AbstractBackgroundNeurodegenerative Tauopathies, including Alzheimer’s disease (AD), are characterized by the appearance of extracellular lesions composed of aggregated and post‐translationally modified tau proteins. The resulting tau pathology is an established marker for differential disease diagnosis and staging, a surrogate marker for neurodegeneration, a potential vector for disease propagation, and a source of toxicity in biological models. In comparison to the well‐established methods for isolation/preparation and biochemical and structural characterization of stable tau filaments from tauopathy brain, the characterization of smaller and much dynamic oligomeric tau aggregates has not been investigated to the same degree. As a result, tau oligomers that may associate most closely with disease propagation and toxicity as suggested by a large body of research, are not fully established.MethodHerein, we leverage our well‐optimized brain‐derived tau oligomers (BDTOs) from well‐vetted human tauopathy cases of AD, progressive supranuclear palsy (PSP) and dementia with Lewy bodies (DLB). In addition to our rigorous biochemical characterization of the BDTOs by immunological probing, and structural characterization by proteolysis and microscopic analyses, we have optimized Fluorescent Amyloid Multi Emission Spectra (FLAMES) microscopy for cataloging and comparing tau conformational variants across a wide range of samples, from brain tissue sections and BDTOs to in vitro aggregated tau assemblies. For selective immunodetection of the BDTOs, we have used our novel four fully sequenced and epitope mapped Tau Oligomer Monoclonal Antibodies (TOMAs), which recognize non‐continuous sequences on tau. Furthermore, the bioactivity of the BDTOs was assessed by evaluating their seeding propensity in biosensor cells and primary cortical neurons from tauopathy mice and their effects on synaptic function by electrophysiological studies.ResultWe observed that the BDTOs differed in their immunological properties, morphology, and sensibility/stability to proteolysis. The BDTOs were distinguished based on FLAMES spectral signature. Distinct seeding propensity and impairment of long‐term potentiation (LTP) indicated their differences in bioactivity as well.ConclusionOur thorough investigation/characterization of biologically active disease‐relevant tau oligomers will provide new opportunities to diagnose and evaluate therapeutics beyond the current capabilities for AD and related tauopathies by developing specific tau aggregation inhibitors and/or diagnostic probes for pathological tau protein.