ABSTRACT
Mical is a reduction-oxidation (redox) enzyme that functions as an unusual F-actin disassembly factor during Drosophila development. Although three Molecule interacting with CasL (MICAL) proteins exist in vertebrate species, their mechanism of action remains poorly defined and their role in vivo unknown. Here, we report that vertebrate MICAL-1 regulates the targeting of secretory vesicles containing immunoglobulin superfamily cell adhesion molecules (IgCAMs) to the neuronal growth cone membrane through its ability to control the actin cytoskeleton using redox chemistry, thereby maintaining appropriate IgCAM cell surface levels. This precise regulation of IgCAMs by MICAL-1 is essential for the lamina-specific targeting of mossy fibre axons onto CA3 pyramidal neurons in the developing mouse hippocampus in vivo. These findings reveal the first in vivo role for a vertebrate MICAL protein, expand the repertoire of cellular functions controlled through MICAL-mediated effects on the cytoskeleton, and provide insights into the poorly characterized mechanisms underlying neuronal protein cell surface expression and lamina-specific axonal targeting.
Subject(s)
Cytoskeleton/physiology , Hippocampus/physiology , Microtubule-Associated Proteins/physiology , Mixed Function Oxygenases/physiology , Mossy Fibers, Hippocampal/physiology , Secretory Vesicles/physiology , Actins/physiology , Animals , Cell Adhesion Molecules, Neuronal/physiology , Female , Hippocampus/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microfilament Proteins , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Mixed Function Oxygenases/deficiency , Mixed Function Oxygenases/genetics , Models, Animal , Oxidation-Reduction , Synapses/physiology , Tissue Culture TechniquesABSTRACT
MICALs form an evolutionary conserved family of multidomain signal transduction proteins characterized by a flavoprotein monooxygenase domain. MICALs are being implicated in the regulation of an increasing number of molecular and cellular processes including cytoskeletal dynamics and intracellular trafficking. Intriguingly, some of these effects are dependent on the MICAL monooxygenase enzyme and redox signaling, while other functions rely on other parts of the MICAL protein. Recent breakthroughs in our understanding of MICAL signaling identify the ability of MICALs to bind and directly modify the actin cytoskeleton, link MICALs to the docking and fusion of exocytotic vesicles, and uncover MICALs as anti-apoptotic proteins. These discoveries could lead to therapeutic advances in neural regeneration, cancer, and other diseases.
Subject(s)
Apoptosis , Cytoskeletal Proteins/physiology , Cytoskeleton/metabolism , Exocytosis , LIM Domain Proteins/physiology , Amino Acid Motifs , Calcium-Binding Proteins/chemistry , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/metabolism , LIM Domain Proteins/chemistry , LIM Domain Proteins/metabolism , Microfilament Proteins/chemistry , Multigene Family/physiology , Proline/chemistry , Protein Structure, Tertiary , Signal Transduction , CalponinsABSTRACT
Dopaminergic neurons in the mesodiencephalon (mdDA neurons) make precise synaptic connections with targets in the forebrain via the mesostriatal, mesolimbic, and mesoprefrontal pathways. Because of the functional importance of these remarkably complex ascending axon pathways and their implication in human disease, the mechanisms underlying the development of these connections are of considerable interest. Despite extensive in vitro studies, the molecular determinants that ensure the perfect formation of these pathways in vivo remain mostly unknown. Here, we determine the embryonic origin and ontogeny of the mouse mesoprefrontal pathway and use these data to reveal an unexpected requirement for semaphorin 3F (Sema3F) and its receptor neuropilin-2 (Npn-2) during mdDA pathway development using tissue culture approaches and analysis of sema3F(-/-), npn-2(-/-), and npn-2(-/-);TH-Cre mice. We show that Sema3F is a bifunctional guidance cue for mdDA axons, some of which have the remarkable ability to regulate their responsiveness to Sema3F as they develop. During early developmental stages, Sema3F chemorepulsion controls previously uncharacterized aspects of mdDA pathway development through both Npn-2-dependent (axon fasciculation and channeling) and Npn-2-independent (rostral growth) mechanisms. Later on, chemoattraction mediated by Sema3F and Npn-2 is required to orient mdDA axon projections in the cortical plate of the medial prefrontal cortex. This latter finding demonstrates that regulation of axon orientation in the target field occurs by chemoattractive mechanisms, and this is likely to also apply to other neural systems. In all, this study provides a framework for additional dissection of the molecular basis of mdDA pathway development and disease.
Subject(s)
Axons/physiology , Body Patterning/physiology , Diencephalon/embryology , Membrane Proteins/physiology , Nerve Tissue Proteins/physiology , Neuropilin-2/metabolism , Prosencephalon/embryology , Animals , Dopamine/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Neurites/ultrastructure , Neurons/cytology , Protein Transport , Tissue Culture TechniquesABSTRACT
Semaphorins were initially characterized according to their role in repulsive axon guidance but are now recognized as crucial regulators of morphogenesis and homeostasis over a wide range of organ systems. The pleiotropic nature of semaphorin signaling and its implication in human disease has triggered an enormous interest in the receptor and intracellular signaling mechanisms that direct the cell-type-specific and diverse biological effects of semaphorins. Recent breakthroughs in our understanding of semaphorin signaling link integrin and semaphorin signaling pathways, identify novel ligand-receptor interactions and provide insight into the cellular and molecular bases of bifunctional and reverse signaling events. These discoveries could lead to therapeutic advances in axonal regeneration, cancer and other diseases.
Subject(s)
Semaphorins/physiology , Signal Transduction , Animals , Axons/metabolism , Cell Movement , Homeostasis , Humans , Ligands , Models, Biological , Neoplasms/metabolism , Phosphorylation , Protein Structure, Tertiary , Regeneration , Semaphorins/metabolismABSTRACT
PURPOSE: The objective of this investigation was to characterize quantitatively the time-dependent changes in midazolam (MDL) efficacy in the silent period after induction of status epilepticus (SE) in rats. The changes in MDL efficacy were correlated to changes in ex vivo GABA(A)-receptor expression. METHODS: MDL efficacy was quantified by pharmacokinetic-pharmacodynamic (PK-PD) modeling by using the beta-frequency of the EEG as PD end point. Two PK-PD experiments were performed in each animal: the first experiment before and the second experiment at either day 4 or day 14 after SE. SE was induced by repetitive intraperitoneal injections with kainate. GABA(A)-receptor expression was determined by ex vivo autoradiography with [(3)H]flumazenil. RESULTS: The concentration versus EEG effect relation of midazolam was successfully described by the sigmoidal E(max) model. The maximal effect on the beta-frequency of the EEG (E(max)) was reduced to 51.6 +/- 35.6% and 25.8 +/- 33.7% of the original value at 4 and 14 days after induction of SE. The ex vivo study with [(3)H]flumazenil showed that the observed reductions in E(max) were paralleled by a reduction in GABA(A)-receptor density. CONCLUSIONS: The efficacy of MDL is decreased in the silent period after SE, which can be partly accounted for by a reduction in GABA(A)-receptor density.
