ABSTRACT
Cytoplasmic polyadenylation element-binding protein 1 (CPEB1) is an mRNA-binding protein present in both neurons and glia. CPEB1 is capable of both repressing mRNA translation and activating it depending upon its phosphorylation state. CPEB1-bound mRNAs are held in translational dormancy until CPEB1 is phosphorylated, leading to the cytoplasmic polyadenylation of the bound mRNA that triggers translation. Here, we show that CPEB1 can bind to and regulate translation of the mRNA-encoding metadherin (MTDH, also known as AEG-1 and Lyric) in the rat glioblastoma cell line CNS1. MTDH/AEG-1 is being revealed as a critical signaling molecule in tumor progression, playing roles in invasion, metastasis, and chemoresistance. By using a mutant of CPEB1 that cannot be phosphorylated (thereby holding target mRNAs in translational arrest), we show that inhibiting CPEB1-mediated translation blocks MTDH/AEG-1 expression in vitro and inhibits glioblastomas tumor growth in vivo. CPEB1-mediated translation is likely to impact several signaling pathways that may promote tumor progression, but we present evidence suggesting a role in directed cell migration in glioblastoma cells. In addition, reporter mRNA containing CPEB1-binding sites is transported to the leading edge of migrating cells and translated, whereas the same mRNA with point mutations in the binding sites is synthesized perinuclearly. Our findings show that CPEB1 is hyperactive in rat glioblastoma cells and is regulating an important cohort of mRNAs whose increased translation is fueling the progression of tumor proliferation and dispersal in the brain. Thus, targeting CPEB1-mediated mRNA translation might be a sound therapeutic approach.
Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Adhesion Molecules/biosynthesis , Cell Movement/physiology , Glioblastoma/metabolism , Glioblastoma/pathology , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , mRNA Cleavage and Polyadenylation Factors/metabolism , Animals , Astrocytes/metabolism , Brain Neoplasms/genetics , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Growth Processes/physiology , Glioblastoma/genetics , Humans , Male , Membrane Proteins , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Rats , Rats, Inbred Lew , Signal Transduction , Transcription Factors/genetics , Transfection , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
The regulation of protein synthesis in discrete cellular subdomains, or local protein synthesis, has important roles in development as well as brain function. This review will discuss some recent findings that shed new light on mRNA translation regulation and how these layers of regulation may work together to elicit tissue specific and spatially restricted gene expression.
Subject(s)
Protein Biosynthesis , Synapses/metabolism , Animals , Neurons/cytology , Neurons/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Synapses/genetics , Ubiquitin/metabolismABSTRACT
J. Neurochem. (2012) 123, 226-238. ABSTRACT: Fragile X syndrome (FXS), the most common single genetic cause of mental retardation and autistic spectrum disease, occurs when FMR1 gene is mutated. FMR1 encodes fragile X mental retardation protein (FMRP) which regulates translation of mRNAs playing important roles in the development of neurons as well as formation and maintenance of synapses. To examine whether FMRP regulates cell viability, we induced apoptosis in rat primary cortical neurons with glutamate in vitro and with middle cerebral artery occlusion (MCAO) in striatal neurons in vivo. Both conditions elicited a rapid, but transient FMRP expression in neurons. This up-regulated FMRP expression was abolished by pre-treatment with PI3K and Protein Kinase B (Akt) inhibitors: LY294002, Akt inhibitor IV, and VIII. Reduced FMRP expression in vitro or in vivo using small hairpin Fmr1 virus exacerbated cell death by glutamate or MCAO, presumably via hypophosphorylation of Akt and reduced expression of B-cell lymphoma-extra large (Bcl-xL). However, over-expression of FMRP using enhanced green fluorescent protein (eGFP)-FMRP constructs alleviated cell death, increased Akt activity, and enhanced Bcl-xL production. The pro-survival role of Akt-dependent up-regulation of FMRP in glutamate-stimulated cultured neuron as well as in ischemic brain may have a clinical importance in FXS as well as in neurodegenerative disorders and traumatic brain injury.
