RESUMO
FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.
Assuntos
Adenosina/análogos & derivados , Esclerose Lateral Amiotrófica/genética , Núcleo Celular/metabolismo , Metiltransferases/antagonistas & inibidores , Proteína FUS de Ligação a RNA/metabolismo , Adenosina/farmacologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Metilação , Mutação , Agregados Proteicos , Proteína FUS de Ligação a RNA/genética , Frações Subcelulares/metabolismo , Fatores de TempoRESUMO
Z-DNA binding protein 1 (ZBP1) is a cytosolic nucleic acid sensor, functioning as a critical mediator of inflammation and cell death pathways. Since neuroinflammation could occur in response to damage-associated molecular patterns (DAMPs), ZBP1 might be involved in neuroinflammation after stroke. However, the spatiotemporal expression profile of ZBP1 in the post-stroke brain remains to be elucidated. The aim of this study is to demonstrate the spatiotemporal expression patterns of ZBP1 in the post-stroke brain using a mouse photothrombotic stroke model. Real-time PCR assays showed that ZBP1 is induced on days 3-14 post stroke. ZBP1 immunoreactivity was observed in Iba1-positive microglia/macrophages in peri-infarct regions by immunohistochemistry. ZBP1-positive cells were spread in layers surrounding the infarct core by 7-14 days post stroke. Interestingly, ZBP1 immunoreactivity was also detected in CD206-positive border-associated macrophages (BAMs) in the meninges. Furthermore, ZBP1-expressing cells were positive for antibodies against inflammatory mediators such as Toll-like receptor 4 (TLR4), Toll/IL-1R domain-containing adaptor-inducing IFN-ß (TRIF), and receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Morphological analysis with confocal microscopy showed that the co-localization signals of ZBP1 and its adaptor, TRIF, are increased by glucose oxidase (GOx) treatment, which has been reported to induce mitochondrial DNA (mtDNA) release. These results suggest that ZBP1 is induced in peri-infarct microglia/macrophages and may be involved in DAMPs-mediated neuroinflammation involving mtDNA in the post-infarct brain.
Assuntos
Doenças Neuroinflamatórias , Proteínas de Ligação a RNA , Acidente Vascular Cerebral , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Encéfalo/metabolismo , DNA Mitocondrial , Doenças Neuroinflamatórias/metabolismo , Acidente Vascular Cerebral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Camundongos , Modelos Animais de Doenças , Infarto Encefálico/metabolismoRESUMO
BACKGROUND: Chronic pain is a highly refractory and complicated condition that persists even without nociception. Several genome-wide gene expression analyses have shown that the immune response and inflammatory cytokines affect chronic pain establishment in the acute pain phase. However, compared with the acute phase, the chronic phase has a poorly elucidated gene expression profile. This study aimed to determine the gene expression profile in the spinal cord of a neuropathic pain mouse model in the chronic phase to elucidate the chronic pain characteristics. METHODS: We established a sciatic nerve cuff mouse model as a neuropathic pain model by placing a 2-mm section of a split PE-20 polyethylene tube around the sciatic nerve. The spinal cord was harvested at the L4-6 level at 28 postoperative days. Next, we examined differentially expressed genes (DEGs) through RNA sequencing (RNA-seq) compared with the sham group; moreover, we conducted enrichment analyses of the expressed genes. To reveal the chronic pain characteristics, we compared the gene expression profiles of the spinal cord between the acute and chronic phases in the neuropathic pain model. Among the chronic pain-related genes categorized in the dendrites, we focused on cyclin-dependent kinase-like 5 (CDKL5). We analyzed CDKL5 expression and function using real-time polymerase chain reaction (PCR), immunohistochemistry, and neurite extension assay in Neuro 2a (N2a) cells. We used three types of CDKL5 plasmids: wild type, nuclear localization signal-attached, and K42R kinase-dead CDKL5. RESULTS: We identified 403 DEGs, including 104 upregulated and 43 downregulated genes (false discovery rate < 0.01). Rather than inflammation or immune response, the most enriched terms in the chronic phase were "regulation of plasma membrane-bounded cell projection organization" and "dendrite." Real-time PCR assay confirmed increased CDKL5 expression in the ipsilateral dorsal horn. CDKL5 was broadly expressed in the ipsilateral dorsal horn across all layers. The neurite extension assay revealed that the cytoplasmic kinase function of CDKL5 was necessary for neurite outgrowth in N2a cells. CONCLUSION: RNA-seq of the spinal cord revealed that the most enriched genes during the chronic pain phase were involved in regulating axon and dendrite morphogenesis, including CDKL5. Our findings suggest that neural remodeling affects chronic pain establishment. Since patients with CDKL5 mutations have shown reduced pain perception, our findings suggest that CDKL5 in the spinal cord could result in neural remodeling during the chronic pain phase through cytoplasmic kinase activity.
