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1.
J Neurosci ; 2024 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-39266301

RESUMO

Neuroinflammation can positively influence axon regeneration following injury in the central nervous system (CNS). Inflammation promotes the release of neurotrophic molecules and stimulates intrinsic pro-regenerative molecular machinery in neurons, but the detailed mechanisms driving this effect are not fully understood. We evaluated how microRNAs are regulated in retinal neurons in response to intraocular inflammation to identify their potential role in axon regeneration. We found that miR-383-5p is downregulated in retinal ganglion cells in response to zymosan-induced intraocular inflammation. MiR-383-5p downregulation in neurons is sufficient to promote axon growth in vitro, and the intravitreal injection of a miR-383-5p inhibitor into the eye promotes axon regeneration following optic nerve crush. MiR-383-5p directly targets ciliary neurotrophic factor (CNTF) receptor components and miR-383-5p inhibition sensitizes adult retinal neurons to the outgrowth-promoting effects of CNTF. Interestingly, we also demonstrate that CNTF treatment is sufficient to reduce miR-383-5p levels in neurons, constituting a positive-feedback module whereby initial CNTF treatment reduces miR-383-5p levels, which then disinhibits CNTF receptor components to sensitize neurons to the ligand. Additionally, miR-383-5p inhibition de-represses the mitochondrial antioxidant protein peroxiredoxin-3 (PRDX3) which was required for the pro-regenerative effects associated with miR-383-5p loss of function in vitro. We have thus identified a positive feedback mechanism that facilitates neuronal CNTF sensitivity in neurons, and a new molecular signalling module that promotes inflammation-induced axon regeneration.Significance statement Inflammation can both positively and negatively influence the neuronal response to injury. Identifying molecular signalling pathways that mimic pro-regenerative effects of inflammation while bypassing cytotoxic effects is important for our understanding of the precise functions of inflammation in CNS injury and repair. We demonstrate that miR-383-5p is suppressed in neurons in response to inflammatory stimuli and regulates members of the ciliary neurotrophic factor (CNTF) receptor complex, as well as the expression of an antioxidant protein to improve axon regeneration in an optic nerve crush model. These findings identify a new molecular signalling module that promotes axon regeneration and that may bypass detrimental effects of inflammation.

2.
J Neurochem ; 168(10): 3449-3466, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38702968

RESUMO

Ependymal cells form a specialized brain-cerebrospinal fluid (CSF) interface and regulate local CSF microcirculation. It is becoming increasingly recognized that ependymal cells assume a reactive state in response to aging and disease, including conditions involving hypoxia, hydrocephalus, neurodegeneration, and neuroinflammation. Yet what transcriptional signatures govern these reactive states and whether this reactivity shares any similarities with classical descriptions of glial reactivity (i.e., in astrocytes) remain largely unexplored. Using single-cell transcriptomics, we interrogated this phenomenon by directly comparing the reactive ependymal cell transcriptome to the reactive astrocyte transcriptome using a well-established model of autoimmune-mediated neuroinflammation (MOG35-55 EAE). In doing so, we unveiled core glial reactivity-associated genes that defined the reactive ependymal cell and astrocyte response to MOG35-55 EAE. Interestingly, known reactive astrocyte genes from other CNS injury/disease contexts were also up-regulated by MOG35-55 EAE ependymal cells, suggesting that this state may be conserved in response to a variety of pathologies. We were also able to recapitulate features of the reactive ependymal cell state acutely using a classic neuroinflammatory cocktail (IFNγ/LPS) both in vitro and in vivo. Taken together, by comparing reactive ependymal cells and astrocytes, we identified a conserved signature underlying glial reactivity that was present in several neuroinflammatory contexts. Future work will explore the mechanisms driving ependymal reactivity and assess downstream functional consequences.


