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1.
Front Neurosci ; 15: 712261, 2021.
Article in English | MEDLINE | ID: mdl-34616273

ABSTRACT

Perinatal exposure to Bisphenol A (BPA) at a very low dose may modulate the development of synapses of the hippocampus during growth to adulthood. Here, we demonstrate that perinatal exposure to 30 µg BPA/kg per mother's body weight/day significantly altered the dendritic spines of the grownup rat hippocampus. The density of the spine was analyzed by imaging of Lucifer Yellow-injected CA1 glutamatergic neurons in adult hippocampal slices. In offspring 3-month male hippocampus, the total spine density was significantly decreased by BPA exposure from 2.26 spines/µm (control, no BPA exposure) to 1.96 spines/µm (BPA exposure). BPA exposure considerably changed the normal 4-day estrous cycle of offspring 3-month females, resulting in a 4∼5 day estrous cycle with 2-day estrus stages in most of the subjects. In the offspring 3-month female hippocampus, the total spine density was significantly increased by BPA exposure at estrus stage from 2.04 spines/µm (control) to 2.25 spines/µm (BPA exposure). On the other hand, the total spine density at the proestrus stage was moderately decreased from 2.33 spines/µm (control) to 2.19 spines/µm (BPA exposure). Thus, after the perinatal exposure to BPA, the total spine density in males became lower than that in females. Concerning the BPA effect on the morphology of spines, the large-head spine was significantly changed with its significant decrease in males and moderate change in females.

2.
Methods Mol Biol ; 1555: 117-162, 2017.
Article in English | MEDLINE | ID: mdl-28092031

ABSTRACT

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.


Subject(s)
Gene Expression , Proteins/chemistry , Proteins/genetics , Recombinant Fusion Proteins , src Homology Domains , Amino Acid Sequence , Chromatography, Affinity , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Order , Genetic Vectors/genetics , Humans , Protein Binding , Protein Interaction Domains and Motifs , Proteins/isolation & purification
3.
Methods Mol Biol ; 1555: 183-198, 2017.
Article in English | MEDLINE | ID: mdl-28092034

ABSTRACT

Recombinant proteins expressed in bacteria are sometimes insoluble, aggregated, and incorrectly folded. For those Src homology 2 (SH2) domains that are insoluble in bacteria, baculovirus-insect cell expression systems can be an alternative to produce soluble and functionally active proteins. We describe a protocol for cloning and purification of GST-tagged SH2 domains using the Bac-to-Bac baculovirus expression system.


Subject(s)
Baculoviridae/genetics , Gene Expression , Genetic Vectors/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , src Homology Domains , Animals , Cloning, Molecular , Gene Order , Humans , Recombinant Fusion Proteins/chemistry , Sf9 Cells , Transfection , Workflow
4.
Methods Mol Biol ; 1555: 199-223, 2017.
Article in English | MEDLINE | ID: mdl-28092035

ABSTRACT

Recombinant modular protein domains have been a convenient proteomics tool for deciphering protein-protein interactions and elucidating the role of protein modifications in cell signaling. To obtain reliable experimental data, these protein domain probes require sufficient specificity and sensitivity. Since naturally evolved protein domains do not always have optimal biochemical characteristics for in vitro assays, functional alterations such as improved affinity are sometimes needed. In this chapter, we describe preparation of loss-of-function and concatenated (tandem) SH2 domains that should be widely applicable to both high- and low-throughput phosphoproteomics studies.


Subject(s)
Proteins/chemistry , Proteins/metabolism , src Homology Domains , Cloning, Molecular , Gene Expression , Humans , Loss of Function Mutation , Mutagenesis, Site-Directed , Plasmids/genetics , Protein Interaction Domains and Motifs , Proteins/genetics , Recombinant Fusion Proteins , Structure-Activity Relationship , Tandem Repeat Sequences , src Homology Domains/genetics
5.
Elife ; 5: e11835, 2016 Apr 12.
Article in English | MEDLINE | ID: mdl-27071344

ABSTRACT

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.


