Date of Award
Chemistry and Biochemistry
Martin Margittai, Ph.D.
Aggregation, Cell culture, HEK293, Inhibition, MAP2, Tau
Intracellular deposition of aggregated tau is the hallmark of several different tauopathies, the most widespread of these being Alzheimer's disease. Tau is a highly soluble, intrinsically disordered, microtubule associated protein. Tau's native function is to stabilize microtubule formation in the axons of neurons. Post translational modification such as hyperphosphorylation as well as several familial mutations allow tau to nucleate and form fibrils. These fibrils can recruit healthy monomers onto their ends in a fashion described as template-assisted growth. Tau has 6 isoforms that vary by the inclusion or exclusion of two N-terminal repeats and the inclusion or exclusion of the second of four semiconserved repeats within its microtubule binding region. Isoforms containing three repeats are described as 3R isoforms and those containing four repeats are described as 4R isoforms. In Alzheimer's disease the 3rd and 4th microtubule binding repeats form the core of fibrils within Alzheimer's disease. MAP2C is a 3R homologue of tau and MAP2D is 4R homologue and assists in microtubule stabilization. The homology of MAP2 and tau within their microtubule binding repeats gave motivation to investigate whether MAP2 could compete with tau in binding to the ends of tau fibrils and inhibit elongation. Preliminary in-vitro data suggest this to be the case. To further strengthen these findings, two HEK293 cell lines stably expressing two tau constructs were established; hT40P301S-EYFP, a full-length tau construct C-terminally tagged with an Enhanced Yellow Fluorescence Protein, and K18P301S-EYFP, a truncated tau construct C-terminally tagged with an Enhanced Yellow Fluorescence Protein. Monoclonal cell lines of hT40P301S-EYFP were selected and were shown to be capable of forming intracellular puncta when transfected with K18wt seeds. Monoclonal hT40P301S-YFP transfected cells were then used to show in culture inhibition of fibril formation by two truncations (tr) of MAP2, MAP2Ctr and MAP2Dtr. MAP2Ctr was able to decrease the number of cells containing puncta by 33% and MAP2Dtr showed a 54% decrease. This data further supports the findings that MAP2Ctr and MAP2Dtr are capable of inhibiting tau fibril elongation both in vitro and in culture.
Shady, Justin Ray, "Development of a HEK293 Cell Line to Show Inhibition of Tau Aggregation" (2019). Electronic Theses and Dissertations. 1617.
Received from ProQuest
Justin Ray Shady
Available for download on Saturday, August 01, 2020