Date of Award
Doctor of Philosophy (PhD)
Cellular, Molecular and Biomedical Sciences
Christopher L. Berger
Proper coordination of the neuronal cytoskeleton is necessary for the health of the nervous system. Regulation of the microtubule cytoskeleton is achieved, in part, through microtubule associated proteins (MAPs). MAP-Tau, an intrinsically disordered protein highly expressed in axons, functions in signaling cascades, regulation of motor motility, as well as the direct regulation of microtubule dynamics. Tau misregulation and mutations are linked to a class of neurodegenerative diseases called Tauopathies, characterized by the aggregation of Tau. These include progressive supranuclear palsy (PSP), Pick's disease, and Alzheimer's disease. Many of the disease-associated mutations in Tau are found in the microtubule-binding domain, in the C-terminal half of the protein. Effects of C-terminal mutations in Tau have led to the widely accepted disease-state theory that states mutations in Tau reduce Tau:microtubule interactions by either reducing microtubule affinity or increasing Tau propensity to aggregate in solution. This is proposed to reduce microtubule stability, leading to disease. Here, we investigate the effect of an N-terminal arginine to leucine mutation at position 5 in Tau (R5L), associated with PSP, on Tau:microtubule interactions using an in vitro reconstituted system.
Contrary to the canonical disease-state theory, we determine that the R5L mutation does not reduce microtubule affinity using stabilized microtubules and total internal reflection fluorescence (TIRF) microscopy. However, we find that the R5L mutation decreases microtubule bound Tau complexes, or Tau patches, in a concentration and lattice dependent manner. Using Nuclear Magnetic Resonance (NMR), we determine introduction of the R5L mutation results in a local structural change that reduces interactions of the N-terminus in the presence of stabilized microtubules. We determine the R5L mutation influences microtubule stability, by studying Tau-mediated microtubule dynamics. Using Fluorescence Correlation Spectroscopy (FCS), we indicate the R5L mutation does not alter Tau interactions with tubulin. Likewise, we show the R5L mutation has no effect on microtubule growth rate, catastrophe frequency, or rescue frequency via TIRF microscopy. Rather, the R5L mutation increases Tau mediated microtubule shrinkage rate due to the disruption of Tau patches.
These results lead insight into the role of N-terminal domain of Tau on microtubule bound Tau binding behavior. Furthermore, this provides a functional role of Tau patches in Tau-mediated microtubule dynamics. Altogether, these results challenge the current paradigm of how mutations in Tau lead to disease, providing a novel mechanism by which mutations may reduce microtubule stability.
Number of Pages
Cario, Alisa, "Characterization Of Tau Disease-Associated Mutation, R5l, On Microtubule Bound Tau Behavoir And Function" (2022). Graduate College Dissertations and Theses. 1614.