ABSTRACT
The region-specific characteristics of the extracellular matrix are crucial for diverse functions in the brain. Pancortins/neuron-specific olfactomedin-related glycoproteins are components of the extracellular matrix. They comprise four alternatively spliced variants, Pancortin-1 to -4, which share a common portion, the B part, in the middle of their structure, have two pairs of alternatively spliced 5' regions, A1 and A2, and 3' regions, C1 and C2. Here we demonstrate that in mice, Pancortin-3 (A2-B-C1) and Pancortin-4 (A2-B-C2), which we have grouped together the A2-Pancortins, were transcribed early during the development of the brain in a region specific manner and were expressed very stably in vivo. They are N-glycosylated and secreted. Furthermore, we examined their ontogenetical expression profiles in the developing thalamus using antiserum against the common B region, since transient expressions of their mRNAs were notable there. In the developing thalami, they lasted long in oligomerized form even after the transcription of their mRNAs decreased to an undetectable level. Further analyses revealed that cysteine residues that are located in the common B part are important for homo- and hetero-oligomer formation of A2-Pancortins. When we substituted cysteine residues 45 and 47 with serine residues in that common B part, oligomerization of the A2-Pancortins was highly disturbed.
Subject(s)
Brain/metabolism , Extracellular Matrix Proteins/chemistry , Extracellular Matrix Proteins/metabolism , Gene Expression Regulation, Developmental/physiology , Glycoproteins/chemistry , Glycoproteins/metabolism , Age Factors , Animals , Animals, Newborn , Blotting, Western/methods , Brain/embryology , Brain/growth & development , Cell Line , Embryo, Mammalian , Extracellular Matrix Proteins/classification , Extracellular Matrix Proteins/genetics , Glycoproteins/classification , Glycoproteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Immunoprecipitation/methods , In Situ Hybridization/methods , Mice , Mice, Inbred ICR , Molecular Sequence Data , Mutagenesis/physiology , Peptide Fragments/metabolism , Protein Structure, Tertiary/physiology , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methodsABSTRACT
PSD-Zip45 (Homer 1c) and PSD-95 are postsynaptic density (PSD) proteins containing distinct protein-interacting motifs. Green fluorescent protein (GFP)-tagged PSD-Zip45 and PSD-95 molecules were targeted to the PSD in hippocampal neurons. We analyzed dynamic behavior of these GFP-tagged PSD proteins by using time-lapse confocal microscopy. In contrast to the less dynamic properties of PSD-95, PSD-Zip45 showed rapid redistribution and a higher steady-state turnover rate. Differential stimulation protocols were found to alter the direction of PSD-Zip45 assembly-disassembly. Transient increases in intracellular Ca(2+) by voltage-dependent Ca(2+) channel activation induced PSD-Zip45 clustering. In contrast, NMDA receptor-dependent Ca(2+) influx resulted in the disassembly of PSD-Zip45 clusters. Thus, neuronal activity differentially redistributes a specific subset of PSD proteins, which are important for localization of both surface receptors and intracellular signaling complexes.
Subject(s)
Calcium Channels/metabolism , Carrier Proteins/metabolism , Neurons/metabolism , Neuropeptides/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synapses/metabolism , Animals , Cadmium/pharmacology , Calcium/metabolism , Carrier Proteins/genetics , Cells, Cultured , Cytoskeleton/metabolism , Disks Large Homolog 4 Protein , Excitatory Amino Acid Antagonists/pharmacology , Green Fluorescent Proteins , Guanylate Kinases , Hippocampus , Homer Scaffolding Proteins , Intracellular Fluid/metabolism , Intracellular Signaling Peptides and Proteins , Luminescent Proteins/genetics , Macromolecular Substances , Membrane Proteins , Mice , Microscopy, Confocal , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Neuropeptides/genetics , Protein Transport/physiology , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/physiology , Stimulation, ChemicalABSTRACT
We have identified a novel mouse gene named pancortin that is expressed dominantly in the mature cerebral cortex. This gene produces four different species of proteins, Pancortin-1-4, sharing a common region in the middle of their structure with two variations at the N-terminal (A1 or A2 part) and C-terminal (C1 or C2 part) sides, respectively. In the present study, we showed that expression of mRNAs for A2-Pancortins (Pancortin species that contain the A2 part, i.e., Pancortin-3 and -4) is more dominant than that of mRNAs for A1-Pancortins (Pancortin species that contain the A1 part, i.e., Pancortin-1 and -2) in the prenatal mouse cerebral neocortex. Using western blot analysis, we found that substantial amounts of both A2-Pancortins were present in the prenatal cerebral neocortex and P19 cells after inducing neuronal differentiation. A2-Pancortins were still present in the cerebral neocortex of the adult, although their mRNAs were hardly detected. In contrast, the amount of A1-Pancortins did not increase after the third postnatal week in spite of their intense gene expression. Furthermore, we showed that recombinant Pancortin-3, one of the A2-Pancortins, was a secreted protein, in contrast to Pancortin-1 (one of the A1-Pancortins). These results suggest that A2-Pancortins are extracellular proteins essential for neuronal differentiation and that their molecular behavior is distinct from that of A1-Pancortins.
Subject(s)
Gene Expression , Glycoproteins/analysis , Glycoproteins/genetics , Neocortex/growth & development , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/genetics , Alkaline Phosphatase/analysis , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Cell Differentiation , Cell Line , Extracellular Matrix Proteins , Female , Glycoproteins/chemistry , In Situ Hybridization , Mice , Mice, Inbred ICR , Molecular Sequence Data , Neocortex/chemistry , Neocortex/embryology , Nerve Tissue Proteins/chemistry , Neurons/chemistry , Neurons/physiology , Peptide Fragments/chemistry , Peptide Fragments/immunology , RNA, Messenger/analysis , Rabbits , TransfectionABSTRACT
We investigated the effect of nitric oxide (NO) on iron-induced neuronal damage. Incubation of PC12 cells after the addition of FeCl2 induced rapid increases (within 1 hr) in lipid peroxidation and a concentration (0.1-2 mM)-dependent decrease in cell viability at 48 hr, both of which were blocked by deferoxamine and 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazine-3-o ne hydrochloride (MCLA) (a superoxide scavenger) but not by mannitol (a hydroxyl radical scavenger). Iron-induced cytotoxicity was also antagonized by superoxide dismutase with catalase. On the other hand, the NO donors S-nitroso-N-acetylpenicillamine (SNAP), 3-¿(+/-)-(E)-ethyl-2'-[(E)-hydroxylamino]-5-nitro-3-hexenecarbo moyl¿-pyridine (NOR-4), and 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) decreased cell viability 48 hr after addition without increasing lipid peroxidation. However, when added with 1 mM FeCl2, NO donors including NOC-18, SNAP and NOR-4 (0.1-1 mM) inhibited lipid peroxidation in a concentration-dependent manner and suppressed cell death at lower concentrations. Addition of MCLA and NOC-18 also suppressed decreases in iron-induced [3H]thymidine incorporation. In rat brain homogenate, NOC-18 and SNAP both suppressed iron-induced lipid peroxidation. These findings suggest that NO has a dual effect on neuronal viability and can act as an antioxidant which protects neurons from iron-induced damage.
