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1.
Commun Biol ; 4(1): 478, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846535

RESUMO

Mutations in the gene encoding Lamin B receptor (LBR), a nuclear-membrane protein with sterol reductase activity, have been linked to rare human disorders. Phenotypes range from a benign blood disorder, such as Pelger-Huet anomaly (PHA), affecting the morphology and chromatin organization of white blood cells, to embryonic lethality as for Greenberg dysplasia (GRBGD). Existing PHA mouse models do not fully recapitulate the human phenotypes, hindering efforts to understand the molecular etiology of this disorder. Here we show, using CRISPR/Cas-9 gene editing technology, that a 236bp N-terminal deletion in the mouse Lbr gene, generating a protein missing the N-terminal domains of LBR, presents a superior model of human PHA. Further, we address recent reports of a link between Lbr and defects in X chromosome inactivation (XCI) and show that our mouse mutant displays minor X chromosome inactivation defects that do not lead to any overt phenotypes in vivo. We suggest that our N-terminal deletion model provides a valuable pre-clinical tool to the research community and will aid in further understanding the etiology of PHA and the diverse functions of LBR.


Assuntos
Anomalia de Pelger-Huët/genética , Receptores Citoplasmáticos e Nucleares/genética , Inativação do Cromossomo X/genética , Animais , Camundongos , Camundongos Knockout , Fenótipo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina B
2.
Pain ; 160(10): 2305-2315, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31365468

RESUMO

Nerve growth factor (NGF) and its receptors TrkA and p75 play a key role in the development and function of peripheral nociceptive neurons. Here, we describe novel technology to selectively photoablate TrkA-positive nociceptors through delivery of a phototoxic agent coupled to an engineered NGF ligand and subsequent near-infrared illumination. We demonstrate that this approach allows for on demand and localized reversal of pain behaviors in mouse models of acute, inflammatory, neuropathic, and joint pain. To target peripheral nociceptors, we generated a SNAP-tagged NGF derivative NGF that binds to TrkA/p75 receptors but does not provoke signaling in TrkA-positive cells or elicit pain behaviors in mice. NGF was coupled to the photosensitizer IRDye700DX phthalocyanine (IR700) and injected subcutaneously. After near-infrared illumination of the injected area, behavioral responses to nociceptive mechanical and sustained thermal stimuli, but not innocuous stimuli, were substantially reduced. Similarly, in models of inflammatory, osteoarthritic, and neuropathic pain, mechanical hypersensitivity was abolished for 3 weeks after a single treatment regime. We demonstrate that this loss of pain behavior coincides with the retraction of neurons from the skin which then reinnervate the epidermis after 3 weeks corresponding with the return of mechanical hypersensitivity. Thus NGF-mediated photoablation is a minimally invasive approach to reversibly silence nociceptor input from the periphery, and control pain and hypersensitivity to mechanical stimuli.


Assuntos
Técnicas de Ablação/métodos , Fator de Crescimento Neural/administração & dosagem , Neuralgia/terapia , Nociceptores/efeitos dos fármacos , Medição da Dor/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neuralgia/fisiopatologia , Nociceptores/fisiologia , Células PC12 , Ratos
3.
Nat Biomed Eng ; 3(2): 114-125, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30944432

RESUMO

Itch-a major symptom of many chronic skin diseases-can exacerbate inflammation by provoking scratching and subsequent skin damage. Here, we show that activation, via near infrared illumination, of a phototoxic agent that selectively targets itch-sensing cells can reduce itch-associated behaviours in mice. We generated a SNAP-tagged interleukin-31 (IL-31) ligand derivative (IL-31K138A-SNAP) that selectively binds receptors on itch-associated cells, without evoking IL-31-receptor signalling or scratching, and conjugated it to the photosensitizer IRDye 700DX phthalocyanine. Subcutaneous injection of IL-31K138A-SNAP-IR700 in mice followed by near infrared illumination resulted in the long-term reversal of the scratching behaviour evoked by the pruritogenic IL-31, an effect that was associated with the selective retraction of itch-sensing neurons in the skin. We also show that a topical preparation of IL-31K138A-SNAP-IR700 reversed the behavioural and dermatological indicators of disease in mouse models of atopic dermatitis and of the genetic skin disease familial primary localized cutaneous amyloidosis. Targeted photoablation may enable itch control for the treatment of inflammatory skin diseases.


