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Sigma-1 receptors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1xGTP pathway.

ABSTRACT: Sigma-1 receptors (Sig-1Rs) are endoplasmic reticulum (ER)-resident proteins known to be involved in learning and memory. Dendritic spines in hippocampal neurons play important roles in neuroplasticity and learning and memory. This study tested the hypothesis that Sig-1Rs might regulate denritic spine formation in hippocampal neurons and examined potential mechanisms therein. In rat hippocampal primary neurons, the knockdown of Sig-1Rs by siRNAs causes a deficit in the formation of dendritic spines that is unrelated to ER Ca(2+) signaling or apoptosis, but correlates with the mitochondrial permeability transition and cytochrome c release, followed by caspase-3 activation, Tiam1 cleavage, and a reduction in Rac1.GTP. Sig-1R-knockdown neurons contain higher levels of free radicals when compared to control neurons. The activation of superoxide dismutase or the application of the hydroxyl-free radical scavenger N-acetyl cysteine (NAC) to the Sig-1R-knockdown neurons rescues dendritic spines and mitochondria from the deficits caused by Sig-1R siRNA. Further, the caspase-3-resistant TIAM1 construct C1199DN, a stable guanine exchange factor able to constitutively activate Rac1 in the form of Rac1.GTP, also reverses the siRNA-induced dendritic spine deficits. In addition, constitutively active Rac1.GTP reverses this deficit. These results implicate Sig-1Rs as endogenous regulators of hippopcampal dendritic spine formation and suggest a free radical-sensitive ER-mitochondrion-Rac1.GTP pathway in the regulation of dendritic spine formation in the hippocampus.

SUBMITTER: Tsai SY

PROVIDER: S-EPMC2792161 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.

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Sigma-1 receptors regulate hippocampal dendritic spine formation via a free radical-sensitive mechanism involving Rac1xGTP pathway.

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