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1.
Nat Commun ; 12(1): 3285, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078899

RESUMO

In peripheral nerves, Schwann cells form myelin and provide trophic support to axons. We previously showed that the mitochondrial protein prohibitin 2 can localize to the axon-Schwann-cell interface and is required for developmental myelination. Whether the homologous protein prohibitin 1 has a similar role, and whether prohibitins also play important roles in Schwann cell mitochondria is unknown. Here, we show that deletion of prohibitin 1 in Schwann cells minimally perturbs development, but later triggers a severe demyelinating peripheral neuropathy. Moreover, mitochondria are heavily affected by ablation of prohibitin 1 and demyelination occurs preferentially in cells with apparent mitochondrial loss. Furthermore, in response to mitochondrial damage, Schwann cells trigger the integrated stress response, but, contrary to what was previously suggested, this response is not detrimental in this context. These results identify a role for prohibitin 1 in myelin integrity and advance our understanding about the Schwann cell response to mitochondrial damage.


Assuntos
Nervo Femoral/metabolismo , Mitocôndrias/metabolismo , Proteínas Repressoras/genética , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Nervo Tibial/metabolismo , Animais , Aspartato-Amônia Ligase/genética , Aspartato-Amônia Ligase/metabolismo , Axônios/metabolismo , Axônios/ultraestrutura , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Nervo Femoral/patologia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/deficiência , Células de Schwann/patologia , Nervo Isquiático/patologia , Estresse Fisiológico , Nervo Tibial/patologia , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Proteína 1 de Ligação a X-Box/genética , Proteína 1 de Ligação a X-Box/metabolismo , gama-Glutamilciclotransferase/genética , gama-Glutamilciclotransferase/metabolismo
2.
Nat Commun ; 12(1): 3845, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158506

RESUMO

Atr is a serine/threonine kinase, known to sense single-stranded DNA breaks and activate the DNA damage checkpoint by phosphorylating Chek1, which inhibits Cdc25, causing cell cycle arrest. This pathway has not been implicated in neuroregeneration. We show that in Drosophila sensory neurons removing Atr or Chek1, or overexpressing Cdc25 promotes regeneration, whereas Atr or Chek1 overexpression, or Cdc25 knockdown impedes regeneration. Inhibiting the Atr-associated checkpoint complex in neurons promotes regeneration and improves synapse/behavioral recovery after CNS injury. Independent of DNA damage, Atr responds to the mechanical stimulus elicited during regeneration, via the mechanosensitive ion channel Piezo and its downstream NO signaling. Sensory neuron-specific knockout of Atr in adult mice, or pharmacological inhibition of Atr-Chek1 in mammalian neurons in vitro and in flies in vivo enhances regeneration. Our findings reveal the Piezo-Atr-Chek1-Cdc25 axis as an evolutionarily conserved inhibitory mechanism for regeneration, and identify potential therapeutic targets for treating nervous system trauma.


Assuntos
Axônios/metabolismo , Quinase 1 do Ponto de Checagem/genética , Canais Iônicos/genética , Regeneração Nervosa/genética , Animais , Animais Geneticamente Modificados , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Canais Iônicos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética
3.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073798

RESUMO

Type IIa receptor tyrosine phosphatases (RPTPs) play pivotal roles in neuronal network formation. It is emerging that the interactions of RPTPs with glycans, i.e., chondroitin sulfate (CS) and heparan sulfate (HS), are critical for their functions. We highlight here the significance of these interactions in axon regeneration and synaptogenesis. For example, PTPσ, a member of type IIa RPTPs, on axon terminals is monomerized and activated by the extracellular CS deposited in neural injuries, dephosphorylates cortactin, disrupts autophagy flux, and consequently inhibits axon regeneration. In contrast, HS induces PTPσ oligomerization, suppresses PTPσ phosphatase activity, and promotes axon regeneration. PTPσ also serves as an organizer of excitatory synapses. PTPσ and neurexin bind one another on presynapses and further bind to postsynaptic leucine-rich repeat transmembrane protein 4 (LRRTM4). Neurexin is now known as a heparan sulfate proteoglycan (HSPG), and its HS is essential for the binding between these three molecules. Another HSPG, glypican 4, binds to presynaptic PTPσ and postsynaptic LRRTM4 in an HS-dependent manner. Type IIa RPTPs are also involved in the formation of excitatory and inhibitory synapses by heterophilic binding to a variety of postsynaptic partners. We also discuss the important issue of possible mechanisms coordinating axon extension and synapse formation.