Subject(s)
Feedback, Physiological/physiology , Fragile X Mental Retardation Protein/physiology , Neurons/physiology , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction/physiology , Animals , Apoptosis/physiology , Cell Death/physiology , Cell Survival/physiology , Cells, Cultured , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Infarction, Middle Cerebral Artery/prevention & control , Male , Neurons/pathology , Rats , Rats, WistarABSTRACT
Astrocytes are the most abundant cells in the brain, playing vital roles in neuronal survival, growth, and function. Understanding the mechanism(s) regulating astrocyte proliferation will have important implications in brain development, response to injury, and tumorigenesis. Cyclin B1 is well known to be a critical regulator of mitotic entry via its interaction with cyclin-dependent kinase 1. In rat astrocytes, we now show that the mRNA binding protein cytoplasmic polyadenylation element binding protein 1 (CPEB1) is associated with cyclin B1 mRNA and that this interaction is enriched at the centrosome. In addition, if growth-arrested astrocytes are stimulated to divide, CPEB1 is phosphorylated and cyclin B1 mRNA is polyadenylated, both hallmarks of CPEB1 activation, resulting in an increase in cyclin B1 protein. CPEB1 binding to mRNA initially inhibits translation; therefore, removing CPEB1 from mRNA should result in an increase in translation due to derepression. Indeed, when we either knocked down CPEB1 protein with siRNA or sequestered it from endogenous mRNA by expressing RNA containing multiple CPEB1 binding sites, cyclin B1 protein was increased and cell proliferation was stimulated. Our data suggest a mechanism wherein CPEB1 is bound and represses cyclin B1 mRNA translation until a signal to proliferate phosphorylates CPEB1, resulting in an increase in cyclin B1 protein and progression into mitosis. Our results demonstrate for the first time a role for CPEB1 in regulating cell proliferation in the brain.
Subject(s)
Astrocytes/cytology , Astrocytes/metabolism , Cyclin B1/biosynthesis , Cyclin B1/genetics , Gene Expression Regulation/physiology , Polyadenylation/genetics , RNA Stability/genetics , RNA-Binding Proteins/metabolism , Animals , Animals, Newborn , Cell Proliferation , Cells, Cultured , Centrosome/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Female , Male , RNA-Binding Proteins/genetics , Rats , Rats, Sprague-DawleyABSTRACT
Neuronal morphogenesis, the growth and arborization of neuronal processes, is an essential component of brain development. Two important but seemingly disparate components regulating neuronal morphology have previously been described. In the hippocampus, neurotrophins, particularly brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3), act to enhance cell growth and branching, while activity-induced branching was shown to be dependent upon intracellular beta-catenin. We now describe a molecular link between NT3 stimulation and beta-catenin increase in developing neurons and demonstrate that this process is required for the NT3-mediated increase in process branching. Here, we show that beta-catenin is rapidly increased specifically in growth cones following NT3 stimulation. This increase in beta-catenin is protein synthesis dependent and requires the activity of cytoplasmic polyadenylation element-binding protein-1 (CPEB1), an mRNA-binding protein that regulates mRNA translation. We find that CPEB1 protein binds beta-catenin mRNA in a CPE-dependent manner and that both localize to growth cones of developing hippocampal neurons. Both the NT3-mediated rapid increase in beta-catenin and process branching are abolished when CPEB1 function is inhibited. In addition, the NT3-mediated increase in beta-catenin in growth cones is dependent upon internal calcium and the activity of CaMKII (calcium/calmodulin-dependent kinase II). Together, these results suggest that CPEB1 regulates beta-catenin synthesis in neurons and may contribute to neuronal morphogenesis.
Subject(s)
Hippocampus/growth & development , Hippocampus/physiology , Neurons/physiology , Neurotrophin 3/metabolism , RNA-Binding Proteins/metabolism , beta Catenin/metabolism , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cells, Cultured , Growth Cones/physiology , Protein Biosynthesis , RNA, Messenger/metabolism , Rats , Sequence Homology, Nucleic Acid , Signal Transduction , beta Catenin/biosynthesis , beta Catenin/geneticsABSTRACT
A crucial step in directed cell migration is the recruitment of cytoskeletal regulatory and signaling proteins to the leading edge of the cell. One protein localized to the leading edge of a migrating astrocyte is beta-catenin. Using an in vitro wound-healing assay, we show that the localization of beta-catenin to the leading edge is dependent upon new protein synthesis at the time of wounding. We examined the mRNA encoding beta-catenin for potential regulatory elements and identified a conserved cytoplasmic polyadenylation element in the 3'-untranslated region (UTR). We now show that the CPE-binding protein (CPEB1) is expressed in astrocytes and that translation of beta-catenin mRNA is regulated by CPEB1. Further, expression of a mutant CPEB1 protein in astrocytes not only blocks beta-catenin protein localization, it also inhibits cell migration. These findings demonstrate a role for CPEB1-mediated protein synthesis in the localization of beta-catenin protein to the leading edge of migrating astrocytes and in regulating directed cell motility.
