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. 2016 Mar 4:6:22685.
doi: 10.1038/srep22685.

Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

Marianna Karina Gorsky  1 , Sylvie Burnouf  1 , Jacqueline Dols  1 , Eckhard Mandelkow  2   3 , Linda Partridge  1
Affiliations

Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo

Marianna Karina Gorsky et al. Sci Rep. .

Abstract

Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer's disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD.

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Figures

Figure 1
Figure 1. Decreased fly survival, impaired climbing ability and increased photoreceptor neurodegeneration in an adult-onset hTau overexpression model.
(a) Survival was drastically reduced in flies overexpressing the full-length 2N4R hTau-wt protein (plain vs. dotted red curve) in the adult nervous system (****p < 0.0001, RU486-induced hTau-wt vs. non-induced controls). Survival of the elavGS driver line control was not affected by RU486 feeding (black curves). (b) Flies overexpressing the hTau-wt protein (plain red curve) presented progressive, drastic climbing defects as compared to both non-induced controls and elavGS flies (****p < 0.0001, RU486-induced hTau-wt vs. non-induced controls at day 10 and day 15, two-way ANOVA). (c) hTau-wt overexpression in adult fly neurons led to progressive photoreceptor neurodegeneration as measured by the percentage of ommatidia lacking rhabdomeres over age (**p < 0.01, one-way ANOVA), while RU486-fed elavGS controls showed no significant neurodegeneration over time (p > 0.05, one-way ANOVA). The inset displays a representative caption of one ommatidium lacking the central rhabdomere (circle), surrounded by several intact ommatidia containing 7 visible rhabdomeres.
Figure 2
Figure 2. hTau transcript levels.
qRT-PCR analysis of hTau mRNA levels in heads of hTau-wt- (red), hTau-K280Q- (grey) and hTau-K280R- (blue) expressing transgenic flies following one (a) and five days (b) of induction in the adult nervous system (elavGS driver) showed no significant difference among the transgenic lines. p > 0.05, one-way ANOVA, n = 5–6/genotype.
Figure 3
Figure 3. hTau-K280Q expression in adult fly neurons exacerbated photoreceptor neurodegeneration.
Quantification of the proportion of ommatidia lacking at least one rhabdomere following the adult-onset expression of hTau-wt (red), hTau-K280Q (grey) and hTau-K280R (blue) in fly neurons (elavGS driver) after 1, 9, 14, 20, 24 and 27 days of induction (Day9 and day24: *p < 0.05, hTau-K280Q vs. hTau-K280R and hTau-wt; day20: **p < 0.01, hTau-K280Q vs. hTau-K280R and hTau-wt; day 27: *p < 0.05, hTau-K280Q vs. hTau-K280R, using one way ANOVA followed by Tukey’s post hoc test).
Figure 4
Figure 4. Pseudo-acetylation at K280 influenced the phosphorylation pattern of hTau proteins.
(a) Illustration of hTau phosphorylation and acetylation sites used in this study. hTau phosphorylation on S262, S202/T205 (AT8) and T212/S214 (AT100) was analysed following pseudo-acetylation or pseudo-deacetylation of hTau at K280 (K280Q and K280R, respectively). (b,c) Western blot analysis (b) and quantification (c) of hTau phosphorylation on S262, S202/T205 (AT8) and T212/S214 (AT100) and total hTau levels using the polyclonal K9JA (Dako) and monoclonal HT7 antibodies, following 14 days of hTau expression in the fly nervous system using the elavGS driver. Results are normalised to both Tubulin and total hTau levels and are expressed relative to levels observed in the hTau-wt transgenic line (*p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 5/genotype).
Figure 5
Figure 5. hTau protein solubility was not affected by K280 acetyl-mimic mutation.
(a) Representative western blots of RAB-soluble and RAB-insoluble/RIPA-1%SDS-soluble hTau fractions retrieved from heads of hTau-wt, hTau-K280Q- and hTau-K280R-expressing flies following 14 days of induction in the nervous system (elavGS driver), using the total hTau K9JA antibody. (b) The proportion of insoluble hTau species was quantified and expressed relative to the ratios observed in the hTau-wt transgenic line, p > 0.05, one-way ANOVA. n = 4/genotype.
Figure 6
Figure 6. hTau clearance was affected by K280 pseudo-acetylation.
Western blot analysis (a) and quantification (b) of total hTau levels retrieved from adult fly head extracts following neuronal (elavGS-driven) expression of hTau-wt, hTau-K280Q or hTau-K280R following either 2 days of RU486 induction (“2d ON”) or 2 days of RU486 induction followed by exposure to RU486-free food for either 2 or 5 days (“2d ON + 2d OFF” and “2d ON + 5d OFF”, respectively). hTau detection was achieved using the polyclonal K9JA antibody (Dako) and Actin was used for normalisation. Results are expressed relative to levels observed in the “2d ON” condition (*p < 0.05, **p < 0.01 and ***p < 0.001, one-way ANOVA followed by Tukey’s post hoc test, n = 4/condition).
Figure 7
Figure 7. The K280R mutation mitigated hTau-induced climbing defects in old flies.
Climbing ability was monitored over time in flies over-expressing either hTau-wt (red), hTau-K280Q (grey) or hTau-K280R (blue) in the adult nervous system (elavGS-driven). Day 13: *p < 0.05, RU486-induced hTau-K280R vs. RU486-induced hTau-K280Q and ****p < 0.0001, RU486-induced hTau-K280R vs. RU486-induced hTau-wt; two-way ANOVA followed by Tukey’s post hoc test.
Figure 8
Figure 8. Mutating K280 to a pseudo-acetylated or pseudo-unacetylated state both equally delayed fly death.
Representative survival curves of transgenic fly lines expressing (plain curves) either hTau-wt (red), hTau-K280Q (grey) or hTau-K280R (blue) in the adult fly nervous system (elavGS driver and RU486 induction). Survival of non-induced lines is shown as colour-matched dotted curves.
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