F-spondin and mindin: two structurally and functionally related genes expressed in the hippocampus that promote outgrowth of embryonic hippocampal neurons

Extracellular matrix (ECM) proteins play an important role in early cortical development, specifically in the formation of neural connections and in controlling the cyto-architecture of the central nervous system. F-spondin and Mindin are a family of matrix-attached adhesion molecules that share structural similarities and overlapping domains of expression. Genes for both proteins contain a thrombospondin type I repeat(s) at the C terminus and an FS1-FS2 (spondin) domain. Both the vertebrate F-spondin and the zebrafish mindins are expressed on the embryonic floor plate. In the current study we have cloned the rat homologue of mindin and studied its expression and activity together with F-spondin in the developing rodent brain. The two genes are abundantly expressed in the developing hippocampus. In vitro studies indicate that both F-spondin and Mindin promote adhesion and outgrowth of hippocampal embryonic neurons. We have also demonstrated that the two proteins bind to a putative receptor(s) expressed on both hippocampal and sensory neurons.

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Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

CNS & Neurological Disorders - Drug Targets ◽

10.2174/1871527319666201026165855 ◽

2020 ◽

Vol 19 ◽

Author(s):

Sumei Li ◽

Jifeng Zhang ◽

Jiaqi Zhang ◽

Jiong Li ◽

Longfei Cheng ◽

...

Keyword(s):

Neurite Outgrowth ◽

Hippocampal Neurons ◽

Neuronal Development ◽

Phosphorylation Site ◽

Microtubule Dynamics ◽

Microtubule Assembly ◽

C Terminus ◽

Mediator Protein ◽

Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

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TrkB deubiquitylation by USP8 regulates receptor levels and BDNF-dependent neuronal differentiation

Journal of Cell Science ◽

10.1242/jcs.247841 ◽

2020 ◽

Vol 133 (24) ◽

pp. jcs247841 ◽

Cited By ~ 1

Author(s):

Carlos Martín-Rodríguez ◽

Minseok Song ◽

Begoña Anta ◽

Francisco J. González-Calvo ◽

Rubén Deogracias ◽

...

Keyword(s):

Neuronal Differentiation ◽

Neurotrophic Factor ◽

Tyrosine Kinases ◽

Hippocampal Neurons ◽

Receptor Tyrosine Kinases ◽

Neurotrophin Receptor ◽

C Terminus ◽

Carboxyl Terminal

ABSTRACTUbiquitylation of receptor tyrosine kinases (RTKs) regulates both the levels and functions of these receptors. The neurotrophin receptor TrkB (also known as NTRK2), a RTK, is ubiquitylated upon activation by brain-derived neurotrophic factor (BDNF) binding. Although TrkB ubiquitylation has been demonstrated, there is a lack of knowledge regarding the precise repertoire of proteins that regulates TrkB ubiquitylation. Here, we provide mechanistic evidence indicating that ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF- and TrkB-dependent neuronal differentiation. USP8 binds to the C-terminus of TrkB using its microtubule-interacting domain (MIT). Immunopurified USP8 deubiquitylates TrkB in vitro, whereas knockdown of USP8 results in enhanced ubiquitylation of TrkB upon BDNF treatment in neurons. As a consequence of USP8 depletion, TrkB levels and its activation are reduced. Moreover, USP8 protein regulates the differentiation and correct BDNF-dependent dendritic formation of hippocampal neurons in vitro and in vivo. We conclude that USP8 positively regulates the levels and activation of TrkB, modulating BDNF-dependent neuronal differentiation.This article has an associated First Person interview with the first author of the paper.

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Interaction of the Endocytic Scaffold Protein Pan1 with the Type I Myosins Contributes to the Late Stages of Endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0436 ◽

2007 ◽

Vol 18 (8) ◽

pp. 2893-2903 ◽

Cited By ~ 29

Author(s):

Sarah L. Barker ◽

Linda Lee ◽

B. Daniel Pierce ◽

Lymarie Maldonado-Báez ◽

David G. Drubin ◽

...

Keyword(s):

Late Stage ◽

Actin Polymerization ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Type I ◽

Reading Frame ◽

Rich Domain ◽

C Terminus

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1ΔPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5–Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.