Subject(s)
Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction , src Homology Domains , Blotting, Far-Western , Cell Line, Tumor , Cell Membrane/metabolism , Epidermal Growth Factor/metabolism , Epithelial Cells/drug effects , Epithelial Cells/physiology , Humans , Mass Spectrometry , Optical Imaging , Protein Binding
6.
BMC Biotechnol ; 15: 60, 2015 Jun 26.
Article in English | MEDLINE | ID: mdl-26112401

ABSTRACT

BACKGROUND: There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. RESULTS: We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 µl lysate without requiring phospho-enrichment. CONCLUSIONS: We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein interaction assays. This feature along with short assay runtime makes this method a useful platform for the development of high throughput assays to determine modular domain-ligand interactions which could have wide-ranging applications in both basic and translational cancer research.


Subject(s)
Binding Sites/genetics , Phosphotyrosine/metabolism , Protein Interaction Domains and Motifs/genetics , Protein Interaction Mapping/methods , Protein-Tyrosine Kinases/metabolism , Real-Time Polymerase Chain Reaction/methods , src Homology Domains/genetics , Antibodies/immunology , ErbB Receptors/immunology
7.
Brain Res ; 1621: 133-46, 2015 Sep 24.
Article in English | MEDLINE | ID: mdl-25498865

ABSTRACT

Rapid modulation of hippocampal synaptic plasticity through synaptic estrogen receptors is an essential topic. We analyzed estradiol-induced modulation of CA1 dendritic spines using adult male ERαKO and ERßKO mice. A 2h treatment of estradiol particularly increased the density of middle-head spines (diameter 0.3-0.4 µm) in wild type mouse hippocampal slices. The enhancement of spinogenesis was completely suppressed by MAP kinase inhibitor. Estradiol-induced increase in middle-head spines was observed in ERßKO mice (which express ERα), but not in ERαKO, indicating that ERα is necessary for the spinogenesis. Direct observation of the dynamic estradiol-induced enhancing effect on rapid spinogenesis was performed using time-lapse imaging of spines in hippocampal live slices from yellow fluorescent protein expressed mice. Both appearance and disappearance of spines occurred, and the number of newly appeared spines was significantly greater than that of disappeared spines, resulting in the net increase of the spine density within 2h. As another type of synaptic modulation, we observed that estradiol rapidly enhanced N-methyl-D-aspartate (NMDA)-induced long-term depression (LTD) in CA1 of the wild type mouse hippocampus. In contrast, estradiol did not enhance NMDA-LTD in ERαKO mice, indicating the involvement of ERα in the estrogen signaling. This article is part of a Special Issue entitled SI: Brain and Memory.


Subject(s)
CA1 Region, Hippocampal/physiology , Dendritic Spines/physiology , Estradiol/physiology , Estrogen Receptor alpha/physiology , Estrogen Receptor beta/physiology , Long-Term Synaptic Depression , Animals , CA1 Region, Hippocampal/cytology , CA1 Region, Hippocampal/metabolism , Estradiol/administration & dosage , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/genetics , Estrogen Receptor beta/metabolism , Long-Term Synaptic Depression/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout
8.
Neuro Endocrinol Lett ; 34(2): 129-34, 2013.
Article in English | MEDLINE | ID: mdl-23645310

ABSTRACT

OBJECTIVE: Low dose exposure to endocrine disrupters (environmental chemicals) may induce hormone-like effects on wildlife and humans. bisphenol A (BPA) might disturb the neuronal signaling regulated by endogenous estrogens. We investigated the rapid modulation effects of 10nM BPA, a typical endocrine disruptor, on long-term depression (LTD) of adult rat hippocampal slices. METHOD: LTD was induced by a transient perfusion of 30 µM NMDA for 3 min. And measured with multielectrode probes. RESULTS: A 30 min perfusion of 10 nM BPA rapidly enhanced LTD in CA1, however, BPA suppressed LTD in dentate gyrus (DG). An ERRγ antagonist, 4-OH-tamoxifen, suppressed LTD in CA1 and DG. Inhibitor of estrogen receptor ICI 182,780 did not disturb BPA effects. On the other hand, tributyltin (TBT), another endocrine disruptor, did not have any effect on LTD in CA1 and DG. CONCLUSION: ERRγ, but not estrogen receptors, is a high affinity BPA receptor in LTD processes, since the effect of BPA on LTD was suppressed by an ERRγ antagonist. A possible mechanisms of BPA-induced enhancement of LTD could be described with ERRγ, MAPK activation and phosphorylation of MMDA receptors.


