACF7 and genetic analyses of the Drosophila spectraplakin Short stop (Shot) suggest an important role during neurogenesis. Using three parallel neuronal culture systems we demonstrate that, like Shot, ACF7 is essential for axon extension and describe, for the first time, their subcellular functions during axonal growth. Firstly, both ACF7 and Shot regulate the organisation of neuronal microtubules, a role dependent on both the F-actin- and microtubule-binding domains. This role in microtubule organisation is probably the key mechanism underlying the roles of Shot and ACF7 in growth cone advance. Secondly, we found a novel role for ACF7 and Shot in regulating the actin cytoskeleton through their ability to control the formation of filopodia. This function in F-actin regulation requires EF-hand motifs and interaction with the translational regulator Krasavietz/eIF5C, indicating that the underlying mechanisms are completely different from those used to control microtubules. Our data provide the basis for the first mechanistic explanation for the role of Shot and ACF7 in the developing nervous system and demonstrate their ability to coordinate the organisation of both actin and microtubule networks during axonal growth.

Neuronal nicotinic acetylcholine receptors (nAChR) can regulate several neuronal processes through Ca 2+ -dependent mechanisms. The versatility of nAChR-mediated responses presumably reflects the spatial and temporal characteristics of local changes in intracellular Ca 2+ arising from a variety of sources. The aim of this study was to analyse the components of nicotine-evoked Ca 2+ signals in SH-SY5Y cells, by monitoring fluorescence changes in cells loaded with fluo-3 AM. Nicotine (30 lM) generated a rapid elevation in cytoplasmic Ca 2+ that was partially and additively inhibited (40%) by a7 and a3b2* nAChR subtype selective antagonists; a3b4* nAChR probably account for the remaining response (60%). A substantial blockade (80%) by CdCl 2 (100 lM) indicates that voltage-operated Ca 2+ channels (VOCC) mediate most of the nicotine-evoked response, although the a7 selective antagonist a-bungarotoxin (40 nM) further decreased the CdCl 2 -resistant component. The elevation of intracellular Ca 2+ levels provoked by nicotine was sustained for at least 10 min and required the persistent activation of nAChR throughout the response. Intracellular Ca 2+ stores were implicated in both the initial and sustained nicotine-evoked Ca 2+ responses, by the blockade observed after ryanodine (30 lM) and the inositoltriphosphate (IP 3 )-receptor antagonist, xestospongin-c (10 lM). Thus, nAChR subtypes are differentially coupled to specific sources of Ca 2+ : activation of nAChR induces a sustained elevation of intracellular Ca 2+ levels which is highly dependent on the activation of VOCC, and also involves Ca 2+ release from ryanodine and IP 3 -dependent intracellular stores. Moreover, the a7, but not a3b2* nAChR, are responsible for a fraction of the VOCC-independent nicotine-evoked Ca 2+ increase that appears to be functionally coupled to ryanodine sensitive Ca 2+ stores.

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also in¯uenced by the serine/ threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 lM) increased the activity of ERK1/2. This effect was Ca 2+ dependent, and prevented by the a7 nAChR antagonist a-bungarotoxin (a-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca 2+ entry to MEK-ERK1/2 activation, inhibitors of Ca 2+ -dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31±8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotinedependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was signi®cantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of a7 nAChR to the activation of ERK1/2, in a Ca 2+ and PKA dependent manner. Neuronal nicotinic acetylcholine receptors (nAChR) are ligand gated cation channels permeable to Na + and Ca 2+ and therefore capable of increasing intracellular Ca 2+ concentrations per se and by activation of voltage-operated Ca 2+ channels (VOCC). As a result, they have the capacity to in¯uence a variety of neuronal activities, such as neurotransmitter release (Wonnacott 1997), cell survival (Donnelly Roberts and Brioni 1999), synaptic plasticity (Ji et al. 2001) and memory processing (Levin and Simon 1998). Numerous subtypes of nAChR occur in the brain, including the two major subtypes comprised of a7 and a4b2 subunits, respectively. The a7 nAChR, in particular, has been implicated in several cellular processes, including long-term potentiation (Mansvelder and McGehee 2000;Matsuyama et al. 2000), neuroprotection (Donnelly Roberts et al. 1996;Dajas-Bailador et al. 2000;Kihara et al. 2001) and learning and memory (Levin et al. 1999). Although the Ca 2+ dependence of some of these a7 nAChR-mediated actions has been demonstrated, the downstream mechanisms that follow nAChR activation, and those attributed to a7 in particular, have not been fully elucidated.One candidate implicated in many of the cellular processes also modulated by nAChR stimulation is the mitogen activated/extracellular signal-regulated protein kinase Abbreviations used: a-Bgt, a-bungarotoxin; CaM, kinase II, Ca 2+ calmodulin dependent kinase II; CREB, cyclic AMP response element binding protein; DMEM, Dulbecco's modi®ed Eagle's medium; ECL, electrochemiluminescence; ERK1/2, extracellular-signal regulated pro...