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1.
J Neurosci ; 37(12): 3181-3191, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213441

RESUMO

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.


Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Ativação Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Ligação Proteica
2.
J Biol Chem ; 289(18): 12922-30, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24644294

RESUMO

Reelin is a secreted glycoprotein that plays essential roles in the brain. Reelin is specifically cleaved at two distinct sites, called N-t and C-t, with the former being the major one. N-t cleavage can occur both in the extracellular space and in the endosomes, although the physiological importance of endosomal N-t cleavage has not been investigated. In this study, we first determined the exact N-t cleavage site catalyzed by a protease secreted by cerebral cortical neurons. Cleavage occurred between Pro-1244 and Ala-1245 within Reelin repeat 3. A Reelin mutant in which Pro-1244 was replaced with aspartate (Reelin-PD) was resistant to a protease secreted by cultured cerebral cortical neurons, and its biological activity stayed active longer than that of wild-type Reelin. Interestingly, Reelin-PD remained in the intracellular compartments longer than wild-type Reelin and persistently activated downstream signaling. Therefore, N-t cleavage of Reelin is required for halting the signaling machinery in the extracellular space as well as within endosomes of target neurons. We established a monoclonal antibody specific to uncleaved Reelin protein and found that it is localized in the vicinity of Reelin-producing cells, whereas the N-terminal fragment diffuses, or is transported, to distant regions. These data demonstrate that N-t cleavage of Reelin plays critical roles in regulating the duration and range of Reelin functions both in the extracellular milieu and in the intracellular compartments.


Assuntos
Ácido Aspártico/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Mutação , Proteínas do Tecido Nervoso/genética , Prolina/genética , Serina Endopeptidases/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Western Blotting , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Endossomos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Espaço Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeo Hidrolases/metabolismo , Prolina/metabolismo , Proteólise , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/metabolismo
3.
FEBS Lett ; 586(19): 3349-53, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22819337

RESUMO

Reelin is a glycoprotein essential for brain development and functions. Reelin is subject to specific proteolysis at two distinct (N-t and C-t) sites, and these cleavages significantly diminish Reelin activity. The decrease of Reelin activity is detrimental for brain function, but the protease that catalyzes specific cleavage of Reelin remains elusive. Here we found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) cleaves Reelin in an isoform-specific manner. Among ADAMTS-4 isoforms, p50 cleaves the N-t site only, while p75 cleaves both sites. This is the first report identifying a protease that can specifically cleave Reelin.


Assuntos
Proteínas ADAM/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/metabolismo , Proteínas ADAM/química , Proteínas ADAM/genética , Proteína ADAMTS4 , Animais , Sequência de Bases , Sítios de Ligação , Encéfalo/embriologia , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/química , Moléculas de Adesão Celular Neuronais/genética , Primers do DNA/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Hibridização In Situ , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Gravidez , Pró-Colágeno N-Endopeptidase/química , Pró-Colágeno N-Endopeptidase/genética , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Endopeptidases/química , Serina Endopeptidases/genética , Especificidade por Substrato , Distribuição Tecidual
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