Subject(s)
Benzhydryl Compounds/pharmacology , Endocrine Disruptors/pharmacology , Estrogens, Non-Steroidal/pharmacology , Hippocampus/drug effects , Long-Term Synaptic Depression/drug effects , Phenols/pharmacology , Receptors, Estrogen/drug effects , Animals , Electrodes , Hippocampus/metabolism , Long-Term Synaptic Depression/physiology , Male , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar
9.
Proc Natl Acad Sci U S A ; 109(35): 14024-9, 2012 Aug 28.
Article in English | MEDLINE | ID: mdl-22886086

ABSTRACT

Receptor tyrosine kinases (RTKs) control a host of biological functions by phosphorylating tyrosine residues of intracellular proteins upon extracellular ligand binding. The phosphotyrosines (p-Tyr) then recruit a subset of ∼100 Src homology 2 (SH2) domain-containing proteins to the cell membrane. The in vivo kinetics of this process are not well understood. Here we use total internal reflection (TIR) microscopy and single-molecule imaging to monitor interactions between SH2 modules and p-Tyr sites near the cell membrane. We found that the dwell time of SH2 modules within the TIR illumination field is significantly longer than predictions based on chemical dissociation rate constants, suggesting that SH2 modules quickly rebind to nearby p-Tyr sites after dissociation. We also found that, consistent with the rebinding model, the effective diffusion constant is negatively correlated with the respective dwell time for different SH2 domains and the dwell time is positively correlated with the local density of RTK phosphorylation. These results suggest a mechanism whereby signal output can be regulated through the spatial organization of multiple binding sites, which will prompt reevaluation of many aspects of RTK signaling, such as signaling specificity, mechanisms of spatial control, and noise suppression.


Subject(s)
Cell Membrane/metabolism , ErbB Receptors/chemistry , ErbB Receptors/metabolism , Models, Chemical , src Homology Domains/physiology , Binding Sites/physiology , Carcinoma, Squamous Cell , Cell Line, Tumor , Diffusion , Humans , Kinetics , Lung Neoplasms , Microscopy/methods , Phosphorylation/physiology , Phosphotyrosine/metabolism , Protein Structure, Tertiary/physiology , Signal Transduction/physiology , src Homology Domains/genetics
10.
FEBS Lett ; 586(17): 2586-96, 2012 Aug 14.
Article in English | MEDLINE | ID: mdl-22710164

ABSTRACT

The ability of modular protein domains to independently fold and bind short peptide ligands both in vivo and in vitro has allowed a significant number of protein-protein interaction studies to take advantage of them as affinity and detection reagents. Here, we refer to modular domain based proteomics as "domainomics" to draw attention to the potential of using domains and their motifs as tools in proteomics. In this review we describe core concepts of domainomics, established and emerging technologies, and recent studies by functional category. Accumulation of domain-motif binding data should ultimately provide the foundation for domain-specific interactomes, which will likely reveal the underlying substructure of protein networks as well as the selectivity and plasticity of signal transduction.


Subject(s)
Proteomics/methods , Amino Acid Motifs , Animals , Binding Sites , Computational Biology/methods , Cross-Linking Reagents/chemistry , Genome , Humans , Ligands , Peptide Library , Proline/chemistry , Protein Interaction Mapping/methods , Protein Structure, Tertiary , Signal Transduction
11.
Mol Cell Endocrinol ; 351(2): 317-25, 2012 Apr 04.
Article in English | MEDLINE | ID: mdl-22281313