Assuntos
Astrócitos , Encefalomielite Autoimune Experimental , Epêndima , Camundongos Endogâmicos C57BL , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Epêndima/metabolismo , Epêndima/patologia , Camundongos , Encefalomielite Autoimune Experimental/patologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Feminino , Doenças Neuroinflamatórias/patologia , Transcriptoma
3.
Int J Mol Sci ; 23(23)2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36499143

RESUMO

Multiple sclerosis (MS) is an autoimmune and neurodegenerative disease driven by inflammation and demyelination in the brain, spinal cord, and optic nerve. Optic neuritis, characterized by inflammation and demyelination of the optic nerve, is a symptom in many patients with MS. The optic nerve is the highway for visual information transmitted from the retina to the brain. It contains axons from the retinal ganglion cells (RGCs) that reside in the retina, myelin forming oligodendrocytes and resident microglia and astrocytes. Inflammation, demyelination, and axonal degeneration are also present in the optic nerve of mice subjected to experimental autoimmune encephalomyelitis (EAE), a preclinical mouse model of MS. Monitoring the optic nerve in EAE is a useful strategy to study the presentation and progression of pathology in the visual system; however, current approaches have relied on sectioning, staining and manual quantification. Further, information regarding the spatial load of lesions and inflammation is dependent on the area of sectioning. To better characterize cellular pathology in the EAE model, we employed a tissue clearing and 3D immunolabelling and imaging protocol to observe patterns of immune cell infiltration and activation throughout the optic nerve. Increased density of TOPRO staining for nuclei captured immune cell infiltration and Iba1 immunostaining was employed to monitor microglia and macrophages. Axonal degeneration was monitored by neurofilament immunolabelling to reveal axonal swellings throughout the optic nerve. In parallel, we developed a convolutional neural network with a UNet architecture (CNN-UNet) called BlebNet for automated identification and quantification of axonal swellings in whole mount optic nerves. Together this constitutes a toolkit for 3-dimensional immunostaining to monitor general optic nerve pathology and fast automated quantification of axonal defects that could also be adapted to monitor axonal degeneration and inflammation in other neurodegenerative disease models.


Assuntos
Aprendizado Profundo , Encefalomielite Autoimune Experimental , Esclerose Múltipla , Doenças Neurodegenerativas , Neurite Óptica , Camundongos , Animais , Camundongos Endogâmicos C57BL , Neurite Óptica/patologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/patologia , Degeneração Neural , Inflamação , Modelos Animais de Doenças
4.
Brain ; 142(10): 2979-2995, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31412103

RESUMO

Multiple sclerosis is a chronic inflammatory, demyelinating, and neurodegenerative disease affecting the brain, spinal cord and optic nerves. Neuronal damage is triggered by various harmful factors that engage diverse signalling cascades in neurons; thus, therapeutic approaches to protect neurons will need to focus on agents that can target multiple biological processes. We have therefore focused our attention on microRNAs: small non-coding RNAs that primarily function as post-transcriptional regulators that target messenger RNAs and repress their translation into proteins. A single microRNA can target many functionally related messenger RNAs making microRNAs powerful epigenetic regulators. Dysregulation of microRNAs has been described in many neurodegenerative diseases including multiple sclerosis. Here, we report that two microRNAs, miR-223-3p and miR-27a-3p, are upregulated in neurons in the experimental autoimmune encephalomyelitis mouse model of CNS inflammation and in grey matter-containing multiple sclerosis lesions. Prior work has shown peripheral blood mononuclear cell conditioned media causes sublethal degeneration of neurons in culture. We find overexpression of miR-27a-3p or miR-223-3p protects dissociated cortical neurons from condition media mediated degeneration. Introduction of miR-223-3p in vivo in mouse retinal ganglion cells protects their axons from degeneration in experimental autoimmune encephalomyelitis. In silico analysis revealed that messenger RNAs involved in glutamate receptor signalling are enriched as miR-27a-3p and miR-223-3p targets. We observe that antagonism of NMDA and AMPA type glutamate receptors protects neurons from condition media dependent degeneration. Our results suggest that miR-223-3p and miR-27a-3p are upregulated in response to inflammation to mediate a compensatory neuroprotective gene expression program that desensitizes neurons to glutamate by targeting messenger RNAs involved in glutamate receptor signalling.


Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/patologia , MicroRNAs/genética , Neurônios/patologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Camundongos , MicroRNAs/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Esclerose Múltipla/patologia , Degeneração Neural/genética , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/genética , Medula Espinal/patologia
5.
J Neurosci ; 38(3): 518-529, 2018 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-29196321

RESUMO

Cell-surface molecules are dynamically regulated at the synapse to assemble and disassemble adhesive contacts that are important for synaptogenesis and for tuning synaptic transmission. Metalloproteinases dynamically regulate cellular behaviors through the processing of cell surface molecules. In the present study, we evaluated the role of membrane-type metalloproteinases (MT-MMPs) in excitatory synaptogenesis. We find that MT3-MMP and MT5-MMP are broadly expressed in the mouse cerebral cortex and that MT3-MMP loss-of-function interferes with excitatory synapse development in dissociated cortical neurons and in vivo We identify Nogo-66 receptor (NgR1) as an MT3-MMP substrate that is required for MT3-MMP-dependent synapse formation. Introduction of the shed ectodomain of NgR1 is sufficient to accelerate excitatory synapse formation in dissociated cortical neurons and in vivo Together, our findings support a role for MT3-MMP-dependent shedding of NgR1 in regulating excitatory synapse development.SIGNIFICANCE STATEMENT In this study, we identify MT3-MMP, a membrane-bound zinc protease, to be necessary for the development of excitatory synapses in cortical neurons. We identify Nogo-66 receptors (NgR1) as a downstream target of MT3-MMP proteolytic activity. Furthermore, processing of surface NgR1 by MT3-MMP generates a soluble ectodomain fragment that accelerates the formation of excitatory synapses. We propose that MT3-MMP activity and NgR1 shedding could stimulate circuitry remodeling in the adult brain and enhance functional connectivity after brain injury.


Assuntos
Córtex Cerebral/metabolismo , Metaloproteinase 16 da Matriz/metabolismo , Neurônios/metabolismo , Receptor Nogo 1/metabolismo , Sinapses/metabolismo , Animais , Metalotioneína 3 , Camundongos , Ratos
6.
J Neuroinflammation ; 16(1): 223, 2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31729981

RESUMO

BACKGROUND: Multiple sclerosis is an autoimmune disease with a distinct female bias, as well as a high prevalence of neuropathic pain in both sexes. The dorsal root ganglia (DRG) contain the primary sensory neurons that give rise to pain, and damage to these neurons may lead to neuropathic pain. Here, we investigate the sex differences of the DRG transcriptome in a mouse model of MS. METHODS: Next-generation sequencing was used to establish RNA and microRNA profiles from the DRG of mice with MOG35-55-induced EAE, a model of CNS inflammation that mimics aspects of MS. Differential expression and multiple meta-analytic approaches were used to compare expression profiles in immunized female and male mice. Differential expression of relevant genes and microRNAs were confirmed by qPCR. RESULTS: Three thousand five hundred twenty genes and 29 microRNAs were differentially expressed in the DRG of female mice with MOG35-55-EAE, while only 189 genes and 3 microRNAs were differentially expressed in males with MOG35-55-EAE. Genes related to the immune system were uniquely regulated in immunized female mice. Direct comparison of sex within disease indicates significant differences in interferon and phagosomal pathways between the sexes. miR-21a-5p is the primary dysregulated microRNA in both sexes, with females having additional dysregulated microRNAs, including miR-122-5p. CONCLUSIONS: This study provides evidence that females are uniquely affected by MOG35-55-EAE and that this difference may result from additional signaling not present in the male. The altered transcriptome of females correlates with other studies finding hyperactivity of pain-sensing neurons and suggests underlying sex-specific pathways for neuropathic pain.


