Engineered Hsp70 chaperones prevent Aβ42-induced memory impairments in a Drosophila model of Alzheimer's disease

Abstract

Proteinopathies constitute a group of diseases in which certain proteins are abnormally folded leading to aggregation and eventual cell failure. Most neurodegenerative diseases belong to protein misfolding disorders and, among them, Alzheimer's disease (AD) is the most prevalent. AD is characterized by accumulation of the amyloid-β42 (Aβ42) peptide in the extracellular space. Hence, we genetically engineered a molecular chaperone that was selectively delivered to this cellular location. It has been reported that the heat shock protein 70 (Hsp70) binds Aβ42 preventing self-aggregation. Here, we employed two isoforms of the Hsp70, cytosolic and extracellular, to evaluate their potential protective effect against the memory decline triggered by extracellular deposition of Aβ42. Both Hsp70 isoforms significantly improved memory performance of flies expressing Aβ42, irrespective of their age or the level of Aβ42 load. Using olfactory classical conditioning, we established a Drosophila model of AD based on Aβ42 neurotoxicity and monitored memory decline through aging. The onset of the memory impairment observed was proportional to the cumulative level of Aβ42 in the Drosophila brain. These data support the use of this Drosophila model of AD to further investigate molecules with a protective activity against Aβ42-induced memory loss, contributing to the development of palliative therapies for AD.

Figure 1

Aβ42 expression level dictates the onset of memory impairments in a Drosophila model of Alzheimer’s disease. (a) Protein extracts from heads of flies raised at 25 °C or 27 °C were immunodetected for Aβ42 and β-Tubulin. (b) Aβ42 levels were quantified and normalized to the amount of β-Tubulin for control flies (UAS-LacZ/+; ok107-Gal4/+ and UAS-Aβ42/+) and flies expressing Aβ42 in the MBs (UAS-Aβ42/+; ok107-Gal4/+) at both temperatures. (c,d) Flies were raised at either 25 °C (c) or 27 °C (d) throughout development and up to day 1, 5, 15 or 30 post-eclosion and then trained using olfactory classical conditioning. Flies were tested immediately after conditioning. Memory performance index (P.I.) is shown for control flies (UAS-LacZ/+; ok107-Gal4/+) and flies expressing Aβ42 in the MB neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+). (c) At 25 °C, control flies show normal memory decay through aging (day 1 vs day 5, p = 0.5202; day 5 vs day 15, p = 0.0005; day 15 vs day 30, p = 0.9215). Expression of the Aβ42 peptide in the MB neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+) enhances this memory decline (day 1 vs day 5, p = 0.1031; day 5 vs day 15, p = < 0.0001; day 15 vs day 30, p = 0.2099), but does not significantly impair memory performance until day 5 (t-test comparison; day 1, p = 0.6056; day 5, p = 0.0123; day 15, p = 0.0071; day 30, p = 0.0015) and then significantly increases with age. (d) At 27 °C, control flies show a memory decline associated with age that is slightly more pronounced at older ages, and this is significantly increased in flies expressing Aβ42 in the MB neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+) at all ages tested (t-test comparison; day 1, p < 0.0001; day 5, p < 0.0001; day 15, p = 0.0002; day 30, p < 0.0001). Error bars indicate SEM; n = 10 per group; ^*p < 0.05, ^**p < 0.001, ^***p < 0.001. (e) Aβ42 immunodetection at day 1, 5 and 15 post-eclosion in protein extracts from flies (UAS-Aβ42/+; ok107-Gal4/+) raised at 25 °C or 27 °C. (f) Aβ42 levels were normalized to the amount of β-Tubulin in the matching groups, observing a correlation between temperature and the level of Aβ42 from day 1 to day 15 (n > 3).

Figure 2

The architecture of the mushroom body is preserved in flies expressing Aβ42. Representative confocal images of the MBs from flies co-expressing CD8::GFP and LacZ (UAS-LacZ/+; UAS-CD8::GFP/+; ok107-Gal4/+) or Aβ42 (UAS-Aβ42/+; UAS-CD8::GFP/+; ok107-Gal4/+) in the MB neurons at day 1 (a–d), 5 (e,f) and 15 (g,h) post-eclosion. The morphology of the MBs was analyzed in three-dimensional reconstructions of brains from flies raised at 25 °C (a,c,e,g) and 27 °C (b,d,f,h). Frontal (i) and dorsal (ii) view of the MB lobes and dorsal (iii) and frontal (iv) view of the MB calyxes. No significant defects were found at the macroscopic level. Scale bar 30 μm.

