Interactions between Tau and α-synuclein augment neurotoxicity in a Drosophila model of Parkinson's disease

Abstract

Clinical and pathological studies have suggested considerable overlap between tauopathies and synucleinopathies. Several genome-wide association studies have identified alpha-Synuclein (SNCA) and Tau (MAPT) polymorphisms as common risk factors for sporadic Parkinson's disease (PD). However, the mechanisms by which subtle variations in the expression of wild-type SNCA and MAPT influence risk for PD and the underlying cellular events that effect neurotoxicity remain unclear. To examine causes of neurotoxicity associated with the α-Syn/Tau interaction, we used the fruit fly as a model. We utilized misexpression paradigms in three different tissues to probe the α-Syn/Tau interaction: the retina, dopaminergic neurons and the larval neuromuscular junction. Misexpression of Tau and α-Syn enhanced a rough eye phenotype and loss of dopaminergic neurons in fly tauopathy and synucleinopathy models, respectively. Our findings suggest that interactions between α-Syn and Tau at the cellular level cause disruption of cytoskeletal organization, axonal transport defects and aberrant synaptic organization that contribute to neuronal dysfunction and death associated with sporadic PD. α-Syn did not alter levels of Tau phosphorylated at the AT8 epitope. However, α-Syn and Tau colocalized in ubiquitin-positive aggregates in eye imaginal discs. The presence of Tau also led to an increase in urea soluble α-Syn. Our findings have important implications in understanding the cellular and molecular mechanisms underlying α-Syn/Tau-mediated synaptic dysfunction, which likely arise in the early asymptomatic phase of sporadic PD.

Figure 1.

Misexpression of α-Syn enhances the Tau-mediated eye phenotype. (A′) Scanning EM of the adult Drosophila eye showing the anterior–posterior axis. Light micrographs (A–H) and three-dimensional reconstructed images (I–P); stacks were generated at 10 μm intervals. Misexpression of GFP did not alter Tau-induced toxicity (A–D; I–L), whereas α-Syn enhances Tau toxicity (E–H; M–P). Genotypes: GMR-GAL4/+ (A, E, I, M), GMR-GAL4/+; +; UAS-GFP/+ (B, J), GMR-GAL4/+; +; UAS-Syn/+ (F, N), GMR-GAL4/+; gl-Tau/+ (C, G, K, O), GMR-GAL4/+; gl-Tau/+; UAS-GFP/+ (D, L), GMR-GAL4/+; gl-Tau/+; UAS-Syn/+ (H, P). Scale bar: 100 µm.

Figure 2.

Misexpression of α-Syn enhances Tau-mediated cleaved caspase-3 activity. (A–D) Z-stacked confocal images of third instar larval eye discs stained with antibody against cleaved caspase 3. (E) Bar graph representing the number of cleaved caspase 3 puncta. Y-axis represents the total number of cleaved Caspase 3 spots/area. Values on the Y-axis represent mean ± SEM. X-axis represents the various genotypes. **P < 0.01; ***P < 0.05 using one-way ANOVA + Fisher LSD multiple comparison test. Genotypes: (A) GMR-GAL4/+ (N = 7, Y-axis value = 0.00038), (B) GMR-GAL4/UAS-lacZ; UAS-Syn/+ (N = 7, Y-axis value = 0.00041), (C) GMR-GAL4, UAS-Tau/+; UAS-lacZ/+ (N = 10, Y-axis value = 0.00111) and (D) GMR-GAL4, UAS-Tau/+; UAS-Syn/+ (N = 10, Y-axis value = 0.00204). Scale bar: 170 µm.

Figure 3.

Misexpression of α-Syn enhances Tau-induced motor dysfunction. Using the pan-neuronal-driver C155-GAL4, both Tau alone and Syn + Tau demonstrate impairments in climbing performance when compared with control and α-Syn flies. However, the extent of this motor impairment is higher (∼3-fold) in the α-Syn/Tau flies. Bar graphs representing single fly motor behavior. The Y-axis represents the distance climbed by a 7-day-old male fly in 10 s. Values on the Y-axis represent mean ± SEM. **P < 0.01; *P < 0.05 using one-way ANOVA + Fisher LSD correction for multiple comparisons. Genotypes: (A) WT: C155-GAL4/+ (N = 9, Y-axis value = 11.26), (B) Syn: C155-GAL4/+; UAS-lacZ/+; UAS-Syn/+ (N = 11, Y-axis value = 11.78), (C) Tau: C155-GAL4/+; UAS-Tau/+; UAS-lacZ/+ (N = 9, Y-axis value = 3.67) and (D) Syn + Tau: C155-GAL4/+; UAS-Tau/+; UAS-Syn/+ (N = 12, Y-axis value = 1.29).