Assuntos
Axônios/metabolismo , Regeneração Nervosa , Polissacarídeos/fisiologia , Proteínas Tirosina Fosfatases Semelhantes a Receptores/fisiologia , Sinapses/metabolismo , Animais , Axônios/fisiologia , Humanos , Polissacarídeos/metabolismo , Proteínas Tirosina Fosfatases Semelhantes a Receptores/metabolismo , Sinapses/fisiologia
4.
ACS Chem Neurosci ; 12(11): 1931-1939, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34018719

RESUMO

Low-intensity pulsed ultrasound is found to be effective in axonal regeneration, while the role of ultrasound in axonal growth guidance is still unclear. This study was performed to explore the neuroprotective role of low-intensity pulsed ultrasound (US) both in vitro and in vivo. Primary cultured rat cortical neurons were subjected to 1.0 MHz ultrasound for 5 min every day at intensity of 0, 0.008, 0.12, and 0.21 W/cm2. Our results demonstrated that low-intensity pulsed ultrasound significantly increased neuronal cell viability and inhibited neuronal apoptosis in vitro as determined by fluorescein diacetate assay (FDA) and a TdT-mediated biotin-dUTP nicked-end labeling (TUNEL) assay. Moreover, low-intensity pulsed ultrasound at 0.12 W/cm2 significantly enhanced the axonal growth guidance by activation of netrin-1 and DCC (deleted in colorectal carcinoma) expression as determined by Western blots assay. More interestingly, we further found that low-intensity pulsed ultrasound treatment at 0.21 W/cm2 promoted the functional restoration of rat injured nerves in vivo, decreased hemorrhage, and reversed the injury process by activating positive netrin-1 expression as seen in the immunohistochemistry (IHC) assay. Thus, our study strongly demonstrated that low-intensity pulsed ultrasound activated netrin-1/DCC signaling and further mediated neurite outgrowth. It would be a new approach to nerve regeneration in the future.


Assuntos
Orientação de Axônios , Neurônios , Animais , Axônios/metabolismo , Células Cultivadas , Receptor DCC/metabolismo , Netrina-1 , Neurônios/metabolismo , Ratos , Transdução de Sinais , Ondas Ultrassônicas
5.
Nat Commun ; 12(1): 2941, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011929

RESUMO

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin's nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method's sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures.


Assuntos
Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/ultraestrutura , Bainha de Mielina/metabolismo , Bainha de Mielina/ultraestrutura , Tomografia Computadorizada por Raios X/métodos , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Sistema Nervoso Central/diagnóstico por imagem , Pré-Escolar , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas da Mielina/metabolismo , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Neuroimagem/métodos , Estudo de Prova de Conceito , Espalhamento a Baixo Ângulo , Medula Espinal/metabolismo , Medula Espinal/ultraestrutura
6.
Nat Commun ; 12(1): 2555, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953205

RESUMO

Transcription factors (TFs) act as powerful levers to regulate neural physiology and can be targeted to improve cellular responses to injury or disease. Because TFs often depend on cooperative activity, a major challenge is to identify and deploy optimal sets. Here we developed a bioinformatics pipeline, centered on TF co-occupancy of regulatory DNA, and used it to predict factors that potentiate the effects of pro-regenerative Klf6 in vitro. High content screens of neurite outgrowth identified cooperative activity by 12 candidates, and systematic testing in a mouse model of corticospinal tract (CST) damage substantiated three novel instances of pairwise cooperation. Combined Klf6 and Nr5a2 drove the strongest growth, and transcriptional profiling of CST neurons identified Klf6/Nr5a2-responsive gene networks involved in macromolecule biosynthesis and DNA repair. These data identify TF combinations that promote enhanced CST growth, clarify the transcriptional correlates, and provide a bioinformatics approach to detect TF cooperation.