Assuntos
Dor Crônica/metabolismo , Perfilação da Expressão Gênica , Neuralgia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Medula Espinal/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Proteínas Serina-Treonina Quinases/genética , Nervo Isquiático/metabolismoRESUMO
The vertical occipital fasciculus (VOF) is an association fiber tract coursing vertically at the posterolateral corner of the brain. It is re-evaluated as a major fiber tract to link the dorsal and ventral visual stream. Although previous tractography studies showed the VOF's cortical projections fall in the dorsal and ventral visual areas, the post-mortem dissection study for the validation remains limited. First, to validate the previous tractography data, we here performed the white matter dissection in post-mortem brains and demonstrated the VOF's fiber bundles coursing between the V3A/B areas and the posterior fusiform gyrus. Secondly, we analyzed the VOF's structural connectivity with diffusion tractography to link vision-associated cortical areas of the HCP MMP1.0 atlas, an updated map of the human cerebral cortex. Based on the criteria the VOF courses laterally to the inferior longitudinal fasciculus (ILF) and craniocaudally at the posterolateral corner of the brain, we reconstructed the VOF's fiber tracts and found the widespread projections to the visual cortex. These findings could suggest a crucial role of VOF in integrating visual information to link the broad visual cortex as well as in connecting the dual visual stream.
Assuntos
Lobo Occipital/anatomia & histologia , Córtex Visual/anatomia & histologia , Córtex Visual/fisiologia , Percepção Visual/fisiologia , Substância Branca/anatomia & histologia , Imagem de Tensor de Difusão , Humanos , Lobo Occipital/fisiologia , Substância Branca/fisiologiaRESUMO
The neuroinflammation in the ischemic brain could occur as sterile inflammation in response to damage-associated molecular patterns (DAMPs). However, its long-term dynamic transcriptional changes remain poorly understood. It is also unknown whether this neuroinflammation contributes to the recovery or just deteriorates the outcome. The purpose of this study is to characterize the temporal transcriptional changes in the post-stroke brain focusing on DAMPs-related genes by RNA-sequencing during the period of 28 days. We conducted the RNA-sequencing on day 1, 3, 7, 14, 28 post-stroke in the mouse photothrombosis model. The gross morphological observation showed the ischemic lesion on the ipsilateral cortex turned into a scar with the clearance of cellular debris by day 28. The transcriptome analyses indicated that post-stroke period of 28 days was classified into four categories (I Baseline, II Acute, III Sub-acute-#1, IV Sub-acute-#2 phase). During this period, the well-known genes for DAMPs, receptors, downstream cascades, pro-inflammatory cytokines, and phagocytosis were transcriptionally increased. The gene ontology (GO) analysis of biological process indicated that differentially expressed genes (DEGs) are genetically programmed to achieve immune and inflammatory pathways. Interestingly, we found the biphasic induction of various genes, including DAMPs and pro-inflammatory factors, peaking at acute and sub-acute phases. At the sub-acute phase, we also observed the induction of genes for phagocytosis as well as regulatory and growth factors. Further, we found the activation of CREB (cAMP-response element binding protein), one of the key players for neuronal plasticity, in peri-ischemic neurons by immunohistochemistry at this phase. Taken together, these findings raise the possibility the recurrent inflammation occurs at the sub-acute phase in the post-stroke brain, which could be involved in the debris clearance as well as neural reorganization.