Subject(s)
Astrocytes/physiology , Cell Migration Inhibition/physiology , Cell Movement/physiology , Protein Biosynthesis/physiology , Transcription Factors/physiology , beta Catenin/biosynthesis , beta Catenin/genetics , mRNA Cleavage and Polyadenylation Factors/physiology , Animals , Astrocytes/cytology , Astrocytes/metabolism , Cells, Cultured , RatsABSTRACT
Many cellular functions require the synthesis of a specific protein or functional cohort of proteins at a specific time and place in the cell. Local protein synthesis in neuronal dendrites is essential for understanding how neural activity patterns are transduced into persistent changes in synaptic connectivity during cortical development, memory storage and other long-term adaptive brain responses. Regional and temporal changes in protein levels are commonly coordinated by an asymmetric distribution of mRNAs. This Review attempts to integrate current knowledge of dendritic mRNA transport, storage and translation, placing particular emphasis on the coordination of regulation and function during activity-dependent synaptic plasticity in the adult mammalian brain.
Subject(s)
Dendrites/metabolism , Protein Biosynthesis/physiology , RNA Transport/physiology , RNA, Messenger/metabolism , Animals , Dendrites/genetics , Humans , Neuronal Plasticity/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/physiologyABSTRACT
The ability of neurons to modify synaptic connections is critical for proper brain development and function in the adult. It is now clear that changes in synaptic strength are often accompanied by changes in synaptic morphology. This synaptic plasticity can be maintained for varying lengths of time depending on the type of neuronal activity that first induced the changes. Long-term synaptic plasticity requires the synthesis of new proteins, and one mechanism for the regulation of experience-induced protein synthesis in neurons involves cytoplasmic polyadenylation element binding protein (CPEB1). CPEB1 can bidirectionally regulate mRNA translation, first repressing translation, and then activating translation after the phosphorylation of two critical residues (T171 and S177). To determine the full extent of CPEB1-mediated protein synthesis in synaptic function, we engineered a line of mice expressing CPEB1 with these phosphorylation sites mutated to alanines (mCPEB1-AA) exclusively in cerebellar Purkinje neurons (PNs). Thus, mRNAs bound by mCPEB1-AA would be held in a translationally dormant state. We show that mCPEB1-AA localizes to synapses in cerebellum and resulted in a loss of protein synthesis-dependent phase of parallel fiber-PN long-term depression. This was accompanied by a change in spine number and spine length that are likely attributable in part to the dysregulation of IRSp53, a protein known to play a role in synaptic structure. Finally, mCPEB1-AA mice displayed a significant impairment of motor coordination and a motor learning delay.
Subject(s)
Cerebellum/cytology , Long-Term Synaptic Depression/physiology , Protein Biosynthesis/physiology , Psychomotor Performance/physiology , Purkinje Cells/metabolism , Transcription Factors/physiology , mRNA Cleavage and Polyadenylation Factors/physiology , Animals , Behavior, Animal , Biotin/analogs & derivatives , Biotin/metabolism , Dendritic Spines/metabolism , Dose-Response Relationship, Radiation , Electric Stimulation , Gene Expression Regulation/genetics , Green Fluorescent Proteins/biosynthesis , Immunoprecipitation/methods , In Vitro Techniques , Indoles/pharmacology , Mice , Mice, Transgenic , Mutation , Patch-Clamp Techniques , Purkinje Cells/ultrastructure , Receptors, Glutamate/metabolism , Sulfonamides/pharmacology , Transcription Factors/genetics , mRNA Cleavage and Polyadenylation Factors/geneticsABSTRACT
Regulation of protein expression in neurons by controlling not only when, but where, mRNAs are translated is likely to play an important role in neuronal function. In this review I focus on the mRNA-binding proteins that control mRNA translation in neurons and how they may participate in local, synaptodendritic protein synthesis.
