anisomycin and 3-4-dihydroxyphenylglycol

Deregulation of synaptic plasticity may contribute to the pathogenesis of developmental cognitive disorders. In particular, exaggerated mGluR-dependent LTD is featured in fragile X syndrome, but the mechanisms that regulate mGluR-LTD remain incompletely understood. We report that conditional knockout of Cdh1, the key regulatory subunit of the ubiquitin ligase Cdh1-anaphase-promoting complex (Cdh1-APC), profoundly impairs mGluR-LTD in the hippocampus. Mechanistically, we find that Cdh1-APC operates in the cytoplasm to drive mGluR-LTD. We also identify the fragile X syndrome protein FMRP as a substrate of Cdh1-APC. Endogenous Cdh1-APC forms a complex with endogenous FMRP, and knockout of Cdh1 impairs mGluR-induced ubiquitination and degradation of FMRP in the hippocampus. Knockout of FMRP suppresses, and expression of an FMRP mutant protein that fails to interact with Cdh1 phenocopies, the Cdh1 knockout phenotype of impaired mGluR-LTD. These findings define Cdh1-APC and FMRP as components of a novel ubiquitin signaling pathway that regulates mGluR-LTD in the brain.

Topics: Animals; Animals, Newborn; Anisomycin; Cdh1 Proteins; Female; Fragile X Mental Retardation Protein; HEK293 Cells; Hippocampus; Humans; In Vitro Techniques; Long-Term Potentiation; Male; Methoxyhydroxyphenylglycol; Mice, Transgenic; Protein Synthesis Inhibitors; Receptors, Metabotropic Glutamate; Signal Transduction; Subcellular Fractions; Synapses; Ubiquitin

We have previously shown that selective activation of group I metabotropic glutamate receptors (mGluRs) results in long-lasting enhancement of synchronized network activity in the hippocampal slice. Data herein suggest that activation of group I mGluRs need not result in this potentially epileptogenic effect. (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (ACPD), a nonselective mGluR agonist, elicits ictaform bursts identical in appearance to those induced by selective agonists, but ACPD-induced bursts do not persist following removal of the agent. Like the bursts induced by selective agonist, the ACPD bursts are blocked with group I mGluR antagonists and are not dependent on activation of either N-methyl-D-aspartate (NMDA) receptors or protein kinase C. However, they differ from the persistent bursts in that they do not require active protein synthesis and they are not suppressed with L-cysteine sulfinic acid, an agonist at a phospholipase D-coupled metabotropic receptor. These novel findings provide evidence that group I mGluR-induced epileptogenesis may be preventable.

Topics: Analysis of Variance; Animals; Anisomycin; Benzophenanthridines; Dioxolanes; Drug Synergism; GABA Antagonists; Guinea Pigs; Hippocampus; In Vitro Techniques; Methoxyhydroxyphenylglycol; Phospholipase D; Picrotoxin; Protein Synthesis Inhibitors; Purines; Receptors, Metabotropic Glutamate

Fragile X syndrome (FXS) is the most common form of inherited mental retardation. The syndrome results from the absence of the fragile X mental retardation protein (FMRP), which is encoded by the fragile X mental retardation 1 (FMR1) gene. FMR1 and its two paralogs, fragile X-related genes 1 and 2 (FXR1 and -2), form the Fmr1 gene family. Here, we examined long-lasting synaptic plasticity in Fmr1 knockout, Fxr2 knockout, and Fmr1/Fxr2 double knockout mice. We found that metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in the hippocampus was affected in Fmr1 knockout, Fxr2 knockout, and Fmr1/Fxr2 double knockout mice at young ages (4-6 wk old). In addition, Fmr1/Fxr2 double knockout mice showed significant deficiencies relative to either Fmr1 or Fxr2 knockout mice in baseline synaptic transmission and short-term presynaptic plasticity, suggesting FMRP and FXR2P may contribute in a cooperative manner to pathways regulating presynaptic plasticity. However, compared with wild-type littermates, late-phase long-term potentiation (L-LTP) was unaltered in all knockout mice at 4-6 mo of age. Interestingly, although Fmr1/Fxr2 double knockout mice exhibited a more robust enhancement in mGluR-LTD compared with that in Fmr1 knockout mice, Fxr2 knockout mice exhibited reduced mGluR-LTD. Furthermore, unlike Fmr1 knockout mice, mGluR-LTD in Fxr2 knockout mice required new protein synthesis, whereas mGluR-LTD in Fmr1/Fxr2 double knockout mice was partially dependent on protein synthesis. These results indicated that both FMRP and FXR2P function in synaptic plasticity and that they likely operate in related but independent pathways.