Assuntos
Alarminas/genética , Isquemia Encefálica/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Inflamação/genética , Acidente Vascular Cerebral/genética , Alarminas/metabolismo , Animais , Encéfalo/patologia , Isquemia Encefálica/complicações , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Ontologia Genética , Inflamação/complicações , Masculino , Camundongos Endogâmicos C57BL , Neuroglia/metabolismo , Neuroglia/patologia , Acidente Vascular Cerebral/complicações , Fatores de Tempo , Transcriptoma/genética , Regulação para Cima/genéticaRESUMO
Although myelin-associated neurite outgrowth inhibitors express their effects through RhoA/Rho-kinase, the downstream targets of Rho-kinase remain unknown. We examined the involvement of myosin II, which is one of the downstream targets of Rho-kinase, by using blebbistatin - a specific myosin II inhibitor - and small interfering RNA targeting two myosin II isoforms, namely, MIIA and MIIB. We found that neurite outgrowth inhibition by repulsive guidance molecule (RGMa) was mediated via myosin II, particularly MIIA, in cerebellar granule neurons. RGMa induced myosin light chain (MLC) phosphorylation by a Rho-kinase-dependent mechanism. After spinal cord injury in rats, phosphorylated MLC in axons around the lesion site was up-regulated, and this effect depends on Rho-kinase activity. Further, RGMa-induced F-actin reduction in growth cones and growth cone collapse were mediated by MIIA. We conclude that Rho-kinase-dependent activation of MIIA via MLC phosphorylation induces F-actin reduction and growth cone collapse and the subsequent neurite retraction/outgrowth inhibition triggered by RGMa.
Assuntos
Miosina Tipo II/fisiologia , Inibição Neural/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Actinas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Cerebelo/citologia , Feminino , Proteínas Ligadas por GPI , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Imunoprecipitação/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Laminectomia/métodos , Glicoproteínas de Membrana/farmacologia , Cadeias Leves de Miosina/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Inibição Neural/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Transfecção/métodos , Quinases Associadas a rho/metabolismoRESUMO
Inflammatory pain, characterized by a decrease in the nociceptive threshold, arises through the actions of inflammatory mediators, and one of the key molecules is nerve growth factor (NGF). Here we report that the administration of neutralizing antibody to the neurotrophin receptor p75 (p75(NTR)) blocks hyperalgesia, which develops with complete Freund's adjuvant (CFA)-induced inflammation or with an intraplantar injection of NGF. Although CFA injection results in the up-regulation of calcitonin gene-related peptide (CGRP) levels in the primary sensory neurons, blocking p75(NTR) abolishes this effect. We further demonstrate that pro-NGF is the predominant ligand of p75(NTR) in vivo. Plasmin treatment, which is intended to decompose pro-NGF, ameliorates CFA-induced hyperalgesia. In addition, an intraplantar injection of pro-NGF induces hyperalgesia. These data together suggest that pro-NGF, as well as mature NGF, binding to p75(NTR) plays an important role in inflammation-induced hyperalgesia. Interference in the binding may provide a therapeutic approach for the treatment of inflammatory pain.
Assuntos
Hiperalgesia/metabolismo , Inflamação/metabolismo , Fator de Crescimento Neural/metabolismo , Nociceptores/metabolismo , Receptor de Fator de Crescimento Neural/metabolismo , Células Receptoras Sensoriais/metabolismo , Animais , Anticorpos/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Modelos Animais de Doenças , Fibrinolisina/farmacologia , Adjuvante de Freund/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/fisiopatologia , Hiperalgesia/induzido quimicamente , Hiperalgesia/fisiopatologia , Inflamação/induzido quimicamente , Inflamação/fisiopatologia , Mediadores da Inflamação/farmacologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fator de Crescimento Neural/farmacologia , Nociceptores/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Wistar , Receptor de Fator de Crescimento Neural/agonistas , Receptor de Fator de Crescimento Neural/antagonistas & inibidores , Células Receptoras Sensoriais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Ischemic stroke is a devastating neural event as currently no therapies other than physical rehabilitation are available to enhance recovery after stroke. To identify endogenous mediators to repair stroke brain, we performed the expression profiling analysis of transcripts in the mouse photothrombotic stroke brain. Based on real-time PCR analysis, we found VGF, identified as a nerve growth factor (NGF)-regulated transcript, was induced transcriptionally in stroke brain at 1-7 days after insult. The immunoreactivites of VGF were observed in the neurons around the ischemic core of stroke brain. Experiments with various inhibitors and plasmid transfections indicated that cAMP response element binding protein-mediated complex signaling pathways are possibly implicated in the NGF-mediated VGF expressions in vitro. Furthermore, the over-expression of VGF promoted neurite extensions and conferred protections from ischemic stress in vitro. These findings raise the possibility the application of VGF could be one of the promising therapeutic strategies to enhance recovery after stroke.
