Tubulin post-translational modifications include detyrosination, delta2-tubulin generation, acetylation, glutamylation, and glycylation which occur on the C-terminal domains of tubulin. The C-terminus of the
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tubulin is the major binding sites of microtubule-based molecular motors (kinesins and dynesins) and microtubule-associated proteins (MAPs). The glutamylation and glycylation enzymes are known to be members of the tubulin tyrosine ligase-like (TTLL) family with a tubulin-tyrosine ligase (TTL) homology domain which catalyze ligations of different amino acids to tubulins. Downregulation of dTTLL1, dTTLL3A, dTTLL3B, dTTLL6, dTTLL12, and dTPGS2 enhanced Tau induced toxicity (Fig. 6A-6I). Protein sequence alignment between dTTLL1 and hTTLL1 shows high identity (53.9%) and similarity (65.3%) (Fig. 6J). To study the interaction between hTTLL1 and Tau, we first tested that overexpression of hTTLL1 has no effect itself when expressed by Eq-gal4 (Fig. 7A, 7B, and 7E), but it did potentiate Tau induced toxicity (Fig. 7C, 7D, and 7E). To further validate if the beneficial effect of hTTLL1 would act in neuronal tissues, we conducted survivorship assays. The lifespan of tauopathy flies (average lifespan: 44.83 days, n=120) was
significantly reduced when compared with the control flies (average lifespan: 67.35 days, n=117). Knocking down the expression of dTTLL1 does not shorten the lifespan of flies (average lifespan:
70.71 days, n=115), but it significantly decreased the survivorship of tauopathy flies (average lifespan: 39.71 days, n=120).
Overexpression of hTTLL1 does not shorten the lifespan of flies (average lifespan: 70.44 days, n=113) and significantly increased the survivorship of tauopathy flies (average lifespan：56.26 days, n=119)
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(Fig. 7F). Overexpression of hTTLL1 attenuates Tau mediated toxicity in Drosophila.
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Discussion
To access the tauopathy, many disease fly models have been
generated of which assisted greatly in dissecting the pathomechanisms of tauopathy [22,25,26,33]. These tauopathy fly models have also been used in genetic modifier screens to reveal the genetic pathways controlling Tau toxicity [27,28,30-32]. In most screenings, Drosophila eyes were used as an assessment tool, and the modifiers were identified based on the change in eyes’ roughness, size or volume [24,28,31,32]. In our previous study, we found that Drosophila notal bristle is a versatile system for
quantitatively assessing Tau toxicity [33]. To further test the versatility of our system, we conducted a genetic modifier screen using the notal bristle as an assessment tool. For comparison, the eyes of flies were also used in parallel in the screen. We found that both systems gave us similar results (Fig. 1, 2 and Table 1). However, the notal bristle system seemed more sensitive because many modifiers were recovered by using notal bristle system but not by the eye system (Table 1). In most cases, the phenotypic changes in notal bristles were stronger and easier to be identified (Fig. 1 and Fig. 2).
Abnormal accumulation of misfolded Tau in neurons is considered to be the primary cause of tauopathies [44]. Since molecular chaperons assist correct folding of proteins and target misfolded proteins for
degradation, an array of chaperones were tested in the tauopathy modifier screen. We found that different chaperones exhibited differential effects in tauopathy flies. For instance, overexpression of human Hsp70
(Hspa1L), had no effect in modulating Tau toxicity (Fig. 1H, 1H’ and
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Table 1). Consistently, expression of a dominant-negative mutant form of the constitutively expressed Hsp70 (Hsc70-4K71S) also did not alter the Tau induced bristle loss and rough eye phenotypes in flies (Fig. 1I, 1I’
and Table 1). It has been shown that Hsp70 more effective at inhibiting aggregation formed by 3-repeat Tau isoform [45]. Because the tauopathy fly model was generated by overexpression of the 0N4R Tau isoform, this probably explains why Hsp70 can not attenuate Tau toxicity in our screen [26,46].
Of all chaperones, we found that Hdj1 exhibited the strongest suppressive effect on Tau toxicity in our screen (Fig. 1M, 1M’, 1V and Table 1). This finding is in direct contrast with a previously study in which overexpression of Hdj1 enhanced toxicity of TauV337M, a mutant Tau associated with FTDP-17 [30]. The discrepancy could be due to the differences in Tau variants used in the modifier screens. Indeed, it was reported that overexpression of Hdj1 had no effect on eye phenotype induced by wild type Tau in the same study [30]. In our study, we observed that the size and structure of the ommatidia in flies
co-expressing Hdj1 and Tau were consistently larger and better organized than those of flies expressing Tau alone (Fig. 1B vs. 1M). As discussed above the eye phenotype seemed less sensitive for identifying genetic modifier of tauopathy. To prevent the experimental bias, we further showed that Hdj1 increased notal bristles significantly in Eq>Tau flies (Fig. 1M’, 1V and Table 1). Since both systems gave us consistent results, we concluded that Hdj1 is a suppressor of tauopathy in flies.
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Although the scale of our modifier screen was small and only focused on components of certain pathways, we were able to identify novel modifiers of tauopathy. Notably, we found that co-expression of yu strikingly suppressed toxicity of Tau, whereas, down-regulation of yu expression enhanced Tau toxicity (Fig. 1Q’, 1R’ and Table 1). yu had been identified as a suppressor of SCA8 [47], indicating that common pathomechanism may operate in both neurodegenerations. The yu gene encodes a dual-specificity A-kinase anchor protein (AKAP) and plays roles in oogenesis and long-term memory formation in Drosophila [48,49]. It is interesting to find that the mammalian AKAP also plays a role in learning memory through regulating synaptic plasticity [50]. Since one of the major pathological manifestations of tauopathies is deficits in learning and memory, it will be interested to test whether AKAP would suppress Tau induced cognitive dysfunction.
Reducing the expression of rpi exhibited the strongest suppressive effect against Tau induced eye degeneration and notal bristle loss (Fig. 1T and 1T’), suggesting that rpi is a good therapeutic target for tauopathy.
Indeed, neuronal downregulation of rpi improved motor function and extended lifespan of the tauopathy flies (Fig. 3). To investigate the molecular mechanisms underlying the neuroprotective effect of reduced rpi, we found that the NADPH and reduced GSH levels were increased in tauopathy flies when rpi was downregulated (Fig. 5). Since oxidative insults is a critical pathogenic factor of tauopathy and GSH is the major brain antioxidant [18,19], the increasing in NADPH and GSH contents is likely to protect neurons from Tau toxicity. Consistent with our findings,
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upregulation of antioxidant defenses genes, such as thioredoxin peroxidase (Tpx) and superoxide dismutase 2 (Sod2), or antioxidant treatment, significantly suppressed Tau toxicity in flies [19]. Moreover, cellular oxidative status did not affect Tau phosphorylation because phosphor Tau was not altered when antioxidant defenses genes were downregulated [19]. This may also explain why reduced rpi did not change the phosphorylation of Tau in tauopathy flies (Fig. 4).
Downregulation of tubulin tyrosine ligase-like (TTLL) genes expression enhance Tau induced toxicity. dTTLL1 and hTTLL1 show high identity and similarity (Fig. 6J). The hTTLL1 protein maybe the catalytic subunit of neural tubulin polyglutamylase and polyglutamylate α-tubulin. The interaction between hTTLL1 and Tau, we observed initial result that overexpression of hTTLL1 attenuates Tau toxicity and extends the lifespan of tauopathy flies (Fig. 7). But the relationship between hTTLL1 and Tau are worthy of further investigation.
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Appendix
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