Assuntos
Encefalomielite Autoimune Experimental/genética , Gânglios Espinais/metabolismo , MicroRNAs/biossíntese , Caracteres Sexuais , Transcriptoma , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética
7.
Dev Dyn ; 247(1): 18-23, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28643358

RESUMO

The failure of damaged axons to regrow underlies disability in central nervous system injury and disease. Therapies that stimulate axon repair will be critical to restore function. Extensive axon regeneration can be induced by manipulation of oncogenes and tumor suppressors; however, it has been difficult to translate this into functional recovery in models of spinal cord injury. The current challenge is to maximize the functional integration of regenerating axons to recover motor and sensory behaviors. Insights into axonal growth and wiring during nervous system development are helping guide new approaches to boost regeneration and functional connectivity after injury in the mature nervous system. Here we discuss our current understanding of axonal behavior after injury and prospects for the development of drugs to optimize axon regeneration and functional recovery after CNS injury. Developmental Dynamics 247:18-23, 2018. © 2017 Wiley Periodicals, Inc.


Assuntos
Axônios/fisiologia , Sistema Nervoso Central/lesões , Regeneração Nervosa/fisiologia , Neurogênese/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Sistema Nervoso Central/fisiopatologia , Humanos
8.
J Neurosci ; 36(3): 979-87, 2016 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-26791225

RESUMO

CNS injury may lead to permanent functional deficits because it is still not possible to regenerate axons over long distances and accurately reconnect them with an appropriate target. Using rat neurons, microtools, and nanotools, we show that new, functional neurites can be created and precisely positioned to directly (re)wire neuronal networks. We show that an adhesive contact made onto an axon or dendrite can be pulled to initiate a new neurite that can be mechanically guided to form new synapses at up to 0.8 mm distance in <1 h. Our findings challenge current understanding of the limits of neuronal growth and have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration. Significance statement: Brain and spinal cord injury may lead to permanent disability and death because it is still not possible to regenerate neurons over long distances and accurately reconnect them with an appropriate target. Using microtools and nanotools we have developed a new method to rapidly initiate, elongate, and precisely connect new functional neuronal circuits over long distances. The extension rates achieved are ≥60 times faster than previously reported. Our findings have direct implications for the development of new therapies and surgical techniques to achieve functional regeneration after trauma and in neurodegenerative diseases. It also opens the door for the direct wiring of robust brain-machine interfaces as well as for investigations of fundamental aspects of neuronal signal processing and neuronal function.


Assuntos
Córtex Cerebral/fisiologia , Hipocampo/fisiologia , Rede Nervosa/fisiologia , Regeneração Nervosa/fisiologia , Neurônios/fisiologia , Animais , Axônios/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Feminino , Hipocampo/citologia , Masculino , Rede Nervosa/citologia , Neuritos/fisiologia , Técnicas de Patch-Clamp/métodos , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
9.
J Biol Chem ; 291(9): 4589-602, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26710849

RESUMO

The receptor deleted in colorectal cancer (DCC) mediates the attraction of growing axons to netrin-1 during brain development. In response to netrin-1 stimulation, DCC becomes a signaling platform to recruit proteins that promote axon outgrowth and guidance. The Ras GTPase-activating protein (GAP) p120RasGAP inhibits Ras activity and mediates neurite retraction and growth cone collapse in response to repulsive guidance cues. Here we show an interaction between p120RasGAP and DCC that positively regulates netrin-1-mediated axon outgrowth and guidance in embryonic cortical neurons. In response to netrin-1, p120RasGAP is recruited to DCC in growth cones and forms a multiprotein complex with focal adhesion kinase and ERK. We found that Ras/ERK activities are elevated aberrantly in p120RasGAP-deficient neurons. Moreover, the expression of p120RasGAP Src homology 2 (SH2)-SH3-SH2 domains, which interact with the C-terminal tail of DCC, is sufficient to restore netrin-1-dependent axon outgrowth in p120RasGAP-deficient neurons. We provide a novel mechanism that exploits the scaffolding properties of the N terminus of p120RasGAP to tightly regulate netrin-1/DCC-dependent axon outgrowth and guidance.


