Procedimiento seleccionado para la elaboración del Plan Estratégico en la

Crossing mutant 2N4R tau lines with B2WT5 demonstrated the importance of phosphorylation at T212, T231 and S262 to the 2N4R/BRSK2 phenotype, as demonstrated by Figures 3.7 through 3.15.

At T212, DC values for the non-phosphorylatable alanine mutant were significantly reduced by BRSK2 co-expression, whereas those of the T212D mutant were not. The

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T212D/BRSK2 result can be explained if we posit that the reduction in toxicity caused by the T212D mutant, which should be mimicking part of the

phosphorylation caused by BRSK2, represents the major part of the effect of

BRSK2. This is reinforced by the lack of difference between 2N4R WT/BRSK2 and T212D/BRSK2 when compared by Mann Whitney U test (P=0.23). The ability of BRSK2 to reduce toxicity in the T212A mutant however, especially in this light, is surprising – if T212D is essential for BRSK2-induced protection, how can it still have had such an effect on this non-phosphorylatable mutant? Indeed, if we assume that T212 and S262 are the only important residues affected by this kinase, the effect of BRSK2 would be reversed if S262 was its sole target, as shown by the S262D mutant in Chapter 3. This contradiction is perhaps partially explained by T231. The T231D mutant is predictably affected by BRSK2, with a significant reduction in DC values upon co-expression. However, any effect of BRSK2 is apparently entirely eliminated by T231A mutation. This could be due to a reduction in affinity for BRSK2 caused by this mutation, removing any interaction completely. Alternatively, this could be evidence of yet another target residue of BRSK2. As has previously been observed, the proline-directed T231 was not investigated in the only major screen of tau phosphorylation by AMPK-related kinases (Yoshida and Goedert, 2011), and has been shown to be phosphorylated by AMPK itself (Thornton et al., 2011). Given the complexity of the tau phosphorylation code, it is possible that some level of T231 phosphorylation is a prerequisite of the reduction in tau toxicity

observed upon introduction of BRSK2, potentially even (at endogenous levels) of the T212D S262D double mutation. Endogenous levels of T231 phosphorylation are clearly not sufficient alongside S262D pseudo-phosphorylation to produce such protection, otherwise the S262D mutant would exhibit such an effect alone; thus, it can only explain the ability of BRSK2 to reduce toxicity in the T212A mutant if the kinase increases phosphorylation at this site beyond endogenous levels. One

additional possibility is that there is at least one other target of BRSK2 on 2N4R tau, which together with T212 (and potentially T231) creates some redundancy.

Toxicity caused by both S262 mutants can be reduced significantly by BRSK2, which would indicate a lack of importance of this residue in the BRSK2 phenotypic changes. This is perhaps supported by the similarity in phenotype between the T212D mutant and the T212D S262D double mutant. However, direct comparison of

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hemizygous DC values between these two do show a significant reduction in the double mutant (P=0.026), which is eliminated after addition of BRSK2 to T212D (P=0.27) so there would appear to be some extra effect in the double mutant. Together, these studies suggest a much more complex phosphorylation profile underlying the 2N4R tau/BRSK2 phenotype than that represented by the double phospho-mimetic mutant already generated. To summarise, BRSK2 leads to a phenotype suggestive of T212 pseudophosphorylation and recapitulated by a

T212D/S262D mutant, but which is independent of T212 and S262 phosphorylation status, whilst being dependant on T231 phosphorylation. These data suggest that the similarity between the T212D S262D phenotype and that of 2N4R WT/BRSK2 is not evidence of these mutations representing the entirety of the effect of BRSK2. Perhaps inevitably given the complex system being investigated, there appear to be multiple paths to reaching the same result (and degree) of reduced toxicity, with far more combinations than measurable potential outcomes. If the effect of BRSK2 is

indeed due to phosphorylation, and if the 1N4R tau phosphorylation data in vitro are

applicable to this 2N4R model in vivo, then at the very least there must be some

redundancy in this phosphorylation code. This would require phosphorylation of tau at other residues, likely T231 and others. A simple initial set of experiments would be to probe for changes in phosphorylation at T212, T231 and S262 in 2N4R tau in samples from flies co-expressing BRSK2. This would give an indication of relevance

of the in vitro work as well as examining the relationship between T231 and BRSK2.

Ultimately though, such small scale screens are likely to lead to uncertain

conclusions given the number of potential residues of interest. Equally, expansion of the tau mutant library can only be effective with enough information to direct

targeted mutation of residues of interest – the process is too slow and costly to explore every possible combination, even among the few sites listed here. This is rendered all the more pressing by the unearthed differences between tau isoforms and potential interest in replicating the library in 0N4R tau. One potential method of exploring BRSK2-induced phosphorylation could come from phosphoproteomics. Advances in the field of mass spectrometry over the past decade have begun to allow quantitative evaluation of the phosphorylation state of proteins. For example, SILAC (stable isotope labelling with amino acids in cell culture) is a method which uses heavy isotope-labelled growth media to differentiate between cell populations,

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before aligning cleaved peptides by liquid chromatography prior to analysis (Ong et al., 2002). This method has been used proteome-wide to explore Cdk1(cyclin- dependent kinase 1) phosphorylation (Holt et al., 2009). Less expensive stable isotope labelling methods have also been developed, in which deuterium labelling occurs at the peptide level, following digestion which would likely be more practical for study of a single protein (Boersema et al., 2009). Given the number of post- translational modifications tau is subject to, such comprehensive data would be very useful for informing any future addition to the phosphorylation library or phospho- antibody collection.

The other possible explanation for the discrepancy between the predicted importance of BRSK2 phosphorylation and the apparent reality would be for the kinase to play an entirely different role in determining tau toxicity.