Assuntos
Axônios/metabolismo , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Biologia Computacional , DNA , Reparo do DNA , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Fator 6 Semelhante a Kruppel/genética , Fator 6 Semelhante a Kruppel/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Tratos Piramidais/metabolismo , Ratos Sprague-Dawley , Receptores Citoplasmáticos e Nucleares/metabolismo , Transcriptoma
7.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810342

RESUMO

The retinal ganglion cells (RGC) may be considered an easily accessible pathophysiological site of degenerative processes in neurological diseases, such as the RGC damage detectable in multiple sclerosis (MS) patients with (HON) and without a history of optic neuritis (NON). We aimed to assess and interrelate RGC functional and structural damage in different retinal layers and retinal sites. We included 12 NON patients, 11 HON patients and 14 healthy controls for cross-sectional multifocal pattern electroretinography (mfPERG) and optical coherence tomography (OCT) measurements. Amplitude and peak times of the mfPERG were assessed. Macula and disc OCT scans were acquired to determine macular retinal layer and peripapillary retinal nerve fiber layer (pRNFL) thickness. In both HON and NON patients the foveal N2 amplitude of the mfPERG was reduced compared to controls. The parafoveal P1 peak time was significantly reduced in HON only. For OCT, parafoveal (pfGCL) and perifoveal (pGCL) ganglion cell layer thicknesses were decreased in HON vs. controls, while pRNFL in the papillomacular bundle sector (PMB) showed reductions in both NON and HON. As the mfPERG derived N2 originates from RGC axons, these findings suggest foveal axonal dysfunction not only in HON, but also in NON patients.


Assuntos
Esclerose Múltipla/metabolismo , Neurite Óptica/metabolismo , Células Ganglionares da Retina/metabolismo , Adulto , Algoritmos , Axônios/metabolismo , Estudos de Casos e Controles , Eletrorretinografia , Feminino , Humanos , Macula Lutea/metabolismo , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/diagnóstico por imagem , Disco Óptico/diagnóstico por imagem , Estudos Prospectivos , Recidiva , Relação Estrutura-Atividade , Tomografia de Coerência Óptica , Adulto Jovem
8.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810425

RESUMO

Motor neurons and their axons reaching the skeletal muscle have long been considered as the best characterized targets of the degenerative process observed in amyotrophic lateral sclerosis (ALS). However, the involvement of glial cells was also more recently reported. Although oligodendrocytes have been underestimated for a longer time than other cells, they are presently considered as critically involved in axonal injury and also conversely constitute a target for the toxic effects of the degenerative neurons. In the present review, we highlight the recent advances regarding oligodendroglial cell involvement in the pathogenesis of ALS. First, we present the oligodendroglial cells, the process of myelination, and the tight relationship between axons and myelin. The histological abnormalities observed in ALS and animal models of the disease are described, including myelin defects and oligodendroglial accumulation of pathological protein aggregates. Then, we present data that establish the existence of dysfunctional and degenerating oligodendroglial cells, the chain of events resulting in oligodendrocyte degeneration, and the most recent molecular mechanisms supporting oligodendrocyte death and dysfunction. Finally, we review the arguments in support of the primary versus secondary involvement of oligodendrocytes in the disease and discuss the therapeutic perspectives related to oligodendrocyte implication in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Doenças Desmielinizantes/fisiopatologia , Oligodendroglia/citologia , Animais , Astrócitos/citologia , Axônios/metabolismo , Morte Celular , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/química , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Neurônios Motores/metabolismo , Bainha de Mielina/metabolismo , Neuroglia/metabolismo , Oxigênio/química , Prosencéfalo/metabolismo , Ratos , Medula Espinal/metabolismo , Superóxido Dismutase-1/metabolismo
9.
Front Immunol ; 12: 533423, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33815353

