RESUMEN
Tau aggregation assays detect and quantify the conversion of soluble tau monomers into species having filamentous or oligomeric structure. Assays for filamentous aggregates in cross-ß-sheet conformation leverage optical, biochemical, or biophysical methods, each with their own advantages and throughput capacity. Here we provide protocols for two medium-throughput assays based on sedimentation and laser light scattering and compare their performance, their utility for characterizing tau aggregation dynamics, and their limitations relative to other approaches. Additionally, a protocol for transmission electron microscopy analysis is updated so as to be compatible with the truncated tau variants that have emerged as powerful tools for interrogating the structural basis of tau polymorphism. Together these methods contribute to a rich tool kit for interrogating tau aggregation kinetics and propensity over a wide range of experimental conditions.
Asunto(s)
Rayos Láser , Proteínas tau , Proteínas tau/metabolismo , Microscopía Electrónica de TransmisiónRESUMEN
Tau aggregate-bearing lesions are pathological markers and potential mediators of tauopathic neurodegenerative diseases, including Alzheimer's disease. The molecular chaperone DJ-1 colocalizes with tau pathology in these disorders, but it has been unclear what functional link exists between them. In this study, we examined the consequences of tau/DJ-1 interaction as isolated proteins in vitro. When added to full-length 2N4R tau under aggregation-promoting conditions, DJ-1 inhibited both the rate and extent of filament formation in a concentration-dependent manner. Inhibitory activity was low affinity, did not require ATP, and was not affected by substituting oxidation incompetent missense mutation C106A for wild-type DJ-1. In contrast, missense mutations previously linked to familial Parkinson's disease and loss of α-synuclein chaperone activity, M26I and E64D, displayed diminished tau chaperone activity relative to wild-type DJ-1. Although DJ-1 directly bound the isolated microtubule-binding repeat region of tau protein, exposure of preformed tau seeds to DJ-1 did not diminish seeding activity in a biosensor cell model. These data reveal DJ-1 to be a holdase chaperone capable of engaging tau as a client in addition to α-synuclein. Our findings support a role for DJ-1 as part of an endogenous defense against the aggregation of these intrinsically disordered proteins.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , alfa-Sinucleína/química , Enfermedad de Parkinson/metabolismo , Proteínas tau/genética , Chaperonas Moleculares/genética , Proteína Desglicasa DJ-1/genéticaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer's disease mechanistic and drug discovery studies.