RESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.
Assuntos
Citometria de Fluxo/métodos , Teste de Complementação Genética/métodos , Luciferases de Renilla/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglia/metabolismo , Receptores Imunológicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Linhagem Celular , Humanos , Lipodistrofia/genética , Lipodistrofia/metabolismo , Glicoproteínas de Membrana/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda/genética , Panencefalite Esclerosante Subaguda/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismoRESUMO
Brain aging is central to late-onset Alzheimer's disease (LOAD), although the mechanisms by which it occurs at protein or cellular levels are not fully understood. Alzheimer's disease is the most common proteopathy and is characterized by two unique pathologies: senile plaques and neurofibrillary tangles, the former accumulating earlier than the latter. Aging alters the proteostasis of amyloid-ß peptides and microtubule-associated protein tau, which are regulated in both autonomous and non-autonomous manners. Microglia, the resident phagocytes of the central nervous system, play a major role in the non-autonomous clearance of protein aggregates. Their function is significantly altered by aging and neurodegeneration. This is genetically supported by the association of microglia-specific genes, TREM2 and CD33, and late onset Alzheimer's disease. Here, we propose that the functional characterization of microglia, and their contribution to proteopathy, will lead to a new therapeutic direction in Alzheimer's disease research.