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Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy.
Assuntos
Doença de Charcot-Marie-Tooth , Proteínas rab de Ligação ao GTP , Animais , Camundongos , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7 , Doença de Charcot-Marie-Tooth/metabolismo , Células Receptoras Sensoriais/metabolismo , Mutação , Mitocôndrias/metabolismo , Lisossomos/metabolismo , Guanosina Trifosfato/metabolismoRESUMO
Chemokines such as stromal derived factor 1 and their G protein coupled receptors are well-known regulators of the development and functions of numerous tissues. C-X-C motif chemokine ligand 12 (CXCL12) has two receptors: C-X-C chemokine motif receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3). ACKR3 has been described as an atypical "biased" receptor because it does not appear to signal through G proteins and, instead, signals solely through the ß-arrestin pathway. In support of this conclusion, we have shown that ACKR3 is unable to signal through any of the known mammalian G α isoforms and have generated a comprehensive map of the G α activation by CXCL12/CXCR4. We also synthesized a series of small molecule ligands which acted as selective agonists for ACKR3 as assessed by their ability to recruit ß-arrestin to the receptor. Using select point mutations, we studied the molecular characteristics that determine the ability of small molecules to activate ACKR3 receptors, revealing a key role for the deeper binding pocket composed of residues in the transmembrane domains of ACKR3. The development of more selective ACKR3 ligands should allow us to better appreciate the unique roles of ACKR3 in the CXCL12/CXCR4/ACKR3-signaling axis and better understand the structural determinants for ACKR3 activation. SIGNIFICANCE STATEMENT: We are interested in the signaling produced by the G protein coupled receptor atypical chemokine receptor 3 (ACKR3), which signals atypically. In this study, novel selective ligands for ACKR3 were discovered and the site of interactions between these small molecules and ACKR3 was defined. This work will help to better understand the unique signaling roles of ACKR3.
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Quimiocina CXCL12 , Receptores CXCR4 , Animais , Quimiocina CXCL12/metabolismo , Ligantes , Mamíferos/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismoRESUMO
BACKGROUND: Small fiber neuropathy is a well-recognized complication of type 2 diabetes and has been shown to be responsible for both neuropathic pain and impaired wound healing. In previous studies, we have demonstrated that ganglioside GM3 depletion by knockdown of GM3 synthase fully reverses impaired wound healing in diabetic mice. However, the role of GM3 in neuropathic pain and small fiber neuropathy in diabetes is unknown. PURPOSE: Determine whether GM3 depletion is able to reverse neuropathic pain and small fibers neuropathy and the mechanism of the reversal. RESULTS: We demonstrate that GM3 synthase knockout and the resultant GM3 depletion rescues the denervation in mouse footpad skin and fully reverses the neuropathic pain in diet-induced obese diabetic mice. In cultured dorsal root ganglia from diet-induced diabetic mice, GM3 depletion protects against increased intracellular calcium influx in vitro. CONCLUSIONS: These studies establish ganglioside GM3 as a new candidate responsible for neuropathic pain and small fiber neuropathy in diabetes. Moreover, these observations indicate that systemic or topically applied interventions aimed at depleting GM3 may improve both the painful neuropathy and the wound healing impairment in diabetes by protecting against nerve end terminal degeneration, providing a disease-modifying approach to this common, currently intractable medical issue.
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Diabetes Mellitus Tipo 2/fisiopatologia , Dor/etiologia , Dor/metabolismo , Doenças do Sistema Nervoso Periférico/etiologia , Doenças do Sistema Nervoso Periférico/metabolismo , Sialiltransferases/deficiência , Neuropatia de Pequenas Fibras/etiologia , Neuropatia de Pequenas Fibras/metabolismo , Animais , Glicemia/genética , Glicemia/metabolismo , Células Cultivadas , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Gangliosídeo G(M3)/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Resistência à Insulina/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Dor/genética , Medição da Dor , Doenças do Sistema Nervoso Periférico/genética , Estimulação Física/efeitos adversos , Nervo Isquiático/metabolismo , Sialiltransferases/genética , Pele/inervaçãoRESUMO
We identified a previously unknown neurogenic region at the dorsal surface of the hippocampus; (the "subhippocampal zone," SHZ) in the adult brain. Using a reporter mouse in which SHZ cells and their progeny could be traced through the expression of EGFP under the control of the CXCR4 chemokine receptor promoter we observed the presence of a pool of EGFP expressing cells migrating in direction of the dentate gyrus (DG), which is maintained throughout adulthood. This population appeared to originate from the SHZ where cells entered a caudal migratory stream (aCMS) that included the fimbria, the meninges and the DG. Deletion of CXCR4 from neural stem cells (NSCs) or neuroinflammation resulted in the appearance of neurons in the DG, which were the result of migration of NSCs from the SHZ. Some of these neurons were ectopically placed. Our observations indicate that the SHZ is a neurogenic zone in the adult brain through migration of NSCs in the aCMS. Regulation of CXCR4 signaling in these cells may be involved in repair of the DG and may also give rise to ectopic granule cells in the DG in the context of neuropathology.
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Movimento Celular/fisiologia , Quimiocina CXCL12/fisiologia , Hipocampo/citologia , Neurogênese/fisiologia , Receptores CXCR4/fisiologia , Animais , Giro Denteado/citologia , Corantes Fluorescentes , Proteínas de Fluorescência Verde , Camundongos , Camundongos Knockout , Células-Tronco NeuraisRESUMO
Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2-null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2-null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.
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Artrite Experimental/imunologia , Artrite Experimental/fisiopatologia , Osteoartrite/imunologia , Osteoartrite/fisiopatologia , Receptores CCR2/fisiologia , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/fisiologia , Modelos Animais de Doenças , Gânglios Espinais/imunologia , Gânglios Espinais/patologia , Gânglios Espinais/fisiopatologia , Humanos , Hiperalgesia/genética , Hiperalgesia/imunologia , Hiperalgesia/fisiopatologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/genética , Osteoartrite/patologia , Dor/genética , Dor/imunologia , Dor/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR2/deficiência , Receptores CCR2/genética , Transdução de SinaisRESUMO
BACKGROUND: Painful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN. RESULTS: We demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice. CONCLUSIONS: These data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN.
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Diabetes Mellitus Tipo 2/complicações , Dor/etiologia , Dor/metabolismo , Receptores CXCR4/metabolismo , Transdução de Sinais/fisiologia , Animais , Benzilaminas , Células Cultivadas , Ciclamos , Diabetes Mellitus Tipo 2/etiologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Fatores de Troca do Nucleotídeo Guanina/deficiência , Fatores de Troca do Nucleotídeo Guanina/genética , Compostos Heterocíclicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dor/tratamento farmacológico , Medição da Dor , Limiar da Dor/efeitos dos fármacos , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/genética , Receptores para Leptina/genética , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
ABSTRACT: Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, axonal degeneration, and changes in cutaneous innervation. However, the complete molecular profile underlying the hyperexcitable cellular phenotype of DRG nociceptors in PDN has not been elucidated. This gap in our knowledge is a critical barrier to developing effective, mechanism-based, and disease-modifying therapeutic approaches that are urgently needed to relieve the symptoms of PDN. Using single-cell RNA sequencing of DRGs, we demonstrated an increased expression of the Mas-related G protein-coupled receptor d (Mrgprd) in a subpopulation of DRG neurons in the well-established high-fat diet (HFD) mouse model of PDN. Importantly, limiting Mrgprd signaling reversed mechanical allodynia in the HFD mouse model of PDN. Furthermore, in vivo calcium imaging allowed us to demonstrate that activation of Mrgprd-positive cutaneous afferents that persist in diabetic mice skin resulted in an increased intracellular calcium influx into DRG nociceptors that we assess in vivo as a readout of nociceptors hyperexcitability. Taken together, our data highlight a key role of Mrgprd-mediated DRG neuron excitability in the generation and maintenance of neuropathic pain in a mouse model of PDN. Hence, we propose Mrgprd as a promising and accessible target for developing effective therapeutics currently unavailable for treating neuropathic pain in PDN.
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Diabetes Mellitus Experimental , Neuropatias Diabéticas , Hiperalgesia , Neuralgia , Receptores Acoplados a Proteínas G , Animais , Camundongos , Cálcio/metabolismo , Diabetes Mellitus Experimental/complicações , Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/metabolismo , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Hipersensibilidade/genética , Neuralgia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Hiperalgesia/genética , Hiperalgesia/fisiopatologiaRESUMO
Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies. .