Figure 4.

Coexpression of Tau and α-Syn leads to cytoskeletal disorganization and abnormal microtubule organization. Confocal images of retinal longitudinal sections stained with phalloidin (A–D) and tubulin antibody (E–H). Genotypes: GMR-GAL4/+ (WT: A, E), GMR-GAL4/+; +; UAS-Syn/+ (Syn: B, F), GMR-GAL4/+; gl-Tau/+ (Tau: C, G), GMR-GAL4/+; gl-Tau/+; UAS-Syn/+ (Syn + Tau: D, H). (I–P) Representative confocal images of synapses on muscle 4 of OK6-GAL4/+ (I, M), OK6-GAL4/UAS-lacZ; UAS-Syn/+ (J, N), OK6-GAL4/UAS-Tau; UAS-lacZ/+ (K, O) and OK6-GAL4/UAS-Tau; UAS-Syn/+ (L, P) animals stained with anti-Futsch (22C10). Anti-Futsch labels stable presynaptic microtubules. (M)–(P) represent zoomed images of the boxed regions in (I)–(L), respectively. (Q) Quantitation of microtubule morphology assessed using anti-Futsch. The percentage of boutons of the muscle 4 synapse exhibiting disorganized microtubule phenotypes (abnormal boutons) was calculated. Boutons with unorganized microtubules have been represented with white asterisks in (O) and (P) and boutons with organized microtubules have been marked by white arrowheads in (M) and (N). Y-axis on the bar graph represents percentage of abnormal boutons. [Y-axis values − WT (OK6-GAL4/+): 11%, Syn (OK6-GAL4/UAS-lacZ; UAS-Syn/+): 21%, Tau (OK6-GAL4/UAS-Tau; UAS-lacZ/+): 35% and Syn + Tau (OK6-GAL4/UAS-Tau; UAS-Syn/+): 60%]. N = 12 synapses across six animals; ***P < 0.001; *P < 0.01 using one-way ANOVA + Fisher LSD test. Scale bar: (A)–(L), 20 µm; (I)–(L) and (M)–(P), 5 µm.

Figure 5.

Misexpression of α-Syn enhances Tau-mediated axonal transport defects. (A–D) Immunostaining of larval segmental nerves with antibody against DVGLUT. Confocal images of axons in the distal (with respect to the ventral nerve chord)/posterior regions of the segmental nerve (A5–A6 segments) showing accumulation of DVGLUT-positive axonal aggregates (arrows). The extent of accumulation of DVGLUT aggregates is higher in the VGLUT^OK371-GAL4/UAS-Tau; UAS-Syn/+ animals in comparison with the VGLUT^OK371-GAL4/UAS-Tau; UAS-lacZ/+ animals. VGLUT^OK371-GAL4/UAS-lacZ; UAS-Syn/+ animals show few axonal aggregates and are indistinguishable from controls. (E) Bar graph representing DVGLUT accumulation in axons. Y-axis: number of DVGLUT aggregates of >1.7 µm² along 100 µm of axonal length. Number of animals tested: 7 per genotype. Genotypes: (A) WT: VGLUT^OK371-GAL4/+ [Y-value: 0 aggregates per 100 µm], (B) Syn: VGLUT^OK371-GAL4/UAS-lac Z; UAS-Syn/+ [Y-value : ∼1 aggregate per 100 µm], (C) Tau: VGLUT^OK371-GAL4/UAS-Tau; UAS-lacZ [Y-value : 2 aggregates per 100 µm] and (D) Syn + Tau: VGLUT^OK371-GAL4/UAS-Tau; UAS-Syn/+ [Y-value: 4 aggregates per 100 µm]. ***P < 0.001; **P < 0.01; one-way ANOVA + Fisher test. Scale bar: (A–D), 20 µm.