RESUMO

Voltage gated sodium (Nav) channels contribute to axonal damage following demyelination in experimental autoimmune encephalomyelitis (EAE), a rodent model of multiple sclerosis (MS). The Nav1.6 isoform has been implicated as a primary contributor in this process. However, the role of Nav1.6 in immune processes, critical to the pathology of both MS and EAE, has not been extensively studied. EAE was induced with myelin oligodendrocyte (MOG35-55) peptide in Scn8admu/+ mice, which have reduced Nav1.6 levels. Scn8admu/+ mice demonstrated improved motor capacity during the recovery and early chronic phases of EAE relative to wild-type animals. In the optic nerve, myeloid cell infiltration and the effects of EAE on the axonal ultrastructure were also significantly reduced in Scn8admu/+ mice. Analysis of innate immune parameters revealed reduced plasma IL-6 levels and decreased percentages of Gr-1high/CD11b+ and Gr-1int/CD11b+ myeloid cells in the blood during the chronic phase of EAE in Scn8admu/+ mice. Elevated levels of the anti-inflammatory cytokines IL-10, IL-13, and TGF-ß1 were also observed in the brains of untreated Scn8admu/+ mice. A lipopolysaccharide (LPS) model was used to further evaluate inflammatory responses. Scn8admu/+ mice displayed reduced inflammation in response to LPS challenge. To further evaluate if this was an immune cell-intrinsic difference or the result of changes in the immune or hormonal environment, mast cells were derived from the bone marrow of Scn8admu/+ mice. These mast cells also produced lower levels of IL-6, in response to LPS, compared with those from wild type mice. Our results demonstrate that in addition to its recognized impact on axonal damage, Nav1.6 impacts multiple aspects of the innate inflammatory response.


Assuntos
Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/genética , Inflamação/genética , Esclerose Múltipla/genética , Canal de Sódio Disparado por Voltagem NAV1.6/genética , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Citocinas/sangue , Citocinas/metabolismo , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Expressão Gênica , Heterozigoto , Humanos , Inflamação/metabolismo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Biophys J ; 120(11): 2085-2101, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33812847

RESUMO

Neural function depends on continual synthesis and targeted trafficking of intracellular components, including ion channel proteins. Many kinds of ion channels are trafficked over long distances to specific cellular compartments. This raises the question of whether cargo is directed with high specificity during transit or whether cargo is distributed widely and sequestered at specific sites. We addressed this question by experimentally measuring transport and expression densities of Kv4.2, a voltage-gated transient potassium channel that exhibits a specific dendritic expression that increases with distance from the soma and little or no functional expression in axons. In over 500 h of quantitative live imaging, we found substantially higher densities of actively transported Kv4.2 subunits in axons as opposed to dendrites. This paradoxical relationship between functional expression and traffic density supports a model-commonly known as the sushi belt model-in which trafficking specificity is relatively low and active sequestration occurs in compartments where cargo is expressed. In further support of this model, we find that kinetics of active transport differs qualitatively between axons and dendrites, with axons exhibiting strong superdiffusivity, whereas dendritic transport resembles a weakly directed random walk, promoting mixing and opportunity for sequestration. Finally, we use our data to constrain a compartmental reaction-diffusion model that can recapitulate the known Kv4.2 density profile. Together, our results show how nontrivial expression patterns can be maintained over long distances with a relatively simple trafficking mechanism and how the hallmarks of a global trafficking mechanism can be revealed in the kinetics and density of cargo.


Assuntos
Dendritos , Canais de Potássio Shal , Axônios/metabolismo , Transporte Biológico Ativo , Dendritos/metabolismo , Neurônios/metabolismo , Transporte Proteico , Canais de Potássio Shal/metabolismo
11.
Int J Mol Sci ; 22(9)2021 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-33925390

RESUMO

Decussation of axonal tracts is an important hallmark of vertebrate neuroanatomy resulting in one brain hemisphere controlling the contralateral side of the body and also computing the sensory information originating from that respective side. Here, we show that BMP interferes with optic chiasm formation and RGC pathfinding in zebrafish. Experimental induction of BMP4 at 15 hpf results in a complete ipsilateral projection of RGC axons and failure of commissural connections of the forebrain, in part as the result of an interaction with shh signaling, transcriptional regulation of midline guidance cues and an affected optic stalk morphogenesis. Experimental induction of BMP4 at 24 hpf, resulting in only a mild repression of forebrain shh ligand expression but in a broad expression of pax2a in the diencephalon, does not per se prevent RGC axons from crossing the midline. It nevertheless shows severe pathologies of RGC projections e.g., the fasciculation of RGC axons with the ipsilateral optic tract resulting in the innervation of one tectum by two eyes or the projection of RGC axons in the direction of the contralateral eye.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Quiasma Óptico/embriologia , Células Ganglionares da Retina/metabolismo , Animais , Axônios/metabolismo , Proteínas Morfogenéticas Ósseas/fisiologia , Quiasma Óptico/metabolismo , Quiasma Óptico/fisiologia , Nervo Óptico/fisiologia , Retina/metabolismo , Células Ganglionares da Retina/fisiologia , Vias Visuais/fisiologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismo
12.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923565

RESUMO

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.


