RESUMO
Alzheimer's disease (AD) is the most prevalent type of dementia; therefore, there is a high demand for therapeutic medication targeting it. In this context, extensive research has been conducted to identify molecular targets for drugs. AD manifests through two primary pathological signs: senile plaques and neurofibrillary tangles, caused by accumulations of amyloid-beta (Aß) and phosphorylated tau, respectively. Thus, studies concerning the molecular mechanisms underlying AD etiology have primarily focused on Aß generation and tau phosphorylation, with the anticipation of uncovering a signaling pathway impacting these molecular processes. Over the past two decades, studies using not only experimental model systems but also examining human brains have accumulated fragmentary evidences suggesting that REELIN signaling pathway is deeply involved in AD. Here, we explore REELIN signaling pathway and its involvement in memory function within the brain and review studies investigating molecular connections between REELIN signaling pathway and AD etiology. This review aims to understand how the manipulation (activation) of this pathway might ameliorate the disease's etiology.
RESUMO
Dab1 is an intracellular adaptor protein essential for brain formation during development. Tyrosine phosphorylation in Dab1 plays important roles in neuronal migration, dendrite development, and synapse formation by affecting several downstream pathways. Reelin is the best-known extracellular protein that induces Dab1 phosphorylation. However, whether other upstream molecule(s) contribute to Dab1 phosphorylation remains largely unknown. Here, we found that EphA4, a member of the Eph family of receptor-type tyrosine kinases, induced Dab1 phosphorylation when co-expressed in cultured cells. Tyrosine residues phosphorylated by EphA4 were the same as those phosphorylated by Reelin in neurons. The autophosphorylation of EphA4 was necessary for Dab1 phosphorylation. We also found that EphA4-induced Dab1 phosphorylation was mediated by the activation of the Src family tyrosine kinases. Interestingly, Dab1 phosphorylation was not observed when EphA4 was activated by ephrin-A5 in cultured cortical neurons, suggesting that Dab1 is localized in a different compartment in them. EphA4-induced Dab1 phosphorylation may occur under limited and/or pathological conditions in the brain.
Assuntos
Neurônios , Receptor EphA4 , Proteína Reelina , Quinases da Família src , Proteína Reelina/metabolismo , Fosforilação , Animais , Receptor EphA4/metabolismo , Receptor EphA4/genética , Quinases da Família src/metabolismo , Neurônios/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Células HEK293 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Serina Endopeptidases/metabolismo , Serina Endopeptidases/genética , Células Cultivadas , Efrina-A5/metabolismo , Efrina-A5/genética , Camundongos , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/metabolismo , RatosRESUMO
The mammalian neocortex is formed by sequential radial migration of newborn excitatory neurons. Migrating neurons undergo a multipolar-to-bipolar transition at the subplate (SP) layer, where extracellular matrix (ECM) components are abundantly expressed. Here, we investigate the role of the ECM at the SP layer. We show that TGF-ß signaling-related ECM proteins, and their downstream effector, p-smad2/3, are selectively expressed in the SP layer. We also find that migrating neurons express a disintegrin and metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. Knockdown and knockout of Adamts2 suppresses the multipolar-to-bipolar transition of migrating neurons and disturbs radial migration. Time-lapse luminescence imaging of TGF-ß signaling indicates that ADAMTS2 activates this signaling pathway in migrating neurons during the multipolar-to-bipolar transition at the SP layer. Overexpression of TGF-ß2 in migrating neurons partially rescues migration defects in ADAMTS2 knockout mice. Our data suggest that ADAMTS2 secreted by the migrating multipolar neurons activates TGF-ß signaling by ECM remodeling of the SP layer, which might drive the multipolar to bipolar transition.
Assuntos
Proteínas ADAMTS , Movimento Celular , Camundongos Knockout , Neocórtex , Neurônios , Transdução de Sinais , Fator de Crescimento Transformador beta , Animais , Neocórtex/metabolismo , Neocórtex/citologia , Proteínas ADAMTS/metabolismo , Proteínas ADAMTS/genética , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Neurônios/metabolismo , Matriz Extracelular/metabolismoRESUMO
The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.