Assuntos
Comportamento Animal , Epiderme/inervação , Interleucinas/uso terapêutico , Luz , Prurido/patologia , Prurido/terapia , Células Receptoras Sensoriais/patologia , Doença Aguda , Amiloidose Familiar/patologia , Animais , Movimento Celular , Células Dendríticas/patologia , Dermatite Atópica/patologia , Dermatite Atópica/prevenção & controle , Modelos Animais de Doenças , Epiderme/patologia , Indóis/química , Queratinócitos/patologia , Camundongos Endogâmicos C57BL , Psoríase/patologia , Dermatopatias Genéticas/patologia
4.
Nat Commun ; 9(1): 1640, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29691410

RESUMO

Mechanical allodynia is a major symptom of neuropathic pain whereby innocuous touch evokes severe pain. Here we identify a population of peripheral sensory neurons expressing TrkB that are both necessary and sufficient for producing pain from light touch after nerve injury in mice. Mice in which TrkB-Cre-expressing neurons are ablated are less sensitive to the lightest touch under basal conditions, and fail to develop mechanical allodynia in a model of neuropathic pain. Moreover, selective optogenetic activation of these neurons after nerve injury evokes marked nociceptive behavior. Using a phototherapeutic approach based upon BDNF, the ligand for TrkB, we perform molecule-guided laser ablation of these neurons and achieve long-term retraction of TrkB-positive neurons from the skin and pronounced reversal of mechanical allodynia across multiple types of neuropathic pain. Thus we identify the peripheral neurons which transmit pain from light touch and uncover a novel pharmacological strategy for its treatment.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Hiperalgesia/terapia , Terapia a Laser , Glicoproteínas de Membrana/metabolismo , Neuralgia/metabolismo , Neuralgia/terapia , Proteínas Tirosina Quinases/metabolismo , Células Receptoras Sensoriais/efeitos da radiação , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Feminino , Humanos , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Ligantes , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Neuralgia/genética , Neuralgia/fisiopatologia , Proteínas Tirosina Quinases/genética , Células Receptoras Sensoriais/metabolismo , Tato/efeitos da radiação
5.
Mol Neurobiol ; 55(4): 3301-3315, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28488209

RESUMO

MicroRNAs are a class of non-coding RNAs with a growing relevance in the regulation of gene expression related to brain function and plasticity. They have the potential to orchestrate complex phenomena, such as the neuronal response to homeostatic challenges. We previously demonstrated the involvement of miR-135a in the regulation of early stress response. In the present study, we examine the role of miR-135a in stress-related behavior. We show that the knockdown (KD) of miR-135a in the mouse amygdala induces an increase in anxiety-like behavior. Consistently with behavioral studies, electrophysiological experiments in acute brain slices indicate an increase of amygdala spontaneous excitatory postsynaptic currents, as a result of miR-135a KD. Furthermore, we presented direct evidences, by in vitro assays and in vivo miRNA overexpression in the amygdala, that two key regulators of synaptic vesicle fusion, complexin-1 and complexin-2, are direct targets of miR-135a. In vitro analysis of miniature excitatory postsynaptic currents on miR-135a KD primary neurons indicates unpaired quantal excitatory neurotransmission. Finally, increased levels of complexin-1 and complexin-2 proteins were detected in the mouse amygdala after acute stress, accordingly to the previously observed stress-induced miR-135a downregulation. Overall, our results unravel a previously unknown miRNA-dependent mechanism in the amygdala for regulating anxiety-like behavior, providing evidences of a physiological role of miR-135a in the modulation of presynaptic mechanisms of glutamatergic neurotransmission.