Assuntos
Axônios/metabolismo , Magnetossomos/metabolismo , Crescimento Neuronal , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Transporte Biológico , Células Cultivadas , Feminino , Hipocampo/citologia , Magnetospirillum/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Mecânico
13.
Int J Mol Sci ; 22(7)2021 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-33806166

RESUMO

Recovery from axonal injury is extremely difficult, especially for adult neurons. Here, we demonstrate that the activation of G-protein coupled receptor 110 (GPR110, ADGRF1) is a mechanism to stimulate axon growth after injury. N-docosahexaenoylethanolamine (synaptamide), an endogenous ligand of GPR110 that promotes neurite outgrowth and synaptogenesis in developing neurons, and a synthetic GPR110 ligand stimulated neurite growth in axotomized cortical neurons and in retinal explant cultures. Intravitreal injection of GPR110 ligands following optic nerve crush injury promoted axon extension in adult wild-type, but not in gpr110 knockout, mice. In vitro axotomy or in vivo optic nerve injury rapidly induced the neuronal expression of gpr110. Activating the developmental mechanism of neurite outgrowth by specifically targeting GPR110 that is upregulated upon injury may provide a novel strategy for stimulating axon growth after nerve injury in adults.


Assuntos
Axônios/metabolismo , Etanolaminas/farmacologia , Regeneração Nervosa , Receptores Acoplados a Proteínas G/metabolismo , Animais , Feminino , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microfluídica , Simulação de Acoplamento Molecular , Compressão Nervosa , Neurogênese , Neurônios/metabolismo , Nervo Óptico/metabolismo , Retina/metabolismo
14.
Int J Mol Sci ; 22(5)2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33801205

RESUMO

Following an injury, axons of both the central nervous system (CNS) and peripheral nervous system (PNS) degenerate through a coordinated and genetically conserved mechanism known as Wallerian degeneration (WD). Unlike central axons, severed peripheral axons have a higher capacity to regenerate and reinnervate their original targets, mainly because of the favorable environment that they inhabit and the presence of different cell types. Even though many aspects of regeneration in peripheral nerves have been studied, there is still a lack of understanding regarding the dynamics of axonal degeneration and regeneration, mostly due to the inherent limitations of most animal models. In this scenario, the use of zebrafish (Danio rerio) larvae combined with time-lapse microscopy currently offers a unique experimental opportunity to monitor the dynamics of the regenerative process in the PNS in vivo. This review summarizes the current knowledge and advances made in understanding the dynamics of the regenerative process of PNS axons. By using different tools available in zebrafish such as electroablation of the posterior lateral line nerve (pLLn), and laser-mediated transection of motor and sensory axons followed by time-lapse microscopy, researchers are beginning to unravel the complexity of the spatiotemporal interactions among different cell types during the regenerative process. Thus, understanding the cellular and molecular mechanisms underlying the degeneration and regeneration of peripheral nerves will open new avenues in the treatment of acute nerve trauma or chronic conditions such as neurodegenerative diseases.


Assuntos
Axônios/metabolismo , Regeneração Nervosa , Neuroglia/metabolismo , Traumatismos dos Nervos Periféricos/patologia , Peixe-Zebra/fisiologia , Animais , Traumatismos dos Nervos Periféricos/terapia
15.
J Cell Sci ; 134(7)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33674450

RESUMO

The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3' untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.


Assuntos
Axônios , Isoformas de RNA , Axônios/metabolismo , Lipoilação , Regeneração Nervosa , Isoformas de RNA/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo
16.
Front Immunol ; 12: 570425, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33732230

