RESUMO
NMDA receptors (NMDARs) are ionotropic receptors crucial for brain information processing. Yet, evidence also supports an ion-flux-independent signaling mode mediating synaptic long-term depression (LTD) and spine shrinkage. Here, we identify AETA (Aη), an amyloid-ß precursor protein (APP) cleavage product, as an NMDAR modulator with the unique dual regulatory capacity to impact both signaling modes. AETA inhibits ionotropic NMDAR activity by competing with the co-agonist and induces an intracellular conformational modification of GluN1 subunits. This favors non-ionotropic NMDAR signaling leading to enhanced LTD and favors spine shrinkage. Endogenously, AETA production is increased by in vivo chemogenetically induced neuronal activity. Genetic deletion of AETA production alters NMDAR transmission and prevents LTD, phenotypes rescued by acute exogenous AETA application. This genetic deletion also impairs contextual fear memory. Our findings demonstrate AETA-dependent NMDAR activation (ADNA), characterizing AETA as a unique type of endogenous NMDAR modulator that exerts bidirectional control over NMDAR signaling and associated information processing.
RESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease-associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full-length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α-secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α-secretase-mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho-SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid ß-peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9-bound. Moreover, in a mouse model for Alzheimer's disease-related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease-associated state.
Assuntos
Anticorpos Monoclonais/farmacologia , Glicoproteínas de Membrana/imunologia , Microglia , Mieloma Múltiplo , Receptores Imunológicos/imunologia , Peptídeos beta-Amiloides , Animais , Linhagem Celular Tumoral , Feminino , Macrófagos , Camundongos , Microglia/patologia , Ratos , Ratos WistarRESUMO
Alzheimer's disease (AD) is characterized by deposition of amyloid plaques, neurofibrillary tangles, and neuroinflammation. In order to study microglial contribution to amyloid plaque phagocytosis, we developed a novel ex vivo model by co-culturing organotypic brain slices from up to 20-month-old, amyloid-bearing AD mouse model (APPPS1) and young, neonatal wild-type (WT) mice. Surprisingly, co-culturing resulted in proliferation, recruitment, and clustering of old microglial cells around amyloid plaques and clearance of the plaque halo. Depletion of either old or young microglial cells prevented amyloid plaque clearance, indicating a synergistic effect of both populations. Exposing old microglial cells to conditioned media of young microglia or addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) was sufficient to induce microglial proliferation and reduce amyloid plaque size. Our data suggest that microglial dysfunction in AD may be reversible and their phagocytic ability can be modulated to limit amyloid accumulation. This novel ex vivo model provides a valuable system for identification, screening, and testing of compounds aimed to therapeutically reinforce microglial phagocytosis.
Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Modelos Animais de Doenças , Microglia/metabolismo , Placa Amiloide/metabolismo , Animais , Proliferação de Células , Técnicas de Cocultura , Meios de Cultivo Condicionados , Camundongos , Microglia/fisiologia , Técnicas de Cultura de ÓrgãosRESUMO
Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of ß-secretase (ß-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by proteolytic processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III ß-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.
Assuntos
Membrana Celular/enzimologia , Neuregulina-1/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Peptídeo C/metabolismo , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação/genética , Neurônios/metabolismo , Peptídeos/química , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína , Ratos , Esquizofrenia/genética , Especificidade por SubstratoRESUMO
Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-ß peptide. Two principal physiological pathways either prevent or promote amyloid-ß generation from its precursor, ß-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-ß fragments generated by the α- and ß-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (ß-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-ß). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/citologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Neurônios/fisiologia , Proteólise , Proteínas ADAM/metabolismo , Proteína ADAM10 , Doença de Alzheimer/enzimologia , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/líquido cefalorraquidiano , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Sinalização do Cálcio , Modelos Animais de Doenças , Feminino , Hipocampo/enzimologia , Hipocampo/fisiologia , Humanos , Técnicas In Vitro , Potenciação de Longa Duração , Masculino , Metaloproteinases da Matriz Associadas à Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Peso Molecular , Neuritos/enzimologia , Neuritos/metabolismo , Neurônios/enzimologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Placa Amiloide , Processamento de Proteína Pós-Traducional , Análise de Célula ÚnicaRESUMO
Proteolytic shedding of cell surface proteins generates paracrine signals involved in numerous signaling pathways. Neuregulin 1 (NRG1) type III is involved in myelination of the peripheral nervous system, for which it requires proteolytic activation by proteases of the ADAM family and BACE1. These proteases are major therapeutic targets for the prevention of Alzheimer's disease because they are also involved in the proteolytic generation of the neurotoxic amyloid ß-peptide. Identification and functional investigation of their physiological substrates is therefore of greatest importance in preventing unwanted side effects. Here we investigated proteolytic processing of NRG1 type III and demonstrate that the ectodomain can be cleaved by three different sheddases, namely ADAM10, ADAM17, and BACE1. Surprisingly, we not only found cleavage by ADAM10, ADAM17, and BACE1 C-terminal to the epidermal growth factor (EGF)-like domain, which is believed to play a pivotal role in signaling, but also additional cleavage sites for ADAM17 and BACE1 N-terminal to that domain. Proteolytic processing at N- and C-terminal sites of the EGF-like domain results in the secretion of this domain from NRG1 type III. The soluble EGF-like domain is functionally active and stimulates ErbB3 signaling in tissue culture assays. Moreover, the soluble EGF-like domain is capable of rescuing hypomyelination in a zebrafish mutant lacking BACE1. Our data suggest that NRG1 type III-dependent myelination is not only controlled by membrane-retained NRG1 type III, but also in a paracrine manner via proteolytic liberation of the EGF-like domain.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Neurregulinas/metabolismo , Comunicação Parácrina/fisiologia , Proteína ADAM17 , Animais , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Embrião de Mamíferos , Fator de Crescimento Epidérmico/análogos & derivados , Fator de Crescimento Epidérmico/química , Humanos , Imunoprecipitação , Neurregulinas/genética , Neurônios , Fosforilação , Proteólise , RNA Mensageiro/administração & dosagem , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Células de Schwann , Transfecção , Peixe-ZebraRESUMO
Deposition of amyloid ß peptides (Aßs) in extracellular amyloid plaques within the human brain is a hallmark of Alzheimer's disease (AD). Aß derives from proteolytic processing of the amyloid precursor protein (APP) by ß- and γ-secretases. The initial cleavage by ß-secretase results in shedding of the APP ectodomain and generation of APP C-terminal fragments (APP-CTFs), which can then be further processed within the transmembrane domain by γ-secretase, resulting in release of Aß. Here, we demonstrate that accumulation of sphingolipids (SLs), as occurs in lysosomal lipid storage disorders (LSDs), decreases the lysosome-dependent degradation of APP-CTFs and stimulates γ-secretase activity. Together, this results in increased generation of both intracellular and secreted Aß. Notably, primary fibroblasts from patients with different SL storage diseases show strong accumulation of potentially amyloidogenic APP-CTFs. By using biochemical, cell biological, and genetic approaches, we demonstrate that SL accumulation affects autophagic flux and impairs the clearance of APP-CTFs. Thus, accumulation of SLs might not only underlie the pathogenesis of LSDs, but also trigger increased generation of Aß and contribute to neurodegeneration in sporadic AD.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Autofagia , Fibroblastos/metabolismo , Lisossomos/metabolismo , Esfingolipídeos/metabolismo , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Fibroblastos/ultraestrutura , Imuno-Histoquímica , Lisossomos/genética , Lisossomos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Fragmentos de Peptídeos/metabolismo , Esfingolipídeos/genética , TransfecçãoRESUMO
Presenilins (PS) are critical components of the gamma-secretase complex that mediates cleavage of type I membrane proteins including the beta-amyloid precursor protein to generate the amyloid beta-peptide. In addition, PS1 interacts with beta-catenin and facilitates its metabolism. We demonstrate that phosphorylation of serines 353 and 357 by glycogen synthase kinase-3beta (GSK3beta) induces a structural change of the hydrophilic loop of PS1 that can also be mimicked by substitution of the phosphorylation sites by negatively charged amino acids in vitro and in cultured cells. The structural change of PS1 reduces the interaction with beta-catenin leading to decreased phosphorylation and ubiquitination of beta-catenin. The decreased interaction of PS1 with beta-catenin leads to stabilization of beta-catenin thereby increasing its nuclear signaling and the transcription of target genes, including c-MYC. Consistent with increased expression of c-myc, a PS1 mutant that mimics phosphorylated PS1 increased cell proliferation as compared with wild-type PS1. These results indicate a regulatory mechanism in which GSK3beta-mediated phosphorylation induces a structural change of the hydrophilic loop of PS1 thereby negatively modulating the formation of a ternary complex between beta-catenin, PS1, and GSK3beta, which leads to stabilization of beta-catenin.