Testing the therapeutic potential of doxycycline in a Drosophila melanogaster model of Alzheimer disease

Abstract

Therapies for Alzheimer disease that reduce the production of pathogenic amyloid β (Aβ) peptides have been associated with a range of unwanted effects. For this reason, alternative strategies that promote the clearance of the peptide by preventing its aggregation and deposition in the brain have been favored. In this context we have studied doxycycline, a member of the tetracycline family of antibiotics that has shown neuroprotective effects in a number of models of neurodegenerative disease. We investigated the neuroprotective potential of doxycycline in a Drosophila model of Aβ toxicity and sought to correlate any effects with the aggregation state of the peptide. We found that administration of doxycycline to Aβ42-expressing flies did not improve their lifespan but was able to slow the progression of their locomotor deficits. We also measured the rough eye phenotype of transgenic flies expressing the E22G variant of Aβ42 and showed that doxycycline administration partially rescued the toxicity of Aβ in the developing eye. We correlated these in vivo effects with in vitro observations using transmission electron microscopy, dynamic light scattering, and thioflavin T binding. We found that doxycycline prevents Aβ fibrillization and favors the generation of smaller, non-amyloid structures that were non-toxic as determined by the lack of caspase 3 activation in a neuroblastoma cell line. Our confirmation that doxycycline can prevent amyloid β toxicity both in vitro and in vivo supports its therapeutic potential in AD.

FIGURE 1.

Aβ42 peptide expression in the D. melanogaster AD animal model after hatching (day 0). A, immunohistochemistry and confocal microscopy analysis of Aβ42 peptide expression in intact brains from flies expressing the peptide (left panel). Anti-Aβ immunostaining is shown in red, and nuclear counterstaining (TOTO-3) in blue. A few diffuse Aβ aggregates were observed throughout the brain. Fly brain from a control fly showed no Aβ stain (right panel). B, Western blotting was performed on supernatants of soluble (soluble Aβ fraction) and insoluble (insoluble Aβ fraction) extract homogenates and immunodetected by the monoclonal antibody 6E10. An Aβ42 immunoreactive band with a molecular mass of 4 kDa was observed in flies expressing Aβ42, which was absent in flies expressing Aβ40. Total Aβ expression was not reduced in flies treated with 100 μm doxycycline as compared with control, untreated flies. Scale bar = 200 μm.

FIGURE 2.

Doxycycline does not affect longevity of Aβ42 flies. Median survival (days) of control (light gray columns), Aβ40 (black columns), GFP (dark gray columns), and Aβ42 (checked columns) flies were cultured at 25 °C and transferred to fresh food containing 0 μm, 20 μm, 50 μm, and 100 μm of doxycycline every 2–3 days. Aβ42 flies showed a marked reduction in their life span compared with control, Aβ40, and GFP flies. However, doxycycline administration did not affect the longevity of any of the phenotypes analyzed. ***, p < 0.001 compared with control, Aβ40, and GFP flies.

FIGURE 3.

Doxycycline improves locomotion performance of Aβ42 flies. Performance index (climbing %) of control, Aβ40, GFP, and Aβ42 flies at days 5 (light gray bars) and 15 (black bars). The locomotion impairment observed for Aβ42 flies after day 5 is completely rescued by 50 μm doxycycline treatment. ***, p < 0.001 compared with the same concentration at day 5; ###, p < 0.001; ##, p < 0.01 compared with concentration 0 μm for the period of 15 days; &, p < 0.01 compared with concentrations 20 μm for the period of 15 days.

FIGURE 4.

Doxycycline partially rescues the rough eye phenotype given by the Arctic Aβ42 expression. A, analysis of eye phenotype by optical microscopy showing eye size reduction and ommatidial irregularity in Arctic Aβ42 flies (lower panels) when compared with control flies (upper panels) with or without doxycycline administration. B, quantification of total eye areas in control and in Arctic Aβ42 flies, untreated and treated with doxycycline 100 μm, at days 0 and 5, showing that the drug was able to promote partial recovery. **, p < 0.01 compared with 0 μm at day 0; ###, p < 0.001 compared with 0 μm at day 5; ++, p < 0.01 compared with 100 μm at day 0. C, quantification of ommatidia in control and in Arctic Aβ42 flies, untreated and treated with doxycycline 100 μm, at days 0 and 5, showing that the drug was able to promote partial recovery of the irregularity observed at eclosion time. ***, p < 0.001 compared with 0 μm at days 0 and 5; ##, p < 0.01 compared with 100 μm at day 0.

FIGURE 5.

Doxycycline impedes the conformational progression of the Aβ42 peptide. A, Th T binding assay of 50 μm of synthetic Aβ42 incubated alone or with different concentrations of doxycycline (0, 100, and 360 μm), showing the ability of the drug to decrease the amyloidogenic potential of Aβ42. Doxycycline does not interfere with the assay as confirmed by its addition at 360 μm to Aβ42 just prior to fluorescence measurement (360*). ***, p < 0.001 compared with 0 μm and 360* μm for the period of 5 days; ##, p < 0.01 compared with 100 μm for the period of 5 days. B, TEM analyses of Aβ42 alone or in the presence of 100 μm and 360 μm doxycycline at 0 (upper panels) and 5 (lower panels) days, showing that the drug was able to inhibit Aβ fibrillogenesis in a dose-dependent manner. Scale bar = 200 nm. C, DLS analyses of Aβ42 alone (top panels) or incubated with 100 (center panels) and 360 μm (bottom panels) of doxycycline at 0 (left panels) and 5 days (right panels), showing the ability of the drug to reduce the size of Aβ-formed particles.

FIGURE 6.

Doxycycline prevents Aβ inherent toxicity. 10 μm of Aβ42 alone or in the presence of 10 μm of doxycycline incubated at 37 °C for 5 days were added to SH-SY5Y cultured cells and further incubated for 24 h at 37 °C, showing that Aβ species generated upon doxycycline treatment were significantly less toxic than species of Aβ alone. Doxycycline added to aggregated peptide just prior to its addition to cells did not protect against Aβ toxicity. ***, p < 0.001 and **, p < 0.01 compared with untreated cells; #, p < 0.05 compared with cells treated with preaggregated Aβ and doxycycline 10 μm added just prior to addition to cells.