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1.
Hum Mol Genet ; 29(14): 2325-2336, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32543656

RESUMO

Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. ß2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of ß2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether ß2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a ß2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.


Assuntos
Albuterol/farmacologia , Proteínas Musculares/genética , Síndromes Miastênicas Congênitas/tratamento farmacológico , Junção Neuromuscular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/patologia , Junção Neuromuscular/metabolismo , Gravidez , Receptores Colinérgicos/genética , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
2.
Biochem Biophys Res Commun ; 589: 192-196, 2022 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-34922202

RESUMO

A concurrent reduction in muscle mass and strength is frequently observed in numerous conditions, including neuromuscular disease, ageing, and muscle inactivity due to limb immobilization or prolonged bed rest. Thus, identifying the molecular mechanisms that control skeletal muscle mass and strength is fundamental for developing interventions aimed at counteracting muscle loss (muscle atrophy). It was recently reported that muscle atrophy induced by denervation of motor nerves was associated with increased expression of Ca2+/calmodulin-dependent protein serine/threonine kinase II ß (CaMKIIß) in muscle. In addition, treatment with KN-93 phosphate, which inhibits CaMKII-family kinases, partly suppressed denervation-induced muscle atrophy. Therefore, to test a possible role for CaMKIIß in muscle mass regulation, we generated and injected recombinant adeno-associated virus (AAV) vectors encoding wild-type (AAV-WT), inactive (AAV-K43 M), or constitutively active (AAV-T287D) CaMKIIß into the left hindlimb tibialis anterior muscle of mice at three months of age. Although AAV-WT infection induced expression of exogenous CaMKIIß in the hindlimb muscle, no significant changes in muscle mass and strength were observed. By contrast, AAV-K43 M or AAV-T287D infection induced exogenous expression of the corresponding mutants and significantly increased or decreased the muscle mass and strength of the infected hind limb, respectively. Together, these findings demonstrate the potential of CaMKIIß as a novel therapeutic target for enhancing muscle mass and strength.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Dependovirus/metabolismo , Força Muscular/fisiologia , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , Mutação/genética , Animais , Células HEK293 , Membro Posterior/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Tamanho do Órgão
3.
Biochem Biophys Res Commun ; 523(1): 214-219, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-31848047

RESUMO

Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8-week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.


Assuntos
Proteínas Musculares/biossíntese , Músculo Esquelético/metabolismo , Junção Neuromuscular/metabolismo , Transmissão Sináptica , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Junção Neuromuscular/química
4.
Genes Cells ; 24(9): 619-626, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31233664

RESUMO

Inflammatory bowel diseases (IBDs) are characterized by chronic inflammation involving intestinal tissue damage, which include ulcerative colitis and Crohn's disease as major entities. Accumulating evidence suggests that excessive apoptosis of intestinal epithelial cells (IECs) contributes to the development of IBD. It was recently reported that the transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) is involved in inflammation; however, its role in colitis remains unclear. Here, we found that C/EBPδ knockout mice showed enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis, a mouse model of IBD, which was associated with severe colonic inflammation and mucosal damage with increased IEC apoptosis. Additionally, DSS stimulation induced increased expression of pro-apoptotic BH3-only protein Bim in the colon of C/EBPδ knockout mice. Collectively, our findings demonstrate that C/EBPδ plays an essential role in suppressing DSS-induced colitis, likely by attenuating IEC apoptosis.


Assuntos
Apoptose , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Colite Ulcerativa/metabolismo , Mucosa Intestinal/metabolismo , Animais , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Células Cultivadas , Colite Ulcerativa/genética , Deleção de Genes , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL
5.
Biochem Biophys Res Commun ; 498(4): 967-974, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29548825