RESUMO

Peroxisome proliferator-activated receptor (PPAR)-δ is a nuclear receptor that functions to maintain metabolic homeostasis, regulate cell growth, and limit the development of excessive inflammation during immune responses. Previously, we reported that PPAR-δ-deficient mice develop a more severe clinical course of experimental autoimmune encephalomyelitis (EAE); however, it was difficult to delineate the role that microglia played in this disease phenotype since PPAR-δ-deficient mice exhibited a number of immune defects that enhanced CNS inflammation upstream of microglia activation. Here, we specifically investigated the role of PPAR-δ in microglia during EAE by using mice where excision of a floxed Ppard allele was driven by expression of a tamoxifen (TAM)-inducible CX3C chemokine receptor 1 promoter-Cre recombinase transgene (Cx3cr1 CreERT2: Ppard fl/fl). We observed that by 30 days of TAM treatment, Cx3cr1 CreERT2: Ppard fl/fl mice exhibited Cre-mediated deletion primarily in microglia and this was accompanied by efficient knockdown of Ppard expression in these cells. Upon induction of EAE, TAM-treated Cx3cr1 CreERT2: Ppard fl/fl mice presented with an exacerbated course of disease compared to TAM-treated Ppard fl/fl controls. Histopathological and magnetic resonance (MR) studies on the spinal cord and brains of EAE mice revealed increased Iba-1 immunoreactivity, axonal injury and CNS tissue loss in the TAM-treated Cx3cr1 CreERT2: Ppard fl/fl group compared to controls. In early EAE, a time when clinical scores and the infiltration of CD45+ leukocytes was equivalent between Cx3cr1 CreERT2: Ppard fl/fl and Ppard fl/fl mice, Ppard-deficient microglia exhibited a more reactive phenotype as evidenced by a shorter maximum process length and lower expression of genes associated with a homeostatic microglia gene signature. In addition, Ppard-deficient microglia exhibited increased expression of genes associated with reactive oxygen species generation, phagocytosis and lipid clearance, M2-activation, and promotion of inflammation. Our results therefore suggest that PPAR-δ has an important role in microglia in limiting bystander tissue damage during neuroinflammation.


Assuntos
Axônios/metabolismo , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Microglia/imunologia , Microglia/metabolismo , PPAR delta/deficiência , Animais , Axônios/patologia , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/diagnóstico , Ativação Linfocitária/imunologia , Imageamento por Ressonância Magnética , Camundongos , Camundongos Knockout , Microglia/patologia , Índice de Gravidade de Doença , Linfócitos T/imunologia , Linfócitos T/metabolismo
17.
eNeuro ; 8(2)2021.
Artigo em Inglês | MEDLINE | ID: mdl-33771901

RESUMO

Elaboration of neuronal processes is an early step in neuronal development. Guidance cues must work closely with intracellular trafficking pathways to direct expanding axons and dendrites to their target neurons during the formation of neuronal networks. However, how such coordination is achieved remains incompletely understood. Here, we characterize an interaction between fasciculation and elongation protein zeta 1 (FEZ1), an adapter involved in synaptic protein transport, and collapsin response mediator protein (CRMP)1, a protein that functions in growth cone guidance, at neuronal growth cones. We show that similar to CRMP1 loss-of-function mutants, FEZ1 deficiency in rat hippocampal neurons causes growth cone collapse and impairs axonal development. Strikingly, FEZ1-deficient neurons also exhibited a reduction in dendritic complexity stronger than that observed in CRMP1-deficient neurons, suggesting that the former could partake in additional developmental signaling pathways. Supporting this, FEZ1 colocalizes with VAMP2 in developing hippocampal neurons and forms a separate complex with deleted in colorectal cancer (DCC) and Syntaxin-1 (Stx1), components of the Netrin-1 signaling pathway that are also involved in regulating axon and dendrite development. Significantly, developing axons and dendrites of FEZ1-deficient neurons fail to respond to Netrin-1 or Netrin-1 and Sema3A treatment, respectively. Taken together, these findings highlight the importance of FEZ1 as a common effector to integrate guidance signaling pathways with intracellular trafficking to mediate axo-dendrite development during neuronal network formation.


Assuntos
Axônios , Receptores de Superfície Celular , Proteínas Adaptadoras de Transdução de Sinal , Animais , Axônios/metabolismo , Receptor DCC , Cones de Crescimento/metabolismo , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Ratos , Receptores de Superfície Celular/metabolismo
18.
Nat Commun ; 12(1): 1923, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33772011

RESUMO

Chronic demyelination in the human CNS is characterized by an inhibitory microenvironment that impairs recruitment and differentiation of oligodendrocyte progenitor cells (OPCs) leading to failed remyelination and axonal atrophy. By network-based transcriptomics, we identified sulfatase 2 (Sulf2) mRNA in activated human primary OPCs. Sulf2, an extracellular endosulfatase, modulates the signaling microenvironment by editing the pattern of sulfation on heparan sulfate proteoglycans. We found that Sulf2 was increased in demyelinating lesions in multiple sclerosis and was actively secreted by human OPCs. In experimental demyelination, elevated OPC Sulf1/2 expression directly impaired progenitor recruitment and subsequent generation of oligodendrocytes thereby limiting remyelination. Sulf1/2 potentiates the inhibitory microenvironment by promoting BMP and WNT signaling in OPCs. Importantly, pharmacological sulfatase inhibition using PI-88 accelerated oligodendrocyte recruitment and remyelination by blocking OPC-expressed sulfatases. Our findings define an important inhibitory role of Sulf1/2 and highlight the potential for modulation of the heparanome in the treatment of chronic demyelinating disease.