Assuntos
Corpo Celular , Cones de Crescimento , Neurônios/metabolismo , Membrana Celular , Hipocampo , Células CultivadasRESUMO
Reelin is a large secreted protein important for brain development and functions. In both humans and mice, the lack of Reelin gene causes cerebellar hypoplasia and ataxia. Treatment against Reelin deficiency is currently unavailable. Here, we show that the injection of recombinant Reelin protein into the cerebellum of Reelin-deficient reeler mice at postnatal day 3 ameliorates the forelimb coordination and mice are noted to stand up along cage wall more frequently. A mutant Reelin protein resistant to proteases has no better effect than the wild-type Reelin. Such ameliorations were not observed when a mutant Reelin protein that does not bind to Reelin receptors was injected and the injection of Reelin protein did not ameliorate the behavior of Dab1-mutant yotari mice, indicating that its effect is dependent on the canonical Reelin receptor-Dab1 pathway. Additionally, a Purkinje cell layer in reeler mice was locally induced by Reelin protein injection. Our results indicate that the reeler mouse cerebellum retains the ability to react to Reelin protein in the postnatal stage and that Reelin protein has the potential to benefit Reelin-deficient patients.
Assuntos
Proteínas da Matriz Extracelular , Proteína Reelina , Humanos , Camundongos , Animais , Camundongos Mutantes Neurológicos , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Cerebelo , Proteínas do Tecido Nervoso/metabolismoRESUMO
Reelin, a large secreted glycoprotein, plays an important role in neuronal migration during brain development. The C-terminal region (CTR) of Reelin is involved in the efficient activation of downstream signaling and its loss leads to abnormal hippocampal layer formation. However, the molecular mechanism by which Reelin CTR regulates hippocampal development remains unknown. Here, we showed that the migration of late-born, but not early-born, neurons is impaired in the knock-in mice in which Reelin CTR is deleted (ΔC-KI mice). The phosphorylation of cofilin, an actin-depolymerizing protein, was remarkably decreased in the hippocampus of the ΔC-KI mice. Exogenous expression of pseudo-phosphorylated cofilin rescued the ectopic positioning of neurons in the hippocampus of ΔC-KI mice. These results suggest that Reelin CTR is required for the migration of late-born neurons in the hippocampus and that this event involves appropriate phosphorylation of cofilin.
Assuntos
Fatores de Despolimerização de Actina , Proteínas da Matriz Extracelular , Proteína Reelina , Animais , Camundongos , Fatores de Despolimerização de Actina/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Fosforilação , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Proteína Reelina/metabolismoRESUMO
Reelin is a secreted glycoprotein important for brain development and synaptic plasticity in the adult brain. Some reports suggest that Reelin is secreted from the nerve terminals and functions as a neurotransmitter. However, the mechanism of Reelin secretion is unknown. In this study, we visualized Reelin secretion by bioluminescence imaging using a fusion protein of Reelin and Gaussia luciferase (GLase-Reelin). GLase-Reelin expressed in HEK293T cells was correctly processed and secreted. Luminescence signals from the secreted GLase-Reelin of primary cultured neurons were visualized by bioluminescence microscopy. Reelin secretory events were observed at neurites and cell bodies. Bioluminescence imaging was also performed before and after KCl depolarization to compare the secretory events of Reelin and brain-derived neurotrophic factor (BDNF). The secretion of BDNF increased markedly shortly after depolarization. In contrast, the frequency of Reelin secretion did not change significantly by depolarization. Thus, Reelin secretion from neurites might not be regulated in a neuronal activity-dependent manner.
Assuntos
Fator Neurotrófico Derivado do Encéfalo , Neurônios , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Neuritos/metabolismo , Neurônios/metabolismoRESUMO
Reelin, a large extracellular matrix protein, helps to regulate neuronal plasticity and cognitive function. Several studies have shown that Reelin dysfunction, resulting from factors such as mutations in gene RELN or low Reelin expression, is associated with schizophrenia (SCZ). We previously reported that microinjection of Reelin into cerebral ventricle prevents phencyclidine-induced cognitive and sensory-motor gating deficits. However, it remains unclear whether and how Reelin ameliorates behavioral abnormalities in the animal model of SCZ. In the present study, we evaluated the effect of recombinant Reelin microinjection into the medial prefrontal cortex (mPFC) on abnormal behaviors induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Microinjection of Reelin into the mPFC prevented impairment of recognition memory of MK-801-treated mice in the novel object recognition test (NORT). On the other hand, the same treatment had no effect on deficits in sensory-motor gating and short-term memory in the pre-pulse inhibition and Y-maze tests, respectively. To establish the neural substrates that respond to Reelin, the number of c-Fos-positive cells in the mPFC was determined. A significant increase in c-Fos-positive cells in the mPFC of MK-801-treated mice was observed when compared with saline-treated mice, and this change was suppressed by microinjection of Reelin into the mPFC. A K2360/2467A Reelin that cannot bind to its receptor failed to ameliorate MK-801-induced cognitive deficits in NORT. These results suggest that Reelin prevents MK-801-induced recognition memory impairment by acting on its receptors to suppress neural activity in the mPFC of mice.