Assuntos
Axônios/metabolismo , Córtex Cerebral/metabolismo , Fatores de Crescimento Neural/metabolismo , Neurônios/metabolismo , Receptores de Superfície Celular/agonistas , Transdução de Sinais , Proteínas Supressoras de Tumor/agonistas , Proteínas Supressoras de Tumor/metabolismo , Proteína p120 Ativadora de GTPase/metabolismo , Substituição de Aminoácidos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Galinhas , Receptor DCC , Embrião de Mamíferos/citologia , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Proteínas Mutantes/agonistas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fatores de Crescimento Neural/antagonistas & inibidores , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Netrina-1 , Neurônios/citologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Interferência de RNA , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteína p120 Ativadora de GTPase/antagonistas & inibidores , Proteína p120 Ativadora de GTPase/química , Proteína p120 Ativadora de GTPase/genética
10.
Pharmacol Res ; 125(Pt B): 114-121, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28918174

RESUMO

14-3-3s are a family of ubiquitously expressed adaptor proteins that regulate hundreds of functionally diverse 'client proteins.' In humans, there are seven isoforms with conserved structure and function. 14-3-3s typically bind to client proteins at phosphorylated serine/threonine motifs via a linear binding groove. Binding can have a variety of effects on the stability, activity and/or localization of the client protein. 14-3-3s are generating significant interest as potential drug targets for their involvement in cellular homeostasis and disease. They are especially abundant in the central nervous system (CNS) and are implicated in numerous CNS diseases, often through specific interactions with disease-relevant client proteins. Several tool compounds that can modulate 14-3-3 interactions with client proteins to elicit therapeutic effects have recently been described. Here we offer a perspective on the functions of 14-3-3s in neurons and the potential development of drugs to therapeutically target 14-3-3 PPIs for CNS diseases.


Assuntos
Proteínas 14-3-3/metabolismo , Doenças do Sistema Nervoso Central/metabolismo , Proteínas 14-3-3/química , Animais , Axônios/fisiologia , Humanos , Transtornos Mentais/metabolismo , Neuroproteção , Peptídeos/química , Peptídeos/metabolismo , Regeneração
11.
J Biol Chem ; 290(7): 4330-42, 2015 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-25538237

RESUMO

Matrix metalloproteinases and a disintegrin and metalloproteinases are members of the zinc endopeptidases, which cleave components of the extracellular matrix as well as cell surface proteins resulting in degradation or release of biologically active fragments. Surface ectodomain shedding affects numerous biological processes, including survival, axon outgrowth, axon guidance, and synaptogenesis. In this study, we evaluated the role of metalloproteinases in regulating cortical neurite growth. We found that treatment of mature cortical neurons with pan-metalloproteinase inhibitors or with tissue inhibitors of metalloproteinase-3 reduced neurite outgrowth. Through mass spectrometry, we characterized the metalloproteinase-sensitive cell surface proteome of mature cortical neurons. Members of the IgLON family of glycosylphosphatidylinositol-anchored neural cell adhesion molecules were identified and validated as proteins that were shed from the surface of mature cortical neurons in a metalloproteinase-dependent manner. Introduction of two members of the IgLON family, neurotrimin and NEGR1, in early embryonic neurons was sufficient to confer sensitivity to metalloproteinase inhibitors in neurite outgrowth assays. Outgrowth experiments on immobilized IgLON proteins revealed a role for all IgLON family members in promoting neurite extension from cortical neurons. Together, our findings support a role for metalloproteinase-dependent shedding of IgLON family members in regulating neurite outgrowth from mature cortical neurons.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Membrana Celular/metabolismo , Córtex Cerebral/citologia , Metaloproteases/metabolismo , Neurogênese/fisiologia , Neurônios/citologia , Animais , Apoptose , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Córtex Cerebral/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Immunoblotting , Técnicas Imunoenzimáticas , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Proteômica , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
12.
Exp Eye Res ; 146: 304-312, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27072342

