ABSTRACT
Aberrant dopamine D2 receptor (D2R) activity is associated with neuropsychiatric disorders, making those receptors targets for antipsychotic drugs. Here, we report that novel signaling through the intracellularly localized D2R long isoform (D2LR) elicits extracellular signal-regulated kinase (ERK) activation and dendritic spine formation through Rabex-5/platelet-derived growth factor receptor-ß (PDGFRß)-mediated endocytosis in mouse striatum. We found that D2LR directly binds to and activates Rabex-5, promoting early-endosome formation. Endosomes containing D2LR and PDGFRß are then transported to the Golgi apparatus, where those complexes trigger Gαi3-mediated ERK signaling. Loss of intracellular D2LR-mediated ERK activation decreased neuronal activity and dendritic spine density in striatopallidal medium spiny neurons (MSNs). In addition, dendritic spine density in striatopallidal MSNs significantly increased following treatment of striatal slices from wild-type mice with quinpirole, a D2R agonist, but those changes were lacking in D2LR knockout mice. Moreover, intracellular D2LR signaling mediated effects of a typical antipsychotic drug, haloperidol, in inducing catalepsy behavior. Taken together, intracellular D2LR signaling through Rabex-5/PDGFRß is critical for ERK activation, dendritic spine formation and neuronal activity in striatopallidal MSNs of mice.
Subject(s)
Guanine Nucleotide Exchange Factors/metabolism , Receptors, Dopamine D2/metabolism , Animals , Cell Culture Techniques , Corpus Striatum/drug effects , Dendritic Spines/metabolism , Dendritic Spines/physiology , Dopamine Agonists/pharmacology , Endocytosis/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Guanine Nucleotide Exchange Factors/genetics , HEK293 Cells , Haloperidol/pharmacology , Humans , MAP Kinase Signaling System , Male , Mice , Mice, Knockout , Mice, Transgenic , Neurons/metabolism , Phosphorylation , Protein Isoforms , Quinpirole/pharmacology , Receptor, Platelet-Derived Growth Factor beta/metabolism , Receptors, Dopamine D1/metabolism , Signal Transduction/drug effects , Synaptic Transmission/drug effectsABSTRACT
The mycotoxin sterigmatocystin (STC) is produced mainly by some Aspergillus and Penicillium fungi; it naturally contaminates cereals, peanuts, and products derived from these crops, and is both mutagenic and carcinogenic. As an intermediate of aflatoxin (AF) biosynthesis, its structure is similar to that of AF. Although immunoaffinity columns (IACs) are a popular approach to sample clean-up, no IAC is commercially available for STC, but a commercially available IAC for AF shows cross reactivity to STC. We here developed a new method for analyzing STC in grains using such an IAC and liquid chromatography mass spectrometry (LCMS), and validated this method using six different grains. The STC limit of detection (signal-to-noise ratio, S/N = 3) was 2.5 pg (1.0 µg/kg in the product), and the calibration curve was linear in the range of 7.5-375 pg (3.0-150 µg/kg in the product). The within-day recovery of STC from samples spiked with STC at 5.0 and 50 µg/kg was 83.2-102.5% and the RSDr (relative standard deviation of repeatability) of these samples was 1.9-6.5%; the RSDr of STC-pretreated grain samples was 3.1-14.0%. Average recovery of STC from samples spiked with STC in the range of 5.0-100 µg/kg STC was 83.2-102.5%, with an RSDr of 0.24-6.5%; the RSDr of STC-pretreated grain samples was 2.4-14.0%. In an intermediate precision study, the average STC recovery from STC-spiked samples by three analysts was 95.2-107.5%, with RSDRi (intermediate precision) of 4.0-7.1%; the RSDRi of the STC-pretreated samples was 4.8-10.4%. Thus, the proposed method was effective for STC analysis in grains, and holds potential for a novel application of a commercial IAC, intended for AFs, in STC analysis.