Assuntos
Transtornos da Memória/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Proteína Reelina/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Células Cultivadas , Maleato de Dizocilpina , Masculino , Transtornos da Memória/induzido quimicamente , Camundongos Endogâmicos C57BL , Microinjeções , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Córtex Pré-Frontal , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Proteínas Recombinantes/administração & dosagem , Proteína Reelina/genéticaRESUMO
The secreted glycoprotein Reelin plays important roles in both brain development and function. During development, Reelin regulates neuronal migration and dendrite development. In the mature brain, the glycoprotein is involved in synaptogenesis and synaptic plasticity. It has been suggested that Reelin loss or decreased function contributes to the onset and/or deterioration of neuropsychiatric diseases, including schizophrenia and Alzheimer's disease. While the molecular mechanisms underpinning Reelin function remain unclear, recent studies have suggested that the specific proteolytic cleavage of Reelin may play central roles in the embryonic and postnatal brain. In this review, we focus on Reelin proteolytic processing and review its potential physiological roles.
Assuntos
Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Esquizofrenia/metabolismo , Serina Endopeptidases/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/patologia , Humanos , Proteína Reelina , Esquizofrenia/patologiaRESUMO
Reelin, an extracellular matrix protein, is secreted by Cajal-Retzius cells and plays crucial roles in the development of brain structures and neuronal functions. Reductions in Reelin cause the brain dysfunctions associated with mental disorders, such as schizophrenia. A recent genome-wide copy number variation analysis of Japanese schizophrenia patients identified a novel deletion in RELN encoding Reelin. To clarify the pathophysiological role of the RELN deletion, we developed transgenic mice carrying the RELN deletion (Reln-del) and found abnormalities in their brain structures and social behavior. In the present study, we performed an in vitro analysis of Reelin expression, intracellular Reelin signaling, and the morphology of primary cultured cortical neurons from wild-type (WT) and Reln-del mice. Reelin protein levels were lower in Reln-del neurons than in WT neurons. Dab1 expression levels were significantly higher in Reln-del neurons than in WT neurons, suggesting that Reelin signaling was decreased in Reln-del neurons. Reelin was mainly expressed in γ-aminobutyric acid (GABA)-ergic inhibitory neurons, but not in parvalbumin (PV)-positive neurons. A small proportion of Ca2+/calmodulin-dependent protein kinase II α subunit (CaMKIIα)-positive excitatory neurons also expressed Reelin. In comparisons with WT neurons, significant decreases were observed in neurite lengths and branch points as well as in the number of postsynaptic density protein 95 (PSD95) immunoreactive puncta in Reln-del neurons. A disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3) is a protease that inactivates Reelin by cleavage at the N-t site. The knockdown of ADAMTS-3 by short hairpin RNAs suppressed Reelin cleavage in conditioned medium and reduced Dab1 expression, indicating that Reelin signaling was enhanced in the primary cultured cortical neurons of WT and heterozygous Reln-del. Accordingly, the inhibition of ADAMTS-3 may be a potential candidate in the clinical treatment of schizophrenia by enhancing Reelin signaling in the brain.