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Embryonic meninges secrete the chemokine SDF-1/CXCL12 as a chemotactic guide for migrating neural stem cells, but SDF-1 is not known to directly regulate the functions of radial glia. Recently, the developing meninges have been shown to regulate radial glial function, yet the mechanisms and signals responsible for this phenomenon remain unclear. Moreover, as a nonmigratory cell type, radial glia do not conform to traditional models associated with chemokine signaling in the central nervous system. Using fluorescent transgenes, in vivo genetic manipulations and pharmacological techniques, we demonstrate that SDF-1 derived from the meninges exerts a CXCR4-dependent effect on radial glia. Deletion of CXCR4 expression by radial glia influences their morphology, mitosis, and progression through both oligodendroglial and astroglial lineages. Additionally, disruption of CXCR4 signaling in radial glia has a transient effect on the migration of oligodendrocyte progenitors. These data indicate that a specific chemokine signal derived from the meninges has multiple regulatory effects on radial glia.
Assuntos
Células Ependimogliais/citologia , Células Ependimogliais/metabolismo , Células-Tronco Neurais/fisiologia , Receptores CXCR4/deficiência , Receptores CXCR4/fisiologia , Transdução de Sinais/fisiologia , Medula Espinal/embriologia , Medula Espinal/fisiologia , Animais , Linhagem da Célula/fisiologia , Movimento Celular/genética , Células Ependimogliais/fisiologia , Feminino , Camundongos Knockout , Mitose/genética , Técnicas de Cultura de Órgãos , Gravidez , Transdução de Sinais/genética , Medula Espinal/citologia , TransgenesRESUMO
In the present work, we characterized in detail strain CM-3-T8T, which was isolated from the rhizosphere soil of strawberries in Beijing, China, in order to elucidate its taxonomic position. Cells of strain CM-3-T8T were Gram-negative, non-spore-forming, aerobic, short rod. Growth occurred at 25-37 °C, pH 5.0-10.0, and in the presence of 0-8% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CM-3-T8T formed a stable clade with Lysobacter soli DCY21T and Lysobacter panacisoli CJ29T, with the 16S rRNA gene sequence similarities of 98.91% and 98.50%. The average nucleotide identity and digital DNA-DNA hybridization values between strain SG-8 T and the two reference type strains listed above were 76.3%, 79.6%, and 34.3%, 27%, respectively. The DNA G + C content was 68.4% (mol/mol). The major cellular fatty acids were comprised of C15:0 iso (36.15%), C17:0 iso (8.40%), and C11:0 iso 3OH (8.28%). The major quinone system was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine (PE), phosphatidylethanolamine (PME), diphosphatidylglycerol (DPG), and aminophospholipid (APL). On the basis of phenotypic, genotypic, and phylogenetic evidence, strain CM-3-T8T (= ACCC 61714 T = JCM 34576 T) represents a new species within the genus Lysobacter, for which the name Lysobacter changpingensis sp. nov. is proposed.
Assuntos
Fragaria , Lysobacter , Fosfolipídeos/química , Fragaria/genética , Fosfatidiletanolaminas , Lysobacter/genética , Filogenia , Rizosfera , RNA Ribossômico 16S/genética , Solo , DNA Bacteriano/genética , DNA Bacteriano/química , Ácidos Graxos/análise , China , Análise de Sequência de DNA , Técnicas de Tipagem BacterianaRESUMO
Non-opioid targets are needed for addressing osteoarthritis pain, which is mechanical in nature and associated with daily activities such as walking and climbing stairs. Piezo2 has been implicated in the development of mechanical pain, but the mechanisms by which this occurs remain poorly understood, including the role of nociceptors. Here we show that nociceptor-specific Piezo2 conditional knock-out mice were protected from mechanical sensitization associated with inflammatory joint pain in female mice, joint pain associated with osteoarthritis in male mice, as well as both knee swelling and joint pain associated with repeated intra-articular injection of nerve growth factor in male mice. Single cell RNA sequencing of mouse lumbar dorsal root ganglia and in situ hybridization of mouse and human lumbar dorsal root ganglia revealed that a subset of nociceptors co-express Piezo2 and Ntrk1 (the gene that encodes the nerve growth factor receptor TrkA). These results suggest that nerve growth factor-mediated sensitization of joint nociceptors, which is critical for osteoarthritic pain, is also dependent on Piezo2, and targeting Piezo2 may represent a therapeutic option for osteoarthritis pain control.
