ABSTRACT
Silencing of a single gene, FMR1, is linked to a highly prevalent form of mental retardation, characterized by social and cognitive impairments, known as fragile X syndrome (FXS). The FMR1 gene encodes fragile X mental retardation protein (FMRP), which negatively regulates translation. Knockout of Fmr1 in mice results in enhanced long-term depression (LTD) induced by metabotropic glutamate receptor (mGluR) activation. Despite the evidence implicating FMRP in LTD, the role of FMRP in long-term potentiation (LTP) is less clear. Synaptic strength can be augmented heterosynaptically through the generation and sequestration of plasticity-related proteins, in a cell-wide manner. If heterosynaptic plasticity is altered in Fmr1 knockout (KO) mice, this may explain the cognitive deficits associated with FXS. We induced homosynaptic plasticity using the ß-adrenergic receptor (ß-AR) agonist, isoproterenol (ISO), which facilitated heterosynaptic LTP that was enhanced in Fmr1 KO mice relative to wild-type (WT) controls. To determine if enhanced heterosynaptic LTP in Fmr1 KO mouse hippocampus requires protein synthesis, we applied a translation inhibitor, emetine (EME). EME blocked homo- and heterosynaptic LTP in both genotypes. We also probed the roles of mTOR and ERK in boosting heterosynaptic LTP in Fmr1 KO mice. Although heterosynaptic LTP was blocked in both WT and KOs by inhibitors of mTOR and ERK, homosynaptic LTP was still enhanced following mTOR inhibition in slices from Fmr1 KO mice. Because mTOR will normally stimulate translation initiation, our results suggest that ß-AR stimulation paired with derepression of translation results in enhanced heterosynaptic plasticity.
Subject(s)
Excitatory Postsynaptic Potentials/genetics , Fragile X Mental Retardation Protein/metabolism , Hippocampus/cytology , Neuronal Plasticity/genetics , Neurons/physiology , Adrenergic beta-Agonists/pharmacology , Animals , Bicuculline/pharmacology , Biophysics , Dose-Response Relationship, Drug , Electric Stimulation/methods , Emetine/pharmacology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Flavonoids/pharmacology , Fragile X Mental Retardation Protein/genetics , GABA-A Receptor Antagonists/pharmacology , Hippocampus/physiology , Immunosuppressive Agents/pharmacology , In Vitro Techniques , Isoproterenol/pharmacology , Long-Term Potentiation/drug effects , Long-Term Potentiation/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuronal Plasticity/drug effects , Patch-Clamp Techniques , Pyridines/pharmacology , Sirolimus/pharmacology , Time Factors , rap GTP-Binding Proteins/pharmacologyABSTRACT
Low-density lipoprotein receptor-related protein (LRP-1) is an endocytic receptor for diverse proteins, including matrix metalloproteinase-9 (MMP-9), and a cell-signaling receptor. In the peripheral nervous system (PNS), LRP-1 is robustly expressed by Schwann cells only after injury. Herein, we demonstrate that MMP-9 activates extracellular-signal-regulated kinase (ERK1/2) and Akt in Schwann cells in culture. MMP-9 also promotes Schwann cell migration. These activities require LRP-1. MMP-9-induced cell signaling and migration were blocked by inhibiting MMP-9-binding to LRP-1 with receptor-associated protein (RAP) or by LRP-1 gene silencing. The effects of MMP-9 on Schwann cell migration also were inhibited by blocking the cell-signaling response. An antibody targeting the hemopexin domain of MMP-9, which mediates the interaction with LRP-1, blocked MMP-9-induced cell signaling and migration. Furthermore, a novel glutathione-S-transferase fusion protein (MMP-9-PEX), which includes only the hemopexin domain of MMP-9, replicated the activities of intact MMP-9, activating Schwann cell signaling and migration by an LRP-1-dependent pathway. Constitutively active MEK1 promoted Schwann cell migration; in these cells, MMP-9-PEX had no further effect, indicating that ERK1/2 activation is sufficient to explain the effects of MMP-9-PEX on Schwann cell migration. Injection of MMP-9-PEX into sciatic nerves, 24 h after crush injury, robustly increased phosphorylation of ERK1/2 and Akt. This response was inhibited by RAP. MMP-9-PEX failed to activate cell signaling in uninjured nerves, consistent with the observation that Schwann cells express LRP-1 at significant levels only after nerve injury. These results establish LRP-1 as a cell-signaling receptor for MMP-9, which may be significant in regulating Schwann cell migration and physiology in PNS injury.
Subject(s)
Cell Movement/drug effects , Hemopexin/metabolism , LDL-Receptor Related Protein-Associated Protein/physiology , Matrix Metalloproteinase 9/pharmacology , Schwann Cells/cytology , Schwann Cells/physiology , Signal Transduction/drug effects , Animals , Animals, Newborn , Cell Death/drug effects , Cell Movement/physiology , Cells, Cultured , Drug Interactions , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Matrix Metalloproteinase 9/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neuregulin-1/pharmacology , Oncogene Protein v-akt/metabolism , Protein Interaction Domains and Motifs/drug effects , Protein Interaction Domains and Motifs/physiology , RNA, Small Interfering/pharmacology , Rats , Sciatic Nerve/cytology , Sciatic Neuropathy/metabolism , Signal Transduction/physiology , rap GTP-Binding Proteins/pharmacologyABSTRACT
Glutamate is the main excitatory neurotransmitter of the CNS. Tissue-type plasminogen activator (tPA) is recognized as a modulator of glutamatergic neurotransmission. This attribute is exemplified by its ability to potentiate calcium signaling following activation of the glutamate-binding NMDA receptor (NMDAR). It has been hypothesized that tPA can directly cleave the NR1 subunit of the NMDAR and thereby potentiate NMDA-induced calcium influx. In contrast, here we show that this increase in NMDAR signaling requires tPA to be proteolytically active, but does not involve cleavage of the NR1 subunit or plasminogen. Rather, we demonstrate that enhancement of NMDAR function by tPA is mediated by a member of the low-density lipoprotein receptor (LDLR) family. Hence, this study proposes a novel functional relationship between tPA, the NMDAR, a LDLR and an unknown substrate which we suspect to be a serpin. Interestingly, whilst tPA alone failed to cleave NR1, cell-surface NMDARs did serve as an efficient and discrete proteolytic target for plasmin. Hence, plasmin and tPA can affect the NMDAR via distinct avenues. Altogether, we find that plasmin directly proteolyses the NMDAR whilst tPA functions as an indirect modulator of NMDA-induced events via LDLR engagement.