Assuntos
Moléculas de Adesão Celular Neuronais/deficiência , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/deficiência , Deleção de Genes , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Esquizofrenia/metabolismo , Serina Endopeptidases/deficiência , Animais , Moléculas de Adesão Celular Neuronais/biossíntese , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/biossíntese , Proteínas da Matriz Extracelular/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Proteína Reelina , Esquizofrenia/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Transdução de Sinais/fisiologiaRESUMO
The large, secreted glycoprotein reelin regulates embryonic brain development as well as adult brain functions. Although reelin binds to its receptors via its central part, the N-terminal region directs multimer formation and is critical for efficient signal transduction. In fact, the inhibitory antibody CR-50 interacts with the N-terminal region and prevents higher-order multimerization and signalling. Reelin is a multidomain protein in which the central part is composed of eight characteristic repeats, named reelin repeats, each of which is further divided by insertion of a epidermal growth factor (EGF) module into two subrepeats. In contrast, the N-terminal region shows unique 'irregular' domain architecture since it comprises three consecutive subrepeats without the intervening EGF module. Here, we determined the crystal structure of the murine reelin fragment named RX-R1 including the irregular region and the first reelin repeat at 2.0-Å resolution. The overall structure of RX-R1 has a branched Y-shaped form. Interestingly, two incomplete subrepeats cooperatively form one entire subrepeat structure, though an additional subrepeat is inserted between them. We further reveal that Arg335 of RX-R1 is crucial for binding CR-50. A possible self-association mechanism via the N-terminal region is proposed based on our results.
Assuntos
Moléculas de Adesão Celular Neuronais/química , Proteínas da Matriz Extracelular/química , Proteínas do Tecido Nervoso/química , Multimerização Proteica , Serina Endopeptidases/química , Animais , Anticorpos Monoclonais/química , Moléculas de Adesão Celular Neuronais/genética , Cristalografia por Raios X , Proteínas da Matriz Extracelular/genética , Camundongos , Proteínas do Tecido Nervoso/genética , Domínios Proteicos , Proteína Reelina , Sequências Repetitivas de Aminoácidos , Serina Endopeptidases/genéticaRESUMO
Reelin plays versatile roles in neocortical development. The C-terminal region (CTR) of Reelin is required for the correct formation of the superficial structure of the neocortex; however, the mechanisms by which this position-specific effect occurs remain largely unknown. In this study, we demonstrate that Reelin with an intact CTR binds to neuropilin-1 (Nrp1), a transmembrane protein. Both male and female mice were used. Nrp1 is localized with very-low-density lipoprotein receptor (VLDLR), a canonical Reelin receptor, in the superficial layers of the developing neocortex. It forms a complex with VLDLR, and this interaction is modulated by the alternative splicing of VLDLR. Reelin with an intact CTR binds more strongly to the VLDLR/Nrp1 complex than to VLDLR alone. Knockdown of Nrp1 in neurons leads to the accumulation of Dab1 protein. Since the degradation of Dab1 is induced by Reelin signaling, it is suggested that Nrp1 augments Reelin signaling. The interaction between Reelin and Nrp1 is required for normal dendritic development in superficial-layer neurons. All of these characteristics of Reelin are abrogated by proteolytic processing of the six C-terminal amino acid residues of Reelin (0.17% of the whole protein). Therefore, Nrp1 is a coreceptor molecule for Reelin and, together with the proteolytic processing of Reelin, can account for context-specific Reelin function in brain development.SIGNIFICANCE STATEMENT Reelin often exhibits a context-dependent function during brain development; however, its underlying mechanism is not well understood. We found that neuropilin-1 (Nrp1) specifically binds to the CTR of Reelin and acts as a coreceptor for very-low-density lipoprotein receptor (VLDLR). The Nrp1/VLDLR complex is localized in the superficial layers of the neocortex, and its interaction with Reelin is essential for proper dendritic development in superficial-layer neurons. This study provides the first mechanistic evidence of the context-specific function of Reelin (>3400 residues) regulated by the C-terminal residues and Nrp1, a component of the canonical Reelin receptor complex.