RESUMO

Bone mass is determined by coordinated acts of osteoblasts and osteoclasts, which control bone formation and resorption, respectively. Osteoclasts are multinucleated, macrophage/monocyte lineage cells from bone marrow. The Dok-family adaptors Dok-1, Dok-2 and Dok-3 are expressed in the macrophage/monocyte lineage and negatively regulate many signaling pathways, implying roles in osteoclastogenesis. Indeed, mice lacking Dok-1 and Dok-2, the closest homologues with redundant functions, develop osteopenia with increased osteoclast counts compared to the wild-type controls. Here, we demonstrate that Dok-3 knockout (KO) mice also develop osteopenia. However, Dok-3 KO, but not Dok-1/-2 double-KO (DKO), mice develop larger osteoclasts within the normal cell-count range, suggesting a distinctive role for Dok-3. Indeed, Dok-3 KO, but not Dok-1/-2 DKO, bone marrow-derived cells (BMDCs) generated larger osteoclasts with more nuclei due to augmented cell-to-cell fusion in vitro. In addition, while Dok-1/-2 DKO BMDCs generated more osteoclasts, Dok-1/-2/-3 triple-KO (TKO) BMDCs generated osteoclasts increased in both number and size. Furthermore, Dok-1/-2/-3 TKO mice showed the combined effects of Dok-3 and Dok-1/-2 deficiency: severe osteopenia with more and larger osteoclasts. Together, our findings demonstrate that Dok-3 and Dok-1/-2 play distinctive but cooperative roles in osteoclastogenesis and protect mice from osteopenia, providing physiological and pathophysiological insight into bone homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Doenças Ósseas Metabólicas/prevenção & controle , Proteínas de Ligação a DNA/fisiologia , Osteoclastos/citologia , Osteogênese , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células da Medula Óssea/citologia , Contagem de Células , Técnicas de Cultura de Células , Fusão Celular , Proliferação de Células , Tamanho Celular , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética
6.
Clin Calcium ; 28(3): 360-366, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-29512527

RESUMO

The neuromuscular junction(NMJ)is the synapse between a motor neuron and the skeletal muscle that is essential for muscle contraction. Impairments at the NMJ lead to neuromuscular-transmission pathologies characterized by fatigable muscle weakness. Muscle-specific receptor tyrosine kinase MuSK plays key roles in NMJ formation. Over the past decade, studies examining the NMJ formation signals have identified molecules involved in the signaling pathways and have promoted a better understanding of characteristic molecular mechanisms for MuSK activation. Unlike many other receptor tyrosine kinases, MuSK is regulated by the cytoplasmic activator Dok-7 in addition to the extracellular activator agrin. It is well established that all of these molecules are indispensable in the formation and maintenance of the NMJ. In this chapter, we review molecular signaling, particularly MuSK signaling, in the formation of the NMJ and the altered molecular signaling associated with neuromuscular disorders.


Assuntos
Homeostase , Doenças Musculoesqueléticas/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , Humanos
7.
Genes Cells ; 21(6): 670-6, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27091576

RESUMO

The neuromuscular junction (NMJ) is a synapse between a motor neuron and skeletal muscle and is required for muscle contraction. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We previously showed that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK. Indeed, mice lacking either Dok-7 or MuSK form no NMJs, and defects in the human DOK7 gene underlie a congenital myasthenic syndrome (an NMJ disorder). However, it remains unproven whether Dok-7 is required for the postnatal maintenance of NMJs. In this study, we generated recombinant adeno-associated virus (AAV) vectors encoding short hairpin RNAs targeting the mouse dok-7 gene (AAV-shD7). Systemic administration of AAV-shD7 into 2-week-old mice down-regulated dok-7 expression in muscle and induced myasthenic symptoms including reduction in body weight and motor function. Moreover, AAV-shD7 treatment suppressed MuSK-dependent gene expression of NMJ components and reduced the size of NMJs. These results demonstrate that correct, physiological levels of dok-7 expression are required for the postnatal maintenance of NMJs.


Assuntos
Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miastenia Gravis/genética , Junção Neuromuscular/metabolismo , Animais , Técnicas de Silenciamento de Genes , Vetores Genéticos , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis/metabolismo , Miastenia Gravis/patologia , RNA Interferente Pequeno/metabolismo
8.
Proc Natl Acad Sci U S A ; 111(46): 16556-61, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25368159

RESUMO

The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.