ABSTRACT

We demonstrated the rapid effects of 10nM bisphenol A (BPA) on the spinogenesis of adult rat hippocampal slices. The density of spines was analyzed by imaging Lucifer Yellow-injected CA1 neurons in slices. Not only the total spine density but also the head diameter distribution of spine was quantitatively analyzed. Spinogenesis was significantly enhanced by BPA within 2h. In particular, the density of middle-head spine (with head diameter of 0.4-0.5µm) was significantly increased. Hydroxytamoxifen, an antagonist of both estrogen-related receptor gamma (ERRγ) and estrogen receptors (ERα/ERß), blocked the BPA-induced enhancement of the spine density. However, ICI 182,780, an antagonist of ERα/ERß, did not suppress the BPA effects. Therefore, ERRγ is deduced to be a high affinity receptor of BPA, responsible for modulation of spinogenesis. The BPA-induced enhancement of spinogenesis was also suppressed by MAP kinase inhibitor, PD98059, and the blocker of NMDA receptors, MK-801. Washout of BPA for additional 2h after 2h BPA treatment abolished the BPA-induced enhancement of spinogenesis, suggesting that the BPA effect was reversible. ERRγ was localized at synapses as well as cell bodies of principal neurons. ERRγ at synapses may contribute to the observed rapid effect. The level of BPA in the hippocampal slices was determined by mass-spectrometric analysis.


Subject(s)
Dendritic Spines/drug effects , Dendritic Spines/metabolism , Hippocampus/drug effects , Neurons/drug effects , Phenols/pharmacology , Animals , Benzhydryl Compounds , Dendritic Spines/ultrastructure , Dizocilpine Maleate/pharmacology , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor beta/antagonists & inhibitors , Flavonoids/pharmacology , Fulvestrant , Hippocampus/ultrastructure , Humans , Male , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Neurons/metabolism , Neurons/ultrastructure , Phenols/administration & dosage , Rats , Rats, Wistar , Receptors, Estrogen/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology
12.
J Steroid Biochem Mol Biol ; 131(1-2): 37-51, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22075082

ABSTRACT

The hippocampus synthesizes estrogen and androgen in addition to the circulating sex steroids. Synaptic modulation by hippocampus-derived estrogen or androgen is essential to maintain healthy memory processes. Rapid actions (1-2h) of 17ß-estradiol (17ß-E2) occur via synapse-localized receptors (ERα or ERß), while slow genomic E2 actions (6-48h) occur via classical nuclear receptors (ERα or ERß). The long-term potentiation (LTP), induced by strong tetanus or theta-burst stimulation, is not further enhanced by E2 perfusion in adult rats. Interestingly, E2 perfusion can rescue corticosterone (stress hormone)-induced suppression of LTP. The long-term depression is modulated rapidly by E2 perfusion. Elevation of the E2 concentration changes rapidly the density and head structure of spines in neurons. ERα, but not ERß, drives this enhancement of spinogenesis. Kinase networks are involved downstream of ERα. Testosterone (T) or dihydrotestosterone (DHT) also rapidly modulates spinogenesis. Newly developed Spiso-3D mathematical analysis is used to distinguish these complex effects by sex steroids and kinases. It has been doubted that the level of hippocampus-derived estrogen and androgen may not be high enough to modulate synaptic plasticity. Determination of the accurate concentration of E2, T or DHT in the hippocampus is enabled by mass-spectrometric analysis in combination with new steroid-derivatization methods. The E2 level in the hippocampus is approximately 8nM for the male and 0.5-2nM for the female, which is much higher than that in circulation. The level of T and DHT is also higher than that in circulation. Taken together, hippocampus-derived E2, T, and DHT play a major role in modulation of synaptic plasticity.


Subject(s)
Estradiol/physiology , Hippocampus/metabolism , Neuronal Plasticity , Testosterone/physiology , Animals , Dendritic Spines/drug effects , Dendritic Spines/physiology , Dihydrotestosterone/metabolism , Estrogen Receptor alpha/physiology , Estrogen Receptor beta/physiology , Female , Long-Term Potentiation/drug effects , Long-Term Synaptic Depression/drug effects , Male , Memory/drug effects , Memory/physiology , Neuronal Plasticity/drug effects , Rats
13.
Horm Mol Biol Clin Investig ; 7(2): 361-75, 2011 Feb 01.
Article in English | MEDLINE | ID: mdl-25961274

