Since the pro-aggregant animals depicted a mitochondrial mislocalization phenotype, the next step was to examine if the axonal transport of mitochondria is affected. The strain jsIs609 expresses GFP with a mitochondrial localization signal (MLS) in the six mechanosensory neurons of C. elegans. The strain was crossed with our Tau mutants, creating the pro-aggregant and the anti-aggregant mitochondrial marker strains. Mitochondrial movements were quantified by performing single plane live imaging of the middle axonal segment of the PLML or PLMR neurons in immobilized worms. The PLM axon closer to the microscope objective was selected
Figure 3.19: Pro-aggregant Tau transgenic worms show defective mitochondrial transport in the axons of mechanosensory neurons. Representative kymographs of PLM axons of pro-, anti-aggregant animals and wild type derived from time-lapse imaging are shown. Vertical lines represent stationary/docked mitochondria and oblique lines (labeled by arrowheads) represent the tracks of moving mitochondria. The slope of this track is an indicator of velocity. Anterograde movements: slope declines to the right, in retrograde movements to the left. The kymograph space and time scale is shown at the bottom. (A): A kymograph recorded from a day 1 adult wild type animal. (B): A kymograph recorded from a day 1 adult animal of the anti-aggregant strain. Note the oblique line in the middle which shows the track of a fast moving mitochondrial particle (arrowhead). (C): A kymograph recorded from a day 1 adult animal of the pro-aggregant strain. Note that the two particles in motion make long pauses (oblique lines become vertical, labeled by yellow arrowheads) and their track displacement is rather limited. (D): Scatter plot of the mean velocities of mitochondrial particles that were manually tracked from 20 time lapse videos that were analyzed for each strain. Instantaneous velocity values that were < 10 nm/sec were not included in the calculation of the means for this plot, as these frames were classified as pause events that were separately analyzed (see next graph). (E): Bar diagram for the quantification of pausing frequency in relation to time and distance parameter. The number of pausing events is significantly increased in the pro-aggregant strain.
Error bars account for standard error of the mean (s.e.m).In summary, the axonal transport of mitochondria in pro-aggregant worms does not run smoothly due to increased pause events, although instantaneous velocities per se are not substantially different from those seen in the anti-aggregant worms (adapted from
for analysis. At least 20 time lapse acquisitions were collected for each strain, the representative examples of each strain are shown as kymographs in Figures 3.19A-C.
The mean velocity of the continuously moving particles was calculated by manually tracking each moving GFP particle with ImageJ (Fig 3.19 D). The pro-aggregant strain showed lower mitochondrial velocity than the wild type (mean ± s.d. = 171 ± 111 nm/sec versus 256 ± 117 nm/sec), whereas the anti-aggregant strain (194 ± 131 nm/sec) did not substantially differ from wild type (Figure 3.19 D). However, there was a striking difference in the mitochondrial pausing frequency between pro-aggregant and the other two strains. The pro-pro-aggregant strain showed approximately four times more frequent pause events compared to the anti-aggregant (Fig 3.19 E). The data suggests that there is a reduced mitochondrial transport in the axons of the pro-aggregant strain. In summary, our Tau transgenic strains recapitulate most of the phenotypic defects found in the mammalian neurodegenerative models and further consolidates the notion that the amyloidogenic properties of Tau can block axonal transport of mitochondria.
3.17 Compound treatment
Compounds had no effect on the wild-type N2 strain
Having developed a worm model with a well characterised phenotype very early in its life-span, which shows pathology reminiscent of the cellular pathology, observed in patients with tauopathy, we next set out to test various compounds which are known to have anti-aggregation properties. Some of these compounds have already been tested in vitro (Ballatore et al., 2007; Bulic et al., 2010; Bulic et al., 2009;
Pickhardt et al., 2007). These compounds were tested by supplying them in the liquid culture so as to facilitate their entry into the worm through all three entry routes reported so far namely: ingestion, uptake through the skin and uptake via exposed sensory neuronal endings (Choy et al., 2006; Perkins et al., 1986; Smith and Campbell, 1996). Given the fact that drugs can have off-target effects when studied within the context of a whole organism, we tested these compounds first on the wild type N2 strains in order to make sure that there are no off-target effects and any
amelioration in the phenotype could be attributed to the anti-aggregation quality of these drugs.
