Post-transcriptional regulation and localization of the activity-regulated cytoskeleton-associated protein Arc/Arg3.1

Activity-regulated cytoskeleton-associated protein (Arc), also known as Arg3.1, is an immediate early gene whose expression is induced in the brain by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, shares homology with alpha-spectrin, and has been implicated in AMPA receptor trafficking. However, Arc's molecular function remains incompletely understood. Whereas pathways regulating Arc transcription have been extensively investigated, less is known about the role of post-transcriptional mechanisms in Arc expression. In order to study the post-transcriptional regulation of Arc protein expression and further elucidate Arc's molecular function, experiments were performed to determine both its protein level and localization in cultured neurons. Fluorescence microscopy experiments revealed that agents that stimulate the cAMP/PKA pathway increased Arc protein levels. These agents included agonists of the NMDA receptor and Gs-coupled dopamine and beta-adrenergic receptors, all of which are involved in LTP, learning, and memory. Costimulation by both NMDA and the Gs-coupled receptor agonists increased Arc protein expression additively. In addition, Gs-coupled receptor stimulation of Arc expression was ineffective in the presence of an NMDA receptor antagonist, suggesting calcium influx through the NMDA receptor plays a gating role in this pathway. Stimulation of the cAMP/PKA pathway did not affect exogenous Arc mRNA levels or protein stability, thereby identifying translational efficacy as the main determinant of Arc protein level. Next, the subcellular localization of Arc protein was studied in both cultured neurons and HEK 293T cells. Arc was found primarily in the nucleus where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and directly interacted with the betaIV-spectrin splice variant betaSpIVSigma5, which is associated with PML bodies and the nuclear matrix. Finally, co-expression of Arc and betaSpIVSigma5 synergistically increased the number of endogenous PML bodies. Together, these results show that Arc translation is regulated by the same pathways that regulate LTP, learning, and memory, and that Arc functions as a spectrin-binding protein, forming a complex in the nucleus that may function where transcription is regulated.