Assuntos
Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Ansiedade/genética , Ansiedade/fisiopatologia , Comportamento Animal , MicroRNAs/metabolismo , Transmissão Sináptica/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Tonsila do Cerebelo/patologia , Animais , Linhagem Celular Tumoral , Potenciais Pós-Sinápticos Excitadores , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Hipocampo/patologia , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Estresse Fisiológico/genética
6.
PLoS Negl Trop Dis ; 10(8): e0004928, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27518281

RESUMO

BACKGROUND: Schistosomiasis, one of the world's greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus Schistosoma. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed. METHODS AND FINDINGS: Following the screening of a small compound library with an ATP-based luminescent assay on Schistosoma mansoni schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, in vitro, has a marked lethal effect on all S. mansoni parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The in vivo study in S. mansoni-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model. CONCLUSIONS/SIGNIFICANCE: Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.


Assuntos
Genitália/efeitos dos fármacos , Perexilina/análogos & derivados , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/ultraestrutura , Esquistossomose mansoni/tratamento farmacológico , Esquistossomicidas/farmacologia , Animais , Modelos Animais de Doenças , Resistência a Medicamentos , Feminino , Meia-Vida , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Perexilina/farmacologia , Praziquantel/farmacologia
7.
Cell Rep ; 15(12): 2608-15, 2016 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-27332874

RESUMO

Neuropathic pain is a widespread chronic pain state that results from injury to the nervous system. Spinal microglia play a causative role in the pathogenesis of neuropathic pain through secretion of growth factors and cytokines. Here, we investigated the contribution of TMEM16F, a protein that functions as a Ca(2+)-dependent ion channel and a phospholipid scramblase, to microglial activity during neuropathic pain. We demonstrate that mice with a conditional ablation of TMEM16F in microglia do not develop mechanical hypersensitivity upon nerve injury. In the absence of TMEM16F, microglia display deficits in process motility and phagocytosis. Moreover, loss of GABA immunoreactivity upon injury is spared in TMEM16F conditional knockout mice. Collectively, these data indicate that TMEM16F is an essential component of the microglial response to injury and suggest the importance of microglial phagocytosis in the pathogenesis of neuropathic pain.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Medula Espinal/metabolismo , Animais , Anoctaminas , Movimento Celular , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Macrófagos/metabolismo , Camundongos Knockout , Microglia/patologia , Neuralgia/patologia , Fagocitose
8.
EMBO Rep ; 17(4): 585-600, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26929027

RESUMO

Itch, the unpleasant sensation that elicits a desire to scratch, is mediated by specific subtypes of cutaneous sensory neuron. Here, we identify a subpopulation of itch-sensing neurons based on their expression of the receptor tyrosine kinase Ret. We apply flow cytometry to isolate Ret-positive neurons from dorsal root ganglia and detected a distinct population marked by low levels of Ret and absence of isolectin B4 binding. We determine the transcriptional profile of these neurons and demonstrate that they express neuropeptides such as somatostatin (Sst), the NGF receptor TrkA, and multiple transcripts associated with itch. We validate the selective expression of Sst using an Sst-Cre driver line and ablated these neurons by generating mice in which the diphtheria toxin receptor is conditionally expressed from the sensory neuron-specific Avil locus. Sst-Cre::Avil(iDTR) mice display normal nociceptive responses to thermal and mechanical stimuli. However, scratching behavior evoked by interleukin-31 (IL-31) or agonist at the 5HT1F receptor is significantly reduced. Our data provide a molecular signature for a subpopulation of neurons activated by multiple pruritogens.


Assuntos
Gânglios Espinais/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Prurido/genética , Células Receptoras Sensoriais/metabolismo , Somatostatina/genética , Animais , Perfilação da Expressão Gênica , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Hibridização In Situ , Lectinas/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Neurônios Aferentes/metabolismo , Neuropeptídeos/metabolismo , Receptor de Fator de Crescimento Neural/genética
9.
Blood ; 116(23): 4894-905, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-20716772

