RESUMEN
Aggregation and accumulation of amyloid-ß peptide (Aß) are a critical trigger for the onset of Alzheimer's disease (AD). While the plaques are the most outstanding Aß pathological feature, much of the recent research emphasis has been on soluble Aß species because of their diffusible, proinflammatory, and toxic properties. The focus on soluble aggregated Aß species has also increased the interest in antibodies that are selective for different Aß conformations. In the current study, we developed and characterized a new class of monoclonal antibodies (referred to as mAbSL) that are selective for Aß protofibrils. Cloning and sequencing of the heavy and light chain variable regions for multiple antibodies identified sequence characteristics that may impart the conformational selectivity by the antibodies. Transfection of FreeStyle 293F cells with the plasmids permitted in-house expression and purification of mAbSL antibodies along with non-conformation-selective Aß monoclonal antibodies (Aß mAbs). Several of the purified mAbSL antibodies demonstrated significant affinity and selectivity for Aß42 protofibrils compared with Aß42 monomers and Aß42 fibrils. Competition ELISA assays assessing the best overall antibody, mAbSL 113, yielded affinity constants of 7 nM for the antibody-Aß42 protofibril interaction, while the affinity for either Aß42 monomers or Aß42 fibrils was roughly 80 times higher. mAbSL 113 significantly inhibited Aß42 monomer aggregation by a unique mechanism compared with the inhibition displayed by Aß mAb 513. Aß42 protofibril dynamics were also markedly altered in the presence of mAbSL 113, whereby insoluble complex formation and protofibril deposition were stimulated by the antibody at low substoichiometric molar ratios. As the field contemplates the therapeutic effectiveness of Aß conformation-selective antibodies, the findings presented here demonstrate new information on a monoclonal antibody that selectively targets Aß protofibrils and impacts Aß dynamics.
Asunto(s)
Enfermedad de Alzheimer , Anticuerpos Monoclonales , Humanos , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/metabolismo , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
This review discusses the profound connection between microglia, neuroinflammation, and Alzheimer's disease (AD). Theories have been postulated, tested, and modified over several decades. The findings have further bolstered the belief that microglia-mediated inflammation is both a product and contributor to AD pathology and progression. Distinct microglia phenotypes and their function, microglial recognition and response to protein aggregates in AD, and the overall role of microglia in AD are areas that have received considerable research attention and yielded significant results. The following article provides a historical perspective of microglia, a detailed discussion of multiple microglia phenotypes including dark microglia, and a review of a number of areas where microglia intersect with AD and other pathological neurological processes. The overall breadth of important discoveries achieved in these areas significantly strengthens the hypothesis that neuroinflammation plays a key role in AD. Future determination of the exact mechanisms by which microglia respond to, and attempt to mitigate, protein aggregation in AD may lead to new therapeutic strategies.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Inflamación/inmunología , Microglía/inmunología , Degeneración Nerviosa/inmunología , Enfermedad de Alzheimer/patología , Animales , Humanos , Microglía/metabolismo , Degeneración Nerviosa/patologíaRESUMEN
Hydraphiles are synthetic amphiphiles that form ion-conducting pores in liposomal membranes. These pores exhibit open-close behavior when studied by planar bilayer conductance techniques. In previous work, we showed that when co-administered with various antibiotics to the DH5α strain of Escherichia coli, they enhanced the drug's potency. We report here potency enhancements at low concentrations of hydraphiles for the structurally and mechanistically unrelated antibiotics erythromycin, kanamycin, rifampicin, and tetracycline against Gram negative E. coli (DH5α and K-12) and Pseudomonas aeruginosa, as well as Gram positive Bacillus subtilis. Earlier work suggested that potency increases correlated to ion transport function. The data presented here comport with the function of hydraphiles to enhance membrane permeability in addition to, or instead of, their known function as ion conductors.
Asunto(s)
Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Permeabilidad/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Tensoactivos/química , Tensoactivos/farmacología , Antibacterianos/farmacocinética , Bacillus subtilis/metabolismo , Eritromicina/farmacocinética , Eritromicina/farmacología , Escherichia coli/metabolismo , Infecciones por Escherichia coli/tratamiento farmacológico , Humanos , Kanamicina/farmacocinética , Kanamicina/farmacología , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/metabolismo , Rifampin/farmacocinética , Rifampin/farmacología , Tetraciclina/farmacocinética , Tetraciclina/farmacologíaRESUMEN
The NLRP3 inflammasome is a key intracellular component of the innate immune response. It is a three-protein complex essential for the production of mature interleukin 1-ß. The complex, which is comprised of three proteins, NLRP3, ASC, and pro-caspase-1, has been implicated in the physiological response to pathogenic elements of cardiovascular disease and Alzheimer's disease. Investigations into the properties of the three proteins can be aided by larger-scale recombinant expression to produce adequate amounts. In the current study, a variety of NLRP3 inflammasome proteins were expressed in the ExpiCHO-S mammalian cell system with a particular focus on ASC. ASC fusion proteins with glutathione-S transferase, maltose-binding protein, and SUMO increased solubility and aided in determining the stability and oligomerization propensity of individual ASC domains and full-length ASC. ASC oligomerization was highly sensitive to protein concentration, ionic strength, and mutation. These observations provided strategic ways to enhance protein purification and characterize ASC oligomerization. The ExpiCHO-S expression system consistently produced high-yield recombinant NLRP3 inflammasome proteins which led to a further understanding of ASC oligomerization.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-1beta , Mamíferos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Microvesicles (MVs) and exosomes comprise a class of cell-secreted particles termed extracellular vesicles (EVs). These cargo-holding vesicles mediate cell-to-cell communication and have recently been implicated in neurodegenerative diseases such as Alzheimer's disease (AD). The two types of EVs are distinguished by the mechanism of cell release and their size, with the smaller exosomes and the larger MVs ranging from 30 to 100 nm and 100 nm to 1 µm in diameter, respectively. MV numbers are increased in AD and appear to interact with amyloid-ß peptide (Aß), the primary protein component of the neuritic plaques in the AD brain. Because microglial cells play such an important role in AD-linked neuroinflammation, we sought to characterize MVs shed from microglial cells, better understand MV interactions with Aß, and determine whether internalized Aß may be incorporated into secreted MVs. Multiple strategies were used to characterize MVs shed from BV-2 microglia after ATP stimulation. Confocal images of isolated MVs bound to fluorescently labeled annexin-V via externalized phosphatidylserine revealed a polydisperse population of small spherical structures. Dynamic light scattering measurements yielded MV diameters ranging from 150 to 600 nm. Electron microscopy of resin-embedded MVs cut into thin slices showed well-defined uranyl acetate-stained ring-like structures in a similar diameter range. The use of a fluorescently labeled membrane insertion probe, NBD C6-HPC, effectively tracked MVs in binding experiments, and an Aß ELISA confirmed a strong interaction between MVs and Aß protofibrils but not Aß monomers. Despite the lesser monomer interaction, MVs had an inhibitory effect on monomer aggregation. Primary microglia rapidly internalized Aß protofibrils, and subsequent stimulation of the microglia with ATP resulted in the release of MVs containing the internalized Aß protofibrils. The role of MVs in neurodegeneration and inflammation is an emerging area, and further knowledge of MV interaction with Aß may shed light on extracellular spread and influence on neurotoxicity and neuroinflammation.