We report a sensitive LC (liquid chromatography)/MS/MS assay using selected reaction monitoring to quantify RA (retinoic acid), which is applicable to biological samples of limited size (10-20 mg of tissue wet weight), requires no sample derivatization, provides mass identification and resolves atRA (all-trans-RA) from its geometric isomers. The assay quantifies over a linear range of 20 fmol to 10 pmol, and has a 10 fmol limit of detection at a signal/noise ratio of 3. Coefficients of variation are: instrumental, 0.5-2.9%; intra-assay, 5.4+/-0.4%; inter-assay 8.9+/-1.0%. An internal standard (all-trans-4,4-dimethyl-RA) improves accuracy by confirming extraction efficiency and revealing handling-induced isomerization. Tissues of 2-4-month-old C57BL/6 male mice had atRA concentrations of 7-9.6 pmol/g and serum atRA of 1.9+/-0.6 pmol/ml (+/-S.E.M.). Tissue 13-cis-RA ranged from 2.9 to 4.2 pmol/g, and serum 13-cis-RA was 1.2+/-0.3 pmol/ml. CRBP (cellular retinol-binding protein)-null mouse liver had atRA approximately 30% lower than wild-type (P<0.05), but kidney, testis, brain and serum atRA were similar to wild-type. atRA in brain areas of 12-month-old female C57BL/6 mice were (+/-S.E.M.): whole brain, 5.4+/-0.4 pmol/g; cerebellum, 10.7+/-0.3 pmol/g; cortex, 2.6+/-0.4 pmol/g; hippocampus, 8.4+/-1.2 pmol/g; striatum, 15.3+/-4.7 pmol/g. These data provide the first analytically robust quantification of atRA in animal brain and in CRBP-null mice. Direct measurements of endogenous RA should have a substantial impact on investigating target tissues of RA, mechanisms of RA action, and the relationship between RA and chronic disease.

Differentiation and patterning in the developing nervous system require the vitamin A metabolite all-trans-retinoic acid (atRA). Recent data suggest that higher cognitive functions, such as creation of hippocampal memory, also require atRA and its receptors, RAR, through affecting synaptic plasticity. Here we show that within 30 min atRA increased dendritic growth approximately 2-fold, and PSD-95 and synaptophysin puncta intensity approximately 3-fold, in cultured mouse hippocampal neurons, suggesting increased synapse formation. atRA (10 nM) increased ERK1/2 phosphorylation within 10 min. In synaptoneurosomes, atRA rapidly increased phosphorylation of ERK1/2, its target 4E-BP, and p70S6K, and its substrate, ribosome protein S6, indicating activation of MAPK and mammalian target of rapamycin (mTOR). Immunofluorescence revealed intense dendritic expression of RARalpha in the mouse hippocampus and localization of RARalpha on the surfaces of primary cultures of hippocampal neurons, with bright puncta along soma and neurites. Surface biotinylation confirmed the locus of RARalpha expression. Knockdown of RARalpha by shRNA impaired atRA-induced spine formation and abolished dendritic growth. Prolonged atRA stimulation reduced surface/total RARalpha by 43%, suggesting internalization, whereas brain-derived nerve growth factor or bicuculline increased the ratio by approximately 1.8-fold. atRA increased translation in the somatodendritic compartment, similar to brain-derived nerve growth factor. atRA specifically increased dendritic translation and surface expression of the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor (AMPAR) subunit 1 (GluR1), without affecting GluR2. These data provide mechanistic insight into atRA function in the hippocampus and identify a unique membrane-associated RARalpha that mediates rapid induction of neuronal translation by atRA.

All-trans-retinoic acid stimulates dendritic growth in hippocampal neurons within minutes by activating mitogen-activated protein kinase and mTOR and increasing dendritic translation of calcium calmodulin-dependent protein kinase II ␣ and the ␣-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunit GluR1. Hippocampal neurons express RAR␣ in dendrites, and knocking down RAR␣ prevents all-trans-retinoic acid effects on dendritic growth. Here we show, by liquid chromatography/mass spectrometry analysis of immunoaffinity isolates of hippocampal neurons, that RAR␣ partners with many RNA-binding proteins and translation factors conveyed in dendritic RNA transport granules, including the purine-rich element-binding protein, Pur ␣. The interaction of RAR␣ with Pur ␣, an RNA-binding protein required for dendritic RNA transport, and other RNA-binding proteins was confirmed by tandem affinity purification. Confocal microscopy confirmed localization of neuronal RAR␣ in dendritic RNA granules with Pur ␣ and FMRP (the fragile ؋ mental retardation protein). Hippocampal RAR␣ also associates with mRNA, e.g. encoding GluR1 and calcium calmodulin-dependent protein kinase II ␣. Consistent with a granule function of conveying translationally silenced mRNA, RAR␣ inhibits translation initiation, independent of 7-methylguanylate cap or poly(A) tail, and prompts mRNA redistribution to silencing ribonucleoprotein particles. These data afford a mechanism for rapid stimulation of dendritic growth by all-trans-retinoic acid and reveal that the liganddependent transcription factor RAR␣ also regulates translation.The developed nervous system relies on atRA 2 for synaptic plasticity that underlies hippocampus-dependent spatial learning (1, 2). Restricting the dietary atRA precursor retinol (vitamin A) or genetically impairing atRA signaling impairs hippocampus-dependent learning (3-6). Retinoid-compromised rodents err markedly more frequently in tests related to hippocampal function, such as the radial arm and Morris water mazes, and have diminished long term potentiation and abolished long term depression. Conversely, atRA dosing reduces frequency of relational memory errors in older mice relative to young mice (7).Synaptic plasticity relies on protein synthesis in dendrites (8, 9). Large ribonucleoparticles or RNA granules convey translationally silenced mRNA from neuronal cell bodies to dendrites. These RNA encode diverse classes of postsynaptic proteins, including ionotropic glutamate receptors and kinases such as CaMKII ␣ (10). Synaptic activity triggers translation of dendritic mRNA to support rapid synaptic structure and efficacy modifications (10, 11). For example, the ␣-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunits GluR1 and GluR2 insert into synaptic membranes after local translation to enhance the number, strength, and stability of synapses (12).atRA stimulates dendritic growth in primary mouse hippocampal neurons; siRNA knockdown and small hairpin RNA knockdown indicate a central function for RAR␣ in...

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