Assuntos
Dendritos/fisiologia , Neocórtex/citologia , Neocórtex/crescimento & desenvolvimento , Neuropilina-1/fisiologia , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , DNA/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos ICR , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neuropilina-1/genética , Receptores de LDL/metabolismo , Proteína Reelina , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
Oligodendrocyte (OL) progenitor cells (OPCs) are generated, proliferate, migrate, and differentiate in the developing brain. Although the development of OPCs is prerequisite for normal brain function, the molecular mechanisms regulating their development in the neocortex are not fully understood. Several molecules regulate the tangential distribution of OPCs in the developing neocortex, but the cue molecule(s) that regulate their radial distribution remains unknown. Here, we demonstrate that the secreted glycoprotein Reelin suppresses the proliferation of OPCs and acts as a repellent for their migration in vitro These functions rely on the binding of Reelin to its receptors and on the signal transduction involving the intracellular protein Dab1. In the late embryonic neocortex of mice with attenuated Reelin signaling [i.e., Reelin heterozygote-deficient, Dab1 heterozygote-deficient mutant, or very low-density lipoprotein receptor (VLDLR)-deficient mice], the number of OPCs increased and their distribution shifted toward the superficial layers. In contrast, the number of OPCs decreased and they tended to distribute in the deep layers in the neocortex of mice with abrogated inactivation of Reelin by proteolytic cleavage, namely a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3)-deficient mice and cleavage-resistant Reelin knock-in mice. Both male and female animals were used. These data indicate that Reelin-Dab1 signaling regulates the proliferation and radial distribution of OPCs in the late embryonic neocortex and that the regulation of Reelin function by its specific proteolysis is required for the normal development of OPCs.SIGNIFICANCE STATEMENT Here, we report that Reelin-Dab1 signaling regulates the proliferation and radial distribution of OPCs in the late embryonic mouse neocortex. Oligodendrocyte (OL) progenitor cells (OPCs) express Reelin signaling molecules and respond to Reelin stimulation. Reelin-Dab1 signaling suppresses the proliferation of OPCs both in vitro and in vivo Reelin repels OPCs in vitro, and the radial distribution of OPCs is altered in mice with either attenuated or augmented Reelin-Dab1 signaling. This is the first report identifying the secreted molecule that plays a role in the radial distribution of OPCs in the late embryonic neocortex. Our results also show that the regulation of Reelin function by its specific proteolysis is important for the normal development of OPCs.
Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Oligodendroglia/metabolismo , Serina Endopeptidases/metabolismo , Proteínas ADAMTS/metabolismo , Animais , Moléculas de Adesão Celular Neuronais/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neocórtex/citologia , Neocórtex/embriologia , Proteínas do Tecido Nervoso/genética , Células-Tronco Neurais/citologia , Oligodendroglia/citologia , Ligação Proteica , Receptores de LDL/metabolismo , Proteína Reelina , Serina Endopeptidases/genéticaRESUMO
In the majority of schizophrenia patients, chronic atypical antipsychotic administration produces a significant reduction in or even complete remission of psychotic symptoms such as hallucinations and delusions. However, these drugs are not effective in improving cognitive and emotional deficits in patients with schizophrenia. Atypical antipsychotic drugs have a high affinity for the dopamine D2 receptor, and a modest affinity for the serotonin 5-HT2A receptor. The cognitive and emotional deficits in schizophrenia are thought to involve neural networks beyond the classical dopaminergic mesolimbic pathway, however, including serotonergic systems. For example, mutations in the RELN gene, which encodes Reelin, an extracellular matrix protein involved in neural development and synaptic plasticity, are associated with neurodevelopmental disorders such as schizophrenia and autism spectrum disorder. Furthermore, hippocampal Reelin levels are down-regulated in the brains of both schizophrenic patients and in rodent models of schizophrenia. In the present study, we investigated the effect of Reelin microinjection into the mouse hippocampus on behavioral phenotypes to evaluate the role of Reelin in neurodevelopmental disorders and to test a therapeutic approach that extends beyond classical monoamine targets. To model the cognitive and emotional deficits, as well as histological decreases in Reelin-positive cell numbers and hippocampal synaptoporin distribution, a synaptic vesicle protein, offspring that were prenatally exposed to maternal immune activation were used. Microinjections of recombinant Reelin protein into the hippocampus rescued impairments in object memory and anxiety-like behavior and recruited synaptoporin in the hippocampus in offspring exposed to antenatal inflammation. These results suggest that Reelin supplementation has the potential to treat cognitive and emotional impairments, as well as synaptic disturbances, in patients with neurodevelopmental disorders such as schizophrenia.