Assuntos
Encéfalo/patologia , Fatores de Crescimento Neural/metabolismo , Neuritos/fisiologia , Acidente Vascular Cerebral/metabolismo , Análise de Variância , Animais , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Trombose Intracraniana/complicações , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/farmacologia , Fatores de Crescimento Neural/genética , Células PC12 , Fosfopiruvato Hidratase/metabolismo , RNA Mensageiro/metabolismo , Ratos , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/prevenção & controle , Fatores de Tempo , TransfecçãoRESUMO
Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.
Assuntos
Arginina/metabolismo , Grânulos Citoplasmáticos/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína FUS de Ligação a RNA/química , Proteína FUS de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Arsenitos/toxicidade , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Detergentes , Células HEK293 , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Metilação/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Transporte Proteico/efeitos dos fármacos , Solubilidade , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismoRESUMO
Wnt proteins are a large family of diffusible factors that play important roles in embryonic development, including axis patterning, cell fate specification, proliferation, and axon development. It was recently demonstrated that Ryk (receptor related to tyrosine kinase) is a conserved high-affinity Wnt receptor, and that Ryk-Wnt interactions guide corticospinal axons down the spinal cord during development. Here, we report that the Ryk-Wnt signal mediates the inhibition of corticospinal axon growth in the adult spinal cord. The expression of Wnt-5a is induced in reactive astrocytes around the injury site following a spinal cord injury. In vitro, Wnt-5a inhibits the neurite growth of postnatal cerebellar neurons by activating RhoA/Rho-kinase. In rats with thoracic spinal cord contusion, intrathecal administration of a neutralizing antibody to Ryk resulted in significant axonal growth of the corticospinal tract and enhanced functional recovery. Thus, reexpression of the embryonic repulsive cues in adult tissues contributes to the failure of axon regeneration in the central nervous system.
Assuntos
Axônios/metabolismo , Neurônios/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Recuperação de Função Fisiológica/fisiologia , Traumatismos da Medula Espinal/metabolismo , Proteínas Wnt/metabolismo , Animais , Western Blotting , Células Cultivadas , Feminino , Imuno-Histoquímica , Atividade Motora/fisiologia , Neuritos/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Traumatismos da Medula Espinal/fisiopatologia , Vértebras TorácicasRESUMO
Axonal regeneration in the central nervous system is blocked by many different growth inhibitory factors. Some of these inhibitors act on neurons by activating RhoA and Rho-kinase, an effector of RhoA. Several studies have shown that Rho-kinase inhibition immediately after spinal cord injury enhances axonal sprouting and functional recovery. In this study, we ask whether delayed treatment with Rho-kinase inhibitor is effective in promoting regeneration and functional recovery. We administered Fasudil, a Rho-kinase inhibitor, locally to the injury site 4 weeks or immediately after contusion of the thoracic spinal cord in rats. Although the immediate treatment significantly stimulated axonal sprouting and recovery of hindlimb function, treatment started 4 weeks after surgery had no effect on fiber sprouting or locomotor recovery. Our findings suggest that RhoA/Rho-kinase alone may not account for the irreversible arrest of axon outgrowth in the chronic stage of injury in the central nervous system.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Inibidores Enzimáticos/administração & dosagem , Regeneração Nervosa/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/tratamento farmacológico , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Biotina/análogos & derivados , Dextranos , Modelos Animais de Doenças , Esquema de Medicação , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Atividade Motora/efeitos dos fármacos , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/patologia , Ratos , Ratos Wistar , Estatísticas não Paramétricas , Fatores de Tempo , Quinases Associadas a rhoRESUMO
Local axon degeneration is a common pathological feature of many neurodegenerative diseases, whereas the underlying molecular mechanisms are largely unknown. In this study, we used the degeneration of transected axons, termed "Wallerian degeneration," as a model to examine the possible involvement of Rho. Nogo-66, a myelin-derived inhibitor of axon regeneration, significantly accelerated axon degeneration of the dorsal root ganglion explant in vitro, whereas inhibiting Rho-kinase activity abolished the effect. Rho activation was observed in the distal part of the injured axons after spinal cord injury. We demonstrate that degeneration of the injured cortico-spinal axons was significantly retarded by a Rho-kinase inhibitor in vivo. Our findings suggest that inhibiting the signaling pathway may retard axon degeneration in pathological conditions.