Assuntos
Agrina/fisiologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Agrina/deficiência , Agrina/genética , Processamento Alternativo , Animais , Diafragma/embriologia , Diafragma/crescimento & desenvolvimento , Ativação Enzimática , Feminino , Proteínas Relacionadas a Receptor de LDL , Longevidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/fisiologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/crescimento & desenvolvimento , Doenças da Junção Neuromuscular/enzimologia , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Fosforilação , Densidade Pós-Sináptica/fisiologia , Processamento de Proteína Pós-Traducional , Receptores Colinérgicos/fisiologia , Receptores de LDL/deficiência , Receptores de LDL/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-Rod
9.
Clin Calcium ; 27(3): 413-419, 2017.
Artigo em Japonês | MEDLINE | ID: mdl-28232656

RESUMO

The mammalian neuromuscular junction(NMJ), a cholinergic synapse between a motor neuron and a skeletal muscle fiber, is essential for neural control of muscle contraction. Impaired formation and/or maintenance of NMJs results in disorders of neuromuscular transmission such as myasthenia gravis(MG)and congenital myasthenic syndromes(CMSs). The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase MuSK. Activation of MuSK involves its essential cytoplasmic activator Dok-7, the MuSK co-receptor Lrp4, and the motor neuron-derived MuSK activator agrin. Indeed, CMS-associated mutations are identified in the genes encoding these 4 proteins, and autoantibodies against MuSK, Lrp4, or agrin are found in MG patients, demonstrating the pathophysiological significance of the MuSK activation machinery. However, Lrp4 and agrin also play crucial roles separate from MuSK activation in NMJ formation and maintenance. Based on the finding that forced expression of Dok-7 in muscle activates MuSK and enlarges NMJs, we recently developed DOK7 gene therapy as a therapy aimed at enlarging the NMJ, which has potential for treating various neuromuscular disorders with defective NMJ structure.


Assuntos
Doenças Musculares/tratamento farmacológico , Junção Neuromuscular , Animais , Terapia Genética , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Doenças Musculares/genética , Doenças Musculares/terapia , Junção Neuromuscular/metabolismo
10.
Biochem Biophys Res Commun ; 478(1): 135-142, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27450811

RESUMO

Appropriate immune responses and mucosal barrier functions are required for the maintenance of intestinal homeostasis. Defects in this defense system may lead to inflammatory disorders such as inflammatory bowel disease. Downstream of tyrosine kinases 1 (Dok-1) and its closest homolog, Dok-2, are preferentially expressed in immune cells, and play essential roles in the negative regulation of multiple signaling pathways in both innate and adaptive immunity. However, the function of these proteins in intestinal homeostasis remained unclear. Here we show that Dok-1/-2 double knockout (DKO) mice were highly susceptible to dextran sodium sulfate (DSS)-induced colitis compared with Dok-1 or Dok-2 single KO and wild type (WT) mice. Furthermore, DSS-treated Dok-1/-2 DKO mice exhibited increased colonic tissue damage accompanied by reduced proliferation of the epithelial cells relative to WT controls, suggesting that Dok-1/-2 DKO mice have defects in the repair of intestinal epithelial lesions. In addition, the levels of the Th17 cytokines IL-17A and IL-22, which have protective roles in DSS-induced colitis, were reduced in DSS-treated Dok-1/-2 DKO mice compared with WT mice. Taken together, our results demonstrate that Dok-1 and Dok-2 negatively regulate intestinal inflammation, apparently through the induction of IL-17A and IL-22 expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colite/metabolismo , Colo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Regulação para Baixo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genética , Interleucina 22
11.
J Biol Chem ; 289(8): 5051-60, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24385425

RESUMO

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Resistência ao Cisalhamento , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fibrinogênio/farmacologia , Hemorreologia/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/deficiência , Adesividade Plaquetária/efeitos dos fármacos , Resistência ao Cisalhamento/efeitos dos fármacos , Trombose/metabolismo , Trombose/patologia , Trombose/fisiopatologia , Fatores de Tempo
12.
Genes Cells ; 19(4): 287-96, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24475942