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Nociceptores , Osteoartrite , Animais , Camundongos , Masculino , Feminino , Humanos , Nociceptores/metabolismo , Dor/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Camundongos Knockout , Artralgia , Fatores de Crescimento Neural/metabolismo , Gânglios Espinais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismoRESUMO
The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell-cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.
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ABSTRACT: Painful diabetic neuropathy (PDN) is an intractable complication affecting 25% of diabetic patients. Painful diabetic neuropathy is characterized by neuropathic pain accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability, resulting in calcium overload, axonal degeneration, and loss of cutaneous innervation. The molecular pathways underlying these effects are unknown. Using high-throughput and deep-proteome profiling, we found that mitochondrial fission proteins were elevated in DRG neurons from mice with PDN induced by a high-fat diet (HFD). In vivo calcium imaging revealed increased calcium signaling in DRG nociceptors from mice with PDN. Furthermore, using electron microscopy, we showed that mitochondria in DRG nociceptors had fragmented morphology as early as 2 weeks after starting HFD, preceding the onset of mechanical allodynia and small-fiber degeneration. Moreover, preventing calcium entry into the mitochondria, by selectively deleting the mitochondrial calcium uniporter from these neurons, restored normal mitochondrial morphology, prevented axonal degeneration, and reversed mechanical allodynia in the HFD mouse model of PDN. These studies suggest a molecular cascade linking neuropathic pain to axonal degeneration in PDN. In particular, nociceptor hyperexcitability and the associated increased intracellular calcium concentrations could lead to excessive calcium entry into mitochondria mediated by the mitochondrial calcium uniporter, resulting in increased calcium-dependent mitochondrial fission and ultimately contributing to small-fiber degeneration and neuropathic pain in PDN. Hence, we propose that targeting calcium entry into nociceptor mitochondria may represent a promising effective and disease-modifying therapeutic approach for this currently intractable and widespread affliction. Moreover, these results are likely to inform studies of other neurodegenerative disease involving similar underlying events.
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Diabetes Mellitus , Neuropatias Diabéticas , Doenças Neurodegenerativas , Animais , Canais de Cálcio , Diabetes Mellitus/metabolismo , Neuropatias Diabéticas/metabolismo , Gânglios Espinais/metabolismo , Humanos , Camundongos , Mitocôndrias , Doenças Neurodegenerativas/metabolismoRESUMO
The chemokine BRAK/CXCL14 is an ancient member of the chemokine family whose functions in the brain are completely unknown. We examined the distribution of CXCL14 in the nervous system during development and in the adult. Generally speaking, CXCL14 was not expressed in the nervous system prior to birth, but it was expressed in the developing whisker follicles (E14.5) and subsequently in the hair follicles and skin. Postnatally, CXCL14 was also highly expressed in many regions of the brain, including the cortex, basal ganglia, septum and hippocampus. CXCL14 was also highly expressed in the dorsal root ganglia. We observed that in the hippocampal dentate gyrus (DG) CXCL14 was expressed by GABAergic interneurons. We demonstrated that CXCL14 inhibited GABAergic transmission to nestin-EGFP-expressing neural stem/progenitor cells in the adult DG. CXCL14 inhibited both the tonic and phasic effects of synaptically released GABA. In contrast CXCL12 enhanced the effects of GABA at these same synapses. CXCL14 increased [Ca(2+)](i) in neural stem cells cultured from the postnatal brain indicating that they expressed the CXCL14 receptor. These observations are consistent with the view that CXCL12 and CXCL14 may normally act as positive and negative regulators of the effects of GABA in the adult DG stem cell niche.
