Alzheimer's Disease Associated Genes Ankyrin and Tau Cause Shortened Lifespan and Memory Loss in Drosophila

Abstract

Alzheimer's disease (AD) is the most common form of dementia and is characterized by intracellular neurofibrillary tangles of hyperphosphorylated Tau, including the 0N4R isoform and accumulation of extracellular amyloid beta (Aβ) plaques. However, less than 5% of AD cases are familial, with many additional risk factors contributing to AD including aging, lifestyle, the environment and epigenetics. Recent epigenome-wide association studies (EWAS) of AD have identified a number of loci that are differentially methylated in the AD cortex. Indeed, hypermethylation and reduced expression of the Ankyrin 1 (ANK1) gene in AD has been reported in the cortex in numerous different post-mortem brain cohorts. Little is known about the normal function of ANK1 in the healthy brain, nor the role it may play in AD. We have generated Drosophila models to allow us to functionally characterize Drosophila Ank2, the ortholog of human ANK1 and to determine its interaction with human Tau and Aβ. We show expression of human Tau 0N4R or the oligomerizing Aβ 42 amino acid peptide caused shortened lifespan, degeneration, disrupted movement, memory loss, and decreased excitability of memory neurons with co-expression tending to make the pathology worse. We find that Drosophila with reduced neuronal Ank2 expression have shortened lifespan, reduced locomotion, reduced memory and reduced neuronal excitability similar to flies overexpressing either human Tau 0N4R or Aβ42. Therefore, we show that the mis-expression of Ank2 can drive disease relevant processes and phenocopy some features of AD. Therefore, we propose targeting human ANK1 may have therapeutic potential. This represents the first study to characterize an AD-relevant gene nominated from EWAS.

Keywords: Alzheimer’s disease; Ankyrin; Drosophila; Tau; lifespan; locomotion; memory; neurodegeneration.

FIGURE 1

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R) and Drosophila Ank2 on Drosophila lifespan. (A) Survival curves of pan-neuronal expressing Aβ42 (elav > Aβ42), Tau (elav > Tau), Ank2-RNAi (elav > Ank2-RNAi line A or B), Ank2 (elav > Ank2), (B) Tau with Aβ42 (elav > Tau, Aβ42), Aβ42 with Ank2-RNAi (elav > Aβ42, Ank2-RNAi line A or B), Aβ42 with Ank2 (elav > Aβ42, Ank2), Aβ42 with Ank2 (elav > Aβ42, Ank2), Aβ42 with Ank2 (elav > Aβ42, GFP), Tau with Ank2-RNAi (elav > Tau, Ank2-RNAi line A or B), Tau with Ank2 (elav > Tau, Ank2), Tau with GFP (elav > Aβ42, GFP) compared to wild type control (elav/+) flies kept at 25°C. Misexpression of all Alzheimer’s disease (AD) genes caused a significant reduction in lifespan compared to control using the Kaplan–Meier and log rank test (n > 100 per genotype of flies). (C) Table listing all genotypes characterized with median lifespan (days) and significant reductions in lifespan as determined by Kaplan–Meier and log rank test and indicated as ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001 and used in all subsequent figures.

FIGURE 2

The effect of pan-neuronal expression of Drosophila Ank1 and Ank2 on behavior. (A) The negative geotaxis-climbing reflex was used to quantify motor deficits of 2–5 day old flies. Pan-neuronal reduction of Ank1 (elav-Gal4 > Ank1-RNAi line A or B) was compared to control using one-way ANOVA with Dunnett’s multiple comparison and showed no significant difference. n ≥ 6 groups of 10 flies. (B) One hour memory of 2–5 day old flies was assessed using the olfactory shock-conditioning assay. Pan-neuronal reduction in Ank2 (elav > Ank2-RNAi line A) as opposed to Ank1 (elav > Ank1-RNAi line A) caused a significant reduction in memory compared to control using one-way ANOVA with Dunnett’s multiple comparisons. Error bars are standard error of the mean (SEM). n ≥ 4 groups of ∼50 flies.

