Abstract
Retinoic acid (RA) regulates numerous aspects of central nervous system function through modulation of gene transcription via retinoic acid receptors (RARs). However, RA has important roles independent of gene transcription (non-genomic actions) and in the brain a crucial regulator of homeostatic plasticity is RAR control of glutamate receptor subunit 1 (GluR1) translation. An assay to quantify RAR regulation of GluR1 translation would be beneficial both to study the molecular components regulating this system and screen drugs that influence this critical mechanism for learning and memory in the brain. A bioluminescence reporter assay was developed that expresses firefly luciferase under the control of the GluR1 5′ untranslated region bound by RAR. This assay was introduced into SH-SY5Y cells and used to demonstrate the role of RARα in RA regulation of GluR1 translation. A screen of synthetic RAR and RXR ligands indicated that only a subset of these ligands activated GluR1 translation. The results demonstrate the practicality of this assay to explore the contribution of RARα to this pathway and that the capacity of RAR ligands to activate translation is a quality restricted to a limited number of compounds, with implications for their RAR selectivity and potentially their specificity in drug use.
Highlights
Nuclear receptors are involved in a major set of signalling pathways in the brain, and crucial among these is the retinoic acid receptor (RAR) family [1]
To quantify RARα ligand regulation of glutamate receptor subunit 1 (GluR1) translation, a highly sensitive bioluminescence reporter composed of firefly luciferase under the control of the GluR1 5′ untranslated region (5′ UTR) was employed
The construct was transfected into SH-SY5Y cells, a frequently used model of neuronal cells, employing a SHSY5YRARα variant in which high levels of RARα expression can be induced with tetracycline or the more stable derivative doxycycline [30]
Summary
Nuclear receptors are involved in a major set of signalling pathways in the brain, and crucial among these is the retinoic acid receptor (RAR) family [1]. Pharmacological manipulation studies have shown that an increase in synaptic strength is induced when neural activity is inhibited by tetrodotoxin (TTX; which blocks sodium-gated voltage channels) and an N-methyl-D-aspartate receptor (NMDA) receptor antagonist [10, 11]. This synaptic increase in neural activity is mediated by an increase in local translation and insertion of the glutamate receptor 1 (GluR1, known as GRIA1 and GluA1) subunits of the α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptor [12,13,14].
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