Subject(s)
Gene Expression Regulation/physiology , Gene Silencing/physiology , RNA-Binding Proteins/physiology , Repressor Proteins/physiology , Animals , HumansABSTRACT
Myosin VI (Myo6) is an actin-based motor protein implicated in clathrin-mediated endocytosis in nonneuronal cells, though little is known about its function in the nervous system. Here, we find that Myo6 is highly expressed throughout the brain, localized to synapses, and enriched at the postsynaptic density. Myo6-deficient (Snell's waltzer; sv/sv) hippocampus exhibits a decrease in synapse number, abnormally short dendritic spines, and profound astrogliosis. Similarly, cultured sv/sv hippocampal neurons display decreased numbers of synapses and dendritic spines, and dominant-negative disruption of Myo6 in wild-type hippocampal neurons induces synapse loss. Importantly, we find that sv/sv hippocampal neurons display a significant deficit in the stimulation-induced internalization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptors (AMPARs), and that Myo6 exists in a complex with the AMPAR, AP-2, and SAP97 in brain. These results suggest that Myo6 plays a role in the clathrin-mediated endocytosis of AMPARs, and that its loss leads to alterations in synaptic structure and astrogliosis.
Subject(s)
Endocytosis/physiology , Myosin Heavy Chains/physiology , Receptors, AMPA/metabolism , Synapses/physiology , Adaptor Protein Complex 2/metabolism , Adaptor Proteins, Signal Transducing , Adenosine Triphosphate/pharmacology , Animals , Astrocytes/metabolism , Astrocytes/ultrastructure , Brain/metabolism , Brain/ultrastructure , Brain Chemistry , Dendrites/metabolism , Dendrites/ultrastructure , Dendritic Spines/metabolism , Dendritic Spines/ultrastructure , Discs Large Homolog 1 Protein , Dyneins/genetics , Dyneins/metabolism , Endocytosis/drug effects , Female , Glial Fibrillary Acidic Protein/analysis , Guanylate Kinases , Insulin/pharmacology , Male , Membrane Proteins , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Microfilament Proteins/analysis , Microfilament Proteins/metabolism , Microscopy, Electron , Myosin Heavy Chains/genetics , Myosin Heavy Chains/metabolism , Myosin VIIa , Myosins/genetics , Myosins/metabolism , Nerve Tissue Proteins/analysis , Nerve Tissue Proteins/metabolism , Neurons/physiology , Neurons/ultrastructure , Receptors, AMPA/analysis , Receptors, Glutamate/metabolism , Sucrose/pharmacology , Synapses/ultrastructure , Synaptic Membranes/chemistry , Synaptic Membranes/drug effects , Synaptosomes/chemistry , Synaptosomes/drug effects , Transferrin/metabolism , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/metabolismABSTRACT
Long-term synaptic plasticity is both protein synthesis-dependent and synapse-specific. Therefore, the identity of the newly synthesized proteins, their localization, and mechanism of regulation are critical to our understanding of this process. Tissue plasminogen activator (tPA) is a secreted protease required for some forms of long-term synaptic plasticity. Here, we show tPA activity is rapidly increased in hippocampal neurons after glutamate stimulation. This increase in tPA activity corresponds to an increase in tPA protein synthesis that results from the translational activation of mRNA present at the time of stimulation. Furthermore, the mRNA encoding tPA is present in dendrites and is rapidly polyadenylated after glutamate stimulation. Both the polyadenylation of tPA mRNA and the subsequent increase in tPA protein is dependent on metabotropic glutamate receptor (mGluR) activation. A similar mGluR-dependent increase in tPA activity was detected after stimulation of a synaptic fraction isolated from the hippocampus, suggesting tPA synthesis is occurring in the synaptodendritic region. Finally, we demonstrate that tPA mRNA is bound by the mRNA-binding protein CPEB (cytoplasmic polyadenylation element binding protein-1), a protein known to regulate mRNA translation via polyadenylation. These results indicate that neurons are capable of synthesizing a secreted protein in the synaptic region, that mGluR activation induces mRNA polyadenylation and translation of specific mRNA, and suggest a model for synaptic plasticity whereby translational regulation of an immediate early gene precedes the increase in gene transcription.