RESUMO
Reelin is a secreted protein that plays versatile roles in neuronal development and function. The strength of Reelin signaling is regulated by proteolytic processing, but its importance in vivo is not yet fully understood. Here, we generated Reelin knock-in (PA-DV KI) mice in which the key cleavage site of Reelin was abolished by mutation. As expected, the cleavage of Reelin was severely abrogated in the cerebral cortex and hippocampus of PA-DV KI mice. The amount of Dab1, whose degradation is induced by Reelin signaling, decreased in these tissues, indicating that the signaling strength of Reelin was augmented. The brains of PA-DV KI mice were largely structurally normal, but unexpectedly, the hippocampal layer was disturbed. This phenotype was ameliorated in hemizygote PA-DV KI mice, indicating that excess Reelin signaling is detrimental to hippocampal layer formation. The neuronal dendrites of PA-DV KI mice had more branches and were elongated compared to wild-type mice. These results present the first direct evidence of the physiological importance of Reelin cleavage.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Animais , Encéfalo/metabolismo , Imunofluorescência , Expressão Gênica , Técnicas de Introdução de Genes , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Proteólise , Proteína ReelinaRESUMO
Reelin is a large secreted protein that is essential for the brain development and function. Reelin is negatively regulated by the specific cleavage by a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3) which is also secreted from neurons. It is likely that there are other proteases that can cleave Reelin. This chapter describes the protocol for expression and handling of recombinant Reelin and ADAMTS-3 proteins to facilitate investigation of these proteins.
Assuntos
Proteínas ADAMTS/genética , Moléculas de Adesão Celular Neuronais/genética , Proteínas da Matriz Extracelular/genética , Expressão Gênica , Proteínas do Tecido Nervoso/genética , Pró-Colágeno N-Endopeptidase/genética , Serina Endopeptidases/genética , Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas do Tecido Nervoso/metabolismo , Pró-Colágeno N-Endopeptidase/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/metabolismo , Proteína Reelina , Serina Endopeptidases/metabolismoRESUMO
Proteolytic cleavage of the secreted signaling protein Reelin has been suggested to play causative roles in many neuropsychiatric and neurodegenerative disorders. Therefore, characterization of the proteolytic activity against Reelin is important not only for understanding how the brain works but also for the development of novel therapy for these disorders. Notably, ADAMTS family proteases are the primary suspects of Reelin-cleaving proteases under many, though not all, circumstances. Here we describe how to measure the Reelin-cleaving activity of ADAMTS (or of any other protease that may cleave Reelin), how to purify the Reelin-cleaving protease ADAMTS-3 from the culture supernatant of cortical neurons, and how to detect endogenous Reelin protein and its fragments in the brain.
Assuntos
Proteínas ADAMTS/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/química , Córtex Cerebral/citologia , Proteínas da Matriz Extracelular/química , Proteínas do Tecido Nervoso/química , Pró-Colágeno N-Endopeptidase/metabolismo , Serina Endopeptidases/química , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Células HEK293 , Humanos , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Proteólise , Proteína ReelinaRESUMO
Precise regulation of neuronal migration termination is crucial for the establishment of brain cytoarchitectures. However, little is known about how neurons terminate migration. Here we focused on interactions between migrating cortical neurons and their substrates, radial glial (RG) cells, and analyzed the role of Plexin A2 and A4 (PlxnA2/A4) receptors and their repulsive ligand, Semaphorin 6A (Sema6A), for this process. In both PlxnA2/A4 double-knockout and Sema6A mutant mice, the outermost cortical plate neurons ectopically invade layer 1 at a stage when they should reach their destinations. PlxnA2/A4 proteins are abundantly expressed on their leading processes, whereas Sema6A mRNA is enriched in RG cell somata. Cell-targeted gene expression and conditional knockouts indicate critical roles for these molecules. We hypothesize that the timely appearance of repulsive signaling mediated by Sema6A-PlxnA2/A4 weakens migrating neuron-RG cell interactions, leading to migration termination.
RESUMO
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.
Assuntos
Proteínas ADAMTS/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Proteínas ADAMTS/genética , Animais , Cerebelo/crescimento & desenvolvimento , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Células HEK293 , Hipocampo/crescimento & desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Proteína ReelinaRESUMO
Reelin is a large secreted protein that is essential for the development and function of the central nervous system. Dimerization and/or oligomerization is required for its biological activity, but the underlying mechanism is not fully understood. There are several widely used anti-Reelin antibodies and we noticed that their reactivity to monomeric or dimeric Reelin protein is different. We also found that their reactivity to Reelin in the solution or in fixed brain tissues also differs. Our results provide the information regarding how the N-terminal region of Reelin folds and contributes to the formation of higher order structure. We also provide a caveat that appropriate use of anti-Reelin antibody is necessary for quantitative analyses.