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Reverse Genetic Generation of Recombinant Zaire Ebola Viruses Containing Disrupted IRF-3 Inhibitory Domains Results in Attenuated Virus Growth In Vitro and Higher Levels of IRF-3 Activation without Inhibiting Viral Transcription or Replication

Journal of Virology ◽

10.1128/jvi.00044-06 ◽

2006 ◽

Vol 80 (13) ◽

pp. 6430-6440 ◽

Cited By ~ 72

Author(s):

Amy L. Hartman ◽

Jason E. Dover ◽

Jonathan S. Towner ◽

Stuart T. Nichol

Keyword(s):

Reverse Genetics ◽

Ebola Virus ◽

Viral Transcription ◽

Type I ◽

Virus Growth ◽

Recombinant Viruses ◽

Inhibitory Function ◽

C Terminus ◽

Inhibitory Domain

ABSTRACT The VP35 protein of Zaire Ebola virus is an essential component of the viral RNA polymerase complex and also functions to antagonize the cellular type I interferon (IFN) response by blocking activation of the transcription factor IRF-3. We previously mapped the IRF-3 inhibitory domain within the C terminus of VP35. In the present study, we show that mutations that disrupt the IRF-3 inhibitory function of VP35 do not disrupt viral transcription/replication, suggesting that the two functions of VP35 are separable. Second, using reverse genetics, we successfully recovered recombinant Ebola viruses containing mutations within the IRF-3 inhibitory domain. Importantly, we show that the recombinant viruses were attenuated for growth in cell culture and that they activated IRF-3 and IRF-3-inducible gene expression at levels higher than that for Ebola virus containing wild-type VP35. In the context of Ebola virus pathogenesis, VP35 may function to limit early IFN-β production and other antiviral signals generated from cells at the primary site of infection, thereby slowing down the host's ability to curb virus replication and induce adaptive immunity.

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Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by C Terminus of MRAP2 Protein

10.21203/rs.3.rs-829492/v1 ◽

2021 ◽

Author(s):

Meng Wang ◽

Yue Zhai ◽

Xiaowei Lei ◽

Jing Xu ◽

Bopei Jiang ◽

...

Keyword(s):

Energy Homeostasis ◽

Transmembrane Protein ◽

Melanocortin Receptor ◽

Pharmacological Modulation ◽

C Terminus ◽

Melanin Concentrating Hormone ◽

The Central Nervous System

Abstract Background: Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts at its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate energy homeostasis and body weight. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine pivot of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal appetite and peripheral energy homeostasis. However, the regulatory and relationship between MCHR1 and MRAP2 is unknown.Results: In this study, we show that MRAP2 interacts with MCHR1 and suppresses MCHR1 signaling in vitro. We also identified the C-terminal domains of MRAP2 protein required for pharmacological modulation of intracellular Ca2+ cascades and membrane transport.Conclusions: These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

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Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive.

The Journal of Cell Biology ◽

10.1083/jcb.115.3.779 ◽

1991 ◽

Vol 115 (3) ◽

pp. 779-794 ◽

Cited By ~ 136

Author(s):

A L Calof ◽

A D Lander

Keyword(s):

Neuronal Migration ◽

Type I Collagen ◽

Neuronal Cells ◽

Time Lapse ◽

Type I ◽

Type Iv ◽

The Central Nervous System ◽

Adhesive Effect ◽

Fibronectin Type

Regulation by the extracellular matrix (ECM) of migration, motility, and adhesion of olfactory neurons and their precursors was studied in vitro. Neuronal cells of the embryonic olfactory epithelium (OE), which undergo extensive migration in the central nervous system during normal development, were shown to be highly migratory in culture as well. Migration of OE neuronal cells was strongly dependent on substratum-bound ECM molecules, being specifically stimulated and guided by laminin (or the laminin-related molecule merosin) in preference to fibronectin, type I collagen, or type IV collagen. Motility of OE neuronal cells, examined by time-lapse video microscopy, was high on laminin-containing substrata, but negligible on fibronectin substrata. Quantitative assays of adhesion of OE neuronal cells to substrata treated with different ECM molecules demonstrated no correlation, either positive or negative, between the migratory preferences of cells and the strength of cell-substratum adhesion. Moreover, measurements of cell adhesion to substrata containing combinations of ECM proteins revealed that laminin and merosin are anti-adhesive for OE neuronal cells, i.e., cause these cells to adhere poorly to substrata that would otherwise be strongly adhesive. The evidence suggests that the anti-adhesive effect of laminin is not the result of interactions between laminin and other ECM molecules, but rather an effect of laminin on cells, which alters the way in which cells adhere. Consistent with this view, laminin was found to interfere strongly with the formation of focal contacts by OE neuronal cells.

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Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII)

Biochemical Journal ◽

10.1042/bj3540249 ◽

2001 ◽

Vol 354 (2) ◽

pp. 249-257 ◽

Cited By ~ 12

Author(s):

Mitsunori FUKUDA ◽

Katsuhiko MIKOSHIBA

Keyword(s):