RESUMO

Activation of Wnt/ß-catenin signal in muscle satellite cells (mSCs) of aged mice during myogenic differentiation has been appreciated as an important age-related feature of the skeletal muscles, resulting in impairment of their regenerative ability following muscle injury. However, it remains elusive about molecules involved in this age-related alteration of Wnt/ß-catenin signal in myogenic cells. To clarify this issue, we carried out expression analyses of Wnt receptor genes using real-time RT-PCR in mSCs isolated from the skeletal muscles of young and aged mice. Here, we show that expression of Frizzled1 (Fzd1) was detected at high levels in mSCs of aged mice. Higher expression levels of Fzd1 were also detected in mSC-derived myogenic cells from aged mice and associated with activation of Wnt/ß-catenin signal during their myogenic differentiation in vitro. We also provide evidence that suppressed expression of Fzd1 in myogenic cells from aged mice results in a significant increase in myogenic differentiation, and its forced expression in those from young mice results in its drastic inhibition. These findings indicate the critical role of Fzd1 in altered myogenic differentiation associated with aging.


Assuntos
Diferenciação Celular/fisiologia , Receptores Frizzled/metabolismo , Células Satélites de Músculo Esquelético/citologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Fatores Etários , Envelhecimento , Animais , Células Cultivadas , Masculino , Camundongos Endogâmicos ICR , Células Satélites de Músculo Esquelético/metabolismo , Transdução de Sinais
13.
Genes Cells ; 18(1): 56-65, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23205702

RESUMO

Asthma is a chronic inflammatory disease of the lung with airflow obstruction and bronchospasm, characterized by pulmonary eosinophilia, airway remodeling, increased airway hyperresponsiveness to environmental stimuli, and excessive Th2-type cytokine production. Recent studies indicate that crosstalk between the innate and adaptive immune systems is crucial for this disease. We and others have showed that the Dok (downstream of tyrosine kinases) family adaptors, Dok-1, Dok-2, and Dok-3, play essential roles in negative regulation of a wide variety of signaling pathways in both innate and adaptive immunities. Here, histopathology and bronchoalveolar lavage fluid (BALF) cellularity showed spontaneous pulmonary inflammation in Dok-1-/- Dok-2-/- Dok-3-/- (TKO) mice, but not in Dok-1-/- Dok-2-/- or Dok-3-/- mice, with hallmarks of asthma, including eosinophilia, goblet cell hyperplasia, and subepithelial fibrosis. Consistently, TKO mice, but not the other mutants, showed increased airway hyperresponsiveness to methacholine inhalation. In addition, Th2-type cytokine concentrations in BALF were increased in TKO mice. These findings provide strong evidence that Dok-1, Dok-2, and Dok-3 cooperatively play critical anti-inflammatory roles in lung homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Asma/patologia , Pulmão/patologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Asma/genética , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Broncoconstritores/farmacologia , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Fibrose/patologia , Homeostase/genética , Hiperplasia/patologia , Inflamação/genética , Inflamação/patologia , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Pneumonia/genética , Pneumonia/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Mucosa Respiratória/patologia , Células Th2/metabolismo
14.
Brain Nerve ; 76(1): 61-67, 2024 Jan.
Artigo em Japonês | MEDLINE | ID: mdl-38191141

RESUMO

Skeletal muscle is an essential organ for the motor functions and its defects are associated with functional impairments of other organs including brain. Muscle contraction is the fundamental skeletal muscle function and is strictly controlled by motor neuron, which requires neuromuscular junction (NMJ), a chemical synapse between motor nerve terminal and myotube (myofiber). Defects in NMJ cause various functional abnormalities of skeletal muscle including muscle weakness. This review presents an overview of the current understanding of signaling in NMJ formation and maintenance in skeletal muscle and the development of NMJ-targeted therapeutics.


