Conclusiones del tercer capítulo

In order to explore the post-developmental toxicity of tau in neuronal tissue other

than the eye, a recombinant ELAV-GAL4 GAL80TS line (hereafter referred to as

ELAV) was crossed with transgenes described in the previous two Chapters. These crosses were performed and the progeny maintained at 18°C in order to suppress transgene expression. Adults of up to one week of age were then transferred to the DAM2 monitoring system at 29°C for transgene induction and activity recording. These experiments were performed under conditions suitable for study of a free- running period to enable retrospective examination of circadian rhythm changes, and were consequently exposed to equal light/dark cycles for 5 full days before onset of constant darkness for the remainder of the experiment.

5.2.1.1 0N4R tau and BRSK2

The first experiments performed were attempts to examine the effect of 0N4R tau and BRSK2 transgenes on behavioural activity. It was expected that expression of these transgenes would lead to measurable effects on total activity due to morbidity or mortality if they had any toxic effect in the CNS. Crosses were thus performed between the ELAV line and the 0N4R and B2WT5 lines, as well as the recombinant 0N4R/B2WT5 line introduced in Section 4.2.1. Recording by the DAM2 system sorted data into 1 minute bins which, upon completion of the recording, could be re- binned into 30 minute intervals, using the software DAM Filescan 1.07. These bins were then summed into values for each 24 hours of activity, using a simple

spreadsheet in Origin 7. All measurements of total daily activity in this Chapter underwent this re-binning procedure. In Figure 5.1, the average daily activity over a three week time course of each of these lines, as well as ELAV controls, is plotted. Data from individuals were discarded if they did not survive past the first week in order to remove particularly sickly animals from the analysis. In order to determine whether the activity levels of each of the genotypes evolved differently over the period studied, a two-way ANOVA with repeated measures was performed in

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GraphPad Prism 6. Both the genotype (P=0.0062) and day (P<0.0001) were found to be significant sources of variation in total daily activity. In addition, the interaction of these two variables was also found to be significant (P<0.0001), indicating that the rate of change in daily activity is dependent on the genotype. This was still found to be significant if the first week of data was removed from the analysis to avoid taking into account the early acclimatisation period and any possible increase in variance it may have caused: this was the case for genotype (P=0.0048), day (P<0.0001) and their interaction (P<0.0001). This also removes the earliest periods of expression, likely with the lowest transgenic protein levels of the experiment. When compared individually, 0N4R tau was not found to lead to a significant change in activity over the final two weeks of data (genotype/day interaction P=0.1231). BRSK2 expression was found to cause significant changes in activity (P=0.0271 under the same conditions), as was the recombinant 0N4R/B2WT5 line (P=0.001). Both B2WT5 and 0N4R/B2WT5 differed significantly from 0N4R tau alone (P=0.0007 and P=0.0011, respectively). 0N4R/B2WT5 differed significantly from B2WT5 if all three weeks of data were compared (interaction P=0.0025), but not if the first week was omitted (P=0.12). Flies expressing both 0N4R tau and BRSK2 die off earliest, with 100% lethality by day 12. There is an increase in 0N4R-induced activity decline compared with ELAV after day 11, but there are still some survivors after 21 days. As had been suspected after previous unpublished data revealed the lethality of ELAV-controlled developmental BRSK2 expression, the offspring of the ELAV cross to B2WT5 exhibit a severe decline in activity, with 100% lethality by day 20. It would seem that BRSK2 is toxic in at least some vital tissues of the CNS. It was also noted that the high temperatures used here were having an effect on behaviour, with animals becoming predominantly nocturnal in response – this was not surprising given recorded effects of temperature on periodicity (Sawyer et al., 1997) diurnal activity levels (Tomioka et al., 1998). It was also likely responsible for the shortened lifespan of control animals (Miquel et al., 1976).