Figure 6.

Tau misexpression enhances α-Syn-mediated dopaminergic cell loss. (A–D) Confocal images from 6-week-old adult fly brains immunostained with anti-TH (tyrosine hydroxylase) to mark DA neurons. The images represent the dopaminergic neurons in the DL (dorsolateral) cluster. (E) Bar graph representing the total number of TH-positive neurons on the Y-axis. Genotypes: (A/WT): TH-GAL4/+ [Y-axis value: 13]; (B/Tau): UAS-Tau/+; TH-GAL4/+ [Y-axis value: 13]; (C/Syn): TH-GAL4, UAS-Syn/+; TH-GAL4, UAS-Syn/+ [Y-axis value, 11] ; (D/Syn + Tau): TH-GAL4, UAS-Syn/UAS-Tau; TH-GAL4, UAS-Syn/+ [Y-axis value: 9]. N = 11. *P < 0.05; **P < 0.01; one-way ANOVA + Fisher test. Scale bar: (A–D), 15 µm.

Figure 7.

Coexpression of α-Syn and Tau decreases Synapsin in boutons and leads to synaptic apposition defects. (A–D) Confocal images of boutons at muscle 4 stained with anti-HRP (red; labels the presynaptic membrane) and 3C11 (green; anti-synapsin). (E–H) Confocal images of muscle 4 boutons showing the synapsin staining in gray scale. In comparison to the wild-type (OK6-GAL4/+) animals, there is a marked decrease in the levels of synapsin in the OK6-GAL4/UAS-lacZ; UAS Syn/+ and the OK6-GAL4/UAS-Tau; UAS-lacZ/+ animals. The decrease in synapsin is enhanced in the OK6-GAL4/UAS-Tau; UAS-Syn/+ animals. (I) Histogram showing synapsin levels. Y-axis, synapsin intensity/HRP intensity. N = total number of boutons of muscle four A3 hemisegment across 7–8 animals. Genotypes: (A) WT: OK6-GAL4-/+ (N = 140, Y-axis value = 0.388), (B) Syn: OK6-GAL4/UAS-lacZ; UAS-Syn/+ (N = 144, Y-axis value = 0.276), (C) Tau: OK6-GAL4/UAS-Tau; UAS-lacZ/+ (N = 165, Y-axis value = 0.278) and (D) Syn + Tau: OK6-GAL4/UAS-Tau; UAS-Syn/+ (N = 154, Y-axis value = 0.116). (J–M) Type 1b boutons of muscle four stained with antibodies to BRP (green) and the glutamate receptor DGluRIII (red). (J) BRP and glutamate receptor stained puncta either colocalize or are tightly apposed to each other at almost all synapses in wild-type animals (WT). In contrast, Syn (K) or Tau-expressing animals (L) show both colocalized BRP and DGluRIII as well as a few glutamate receptors unapposed to BRP (white arrowheads). DGluRIII unapposed to BRP is greatly increased in the Tau and Syn coexpressing animals. (N) A histogram showing the percentage of unapposed glutamate receptors in all four genotypes. Y-axis represents the percentage of unapposed glutamate receptors in a single bouton. N = total number of boutons of the muscle 4′s A3 hemisegment across 5–6 animals. Genotypes: (A) WT: OK6-GAL4/+ (N = 100, Y-axis value = 5.02), (B) Syn: OK6-GAL4/UAS-lacZ; UAS-Syn/+ (N = 100, Y-axis value = 15.69), (C) Tau: OK6-GAL4/UAS-Tau; UAS-lacZ/+ (N = 100, Y-axis value = 11.38), (D) Syn + Tau: OK6-GAL4/UAS-Tau/+; UAS-Syn/+ (N = 105, Y-axis value = 31.39). Values on the Y-axis represent mean ± SEM. ***P < 0.001; **P < 0.01 using one-way ANOVA + Fisher LSD. Scale bar: 5 µm.

Figure 8.