RESUMO

This protocol outlines the preparation of embryonic mouse retinal explants, which provides an effective technique to analyze neurite outgrowth in central nervous system (CNS) neurons. This validated ex vivo system, which displays limited neuronal death, is highly reproducible and particularly amenable to manipulation. Our previously published studies involving embryonic chick or adult mouse retinal explants were instrumental in the preparation of this protocol; aspects of these previous techniques were combined, adopted and optimized. This protocol thus permits more efficient analysis of neurite growth. Briefly, the retina is dissected from the embryonic mouse eye using precise techniques that take into account the small size of the embryonic eye. The approach applied ensures that the retinal ganglion cell (RGC) layer faces the adhesion substrate on coated cover slips. Neurite growth is clear, well-delineated and readily quantifiable. These retinal explants can therefore be used to examine the neurite growth effects elicited by potential therapeutic agents.


Assuntos
Neuritos/patologia , Retina/embriologia , Células Ganglionares da Retina/citologia , Técnicas de Cultura de Tecidos/métodos , Análise de Variância , Animais , Modelos Animais de Doenças , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuritos/efeitos dos fármacos , Neurogênese , Retina/efeitos dos fármacos , Doenças Retinianas/tratamento farmacológico
13.
J Biol Chem ; 289(43): 30133-43, 2014 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-25225289

RESUMO

Coordinated control of the growth cone cytoskeleton underlies axon extension and guidance. Members of the collapsin response mediator protein (CRMP) family of cytosolic phosphoproteins regulate the microtubule and actin cytoskeleton, but their roles in regulating growth cone dynamics remain largely unexplored. Here, we examine how CRMP4 regulates the growth cone cytoskeleton. Hippocampal neurons from CRMP4-/- mice exhibited a selective decrease in axon extension and reduced growth cone area, whereas overexpression of CRMP4 enhanced the formation and length of growth cone filopodia. Biochemically, CRMP4 can impact both microtubule assembly and F-actin bundling in vitro. Through a structure function analysis of CRMP4, we found that the effects of CRMP4 on axon growth and growth cone morphology were dependent on microtubule assembly, whereas filopodial extension relied on actin bundling. Intriguingly, anterograde movement of EB3 comets, which track microtubule protrusion, slowed significantly in neurons derived from CRMP4-/- mice, and rescue of microtubule dynamics required CRMP4 activity toward both the actin and microtubule cytoskeleton. Together, this study identified a dual role for CRMP4 in regulating the actin and microtubule growth cone cytoskeleton.


Assuntos
Citoesqueleto de Actina/metabolismo , Cones de Crescimento/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Axônios/metabolismo , Tamanho Celular , Feminino , Hipocampo/citologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/deficiência , Estrutura Terciária de Proteína , Tubulina (Proteína)/metabolismo
14.
J Neurochem ; 129(2): 206-12, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24147810

RESUMO

During development, dynamic changes in the axonal growth cone and dendrite are necessary for exploratory movements underlying initial axo-dendritic contact and ultimately the formation of a functional synapse. In the adult central nervous system, an impressive degree of plasticity is retained through morphological and molecular rearrangements in the pre- and post-synaptic compartments that underlie the strengthening or weakening of synaptic pathways. Plasticity is regulated by the interplay of permissive and inhibitory extracellular cues, which signal through receptors at the synapse to regulate the closure of critical periods of developmental plasticity as well as by acute changes in plasticity in response to experience and activity in the adult. The molecular underpinnings of synaptic plasticity are actively studied and it is clear that the cytoskeleton is a key substrate for many cues that affect plasticity. Many of the cues that restrict synaptic plasticity exhibit residual activity in the injured adult CNS and restrict regenerative growth by targeting the cytoskeleton. Here, we review some of the latest insights into how cytoskeletal remodeling affects neuronal plasticity and discuss how the cytoskeleton is being targeted in an effort to promote plasticity and repair following traumatic injury in the central nervous system.


Assuntos
Citoesqueleto/fisiologia , Regeneração Nervosa/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Citoesqueleto de Actina/fisiologia , Actinas/fisiologia , Humanos
15.
Mol Cell Neurosci ; 49(1): 68-76, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21971580

RESUMO

Axonal damage can occur in the central nervous system following trauma, during the course of autoimmune and neurodegenerative disease and during viral and bacterial infections. The degree of axonal damage and absence of spontaneous repair are major determinants of long-term clinical outcome. While inflammation is a common feature of these conditions, the impact of particular immune cell subsets and their products on injured axons is not fully known. To investigate the impact of immune cells on neuronal viability and axonal repair, we developed an in vitro culture system in which neurons are exposed to mixed or distinct immune cell subsets. We find that total peripheral blood mononuclear cells (PBMCs) have a significant inhibitory effect on neurite outgrowth that is independent of apoptosis. Using isolated immune cells subsets, we demonstrate that activated CD4+ T cells enhance neurite outgrowth while activated NK cells and CD8+ T cells inhibit neurite outgrowth. We find that NK cell inhibition of neuronal outgrowth is dependent on MAPK activity. Our findings describe heterogeneous effects of individual immune cell subsets on neuronal growth and offer important insights into the cellular and molecular mechanisms that may impact axonal repair in inflammatory CNS conditions.


Assuntos
Células Matadoras Naturais/imunologia , Neuritos/fisiologia , Linfócitos T/imunologia , Adulto , Animais , Axônios/fisiologia , Células Cultivadas , Sistema Nervoso Central/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células Matadoras Naturais/fisiologia , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/imunologia , Neurônios/citologia , Ratos , Linfócitos T/fisiologia
16.
WIREs Mech Dis ; 15(2): e1594, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36600404

RESUMO

Central nervous system (CNS) inflammation is a key factor in multiple sclerosis (MS). Invasion of peripheral immune cells into the CNS resulting from an unknown signal or combination of signals results in activation of resident immune cells and the hallmark feature of the disease: demyelinating lesions. These lesion sites are an amalgam of reactive peripheral and central immune cells, astrocytes, damaged and dying oligodendrocytes, and injured neurons and axons. Sustained inflammation affects cells directly located within the lesion site and further abnormalities are apparent diffusely throughout normal-appearing white matter and grey matter. It is only relatively recently, using animal models, new tissue sampling techniques, and next-generation sequencing, that molecular changes occurring in CNS resident cells have been broadly captured. Advances in cell isolation through Fluorescence Activated Cell Sorting (FACS) and laser-capture microdissection together with the emergence of single-cell sequencing have enabled researchers to investigate changes in gene expression in astrocytes, microglia, and oligodendrocytes derived from animal models of MS as well as from primary patient tissue. The contribution of some dysregulated pathways has been followed up in individual studies; however, corroborating results often go unreported between sequencing studies. To this end, we have consolidated results from numerous RNA-sequencing studies to identify and review novel patterns of differentially regulated genes and pathways occurring within CNS glial cells in MS. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.