Transmembrane Domain ◽

Amino Acid Level ◽

Subcellular Fractionation ◽

Binding Activity ◽

Type I ◽

C2 Domains ◽

C Terminus ◽

Common Receptor ◽

Antibody Uptake

Synaptotagmin (Syt) belongs to a family of type-I membrane proteins and is a protein that consists of a short extracellular N-terminus, a single transmembrane domain, two C2 domains and a short C-terminus. Here, we cloned and characterized a mouse orthologue of human KIAA1427 protein as an atypical Syt (named Syt XIII). Subcellular fractionation and antibody-uptake experiments indicate that Syt XIII is indeed a type-I membrane protein, but, unlike other Syt isoforms, lacks an N-terminal extracellular domain. Syt XIII C2 domains show relatively little similarity to Syt I (less than 35% identity at the amino acid level), and lack key amino acids responsible for Ca2+ binding. Because of these substitutions, the Syt XIII C2 domains did not show Ca2+-dependent phospholipid-binding activity, and Syt XIII is thus classified as a Ca2+-independent isoform. By contrast, the Syt XIII C-terminal domain is highly homologous with other Syt isoforms and can function as a common receptor for neurexin Iα in vitro. Since Syt XIII is expressed in various tissues outside the brain, Syt XIII may be involved in constitutive vesicle transport.

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Imaging Molecular Targeting In Living Neural Cultures

Microscopy and Microanalysis ◽

10.1017/s1431927600019462 ◽

1999 ◽

Vol 5 (S2) ◽

pp. 1228-1229

Author(s):

Christopher S. Wallace ◽

Michael A. Silverman ◽

Michelle A. Burack ◽

Janis E. Lochner ◽

Richard G. Allen ◽

...

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Hippocampal Neurons ◽

Protein Targeting ◽

Fluorescent Protein ◽

Complex Structure ◽

Time Lapse ◽

The Central Nervous System

Recent technical advances in the ability to attach an endogenously fluorescent protein sequence—i.e., green fluorescent protein or GFP and its derivatives--to any protein of experimental interest promises to mark a new era of progress in the study of protein targeting. Bringing these new tools to bear on neurons of the central nervous system has been challenging, however, because they have a very complex structure and are relatively difficult to transfect because they are post-mitotic.We use two cell culture approaches to characterize protein trafficking within neurons of the central nervous system in vitro. The first is a dissociated culture of hippocampal neurons from embryonic (El8) rats which is especially suited to analysis by conventional light microscopy because these neurons are grown on glass coverslips at low density. Neurons cultured in this way develop a morphology comparable to that seen in vivo and permit the establishment of axons and dendrites to be analyzed by time-lapse microscopy.

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Synaptic and Extrasynaptic NMDA Receptor NR2 Subunits in Cultured Hippocampal Neurons

Journal of Neurophysiology ◽

10.1152/jn.00771.2005 ◽

2006 ◽

Vol 95 (3) ◽

pp. 1727-1734 ◽

Cited By ~ 186

Author(s):

Christopher G. Thomas ◽

Ashleigh J. Miller ◽

Gary L. Westbrook

Keyword(s):

Nmda Receptors ◽

Hippocampal Neurons ◽

Synapse Formation ◽

Whole Cell ◽

Extrasynaptic Receptors ◽

C Terminus ◽

Specific Antagonist ◽

Nr2 Subunits ◽

Cultured Hippocampal Neurons

Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 ± 2.4% [6 days in vitro (DIV)] to 54.9 ± 2.6% (8 DIV), before reaching a plateau in the second week (42.5 ± 2%, 12–19 DIV). In NR2A−/− neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; ≥12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A−/− neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.

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Determination of the Interaction and Pharmacological Modulation of MCHR1 Signaling by C Terminus of MRAP2 Protein

10.21203/rs.3.rs-829492/v2 ◽

2021 ◽

Author(s):

Meng Wang ◽

Yue Zhai ◽

Xiaowei Lei ◽

Jing Xu ◽

Bopei Jiang ◽

...

Keyword(s):

Energy Homeostasis ◽

Transmembrane Protein ◽

Melanocortin Receptor ◽

Pharmacological Modulation ◽

C Terminus ◽

Melanin Concentrating Hormone ◽

The Central Nervous System

Abstract Background: Melanin concentrating hormone (MCH), an orexigenic neuropeptide, is primarily secreted by the hypothalamus and acts at its receptor, the melanin-concentrating hormone receptor 1 (MCHR1), to regulate energy homeostasis and body weight. The Melanocortin Receptor Accessory Protein 2 (MRAP2), a small single transmembrane protein broadly expressed in multiple tissues, has been defined as a vital endocrine pivot of five melanocortin receptors (MC1R-MC5R) and several other GPCRs in the regulation of central neuronal appetite and peripheral energy homeostasis. However, the regulatory and relationship between MCHR1 and MRAP2 is unknown.Results: In this study, we show that MRAP2 interacts with MCHR1 and suppresses MCHR1 signaling in vitro. We also identified the C-terminal domains of MRAP2 protein required for pharmacological modulation of intracellular Ca2+ cascades and membrane transport. Conclusions: These findings elucidated the broad regulatory profile of MRAP2 protein in the central nervous system and may provide implications for the modulation of central MCHR1 function in vivo.

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