Imagine you’re a detective investigating a crime syndicate. You suspect that one criminal, let’s call them Proteopathic Tau, is recruiting innocent bystanders (naïve tau) and turning them into criminals as well. This process is believed to be the reason behind the spread of Alzheimer’s disease. To gather evidence, scientists have developed two simple methods called assays to assess this ‘seeding activity’ of tau. One method involves capturing proteopathic tau onto a membrane and determining its presence by using special antibodies. The other method utilizes cells in a lab dish, where they introduce proteopathic tau to tau-expressing cells and analyze the resulting aggregated tau. They tested these assays using brain samples from Alzheimer’s patients and healthy individuals, discovering that only Alzheimer’s brains showed effective recruitment and aggregation of tau. They also found that areas with more tau abnormalities had higher levels of captured tau or aggregated tau. These assays are both specific and sensitive, making them ideal for regular labs to study Alzheimer’s disease.

The regional distribution of neurofibrillary tangles of hyperphosphorylated tau aggregates is associated with the progression of Alzheimer’s disease (AD). Misfolded proteopathic tau recruits naïve tau and templates its misfolding and aggregation in a prion-like fashion, which is believed to be the molecular basis of propagation of tau pathology. A practical way to assess tau seeding activity is to measure its ability to recruit/bind other tau molecules and to induce tau aggregation. Based on the properties of proteopathic tau, here we report the development of two simple assays to assess tau seeding activity —– capture assay in vitro and seeded-tau aggregation assay in cultured cells. In the capture assay, proteopathic tau was applied onto a nitrocellulose membrane and the membrane was incubated with cell lysate containing HA-tagged tau151-391 (HA-tau151-391). The captured tau on the membrane was determined by immuno-blots developed with anti-HA. For the seeded-tau aggregation assay, HEK-293FT cells transiently expressing HA-tau151-391 were treated with proteopathic tau in the presence of Lipofectamine 2000 and then lysed with RIPA buffer. RIPA-insoluble fraction containing aggregated tau was obtained by ultracentrifugation and analyzed by immuno-blot developed with anti-HA. To validate these two assays, we assessed the seeding activity of tau in the middle frontal gyrus, middle temporal gyrus and basal forebrain of AD and control brains and found that AD, but not control, brain extracts effectively captured and seeded tau151-391 aggregation. Basal forebrain contained less phospho-tau and tau seeding activity. The levels of captured tau or seeded-tau aggregates were positively correlated to the levels of phospho-tau, Braak stages and tangle sores. These two assays are specific and sensitive and can be carried out in a regular biomedical laboratory setting by using routine biochemical techniques.

Read Full Article (External Site)