FIGURE 3

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R), and Drosophila Ank2 on degeneration of photoreceptor neurons. Images of 2–5 day old compound eyes of (A) control fly (GMR-Gal4/+) showing the regular alignment of ommatidia compared to photoreceptors neurons overexpressing, (B) Aβ42 (GMR-Gal4 > Aβ42), (C) Tau (GMR-Gal4 > Tau), and (D) Tau with Aβ42 (GMR-Gal4 > Tau, Aβ42) which were smaller and displayed a “rough eye” phenotype. (E,F) photoreceptors expressing Ank2-RNAi (GMR > Ank2-RNAi line A or B) or (G) Ank2 (GMR > Ank2) appeared normal. (H) Degeneration of photoreceptor neurons was quantified as normalized percentage surface area of the eye of genotypes compared to the mean of the control (GMR/+) which was set at 100%, comparisons were made between to mutant genotypes compared to control using one-way ANOVA with Dunnett’s multiple comparisons. Genotypes that included expression of APP (Aβ42) or MAPT (Tau 0N4R) in the eye showed a significantly reduced size of eye, while those mis-expressing Drosophila Ank2 did not. Co-expression of MAPT (Tau 0N4R) and Ank2 rescued eye size to a level indistinguishable from wild type. Error bars are SEM. n ≥ 7 eyes per genotype.

FIGURE 4

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R) or Drosophila Ank2 on locomotor behavior. The negative geotaxis-climbing reflex was used to quantify locomotor behavior of 2–5 day old flies. About 80% of control (elav-Gal4/+) flies were able to climb to the top of test vial within 10 s; this compared to experimental genotypes expressing AD transgenes throughout their nervous system. Mis-expression of all AD associated genes except for overexpression of Ank2 alone, resulted in a significant reduction in climbing performance using one-way ANOVA with Dunnett’s multiple comparisons. Error bars are SEM. n ≥ 15 groups of 10 flies.

FIGURE 5

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R) and Drosophila Ank2 on memory. One hour memory of 2–5 day old flies was assessed using the olfactory shock-conditioning assay. The performance index of flies expressing AD transgenes throughout their mushroom body (MB) was compared to control (OK107-Gal4/+) and were found to show a significant reduction in memory using one-way ANOVA with Dunnett’s multiple comparisons, except genotypes overexpressing Ank2. Error bars are SEM. n ≥ 4 groups of ∼100 flies.

FIGURE 6

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R) and Drosophila Ank2 on response to negative reinforcement (shock) and olfaction. (A) The response of 2–5 day old flies to negative reinforcement was quantified as % avoidance of shock, with experimental genotypes with AD genes expressed throughout their MB compared to control (OK107/+). The avoidance of these genotypes of flies to 3-octanol (B) and methylcyclohexanol (MCH) (C) odors was expressed as a performance index. All genotypes responded to odors and shock in a manner not significantly different from wild type using one-way ANOVA with Dunnett’s multiple comparisons. n ≥ 4 groups of ∼50 flies.

FIGURE 7

The effect of expression of human mutant APP (Aβ42), MAPT (Tau 0N4R), and Drosophila Ank2 on activation of mushroom body neurons. (A) Exemplary control 2–5 day old fly MB expressing the calcium reporter GCaMP6f shows a big increase in relative fluorescence in response to elevated KCl (100 mM, bath-applied). Images show the same brain before (left) and during (right) KCl application; scale bar 50 μm. (B) The increase in neuronal activity is expressed as the relative change in fluorescence (ΔF/F0) over time showing that high KCl (indicated by bar) causes peak Ca²⁺ influx that returns to baseline after washing. (C) Quantitative analysis of the maximal response for the indicated genotypes shows a reduced responsiveness of all mutants. Data was analyzed with one-way ANOVA with Tukey’s post hoc and error bars are standard deviation, n ≥ 5 brains.

FIGURE 8

Mushroom body morphology is not affected by expression of human mutant APP (Aβ42), MAPT (Tau 0N4R), and Drosophila Ank2 in young flies. Images show representative mushroom bodies of 2–5 day old flies visualized by expression of the Ca²⁺ reporter GCaMP6f of (A) a control fly (OK107/+), (B) OK107 > Tau, (C) OK107 > Aβ42, (D) OK107 > Ank2-RNAi line A, (E) OK107 > Ank2-RNAi line B, and (F) OK107 > Ank2 which all show the regular organization of α, β, γ, α′ and β′ lobes. Scale bar is 50 μm.