Assuntos
Junção Neuromuscular , Sinapses , Humanos , Neurônios Motores , Músculo Esquelético , Encéfalo
15.
Front Cell Dev Biol ; 12: 1363541, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38946797

RESUMO

Introduction: Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding gene that leads to muscle necrosis and degeneration with chronic inflammation during growth, resulting in progressive generalized weakness of the skeletal and cardiac muscles. We previously demonstrated the therapeutic effects of systemic administration of dental pulp mesenchymal stromal cells (DPSCs) in a DMD animal model. We showed preservation of long-term muscle function and slowing of disease progression. However, little is known regarding the effects of cell therapy on the metabolic abnormalities in DMD. Therefore, here, we aimed to investigate the mechanisms underlying the immunosuppressive effects of DPSCs and their influence on DMD metabolism. Methods: A comprehensive metabolomics-based approach was employed, and an ingenuity pathway analysis was performed to identify dystrophy-specific metabolomic impairments in the mdx mice to assess the therapeutic response to our established systemic DPSC-mediated cell therapy approach. Results and Discussion: We identified DMD-specific impairments in metabolites and their responses to systemic DPSC treatment. Our results demonstrate the feasibility of the metabolomics-based approach and provide insights into the therapeutic effects of DPSCs in DMD. Our findings could help to identify molecular marker targets for therapeutic intervention and predict long-term therapeutic efficacy.

16.
iScience ; 27(2): 108997, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38327785

RESUMO

The neuromuscular junction (NMJ) forms centrally in myotubes and, as the only synapse between motor neuron and myotube, are indispensable for motor activity. The midmuscle formation of NMJs, including midmuscle-restricted expression of NMJ-related genes, is governed by the muscle-specific kinase (MuSK). However, mechanisms underlying MuSK-mediated signaling are unclear. Here, we find that the Calcium-binding protein 7 (Cabp7) gene shows midmuscle-restricted expression, and muscle-specific depletion of Cabp7 in mice accelerated age-related NMJ degeneration, muscle weakness/atrophy, and motor dysfunction. Surprisingly, forced expression in muscle of CIP, an inhibitory peptide of the negative regulator of NMJ formation cyclin-dependent kinase 5 (Cdk5), restored NMJ integrity and muscle strength, and healed muscle atrophy in muscle-specific Cabp7-deficient mice, which showed increased muscle expression of the Cdk5 activator p25. These findings together demonstrate that MuSK-mediated signaling induces muscle expression of Cabp7, which suppresses age-related NMJ degeneration likely by attenuating p25 expression, providing insights into prophylactic/therapeutic intervention against age-related motor dysfunction.

17.
Sci Rep ; 14(1): 15053, 2024 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-38956389

RESUMO

Targeting intracellular inhibiting proteins has been revealed to be a promising strategy to improve CD8+ T cell anti-tumor efficacy. Here, we are focusing on intracellular inhibiting proteins specific to TCR signaling: DOK1 and DOK2 expressed in T cells. We hypothesized that depletion of intracellular inhibition checkpoint DOK1 and DOK2 could improve CD8+ T-cell based cancer therapies. To evaluate the role of DOK1 and DOK2 depletion in physiology and effector function of CD8+ T lymphocytes and in cancer progression, we established a transgenic T cell receptor mouse model specific to melanoma antigen hgp100 (pmel-1 TCR Tg) in WT and Dok1/Dok2 DKO (double KO) mice. We showed that both DOK1 and DOK2 depletion in CD8+ T cells after an in vitro pre-stimulation induced a higher percentage of effector memory T cells as well as an up regulation of TCR signaling cascade- induced by CD3 mAbs, including the increased levels of pAKT and pERK, two major phosphoproteins involved in T cell functions. Interestingly, this improved TCR signaling was not observed in naïve CD8+ T cells. Despite this enhanced TCR signaling essentially shown upon stimulation via CD3 mAbs, pre-stimulated Dok1/Dok2 DKO CD8+ T cells did not show any increase in their activation or cytotoxic capacities against melanoma cell line expressing hgp100 in vitro. Altogether we demonstrate here a novel aspect of the negative regulation by DOK1 and DOK2 proteins in CD8+ T cells. Indeed, our results allow us to conclude that DOK1 and DOK2 have an inhibitory role following long term T cell stimulations.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Linfócitos T CD8-Positivos , Proteínas de Ligação a DNA , Memória Imunológica , Camundongos Knockout , Fosfoproteínas , Proteínas de Ligação a RNA , Receptores de Antígenos de Linfócitos T , Transdução de Sinais , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linhagem Celular Tumoral , Camundongos Transgênicos
18.
Immunol Rev ; 232(1): 273-85, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19909370