Localization of Tau and α-Syn in various tissue compartments of the double transgenics. (A–J) Syn and Tau colocalize in the cytoplasm of eye discs. (A–D, F–I) Confocal images of single optical sections of third instar larval eye discs from double transgenics stained with T46 (Tau; green). Syn (AB5038; red) and Elav (Blue). (F–J) Zoomed confocal images of single optical sections of eye discs from the same animals. (D, I) Merged confocal images of single optical section showing the staining pattern of the three antibodies. (E, J) Colocalized pixels (gray) of Tau and α-Syn in a single plane of the eye disc tissue using ImageJ, RG2B Coloc Plugin. (K–N) In random cytoplasmic regions α-Syn and Tau colocalize in ubiquitinated aggregates. (K–N) Confocal images of single sections stained with antibody against Tau (green, K), Syn (red, L) and ubiquitin (blue, M). Ubiquitin immunoreactive punctum is observed (blue) that is largely colocalized with Tau and Syn (N, white arrow). Genotype: GMR-GAL4/UAS-Tau 1.13; UAS Syn/+. (O–S) Synuclein and Tau colocalize in axons of motor neurons: single plane confocal image of segmental nerves of the OK6-GAL4/UAS-Tau; UAS-Syn/+ larvae stained with antibody against total Tau (green, O), Syn (red, P) and HRP (blue, Q). (R) Immunostaining of the OK6-GAL4/UAS-Tau; UAS-Syn/+ larvae shows colocalization of Tau and Syn in the axon (yellow arrow). (S) The colocalized pixels between Tau and Syn are highlighted using ImageJ, RG2B Colocalization PlugIn. Yellow and white arrows represent the intense colocalized signals in the axon of the segmental nerves in R and S. (T–W) Tau and Synuclein do not colocalize in boutons of the larval NMJ: single optical section of muscle four synapses stained with anti-Tau (green, T), anti-Syn (red, U) and HRP (blue, V). Syn is observed in synaptic boutons (U, W) but Tau is absent. Genotype: (O–W) OK6-GAL4/UAS-Tau; UAS-Syn/+. Scale bar: 100 µm (A–E), 6 µm (F–J), 1.5 µm (K–N), 10 µm (O–S) and 5 µm (T–W).

Figure 9.

Mechanisms underlying Syn/Tau-mediated toxicity. Syn misexpression does not affect Tau phosphorylation at the AT8 epitope but Tau increases the formation of highly insoluble or membrane-associated Syn. (A) Immunoblot with phosphotau antibody (AT8), antibody against total Tau (T-46) and antibody against GAPDH (loading control). (B) Histograms representing relative phosphorylation levels of Tau in Tau alone and dual Tau/Syn transgenics. Results shown are derived from densitometry analysis of three separate blots. Each bar represents mean ± SEM (N = 3). P-value = 1; one-way ANOVA + Fisher LSD test). (C) Comparable levels of total Tau protein in Tau alone and dual Tau/Syn transgenics. Immunoblot using head extracts probed with T-46 and antibody against tubulin (loading control). Histograms representing relative levels of Tau in Tau alone and dual Tau/Syn transgenics. Results shown are derived from densitometric analysis and the value on the Y-axis is represented by OD [Tau] OD [Tubulin]. Each bar represents mean ± SEM (N = 3). P-value = 0.4; one-way ANOVA + Fisher multiple comparison test). Genotypes: (A–C) WT: GMR-GAL4/+, Tau: GMR-GAL4/+; gl-Tau/+, Syn + Tau: GMR-GAL4/+; gl-Tau/+; UAS-Syn/+. (D–F) Immunoblot using total Syn (4D6) and GAPDH antibodies (loading control) of PBS soluble (D) and urea soluble fractions (E) obtained after a serial extraction of proteins from fly head extracts in various buffers. Synuclein decreased in the PBS soluble fraction (D) and increased in the urea soluble fraction (E). (F) Histogram representing relative levels of Syn in the PBS soluble and urea soluble fractions of Syn + GFP and Syn + Tau transgenics. Results shown are derived from densitometric analysis of three separate blots. Each bar represents mean ± SEM (N = 3). P-value = 0.002 (PBS fraction) and 0.004 (urea fraction; one-way ANOVA + Fisher test). Genotypes: (D–F) WT: GMR-GAL4/+, Syn + GFP: GMR-GAL4/UAS-GFP; UAS-Syn/+, Syn + Tau: GMR-GAL4/UAS-Tau; UAS-Syn.