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Quimiocinas CXC/metabolismo , Giro Denteado/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Nicho de Células-Tronco/fisiologia , Transmissão Sináptica/fisiologia , 2-Amino-5-fosfonovalerato/farmacologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Quimiocinas CXC/genética , Quimiocinas CXC/farmacologia , Giro Denteado/crescimento & desenvolvimento , Embrião de Mamíferos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutamato Descarboxilase/metabolismo , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Proteínas de Filamentos Intermediários/genética , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Quinoxalinas/farmacologia , RNA Mensageiro/metabolismo , Receptores CXCR/metabolismo , Nicho de Células-Tronco/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genéticaRESUMO
Enhancing the ability of either endogenous or transplanted oligodendrocyte progenitors (OPs) to engage in myelination may constitute a novel therapeutic approach to demyelinating diseases of the brain. It is known that in adults neural progenitors situated in the subventricular zone of the lateral ventricle (SVZ) are capable of generating OPs which can migrate into white matter tracts such as the corpus callosum (CC). We observed that progenitor cells in the SVZ of adult mice expressed CXCR4 chemokine receptors and that the chemokine SDF-1/CXCL12 was expressed in the CC. We therefore investigated the role of chemokine signaling in regulating the migration of OPs into the CC following their transplantation into the lateral ventricle. We established OP cell cultures from Olig2-EGFP mouse brains. These cells expressed a variety of chemokine receptors, including CXCR4 receptors. Olig2-EGFP OPs differentiated into CNPase-expressing oligodendrocytes in culture. To study the migratory capacity of Olig2-EGFP OPs in vivo, we transplanted them into the lateral ventricles of mice. Donor cells migrated into the CC and differentiated into mature oligodendrocytes. This migration was enhanced in animals with Experimental Autoimmune Encephalomyelitis (EAE). Inhibition of CXCR4 receptor expression in OPs using shRNA inhibited the migration of transplanted OPs into the white matter suggesting that their directed migration is regulated by CXCR4 signaling. These findings indicate that CXCR4 mediated signaling is important in guiding the migration of transplanted OPs in the context of inflammatory demyelinating brain disease.
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Encéfalo/citologia , Encéfalo/fisiologia , Células-Tronco Neurais/transplante , Oligodendroglia/transplante , Receptores CXCR4/fisiologia , Transdução de Sinais/fisiologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Células Cultivadas , Quimiocinas/fisiologia , Regulação para Baixo/genética , Encefalomielite Autoimune Experimental/patologia , Imuno-Histoquímica , Ventrículos Laterais/citologia , Camundongos , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , RNA/genética , Receptores CXCR4/genética , Receptores de Quimiocinas/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnicas EstereotáxicasRESUMO
BACKGROUND: C-C chemokine receptor 2 (CCR2) signaling plays a key role in pain associated with experimental murine osteoarthritis (OA) after destabilization of the medial meniscus (DMM). Here, we aimed to assess if CCR2 expressed by intra-articular sensory neurons contributes to knee hyperalgesia in the early stages of the model. METHODS: DMM surgery was performed in the right knee of 10-week-old male wild-type (WT), Ccr2 null, or Ccr2RFP C57BL/6 mice. Knee hyperalgesia was measured using a Pressure Application Measurement device. CCR2 receptor antagonist (CCR2RA) was injected systemically (i.p.) or intra-articularly (i.a.) at different times after DMM to test its ability to reverse knee hyperalgesia. In vivo Ca2+ imaging of the dorsal root ganglion (DRG) was performed to assess sensory neuron responses to CCL2 injected into the knee joint cavity. CCL2 protein in the knee was measured by ELISA. Ccr2RFP mice and immunohistochemical staining for the pan-neuronal marker, protein gene product 9.5 (PGP9.5), or the sensory neuron marker, calcitonin gene-related peptide (CGRP), were used to visualize the location of CCR2 on intra-articular afferents. RESULTS: WT, but not Ccr2 null, mice displayed knee hyperalgesia 2-16 weeks after DMM. CCR2RA administered i.p. alleviated established hyperalgesia in WT mice 4 and 8 weeks after surgery. Intra-articular injection of CCL2 excited sensory neurons in the L4-DRG, as determined by in vivo calcium imaging; responses to CCL2 increased in mice 20 weeks after DMM. CCL2, but not vehicle, injected i.a. rapidly caused transient knee hyperalgesia in naïve WT, but not Ccr2 null, mice. Intra-articular CCR2RA injection also alleviated established hyperalgesia in WT mice 4 and 7 weeks after surgery. CCL2 protein was elevated in the knees of both WT and Ccr2 null mice 4 weeks after surgery. Co-expression of CCR2 and PGP9.5 as well as CCR2 and CGRP was observed in the lateral synovium of naïve mice; co-expression was also observed in the medial compartment of knees 8 weeks after DMM. CONCLUSIONS: The findings suggest that CCL2-CCR2 signaling locally in the joint contributes to knee hyperalgesia in experimental OA, and it is in part mediated through direct stimulation of CCR2 expressed by intra-articular sensory afferents.