RESUMO

The mammalian Dok protein family has seven members (Dok-1-Dok-7). The Dok proteins share structural similarities characterized by the NH2-terminal pleckstrin homology and phosphotyrosine-binding domains followed by SH2 target motifs in the COOH-terminal moiety, indicating an adapter function. Indeed, Dok-1 was originally identified as a 62 kDa protein that binds with p120 rasGAP, a potent inhibitor of Ras, upon tyrosine phosphorylation by a variety of protein tyrosine kinases. Among the Dok family, only Dok-1, Dok-2, and Dok-3 are preferentially expressed in hematopoietic/immune cells. Dok-1 and its closest relative Dok-2 act as negative regulators of the Ras-Erk pathway downstream of many immunoreceptor-mediated signaling systems, and it is believed that recruitment of p120 rasGAP by Dok-1 and Dok-2 is critical to their negative regulation. By contrast, Dok-3 does not bind with p120 rasGAP. However, accumulating evidence has demonstrated that Dok-3 is a negative regulator of the activation of JNK and mobilization of Ca2+ in B-cell receptor-mediated signaling, where the interaction of Dok-3 with SHIP-1 and Grb2 appears to be important. Here, we review the physiological roles and underlying mechanisms of Dok family proteins.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos B/metabolismo , Proteínas de Ligação a DNA/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Ativação Linfocitária , Proteínas de Membrana/química , Fosfoproteínas/química , Fosfoproteínas/imunologia , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/imunologia , Transdução de Sinais , Proteína p120 Ativadora de GTPase/metabolismo
19.
Nat Aging ; 3(8): 1001-1019, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37474791

RESUMO

Protein misfolding is a major factor of neurodegenerative diseases. Post-mitotic neurons are highly susceptible to protein aggregates that are not diluted by mitosis. Therefore, post-mitotic cells may have a specific protein quality control system. Here, we show that LONRF2 is a bona fide protein quality control ubiquitin ligase induced in post-mitotic senescent cells. Under unperturbed conditions, LONRF2 is predominantly expressed in neurons. LONRF2 binds and ubiquitylates abnormally structured TDP-43 and hnRNP M1 and artificially misfolded proteins. Lonrf2-/- mice exhibit age-dependent TDP-43-mediated motor neuron (MN) degeneration and cerebellar ataxia. Mouse induced pluripotent stem cell-derived MNs lacking LONRF2 showed reduced survival, shortening of neurites and accumulation of pTDP-43 and G3BP1 after long-term culture. The shortening of neurites in MNs from patients with amyotrophic lateral sclerosis is rescued by ectopic expression of LONRF2. Our findings reveal that LONRF2 is a protein quality control ligase whose loss may contribute to MN degeneration and motor deficits.


Assuntos
Neurônios Motores , Ubiquitina , Camundongos , Animais , Neurônios Motores/metabolismo , Ubiquitina/metabolismo , Ligases/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas de Ligação a DNA/genética
20.
Ann Neurol ; 69(2): 418-22, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21387385

RESUMO

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction, where acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein (LDL) receptor-related protein 4 (Lrp4) are essential. About 80% and 0% to 10% of patients with generalized MG have autoantibodies to AChR and MuSK, respectively, but pathogenic factors are elusive in others. Here we show that a proportion of AChR antibody-negative patients have autoantibodies to Lrp4. These antibodies inhibit binding of Lrp4 to its ligand and predominantly belong to the immunoglobulin G1 (IgG1) subclass, a complement activator. These findings together indicate the involvement of Lrp4 antibodies in the pathogenesis of AChR antibody-negative MG.


Assuntos
Autoanticorpos/imunologia , Proteínas Relacionadas a Receptor de LDL/imunologia , Miastenia Gravis/imunologia , Humanos , Imunoglobulina G/imunologia , Ensaio de Radioimunoprecipitação , Receptores Colinérgicos/imunologia
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