Assuntos
Esclerose Múltipla , Substância Branca , Animais , Esclerose Múltipla/genética , Microglia/metabolismo , Astrócitos/metabolismo , Substância Branca/metabolismo , Inflamação/genética , RNA/metabolismo , Oligodendroglia/metabolismo
17.
bioRxiv ; 2023 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-37873446

RESUMO

In multiple sclerosis (MS), the invasion of the central nervous system by peripheral immune cells is followed by the activation of resident microglia and astrocytes. This cascade of events results in demyelination, which triggers neuronal damage and death. The molecular signals in neurons responsible for this damage are not yet fully characterized. In MS, retinal ganglion cell neurons (RGCs) of the central nervous system (CNS) undergo axonal injury and cell death. This phenomenon is mirrored in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. To understand the molecular landscape, we isolated RGCs from mice subjected to the EAE protocol. RNA-sequencing and ATAC-sequencing analyses were performed. Pathway analysis of the RNA-sequencing data revealed that RGCs displayed a molecular signature, similar to aged neurons, showcasing features of senescence. Single-nucleus RNA-sequencing analysis of neurons from human MS patients revealed a comparable senescence-like phenotype., which was supported by immunostaining RGCs in EAE mice. These changes include alterations to the nuclear envelope, modifications in chromatin marks, and accumulation of DNA damage. Transduction of RGCs with an Oct4 - Sox2 - Klf4 transgene to convert neurons in the EAE model to a more youthful epigenetic and transcriptomic state enhanced the survival of RGCs. Collectively, this research uncovers a previously unidentified senescent-like phenotype in neurons under pathological inflammation and neurons from MS patients. The rejuvenation of this aged transcriptome improved visual acuity and neuronal survival in the EAE model supporting the idea that age rejuvenation therapies and senotherapeutic agents could offer a direct means of neuroprotection in autoimmune disorders.

18.
J Biol Chem ; 286(36): 31418-24, 2011 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-21768085

RESUMO

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.


Assuntos
Metaloproteinase 16 da Matriz/fisiologia , Proteínas da Mielina/metabolismo , Bainha de Mielina , Receptores de Superfície Celular/metabolismo , Animais , Células Cultivadas , Proteínas Ligadas por GPI/análise , Proteínas Ligadas por GPI/metabolismo , Humanos , Hidrólise , Metalotioneína 3 , Camundongos , Proteínas da Mielina/análise , Neurônios/citologia , Neurônios/fisiologia , Receptor Nogo 1 , Fragmentos de Peptídeos/análise , Ratos , Receptores de Superfície Celular/análise
19.
iScience ; 25(6): 104379, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35620420

RESUMO

The therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.

20.
J Neurosci ; 30(16): 5635-43, 2010 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-20410116

RESUMO

Myelin-associated inhibitors (MAIs) contribute to failed regeneration in the CNS. The intracellular signaling pathways through which MAIs block axonal repair remain largely unknown. Here, we report that the kinase GSK3beta is directly phosphorylated and inactivated by MAIs, consequently regulating protein-protein interactions that are critical for myelin-dependent inhibition. Inhibition of GSK3beta mimics the neurite outgrowth inhibitory effect of myelin. The inhibitory effects of GSK3beta inhibitors and myelin are not additive indicating that GSK3beta is a major effector of MAIs. Consistent with this, overexpression of GSK3beta attenuates myelin inhibition. MAI-dependent phosphorylation and inactivation of GSK3beta regulate phosphorylation of CRMP4, a cytosolic regulator of myelin inhibition, and its ability to complex with RhoA. Introduction of a CRMP4 antagonist attenuates the neurite outgrowth inhibitory properties of GSK3beta inhibitors. We describe the first example of GSK3beta inactivation in response to inhibitory ligands and link the neurite outgrowth inhibitory effects of GSK3beta inhibition directly to CRMP4. These findings raise the possibility that GSK3beta inhibition will not effectively promote long-distance CNS regeneration following trauma such as spinal cord injury.


Assuntos
Axônios/fisiologia , Quinase 3 da Glicogênio Sintase/fisiologia , Proteínas da Mielina/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/fisiologia , Aminofenóis/farmacologia , Animais , Axônios/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Indóis/farmacologia , Maleimidas/farmacologia , Camundongos , Proteínas da Mielina/antagonistas & inibidores , Inibição Neural/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Células PC12 , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Ratos , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP
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