ABSTRACT

Estradiol is synthesized from cholesterol in hippocampal neurons of adult rats by cytochrome P450 and hydroxysteroid dehydrogenase enzymes. These enzymes are expressed in the glutamatergic neurons of the hippocampus. Surprisingly, the concentration of estradiol and androgen in the hippocampus is significantly higher than that in circulation. Locally synthesized estradiol rapidly and potently modulates synaptic plasticity within the hippocampus. E2 rapidly potentiates long-term depression and induces spinogenesis through synaptic estrogen receptors and kinases. The rapid effects of estradiol are followed by slow genomic effects mediated by both estrogen receptors located at the synapse and nucleus, modulating long-term potentiation and promoting the formation of new functional synaptic contacts. Age-related changes in hippocampally derived estradiol synthesis and distribution of estrogen receptors may alter synaptic plasticity, and could potentially contribute to age-related cognitive decline. Understanding factors which regulate hippocampal estradiol synthesis could lead to the identification of alternatives to conventional hormone therapy to protect against age-related cognitive decline.

14.
Article in English | MEDLINE | ID: mdl-22701110

ABSTRACT

Sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. Accumulating evidence shows that hippocampal neurons synthesize both estrogen and androgen. Recently, we also revealed the hippocampal synthesis of corticosteroids. The accurate concentrations of these hippocampus-synthesized steroids are determined by liquid chromatography-tandem mass-spectrometry in combination with novel derivatization. The hippocampal levels of 17ß-estradiol (E2), testosterone (T), dihydrotestosterone (DHT), and corticosterone (CORT), are 5-15 nM, and these levels are sufficient to modulate synaptic plasticity. Hippocampal E2 modulates memory-related synaptic plasticity not only slowly/genomically but also rapidly/non-genomically. Slow actions of E2 occur via classical nuclear receptors (ERα or ERß), while rapid E2 actions occur via synapse-localized or extranuclear ERα or ERß. Nanomolar concentrations of E2 change rapidly the density and morphology of spines in hippocampal neurons. ERα, but not ERß, drives this enhancement/suppression of spinogenesis in adult animals. Nanomolar concentrations of androgens (T and DHT) and CORT also increase the spine density. Kinase networks are involved downstream of ERα and androgen receptor. Newly developed Spiso-3D mathematical analysis is useful to distinguish these complex effects by sex steroids and kinases. Significant advance has been achieved in investigations of rapid modulation by E2 of the long-term depression or the long-term potentiation.

15.
FEBS Lett ; 584(15): 3279-86, 2010 Aug 04.
Article in English | MEDLINE | ID: mdl-20598684

ABSTRACT

Macropinocytosis is regulated by Abl kinase via an unknown mechanism. We previously demonstrated that Abl kinase activity is, itself, regulated by Abi1 subsequent to Abl kinase phosphorylation of Abi1 tyrosine 213 (pY213) [1]. Here we show that blocking phosphorylation of Y213 abrogated the ability of Abl to regulate macropinocytosis, implicating Abi1 pY213 as a key regulator of macropinocytosis. Results from screening the human SH2 domain library and mapping the interaction site between Abi1 and the p85 regulatory domain of PI-3 kinase, coupled with data from cells transfected with loss-of-function p85 mutants, support the hypothesis that macropinocytosis is regulated by interactions between Abi1 pY213 and the C-terminal SH2 domain of p85-thereby linking Abl kinase signaling to p85-dependent regulation of macropinocytosis.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cytoskeletal Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphotyrosine/metabolism , Pinocytosis , Proto-Oncogene Proteins c-abl/metabolism , Signal Transduction , Amino Acid Substitution/genetics , Cell Line, Tumor , Cyclic AMP/analogs & derivatives , Cyclic AMP/metabolism , Humans , Male , Mutation/genetics , Phosphatidylinositol 3-Kinases/chemistry , Phosphopeptides/metabolism , Phosphorylation/drug effects , Pinocytosis/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Signal Transduction/drug effects
16.
Biochim Biophys Acta ; 1800(10): 1030-44, 2010 Oct.
Article in English | MEDLINE | ID: mdl-19909788