Topics: Analysis of Variance; Animals; Anisomycin; Biophysics; Electric Stimulation; Excitatory Postsynaptic Potentials; Female; Fragile X Mental Retardation Protein; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Inbred C57BL; Mice, Knockout; Neural Pathways; Patch-Clamp Techniques; Protein Synthesis Inhibitors; Receptors, Metabotropic Glutamate; RNA-Binding Proteins; Synapses

Fragile X syndrome (FXS), a form of human mental retardation, is caused by loss of function mutations in the fragile X mental retardation gene (FMR1). The protein product of FMR1, fragile X mental retardation protein (FMRP) is an RNA-binding protein and may function as a translational suppressor. Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) in hippocampal area CA1 is a form of synaptic plasticity that relies on dendritic protein synthesis. mGluR-LTD is enhanced in the mouse model of FXS, Fmr1 knockout (KO) mice, suggesting that FMRP negatively regulates translation of proteins required for LTD. Here we examine the synaptic and cellular mechanisms of mGluR-LTD in KO mice and find that mGluR-LTD no longer requires new protein synthesis, in contrast to wild-type (WT) mice. We further show that mGluR-LTD in KO and WT mice is associated with decreases in AMPA receptor (AMPAR) surface expression, indicating a similar postsynaptic expression mechanism. However, like LTD, mGluR-induced decreases in AMPAR surface expression in KO mice persist in protein synthesis inhibitors. These results are consistent with recent findings of elevated protein synthesis rates and synaptic protein levels in Fmr1 KO mice and suggest that these elevated levels of synaptic proteins are available to increase the persistence of LTD without de novo protein synthesis.

Topics: 2-Amino-5-phosphonovalerate; Animals; Anisomycin; Blotting, Western; Disease Models, Animal; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Fragile X Mental Retardation Protein; Fragile X Syndrome; Hippocampus; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Neurons; Protein Biosynthesis; Protein Synthesis Inhibitors; Receptors, Metabotropic Glutamate

Genetic deletion of fragile X mental retardation protein (FMRP) has been shown to enhance mGluR-dependent long-term depression (LTD). Herein, we demonstrate that mGluR-LTD induces a transient, translation-dependent increase in FMRP that is rapidly degraded by the ubiquitin-proteasome pathway. Moreover, proteasome inhibitors abolished mGluR-LTD, and LTD was absent in mice that overexpress human FMRP. Neither translation nor proteasome inhibitors blocked the augmentation of mGluR-LTD in FMRP-deficient mice. In addition, mGluR-LTD is associated with rapid increases in the protein levels of FMRP target mRNAs in wild-type mice. Interestingly, the basal levels of these proteins were elevated and their synthesis was improperly regulated during mGluR-LTD in FMRP-deficient mice. Our findings indicate that hippocampal mGluR-LTD requires the rapid synthesis and degradation of FMRP and that mGluR-LTD triggers the synthesis of FMRP binding mRNAs. These findings indicate that the translation, ubiquitination, and proteolysis of FMRP functions as a dynamic regulatory system for controlling synaptic plasticity.