RESUMO

Inhibition of antigen-dependent B-cell receptor (BCR) signaling is considered a promising therapeutic approach in chronic lymphocytic leukemia (CLL), but experimental in vivo evidence to support this view is still lacking. We have now investigated whether inhibition of BCR signaling with the selective Syk inhibitor fostamatinib disodium (R788) will affect the growth of the leukemias that develop in the Eµ-TCL1 transgenic mouse model of CLL. Similarly to human CLL, these leukemias express stereotyped BCRs that react with autoantigens exposed on the surface of senescent or apoptotic cells, suggesting that they are antigen driven. We show that R788 effectively inhibits BCR signaling in vivo, resulting in reduced proliferation and survival of the malignant B cells and significantly prolonged survival of the treated animals. The growth-inhibitory effect of R788 occurs despite the relatively modest cytotoxic effect in vitro and is independent of basal Syk activity, suggesting that R788 functions primarily by inhibiting antigen-dependent BCR signals. Importantly, the effect of R788 was found to be selective for the malignant clones, as no disturbance in the production of normal B lymphocytes was observed. Collectively, these data provide further rationale for clinical trials with R788 in CLL and establish the BCR-signaling pathway as an important therapeutic target in this disease.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Oxazinas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Transferência Adotiva , Aminopiridinas , Animais , Western Blotting , Separação Celular , Modelos Animais de Doenças , Citometria de Fluxo , Imunofenotipagem , Marcação In Situ das Extremidades Cortadas , Leucemia Linfocítica Crônica de Células B/genética , Camundongos , Camundongos Transgênicos , Morfolinas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Pirimidinas , Receptores de Antígenos de Linfócitos B/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Quinase Syk
10.
Genome Res ; 17(3): 320-7, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17284679

RESUMO

Peptide hormones are small, processed, and secreted peptides that signal via membrane receptors and play critical roles in normal and pathological physiology. The search for novel peptide hormones has been hampered by their small size, low or restricted expression, and lack of sequence similarity. To overcome these difficulties, we developed a bioinformatics search tool based on the hidden Markov model formalism that uses several peptide hormone sequence features to estimate the likelihood that a protein contains a processed and secreted peptide of this class. Application of this tool to an alignment of mammalian proteomes ranked 90% of known peptide hormones among the top 300 proteins. An analysis of the top scoring hypothetical and poorly annotated human proteins identified two novel candidate peptide hormones. Biochemical analysis of the two candidates, which we called spexin and augurin, showed that both were localized to secretory granules in a transfected pancreatic cell line and were recovered from the cell supernatant. Spexin was expressed in the submucosal layer of the mouse esophagus and stomach, and a predicted peptide from the spexin precursor induced muscle contraction in a rat stomach explant assay. Augurin was specifically expressed in mouse endocrine tissues, including pituitary and adrenal gland, choroid plexus, and the atrio-ventricular node of the heart. Our findings demonstrate the utility of a bioinformatics approach to identify novel biologically active peptides. Peptide hormones and their receptors are important diagnostic and therapeutic targets, and our results suggest that spexin and augurin are novel peptide hormones likely to be involved in physiological homeostasis.


Assuntos
Algoritmos , Biologia Computacional/métodos , Hormônios Peptídicos/genética , Hormônios Peptídicos/metabolismo , Proteoma/genética , Proteômica/métodos , Sequência de Aminoácidos , Animais , Linhagem Celular , Primers do DNA , Células Enteroendócrinas/metabolismo , Humanos , Imuno-Histoquímica , Funções Verossimilhança , Cadeias de Markov , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Ratos
11.
Dev Biol ; 278(1): 231-41, 2005 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-15649475

RESUMO

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. Here we show that in mouse the non-muscle-specific gene-n-cofilin-is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. Our data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation.


Assuntos
Proteínas dos Microfilamentos/fisiologia , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Crista Neural/embriologia , Crista Neural/metabolismo , Fatores de Despolimerização de Actina , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , Cofilina 2 , Destrina , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Genes Letais , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Morfogênese/genética , Morfogênese/fisiologia , Sistema Nervoso/citologia , Crista Neural/citologia , Defeitos do Tubo Neural/embriologia , Defeitos do Tubo Neural/etiologia , Defeitos do Tubo Neural/genética
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