Assuntos
Artralgia , Osteoartrite do Joelho , Receptores CCR2 , Animais , Modelos Animais de Doenças , Articulação do Joelho , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor , Receptores CCR2/genética , Células Receptoras SensoriaisRESUMO
CCR2 chemokine receptor signaling has been implicated in the generation of diverse types of neuropathology, including neuropathic pain. For example, ccr2 knock-out mice are resistant to the establishment of neuropathic pain, and mice overexpressing its ligand, monocyte chemoattractant protein-1 (MCP1; also known as CCL2), show enhanced pain sensitivity. However, whether CCR2 receptor activation occurs in the central or peripheral nervous system in states of neuropathic pain has not been clear. We developed a novel method for visualizing CCR2 receptor activation in vivo by generating bitransgenic reporter mice in which the chemokine receptor CCR2 and its ligand MCP1 were labeled by the fluorescent proteins enhanced green fluorescent protein and monomeric red fluorescent protein-1, respectively. CCR2 receptor activation under conditions such as acute inflammation and experimental autoimmune encephalomyelitis could be faithfully visualized by using these mice. We examined the status of CCR2 receptor activation in a demyelination injury model of neuropathic pain and found that MCP1-induced CCR2 receptor activation mainly occurred in the peripheral nervous system, including the injured peripheral nerve and dorsal root ganglia. These data explain the rapid antinociceptive effects of peripherally administered CCR2 antagonists under these circumstances, suggesting that CCR2 antagonists may ameliorate pain by inhibiting CCR2 receptor activation in the periphery. The method developed here for visualizing CCR2 receptor activation in vivo may be extended to G-protein-coupled receptors (GPCRs) in general and will be valuable for studying intercellular GPCR-mediated communication in vivo.
Assuntos
Quimiocina CCL2/metabolismo , Neurônios/metabolismo , Dor/metabolismo , Nervos Periféricos/metabolismo , Receptores CCR2/metabolismo , Animais , Células Cultivadas , Quimiocina CCL2/deficiência , Quimiocina CCL2/genética , Doenças Desmielinizantes/induzido quimicamente , Modelos Animais de Doenças , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Lipopolissacarídeos/administração & dosagem , Lipopolissacarídeos/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Dor/genética , Medição da Dor/métodos , Limiar da Dor/psicologia , Nervos Periféricos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Receptores CCR2/deficiência , Receptores CCR2/genética , Neuropatia Ciática/induzido quimicamente , Transfecção , Proteína Vermelha FluorescenteRESUMO
Mouse skin melanocytes originate from the neural crest and subsequently invade the epidermis and migrate into the hair follicles (HF) where they proliferate and differentiate. Here we demonstrate a role for the chemokine SDF-1/CXCL12 and its receptor CXCR4 in regulating the migration and positioning of melanoblasts during HF formation and cycling. CXCR4 expression by melanoblasts was upregulated during the anagen phase of the HF cycle. CXCR4-expressing cells in the HF also expressed the stem cell markers nestin and LEX, the neural crest marker SOX10 and the cell proliferation marker PCNA. SDF-1 was widely expressed along the path taken by migrating CXCR4-expressing cells in the outer root sheath (ORS), suggesting that SDF-1-mediated signaling might be required for the migration of CXCR4 cells. Skin sections from CXCR4-deficient mice, and skin explants treated with the CXCR4 antagonist AMD3100, contained melanoblasts abnormally concentrated in the epidermis, consistent with a defect in their migration. SDF-1 acted as a chemoattractant for FACS-sorted cells isolated from the anagen skin of CXCR4-EGFP transgenic mice in vitro, and AMD3100 inhibited the SDF-1-induced migratory response. Together, these data demonstrate an important role for SDF-1/CXCR4 signaling in directing the migration and positioning of melanoblasts in the HF.
Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Folículo Piloso/citologia , Melanócitos , Células-Tronco/fisiologia , Animais , Biomarcadores/metabolismo , Quimiocina CXCL12/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Folículo Piloso/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Oxirredutases Intramoleculares/metabolismo , Ligantes , Melanócitos/citologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Transdução de SinaisRESUMO
Stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) are important regulators of the development of the dentate gyrus (DG). Both SDF-1 and CXCR4 are also highly expressed in the adult DG. We observed that CXCR4 receptors were expressed by dividing neural progenitor cells located in the subgranular zone (SGZ) as well as their derivatives including doublecortin-expressing neuroblasts and immature granule cells. SDF-1 was located in DG neurons and in endothelial cells associated with DG blood vessels. SDF-1-expressing neurons included parvalbumin-containing GABAergic interneurons known as basket cells. Using transgenic mice expressing an SDF-1-mRFP1 (monomeric red fluorescence protein 1) fusion protein we observed that SDF-1 was localized in synaptic vesicles in the terminals of basket cells together with GABA-containing vesicles. These terminals were often observed to be in close proximity to dividing nestin-expressing neural progenitors in the SGZ. Electrophysiological recordings from slices of the DG demonstrated that neural progenitors received both tonic and phasic GABAergic inputs and that SDF-1 enhanced GABAergic transmission, probably by a postsynaptic mechanism. We also demonstrated that, like GABA, SDF-1 was tonically released in the DG and that GABAergic transmission was partially dependent on coreleased SDF-1. These data demonstrate that SDF-1 plays a novel role as a neurotransmitter in the DG and regulates the strength of GABAergic inputs to the pool of dividing neural progenitors. Hence, SDF-1/CXCR4 signaling is likely to be an important regulator of adult neurogenesis in the DG.
Assuntos
Quimiocina CXCL12/metabolismo , Giro Denteado/crescimento & desenvolvimento , Neurônios/metabolismo , Células-Tronco/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Giro Denteado/citologia , Giro Denteado/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Interneurônios/metabolismo , Interneurônios/ultraestrutura , Proteínas Luminescentes , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Nestina , Inibição Neural/fisiologia , Vias Neurais/citologia , Vias Neurais/crescimento & desenvolvimento , Vias Neurais/metabolismo , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Receptores CXCR4/metabolismo , Coloração e Rotulagem , Células-Tronco/citologia , Sinapses/metabolismo , Proteína Vermelha FluorescenteRESUMO
Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels initiates key functions such as gene expression and exocytosis of inflammatory mediators. Activation of CRAC channels by store depletion involves the redistribution of the ER Ca(2+) sensor, stromal interaction molecule 1 (STIM1), to peripheral sites where it co-clusters with the CRAC channel subunit, Orai1. However, how STIM1 communicates with the CRAC channel and initiates the subsequent events culminating in channel opening is unclear. Here, we show that redistribution of STIM1 and Orai1 occurs in parallel with a pronounced increase in fluorescence resonance energy transfer (FRET) between STIM1 and Orai1, supporting the idea that activation of CRAC channels occurs through physical interactions with STIM1. Co-expression of Orai1-CFP and Orai1-YFP results in a high degree of FRET in resting cells, indicating that Orai1 exists as a multimer. However, store depletion triggers molecular rearrangements in Orai1 resulting in a decline in Orai1-Orai1 FRET. The decline in Orai1-Orai1 FRET is not seen in the absence of STIM1 co-expression and is abolished in Orai1 mutants with impaired STIM1 interaction. Both the STIM1-Orai1 interaction as well as the molecular rearrangements in Orai1 are altered by two powerful modulators of CRAC channel activity: extracellular Ca(2+) and 2-APB. These studies identify a STIM1-dependent conformational change in Orai1 during the activation of CRAC channels and reveal that STIM1-Orai1 interaction and the downstream Orai1 conformational change can be independently modulated to fine-tune CRAC channel activity.