Topics: Animals; Animals, Newborn; Anisomycin; Benzoates; Blotting, Western; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Fluorescent Antibody Technique; Fragile X Mental Retardation Protein; Glycine; In Vitro Techniques; Leupeptins; Long-Term Synaptic Depression; Male; Methoxyhydroxyphenylglycol; Mice; Mice, Knockout; Microtubule-Associated Proteins; Models, Biological; Proteasome Endopeptidase Complex; Protein Biosynthesis; Protein Synthesis Inhibitors; Pyridines; Receptors, Metabotropic Glutamate; RNA, Messenger; Signal Transduction

The presynaptic and postsynaptic properties of synapses change over the course of postnatal development. Therefore, synaptic plasticity mechanisms would be expected to adapt to these changes to facilitate alterations of synaptic strength throughout ontogeny. Here, we identified developmental changes in long-term depression (LTD) mediated by group 1 metabotropic glutamate receptors (mGluRs) and dendritic protein synthesis in hippocampal CA1 slices (mGluR-LTD). In slices prepared from adolescent rats [postnatal day 21 (P21) to P35], mGluR activation induces LTD and a long-term decrease in AMPA receptor (AMPAR) surface expression, both of which require protein synthesis. In neonatal animals (P8-P15), mGluR-LTD is independent of protein synthesis and is not associated with changes in the surface expression of AMPARs. Instead, mGluR-LTD at neonatal synapses results in large decreases in presynaptic function, measured by changes in paired-pulse facilitation and the rate of blockade by the use-dependent NMDA receptor blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate. Conversely, mGluR-LTD at mature synapses results in little or no change in presynaptic function, suggesting a postsynaptic mechanism of expression. The developmental switch in the synaptic mechanisms of LTD would differentially affect synapse dynamics and perhaps information processing over the course of postnatal development.

Topics: Age Factors; Amino Acids; Animals; Animals, Newborn; Anisomycin; Benzoxazines; Cycloheximide; Dizocilpine Maleate; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Antagonists; Hippocampus; In Vitro Techniques; Long-Term Synaptic Depression; Methoxyhydroxyphenylglycol; Morpholines; Naphthalenes; Neurons; Protein Synthesis Inhibitors; Pyrazoles; Rats; Rats, Long-Evans; Reaction Time; Receptor, Cannabinoid, CB1; Receptors, Metabotropic Glutamate; Synapses; Xanthenes

Hippocampal long-term depression (LTD) comprises an activity-dependent weakening of synaptic strength. In this study we compared persistent LTD induced by the group I mGluR agonist, DHPG, or the group III mGluR agonist, AP4, in the dentate gyrus of freely moving rats. The role of protein translation, using the translation inhibitors, anisomycin and emetine, was also investigated. Potentials were evoked from medial perforant path-dentate gyrus granule cell synapses of male Wistar rats by means of chronically implanted electrodes. Immediately after intracerebral (ventricular) application of DHPG or AP4 robust LTD (>24 h) occurred. Paired-pulse analysis during LTD, and application of mGluR antagonists after stabilisation of depression, supported that LTD genuinely occurred and that the depression was not a consequence of persistence of the agonists at the synapse. Application of a protein synthesis inhibitor 2 h prior to DHPG injection inhibited the expression of LTD (from ca. 6 h post-injection) but did not affect LTD induced by AP4. These data highlight differences in chemical LTD elicited by group I and group III mGluRs. Whereas AP4-induced LTD may arise as a result of modulation of presynaptic glutamate release mechanisms, the protein synthesis dependency of DHPG-induced LTD suggests an additional postsynaptic expression mechanism for this phenomenon.

Topics: Aminobutyrates; Analysis of Variance; Animals; Anisomycin; Dentate Gyrus; Drug Interactions; Electric Stimulation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Long-Term Synaptic Depression; Male; Methoxyhydroxyphenylglycol; Protein Synthesis Inhibitors; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Wakefulness

A hippocampal pyramidal neuron receives more than 10(4) excitatory glutamatergic synapses. Many of these synapses contain the molecular machinery for messenger RNA translation, suggesting that the protein complement (and thus function) of each synapse can be regulated on the basis of activity. Here, local postsynaptic protein synthesis, triggered by synaptic activation of metabotropic glutamate receptors, was found to modify synaptic transmission within minutes.

Topics: Amino Acids; Animals; Anisomycin; Dendrites; Electric Stimulation; Excitatory Amino Acid Antagonists; Hippocampus; Methoxyhydroxyphenylglycol; Nerve Tissue Proteins; Neural Inhibition; Protein Biosynthesis; Protein Synthesis Inhibitors; Rats; Receptors, Metabotropic Glutamate; RNA, Messenger; Synaptic Transmission; Xanthenes