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Figure 5.1: BRSK2 expression in the CNS, with or without 0N4R tau, reduces lifespan

A: Total daily activity measured in number of beam break events. Genotype was shown to control rate of decrease over time by two-way repeated measures ANOVA (P<0.0001). Error bars represent standard error. The black and white bar above the plot indicates the duration of alternating light/dark.

B: Total values normalised to readings of day 7, in order to reduce line-specific differences in baseline activity, and to avoid exaggeration caused by light/dark change. ELAV n=10. 0N4R n=18. B2WT5 n=23. 0N4R/B2WT5 n=10.

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5.2.1.2 2N4R tau and mutants

The 2N4R tau mutant library was also crossed with the ELAV line to determine whether the differences in toxicity found in the eye upon GMR-controlled expression could also be reproduced in the CNS of adults. The 2N4R WT line and all single tau mutations were crossed with ELAV and the progeny studied using the DAM2 system, this time for a longer period of 33 days. Figure 5.2 shows total activity, Figure 5.3 activity relative to day 7. Animals not surviving after the first week were again disregarded from analysis. Two-way repeated measures ANOVA initially indicated a significant interaction between genotype and time point (P<0.0001), but re-analysis of the data obtained under constant dark (after day 7) failed to find a significant relationship (P=0.097). Given that acclimatisation to the conditions of the experiment is likely to lead to increased variation between all individuals over the first week, and that transgene levels would be expected to be lowest during this period, it seems appropriate to consider the latter measure more applicable. It is possible to speculate on some trends, for example comparing S262A to S262D the phospho-mimetic mutant consistently exhibits lower activity levels than its non- phosphorylatable counterpart. The T212A mutant exhibited a long period of hyper- activity before a sharp decline, with maximum lifespan comparable with all other lines. Indeed, upon normalisation of the data in Figure 5.3, it would appear that a certain level of hyperactivity is witnessed in all 2N4R tau and mutant lines. The two T231 mutants produced curves which are particularly close to 2N4R WT, also in line with the smaller range of eye phenotypes produced by mutation at this site.

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Figure 5.2: Absolute activity levels over 33 days do not reveal any 2N4R tau- induced reduction in lifespan

Data from flies expressing 2N4R tau was recorded for 33 days. No significant

difference was found between the curves by two-way ANOVA with repeated measures (P=0.097). The black and white bars above the plots indicate the duration of

alternating light/dark.

A: Data from ELAV controls and WT 2N4R tau-expressing individuals.

B: Data from controls and WT 2N4R, overlaid with data from individuals expressing T212A or T212D mutant tau.

C: Data from controls and WT 2N4R, overlaid with data from individuals expressing T231A or T231D mutant tau.

D: Data from controls and WT 2N4R, overlaid with data from individuals expressing S262A or S262D mutant tau.

ELAV n=9. 2N4R n=9. T212A n=9. T212D n=10. T231A n=8. T231D n=9. S262A n=10. S262D n=10.

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Figure 5.3: Normalised data from Figure 5.2 reveals consistently higher levels of activity after three weeks in 2N4R tau-expressing lines

Data from flies expressing 2N4R tau was recorded for 33 days. Data has been normalised to day 7 values to remove line-specific differences in baseline activity and to avoid additional variation caused by light/dark acclimatisation. The black and white bars above the plots indicate the duration of alternating light/dark. A: Data from ELAV controls and WT 2N4R tau-expressing individuals.

B: Data from controls and WT 2N4R, overlaid with data from individuals expressing T212A or T212D mutant tau.

C: Data from controls and WT 2N4R, overlaid with data from individuals expressing T231A or T231D mutant tau.

D: Data from controls and WT 2N4R, overlaid with data from individuals expressing S262A or S262D mutant tau.

ELAV n=9. 2N4R n=9. T212A n=9. T212D n=10. T231A n=8. T231D n=9. S262A n=10. S262D n=10.

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5.2.2 Effects of PDF-GAL4-controlled transgene expression on activity and