Figure 3.20: Compound treatment of wild type N2 strain. (A): Structure of the compounds. bb14 is a Rhodanine derivative and BSc3094 belongs to the phenylthiazolyl-hydrazide class of compounds while compound 16 (cmp16) belongs to the aminothienopyridazine (ATPZ) class of tau inhibitors (5-Amino-3-(4-chlorophenyl)-N-cyclopropyl-4-oxo-3,4-dihydrothieno[3,4–d]pyridazine-1-carboxamide). (B): Compound treatment in liquid culture was started at the L1 larval stage until day-3 of adulthood and the thrash assay was used as readout. bb14, BSc3094 and cmp16 had no effect on the wild type N2 strain at the three concentrations used.
We first started with bb14, BSc3094 and compound 16. The compound bb14 is a Rhodanine derivative and, BSc3094 is a phenylthiazolyl-hydrazide derivative. They were the two most promising hit compounds obtained in a mammalian cell model of
N-cyclopropyl-4-oxo-3,4-dihydrothieno[3,4-d]pyridazine-1-carboxamide and was the most prominent tau aggregation inhibitor compound published in a recent in vitro screen (see figure 3.20 A for structure). It is also able to cross the blood brain barrier that makes it a good choice for the clinical applications (Ballatore et al., 2010). The worms were allowed to grow in liquid culture supplied with drugs at three different concentrations: 25 µM, 50 µM and 100 µM in DMSO. 1% DMSO was used as a control and thrash assay was used as the readout of the phenotype. The drugs did not have any effect on the thrashes made by wild type worms at any of these concentrations (Fig 3.20 B) which confirms that the drugs have no apparent side-effects.
3.18 bb14, BSc3094 and cmp16 lead to an amelioration of the phenotype in the pro-aggregant strain
Now that the drugs did not have any effect on the wild type strain we finally went on to test these compounds on the pro-aggregant strain in these three concentrations.
Figure 3.21: Treatment of pro-aggregant strain with the anti-aggregation compounds can ameliorate the
either untreated in S medium and 1% DMSO as controls or treated with compounds at three different concentrations. The error bars denote the SEM. bb14 at 50µM (A), BSc3094 at 25µM (B) and compound 16 at 100µM (C) increased the thrash rates by ~ 3, ~ 5 and ~ 3 times respectively. Unpaired t test was implemented for comparisons (*** stands for p<0.001, **** stands for p<0.0001 in the comparisons between untreated pro-aggregant (S medium or 1% DMSO) and compound treated pro-pro-aggregant at the best concentrations.
Figure 3.22: Sequential extraction of Tau from synchronized pro-aggregant animals after treatment with aggregation inhibitor compounds. Soluble Tau fraction (RAB), detergent soluble fraction (RIPA) and detergent insoluble fraction made soluble by 70% formic acid (FA) were immunoblotted using pan Tau antibody, which recognizes both the full-length V337M Tau and the repeat fragments F3ΔK280/ F3ΔK280PP.
Anti-actin antibody was used as an internal control. (A): There is a slight decrease in the insoluble Tau
disappearance of F3∆K280 fragment. Asterisk in red (*) may be a proteolytic fragment of Tau. (B):
Quantification of the total, soluble, detergent soluble and detergent insoluble Tau from the pro-aggregant strain after 50 µM bb14, 25 µM BSc3094 and 100 µM cmp16 treatment. Equal amount of protein was loaded and immunoblotted with K9JA pan tau antibody, using actin as a loading control. For the quantification lane intensities were normalized against the untreated (DMSO). BSc3094 and cmp16 lead to approximately 40%
and 50% decrease in the insoluble Tau respectively (One-Way ANOVA, **p<0.01, ***p<0.001).
Treated pro-aggregant worms showed more thrashes per minute than the control 1%
DMSO treated worms. The compounds ameliorated the phenotype to different extents and at different concentrations. 50µM bb14, 25µM BSc3094 and 100µM cmp16 treated worms produced 3 times, 6 times and 3 times more thrashes per minute respectively than the control 1% DMSO treated counterparts (Fig 3.21). We used these particular concentrations to perform the biochemistry after the compound treatment in order to look at the insoluble tau levels. To perform biochemistry, the culture conditions were extended to 6-well polystyrene plates and allowed to grow the worms in the presence or absence of compounds until day 3 of adulthood before lysing them for protein extraction. At the biochemical level, compound treatment reduced the Tau aggregates. In addition, no F3ΔK280 appeared in the formic acid fraction (Fig 3.22 A). BSc3094 reduced the insoluble tau by ~40% and cmp16 by ~50%
(Fig 3.22 B).