Assuntos
Diferenciação Celular/genética , Microambiente Celular/genética , Doenças Desmielinizantes/genética , Perfilação da Expressão Gênica/métodos , Células Precursoras de Oligodendrócitos/metabolismo , Remielinização/genética , Animais , Axônios/metabolismo , Células Cultivadas , Doenças Desmielinizantes/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Células Precursoras de Oligodendrócitos/citologia , Sulfatases/genética , Sulfatases/metabolismo , Sulfotransferases/genética , Sulfotransferases/metabolismo
19.
Curr Protoc ; 1(3): e64, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33657273

RESUMO

Monitoring Na+ influx in the axon initial segment (AIS) at high spatial and temporal resolution is fundamental to understanding the generation of an action potential (AP). Here, we present protocols to obtain this measurement, focusing on the AIS of layer 5 (L5) somatosensory cortex pyramidal neurons in mouse brain slices. We first outline how to prepare slices for this application, how to select and patch neurons, and how to optimize the image acquisition. Specifically, we describe the preparation of optimal slices, patching and loading of L5 pyramidal neurons with the Na+ indicator ING-2, and Na+ imaging at 100 µs temporal resolution with a pixel resolution of half a micron. Then, we present a data analysis strategy in order to extract information on the kinetics of activated voltage-gated Na+ channels by determining the change in Na+ by compensating for bleaching and calculating the time derivative of the resulting fit. In sum, this approach can be widely applied when investigating the function of Na+ channels during initiation of an AP and propagation under physiological or pathological conditions in neuronal subtypes. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Preparation of cortical slices Basic Protocol 2: Selection, patching, and Na+ fluorescence recording of a neuron Support Protocol: Calibrating Na+ fluorescence Basic Protocol 3: Data analysis.


Assuntos
Segmento Inicial do Axônio , Animais , Segmento Inicial do Axônio/metabolismo , Axônios/metabolismo , Camundongos , Neurônios/metabolismo , Sódio/metabolismo , Canais de Sódio/metabolismo , Córtex Somatossensorial/metabolismo
20.
Nat Genet ; 53(4): 445-454, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33686288

RESUMO

Psychiatric disorders are highly genetically correlated, but little research has been conducted on the genetic differences between disorders. We developed a new method (case-case genome-wide association study; CC-GWAS) to test for differences in allele frequency between cases of two disorders using summary statistics from the respective case-control GWAS, transcending current methods that require individual-level data. Simulations and analytical computations confirm that CC-GWAS is well powered with effective control of type I error. We applied CC-GWAS to publicly available summary statistics for schizophrenia, bipolar disorder, major depressive disorder and five other psychiatric disorders. CC-GWAS identified 196 independent case-case loci, including 72 CC-GWAS-specific loci that were not significant at the genome-wide level in the input case-control summary statistics; two of the CC-GWAS-specific loci implicate the genes KLF6 and KLF16 (from the Krüppel-like family of transcription factors), which have been linked to neurite outgrowth and axon regeneration. CC-GWAS loci replicated convincingly in applications to datasets with independent replication data.


Assuntos
Transtorno Bipolar/genética , Transtorno Depressivo Maior/genética , Frequência do Gene , Loci Gênicos , Fator 6 Semelhante a Kruppel/genética , Fatores de Transcrição Kruppel-Like/genética , Esquizofrenia/genética , Alelos , Axônios/metabolismo , Axônios/patologia , Transtorno Bipolar/metabolismo , Transtorno Bipolar/fisiopatologia , Estudos de Casos e Controles , Conjuntos de Dados como Assunto , Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/fisiopatologia , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Fator 6 Semelhante a Kruppel/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Crescimento Neuronal/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia
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