Axon targeting of the alpha 7 nicotinic receptor in developing hippocampal neurons by Gprin1 regulates growth

Abstract

Cholinergic signaling plays an important role in regulating the growth and regeneration of axons in the nervous system. The α7 nicotinic receptor (α7) can drive synaptic development and plasticity in the hippocampus. Here we show that activation of α7 significantly reduces axon growth in hippocampal neurons by coupling to G protein regulated inducer of neurite outgrowth 1 (Gprin1), which targets it to the growth cone (GC). Knockdown of Gprin1 expression using RNAi is found sufficient to abolish the localization and calcium signaling of α7 at the GC. In particular, α7/Gprin1 interaction appears intimately linked to a Gαo, GAP-43, and CDC42 cytoskeletal regulatory pathway within the developing axon. These findings demonstrate that α7 regulates axon growth in hippocampal neurons, thereby likely contributing to synaptic formation in the developing brain.

Identification and Characterization of a G Protein-binding Cluster in α7 Nicotinic Acetylcholine Receptors

“…␣7 receptor activation and the generation of IP 3 via PLC promotes calcium release via IP 3 Rs in neurons (18,26). We confirmed the involvement of IP 3 Rs in choline-induced calcium transients at the GC of PC12 cells.…”

“…Transfection of PC12 cells with ␣7 siRNA significantly reduces ␣7 nAChR protein expression and confirms the specificity of mAb306 on the Western blot (Fig. 3A) (16,26). Studies in N2a cells indicate that transfection with ␣7 345-348A yields a similar ␣7 nAChR expression as the wild-type (␣7) and supports the finding that mutation at the GPBC does not impact the synthesis of the nAChR in cells.…”

“…2C). Recently we have shown a role for ␣7 nAChRs in the development of hippocampal neurons and PC12 cells via receptor localization at the GC (26). Strong fBgtx labeling was seen at the GC in ␣7, ␣7ϩ, and ␣7 345-348A nAChR expressing cells (Fig.…”

“…Phototoxicity and bleaching were minimized using low-wavelength and neutral density light filters (26). Drugs were applied to the recording chamber via a gravity fed perfusion system at a flow rate of 1 ml/s after 70 frames of baseline recordings.…”

“…Complexes in the Brain-Studies have revealed associations between nAChRs and G proteins in neural and non-neural cells (4,9,18,23,26,28). We surveyed ␣7 nAChR associations with G proteins in the prefrontal cortex, hippocampus, and striatum of the mouse brain.…”