RESUMO
2-Deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) is widely used to study cerebral glucose metabolism. Here, we investigated whether the FDG-PET signal is directly influenced by microglial glucose uptake in mouse models and patients with neurodegenerative diseases. Using a recently developed approach for cell sorting after FDG injection, we found that, at cellular resolution, microglia displayed higher glucose uptake than neurons and astrocytes. Alterations in microglial glucose uptake were responsible for both the FDG-PET signal decrease in Trem2-deficient mice and the FDG-PET signal increase in mouse models for amyloidosis. Thus, opposite microglial activation states determine the differential FDG uptake. Consistently, 12 patients with Alzheimer's disease and 21 patients with four-repeat tauopathies also exhibited a positive association between glucose uptake and microglial activity as determined by 18F-GE-180 18-kDa translocator protein PET (TSPO-PET) in preserved brain regions, indicating that the cerebral glucose uptake in humans is also strongly influenced by microglial activity. Our findings suggest that microglia activation states are responsible for FDG-PET signal alterations in patients with neurodegenerative diseases and mouse models for amyloidosis. Microglial activation states should therefore be considered when performing FDG-PET.
Assuntos
Fluordesoxiglucose F18 , Doenças Neurodegenerativas , Humanos , Glucose , Microglia , Doenças Neurodegenerativas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , CamundongosRESUMO
Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPkappa is cleaved by three proteases: furin, ADAM 10, and gamma-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPkappa. This is followed by gamma-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates beta-catenin, an RPTPkappa substrate. The expression of RPTPkappa suppresses beta-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of beta-catenin signaling. We also found that RPTPmu, another MAM family member, undergoes gamma-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPkappa signaling and implicate PIC in the activation of beta-catenin-mediated transcription.
Assuntos
Proteínas ADAM/metabolismo , Endopeptidases/metabolismo , Furina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , beta Catenina/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Biotinilação , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células COS , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Densitometria , Relação Dose-Resposta a Droga , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Células HCT116 , Humanos , Cinética , Leupeptinas/farmacologia , Luciferases/análise , Luciferases/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Plasmídeos/genética , Testes de Precipitina , Interferência de RNA , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Transcrição Gênica , Trifluoperazina/farmacologiaRESUMO
Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.
Assuntos
Membrana Celular/metabolismo , Endopeptidases/metabolismo , Receptores de Hialuronatos/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Ácido gama-Aminobutírico/análogos & derivados , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Carbamatos/farmacologia , Linhagem Celular , Dipeptídeos/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Receptores de Hialuronatos/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Peptídeos/genética , Presenilina-1 , Presenilina-2 , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Triglicerídeos/farmacologia , Ácido gama-Aminobutírico/farmacologiaRESUMO
The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP.