ABSTRACT
Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.
Subject(s)
Cytoskeletal Proteins/metabolism , Dendrites/metabolism , GTP-Binding Proteins/metabolism , Neurons/metabolism , Neurons/ultrastructure , Animals , Brain/cytology , Brain/metabolism , Cells, Cultured , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Dendrites/ultrastructure , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/genetics , Hippocampus/cytology , Hippocampus/metabolism , RNA Interference , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
The Rap family of small GTPases is implicated in the mechanisms of synaptic plasticity, particularly synaptic depression. Here we studied the role of Rap in neuronal morphogenesis and synaptic transmission in cultured neurons. Constitutively active Rap2 expressed in hippocampal pyramidal neurons caused decreased length and complexity of both axonal and dendritic branches. In addition, Rap2 caused loss of dendritic spines and spiny synapses, and an increase in filopodia-like protrusions and shaft synapses. These Rap2 morphological effects were absent in aspiny interneurons. In contrast, constitutively active Rap1 had no significant effect on axon or dendrite morphology. Dominant-negative Rap mutants increased dendrite length, indicating that endogenous Rap restrains dendritic outgrowth. The amplitude and frequency of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-mediated miniature excitatory postsynaptic currents (mEPSCs) decreased in hippocampal neurons transfected with active Rap1 or Rap2, associated with reduced surface and total levels of AMPA receptor subunit GluR2. Finally, increasing synaptic activity with GABA(A) receptor antagonists counteracted Rap2's inhibitory effect on dendrite growth, and masked the effects of Rap1 and Rap2 on AMPA-mediated mEPSCs. Rap1 and Rap2 thus have overlapping but distinct actions that potentially link the inhibition of synaptic transmission with the retraction of axons and dendrites.
Subject(s)
Cell Differentiation/physiology , Hippocampus/cytology , Neurites/physiology , Neurons/cytology , Synapses/physiology , rap GTP-Binding Proteins/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Action Potentials/radiation effects , Animals , Animals, Newborn , Cell Differentiation/genetics , Cells, Cultured , Diagnostic Imaging/methods , Disks Large Homolog 4 Protein , Embryo, Mammalian , Glutamate Decarboxylase/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunohistochemistry/methods , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Mutagenesis/physiology , Neurites/drug effects , Neurons/classification , Neurons/drug effects , Neurons/physiology , Patch-Clamp Techniques/methods , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Synapses/drug effects , Synaptic Transmission/physiology , Time Factors , Transfection/methods , rap GTP-Binding Proteins/geneticsABSTRACT
Leukocyte common antigen-related (LAR) family receptor protein tyrosine phosphatases (LAR-RPTP) bind to liprin-alpha (SYD2) and are implicated in axon guidance. We report that LAR-RPTP is concentrated in mature synapses in cultured rat hippocampal neurons, and is important for the development and maintenance of excitatory synapses in hippocampal neurons. RNA interference (RNAi) knockdown of LAR or dominant-negative disruption of LAR function results in loss of excitatory synapses and dendritic spines, reduction of surface AMPA receptors, impairment of dendritic targeting of the cadherin-beta-catenin complex, and reduction in the amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). Cadherin, beta-catenin and GluR2/3 are tyrosine phosphoproteins that coimmunoprecipitate with liprin-alpha and GRIP from rat brain extracts. We propose that the cadherin-beta-catenin complex is cotransported with AMPA receptors to synapses and dendritic spines by a mechanism that involves binding of liprin-alpha to LAR-RPTP and tyrosine dephosphorylation by LAR-RPTP.
Subject(s)
Gene Expression Regulation, Developmental/physiology , Hippocampus/cytology , Hippocampus/growth & development , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Protein Tyrosine Phosphatases/metabolism , Receptors, Cell Surface/metabolism , Synapses/physiology , Adaptor Proteins, Signal Transducing , Animals , Animals, Newborn , Blotting, Western/methods , Brain/growth & development , Brain/metabolism , COS Cells , Cells, Cultured , Chlorocebus aethiops , Cytoskeletal Proteins/metabolism , Dendrites/metabolism , Diagnostic Imaging/methods , Enzyme Inhibitors/pharmacology , Excitatory Postsynaptic Potentials/physiology , Genistein/pharmacology , Green Fluorescent Proteins/metabolism , Humans , Immunohistochemistry/methods , Immunoprecipitation/methods , Membrane Potentials/genetics , Membrane Potentials/radiation effects , Molecular Sequence Data , Mutagenesis/physiology , Patch-Clamp Techniques/methods , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/pharmacology , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Proto-Oncogene Proteins c-myc/metabolism , RNA, Antisense/pharmacology , RNA, Small Interfering , Rats , Receptor-Like Protein Tyrosine Phosphatases, Class 2 , Receptors, AMPA/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Time Factors , Trans-Activators/metabolism , Transfection/methods , Tyrosine/metabolism , Vanadates/pharmacology , beta CateninABSTRACT
Removal of synaptic AMPA receptors is important for synaptic depression. Here, we characterize the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization.
