A disintegrin and metalloproteinase with thrombospondin motifs 2 cleaves and inactivates Reelin in the postnatal cerebral cortex and hippocampus, but not in the cerebellum.

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin.

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.

Cleavage within Reelin repeat 3 regulates the duration and range of the signaling activity of Reelin protein.

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.