Cameron, Charles S. (2013) Brain-selective kinase (BRSK) interactions with tau as a cause of neurodegeneration. PhD thesis, University of Warwick.

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Abstract

Tauopathies are a group of neurodegenerative diseases associated with the build-up of intracellular aggregates of the microtubule-associated protein (MAP) tau, called neurofibrillary tangles (NFT). The tau found in NFTs is known to be highly phosphorylated, and as such a number of tau kinases have been examined for their ability to affect tau toxicity in animal models, including members of the AMPK-related kinase family. Two members of this family, the brain-selective kinases (BRSKs), have been shown to phosphorylate tau in vitro and in vivo in an LKB1 and CaMKKα-dependent manner. BRSKs are prevalent in the central nervous system, in which they modulate neuronal polarisation via tau. They have also been shown to dramatically increase 0N4R tau toxicity in a Drosophila model.

In order to determine the importance of phosphorylation to tau toxicity in this model, I have generated a group of 2N4R tau phosphorylation mutants. Based upon in vitro data linking phosphorylation at T212 to tau aggregation, and S262 to microtubule dissociation, these two residues underwent both non-phosphorylatable and phosphomimetic mutation and were inserted into Drosophila. Each of these mutations was found to alter tau toxicity upon expression in the fly eye, as measured by disruption of ommatidia, with the T212D and S262A mutations producing a milder phenotype.

Each of these sites on tau has previously been shown to be a target of phosphorylation by BRSK2 in vitro. Through generation of a T212D S262D double mutant, I was able to investigate whether phenotypic changes caused by BRSK2 were due to phosphorylation. BRSK2 was found to have a different effect on toxicity caused by 2N4R tau, causing a reduction in toxicity. This was found to be comparable to the phenotype of the double mutant. However, examination of the ability of BRSK2 to modulate the toxicity of the single tau mutants demonstrated a more complex phosphorylation profile, also dependent on other residues.

Further experiments examined the consequences of tau and BRSK2 expression in the CNS. This demonstrated the toxicity of BRSK2 overexpression, in the absence of tau, in these tissues. Given that BRSK2 overexpression alone in the eye led to no observable phenotype, this tissue-dependent toxicity of the kinase demonstrates the importance of the choice of model in such studies. In addition, a deficiency screen for modulators of 0N4R tau/BRSK2 toxicity in the eye has generated multiple leads, including the fly homologue of the actin-binding protein profilin.

This project has demonstrated the inequivalence of several phosphorylation sites in vivo, as well as an isoform-dependent aspect of tau toxicity. These data suggest that the influence of tau phosphorylation on toxicity is complex; expansion of the work presented here could help decipher the code of tau phosphorylation.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Nervous system -- Degeneration, Phosphorylation, Drosophila
Official Date:	September 2013
Dates:	Date Event September 2013 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Moffat, Kevin G. ; Frenguelli, Bruno G.
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain)
Extent:	xii, 207 leaves : illustrations, charts
Language:	eng

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