ABSTRACT

The hippocampus is a center for learning and memory as well as a target of Alzheimer's disease in aged humans. Synaptic modulation by estrogen is essential to understand the molecular mechanisms of estrogen replacement therapy. Because the local synthesis of estrogen occurs in the hippocampus of both sexes, in addition to the estrogen supply from the gonads, its functions are attracting much attention. Hippocampal estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly. Slow actions of 17ß-estradiol (17ß-E2) occur via classical nuclear receptors (ERα or ERß), while rapid E2 actions occur via synapse-localized ERα or ERß. Elevation or decrease of the E2 concentration changes rapidly the density and morphology of spines in CA1-CA3 neurons. ERα, but not ERß, drives this enhancement/suppression of spinogenesis. Kinase networks are involved downstream of ERα. The long-term depression but not the long-term potentiation is modulated rapidly by changes of E2 level. Determination of the E2 concentration in the hippocampus is enabled by mass-spectrometry in combination with derivatization methods. The E2 level in the hippocampus is as high as approx. 8 nM for the male and 0.5-2 nM for the female, which is much higher than that in circulation. Therefore, hippocampus-derived E2 plays a major role in modulation of synaptic plasticity. Many hippocampal slice experiments measure the restorative effects of E2 by supplementation of E2 to E2-depleted slices. Accordingly, isolated slice experiments can be used as in vitro models of in vivo estrogen replacement therapy for ovariectomized female animals with depleted circulating estrogen.


Subject(s)
Estradiol/metabolism , Estrogens/metabolism , Hippocampus/metabolism , Neuronal Plasticity/physiology , Neurons/metabolism , Synapses/metabolism , Animals , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Female , Hippocampus/cytology , Humans , Male , Neurons/cytology
17.
Mol Cell Endocrinol ; 290(1-2): 31-43, 2008 Aug 13.
Article in English | MEDLINE | ID: mdl-18541362

ABSTRACT

Estrogen and androgen are synthesized from cholesterol locally in hippocampal neurons of adult animals. These neurosteroids are synthesized by cytochrome P450s and hydroxysteroid dehydrogenases (HSDs) and 5alpha-reductase. The expression levels of enzymes are as low as 1/200-1/50,000 of those in endocrine organs, however these numbers are high enough for local synthesis. Localization of P450(17alpha), P450arom, 17beta-HSD and 5alpha-reductase is observed in principal glutamatergic neurons in CA1, CA3 and the dendate gyrus. Several nanomolar levels of estrogen and androgen are observed in the hippocampus. Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. Rapid action of 17beta-estradiol via membrane receptors is demonstrated for spinogenesis and long-term depression (LTD). The enhancement of LTD by 1-10nM estradiol occurs within 1 h. The density of spine is increased in CA1 pyramidal neurons within 2h after application of estradiol. The density of spine-like structure is, however, decreased by estradiol in CA3 pyramidal neurons. ERalpha, but not ERbeta, induces the same enhancement/suppression effects on both spinogenesis and LTD.


Subject(s)
Brain/metabolism , Estrogens/biosynthesis , Memory , Neuronal Plasticity , Synapses/metabolism , Animals , Brain/ultrastructure , Humans , Synapses/ultrastructure
18.
Brain Res Rev ; 57(2): 363-75, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17822775

ABSTRACT

Estrogen modulates memory-related synaptic plasticity not only slowly but also rapidly in the hippocampus. However, molecular mechanisms of the rapid action are yet largely unknown. We here describe rapid modulation of representative synaptic plasticity, i.e., long-term depression (LTD), long-term potentiation (LTP) and spinogenesis, by 17beta-estradiol, selective estrogen agonists as well as endocrine disrupters. The authors demonstrated that 1-10 nM estradiol induced rapid enhancement of LTD within 1 h in not only CA1 but also CA3 and dentate gyrus (DG). On the other hand, the modulation of LTP by estradiol was not statistically significant. The total density of spines was increased in CA1 pyramidal neurons, within 2 h after application of estradiol. The total density of thorns (postsynaptic spine-like structure) was, however, decreased by estradiol in CA3 pyramidal neurons. Both the increase of spines in CA1 and the decrease of thorns in CA3 were completely suppressed by Erk MAP kinase inhibitor. Only ERalpha agonist PPT induced the same enhancement/suppression effect as estradiol on both LTD and spinogenesis in CA1 and CA3. ERbeta agonist DPN induced completely different results. ERalpha localized in spines and presynapses of principal glutamatergic neurons in CA1, CA3 and DG. The same ERalpha was also located in nuclei and cytoplasm. Identification of ERalpha was successfully performed using purified RC-19 antibody. Non-purified ERalpha antisera, however, reacted significantly with unknown proteins, resulting in wrong immunostaining different from real ERalpha distribution. An issue of 'endocrine disrupters' (1-100 nM low dose of environmental chemicals), which are artificial xenoestrogenic or anti-xenoestrogenic substances, has emerged as a social and environmental problem. Endocrine disrupters were found to significantly modulate LTD and spinogenesis. Bisphenol A (BPA) and diethylstilbestrol (DES) enhanced LTD in CA1 and CA3. The total spine density was significantly increased by BPA and DES in CA1. Most probable receptors for BPA and DES may be Ralpha; however, other receptors might also be involved.


