ABSTRACT
It is widely believed that diet can influence the onset and severity of cognitive aging, but the optimal combination of micronutrients and molecular and cellular mechanisms remain elusive. The purpose of this study was to compare the effects of eight distinct diets, consisting of various concentrations of selected micronutrients, on learning and memory as well as markers of neuronal plasticity, and metabolic and neuro-immune status of the aged hippocampus. Eighteen-month-old male and female C57BL/6J mice were fed the diets for 16 weeks, followed by learning and memory trials on the active avoidance task. Number of immature neurons were measured by immunohistochemical detection of doublecortin (DCX+) in the granule layer of the dentate gyrus. Amount of mitochondrial DNA (mtDNA) and gene expression of molecular markers of mitochondrial biogenesis (Ppargc1α, Sirt1, Tfam), and neuroinflammation (IL-10, Alox15, Ptgs2, IL-1ß, IL-6 and Tnf) were assessed by quantitative real time polymerase chain reaction (qRT-PCR) of hippocampal samples. Tissue levels of selected micronutrients and a number of metabolites were measured by liquid chromatography-mass spectrometry. The diet supplemented with RRR d-alpha tocopheryl acetate, citicholine, 5-methyltetrahydrofolic acid, quercetin and the n-3 fatty acid phosphatidylserine-docosahexaenoic acid, improved performance on the active avoidance learning and memory task compared to all the other less-complex diets. This diet also increased IL-10 expression and attenuated the age-related change in mtDNA content in the hippocampus without affecting metabolite levels. Results suggest cognitive benefits of wholesome diets are partially mediated through combined antioxidant and anti-inflammatory activities of optimized mixtures of micronutrients.
Subject(s)
Aging , Cognition/drug effects , Hippocampus/drug effects , Hippocampus/physiology , Micronutrients/pharmacology , Age Factors , Animals , Avoidance Learning/drug effects , Body Weight/drug effects , Cohort Studies , Cytokines/genetics , Cytokines/metabolism , DNA Copy Number Variations/physiology , DNA, Mitochondrial/genetics , Doublecortin Domain Proteins , Doublecortin Protein , Eating/drug effects , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Lipid Metabolism/drug effects , Male , Metabolome/physiology , Mice , Mice, Inbred C57BL , Microtubule-Associated Proteins/metabolism , Neuropeptides/metabolism , Sex FactorsABSTRACT
Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.
Subject(s)
Myosin Type II/physiology , Proteasome Endopeptidase Complex/physiology , Receptors, N-Methyl-D-Aspartate/physiology , Animals , Blotting, Western , DNA Primers , Densitometry , Electrophoresis, Agar Gel , Endoplasmic Reticulum/metabolism , Fluorescent Antibody Technique , Golgi Apparatus/metabolism , Immunoprecipitation , Microscopy, Confocal , Muscle Contraction/physiology , Myosin Heavy Chains/metabolism , PC12 Cells , RNA/biosynthesis , RNA/isolation & purification , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Ubiquitin/metabolismABSTRACT
Formation of functional N-methyl-D-aspartate (NMDA) receptor channels requires the essential NMDA receptor subunit NR1 and one or more of the modulatory subunits NR2A-D and in some cases an additional subunit NR3A or NR3B. Recent studies indicate that NR1 expression is regulated at translation under both physiological and pathological conditions. The rat pheochromocytoma cell line (PC12) has been used as a model system for NR1 gene expression studies. Characterization of the posttranscriptional regulatory mechanisms suggested the posttranslational degradation and translational regulation of NR1 protein in PC12 cells. In addition a recent study on the translational regulation of NR1 mRNA in intact brain identified two translationally distinct pools of NR1 mRNA. In this review we summarize the evidence for translational regulation of NR1 expression in PC12 cells and the brain.
Subject(s)
Brain/metabolism , Gene Expression Regulation/physiology , Protein Biosynthesis , Receptors, N-Methyl-D-Aspartate/genetics , Animals , Brain/pathology , Mice , PC12 Cells , Rats , Receptors, N-Methyl-D-Aspartate/metabolismABSTRACT
PC12 cells contain NR1 mRNA but lack significant expression of NR1 protein suggesting translational or posttranslational regulation. Translational activity of NR1 mRNA in PC12 cells was examined by sucrose gradient fractionation and by heterologous luciferase NR1 gene expression studies. The cosedimentation and association of NR1 mRNA with large polyribosomes (polysomes) confirmed the translatability of NR1 message in PC12 cells. Possible initiation and/or elongation defects during the translation of NR1 mRNAs were investigated by cyclohexamide treatment. The marked decline in the number of ribosomes associated with NR1 mRNA after prolonged exposure to cyclohexamide suggested that initiation was limiting translation of NR1 mRNA in PC12 cells. Consequently, the effect of the 5' and 3' untranslated regions (UTRs) on translation was examined using fusion constructs consisting of the luciferase coding region fused to either or both the 5' UTR and 3' UTR of NR1. The transfection of PC12 cells with the luciferase NR1-UTR fusion constructs revealed that the 3' UTR of NR1 had a significant inhibitory effect on luciferase expression. In contrast, the 5' UTR of NR1 had no inhibitory effect on mRNA translation in PC12 cells. The results from this study indicate that the translation of NR1 mRNA in PC12 cells may be impeded at initiation and this inhibition may be regulated at least in part through the 3' UTR of NR1.
Subject(s)
Protein Biosynthesis/physiology , Receptors, N-Methyl-D-Aspartate/genetics , 5' Untranslated Regions , Animals , Blotting, Northern , Chemical Fractionation/methods , Embryo, Mammalian , Humans , Kidney , Luciferases/metabolism , PC12 Cells , Polyribosomes , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Rats , Receptors, N-Methyl-D-Aspartate/metabolism , Sequence Alignment , Sucrose , TransfectionABSTRACT
The post-translational fate of N-methyl-D-aspartate receptor (NMDAR) subunit NR1 was characterized in PC12 cells using pulse-chase labeling, block of protein synthesis by cyclohexamide and deglycosylation by endoglycosidase H. Metabolic labeling of NR1 protein indicated a biphasic degradation of NR1 protein with half-lives of 1.6 and 16.1 h for a rapidly (78%) and a slowly (22%) degrading population. Immunoprecipitation of NR1 following the block of protein synthesis by cyclohexamide revealed that the rapidly and slowly degrading pools mainly consisted of the NR1 splice variants NR1-4a and NR1-2a. Sensitivity of NR1 protein to deglycosylation by endoglycosidase H indicated the presence of an immature form of NR1 that was retained in the endoplasmic reticulum. PC12 cells serve as a useful model for the elucidation of translational and post-translational mechanisms of NMDAR expression.