Subject(s)
Endocrine Disruptors/toxicity , Estrogens/pharmacology , Hippocampus/drug effects , Neuronal Plasticity/drug effects , Neurons/drug effects , Animals , Benzhydryl Compounds , Diethylstilbestrol/toxicity , Estrogen Receptor alpha/drug effects , Estrogen Receptor alpha/metabolism , Estrogens/metabolism , Estrogens, Non-Steroidal/toxicity , Hippocampus/physiology , Male , Neuronal Plasticity/physiology , Neurons/physiology , Phenols/toxicity , Rats
19.
Neuroscientist ; 13(4): 323-34, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17644764

ABSTRACT

It is believed that sex hormones are synthesized in the gonads and reach the brain via the blood circulation. In contrast with this view, the authors have demonstrated that sex hormones are also synthesized locally in the hippocampus and that these steroids act rapidly to modulate neuronal synaptic plasticity. The authors demonstrated that estrogens are locally synthesized from cholesterol through dehydroepiandrosterone and testosterone in adult hippocampal neurons. Significant expression of mRNA for P450(17alpha), P450arom, and other steroidogenic enzymes was demonstrated. Localization of P450(17alpha) and P450arom was observed in synapses of principal neurons. In contrast to the slow action of gonadal estradiol, hippocampal neuron-derived estradiol may act locally and rapidly within the neurons. For example, 1 to 10 nM estradiol rapidly enhances long-term depression (LTD). The density of thin spines is selectively increased within two hours upon application of estradiol in pyramidal neurons. Estrogen receptor ERalpha agonist has the same enhancing effect as estradiol on both LTD and spinogenesis. Localization of ERalpha in spines in addition to nuclei of principal neurons implies that synaptic ERalpha is responsible for rapid modulation of synaptic plasticity by endogenous estradiol. Activin A, a peptide sex hormone, may also play a role as a local endogenous modulator of synaptic plasticity.


Subject(s)
Gonadal Steroid Hormones/physiology , Hippocampus/cytology , Neuronal Plasticity/physiology , Neurons/physiology , Synapses/physiology , Animals , Gonadal Steroid Hormones/pharmacology , Humans , Neuronal Plasticity/drug effects , Neurons/drug effects , Synapses/drug effects
20.
J Neurochem ; 100(4): 950-67, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17266735

ABSTRACT

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17beta-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC-19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.


Subject(s)
Dendritic Spines/physiology , Hippocampus/cytology , Long-Term Synaptic Depression/physiology , Neurons/physiology , Receptors, Estrogen/physiology , 6-Cyano-7-nitroquinoxaline-2,3-dione/pharmacology , Animals , Dendritic Spines/drug effects , Dendritic Spines/ultrastructure , Enzyme Inhibitors/pharmacology , Estradiol/analogs & derivatives , Estradiol/pharmacology , Estrogen Antagonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Fulvestrant , In Vitro Techniques , Long-Term Synaptic Depression/drug effects , Male , Mice , Mice, Knockout , Microscopy, Confocal/methods , Microscopy, Immunoelectron/methods , Neurons/ultrastructure , Phenols , Pyrazoles/pharmacology , Rats , Rats, Wistar , Receptors, Estrogen/deficiency , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism
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