Figure 3

Expression of the secreted Hsp70 prevents memory impairments in a Drosophila model of Alzheimer’s disease. Flies were raised at either 25 °C (a) or 27 °C (b–f) throughout development and up to day 1 (c), 5 (d), 15 (e) or 30 (f) post-eclosion and then trained using olfactory classical conditioning. Flies were tested immediately after conditioning. Memory performance index (P.I.) is shown for control flies (UAS-LacZ/+; ok107-Gal4/+), flies expressing Aβ42 and LacZ (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+) or in combination with the secreted form of Hsp70 (UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+) and control flies without carrying the Gal4 driver (UAS-Aβ42/+; UAS-LacZ/+ and UAS-Aβ42/+; UAS-secHsp70/+). (a) At 25 °C, expression of the secreted form of Hsp70 in the MB neurons fully rescues the memory impairments caused by accumulation of Aβ42 in this set of neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+vs UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+; day 1, p = 0.9833; day 5, p = 0.8109; day 15, p = 0.0043; day 30, p = 0.0001); to scores essentially equivalent to those of control flies (UAS-LacZ/+; ok107-Gal4/+ vs UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+; day 1, p = 0.9901; day 5, p = 0.4367; day 15, p = 0.8839; day 30, p = 0.8084). (b) At 27 °C, expression of the secreted Hsp70 chaperone in the MB neurons rescues the memory deficits caused by Aβ42 deposition (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+ vs UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+; day 1, p < 0.0001; day 5, p < 0.0001; day 15, p < 0.0001; day 30, p = 0.0002). Memory performance of flies expressing secHsp70 (UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+) is equivalent to that of control flies (UAS-LacZ/+; ok107-Gal4/+) at all ages tested (day 1, p > 0.9999; day 5, p = 0.7065; day 15, p = 0.0529; day 30, p > 0.9999). (c) One-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+, p < 0.0001) than control flies expressing LacZ alone (UAS-LacZ/+; ok107-Gal4/+). Flies co-expressing the secreted form of Hsp70 and Aβ42 (UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+) performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+, p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p > 0.9999; UAS-Aβ42/+; UAS-LacZ/+, p > 0.9999; UAS-Aβ42/+; UAS-secHsp70/+, p = 0.7506). (d) Five-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0001) than control flies expressing LacZ alone. Flies co-expressing the secreted Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p = 0.7065; UAS-Aβ42/+; UAS-LacZ/+, p = 9283; UAS-Aβ42/+; UAS-secHsp70/+, p = 2109). (e) Fifteen-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0003) than control flies expressing LacZ alone. Flies co-expressing the secreted Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p = 0.0529; UAS-Aβ42/+; UAS-LacZ/+, p = 0.2066; UAS-Aβ42/+; UAS-secHsp70/+, p = 0.3045). (f) Thirty-days-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0001) than control flies expressing LacZ alone. Flies co-expressing the secreted form of Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p = 0.0002) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p > 0.9999; UAS-Aβ42/+; UAS-LacZ/+, p = 0.4083; UAS-Aβ42/+; UAS-secHsp70/+, p = 0.5252). Error bars indicate SEM; n = 10 per group; Tukey’s comparison test: *p < 0.05, ***p < 0.001.

Figure 4

Expression of the cytosolic Hsp70 prevents memory impairments in a Drosophila model of Alzheimer’s disease. Flies were raised at either 25 °C (a) or 27 °C (b–f) throughout development and up to day 1 (c), 5 (d), 15 (e) or 30 (f) post-eclosion and then trained using olfactory classical conditioning. Flies were tested immediately after conditioning. Memory performance index (P.I.) is shown for control flies (UAS-LacZ/+; ok107-Gal4/+), flies expressing Aβ42 and LacZ (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+) or in combination with the cytosolic form of Hsp70 (UAS-Aβ42/+; UAS-cytHsp70/+; ok107-Gal4/+) and control flies without carrying the Gal4 driver (UAS-Aβ42/+; UAS-LacZ/+ and UAS-Aβ42/+; UAS-cytHsp70/+). (a) At 25 °C, expression of the cytosolic Hsp70 in the MB neurons rescues the memory impairments caused by accumulation of Aβ42 in these neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+ vs UAS-Aβ42/+; UAS-cytHsp70/+; ok107-Gal4/+; day 1, p = 0.9991; day 5, p = 0.6834; day 15, p = 0.0083; day 30, p = 0.0039); to scores essentially equivalent to those of control flies (UAS-LacZ/ + ; ok107-Gal4/ + vs UAS-Aβ42/+; UAS-secHsp70/+; ok107-Gal4/+; day 1, p = 0.7899; day 5, p = 0.6424; day 15, p = 0.9373; day 30, p = 0.9784). (b) At 27 °C, expression of the cytosolic Hsp70 in the MB neurons rescues the memory deficits caused by Aβ42 deposition in this set of neurons (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+ vs UAS-Aβ42/+; UAS-cytHsp70/+; ok107-Gal4/+; day 1, p < 0.0001; day 5, p < 0.0001; day 15, p < 0.0001; day 30, p = 0.0002). Memory performance of flies expressing cytHsp70 (UAS-Aβ42/+; UAS-cytHsp70/+; ok107-Gal4/+) is equivalent to that of control flies (UAS-LacZ/+; ok107-Gal4/+) at all ages tested (day 1, p > 0.9999; day 5, p = 0.9963; day 15, p = 0.4423; day 30, p > 0.9999). (c) One-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+, p < 0.0001) than control flies expressing LacZ alone (UAS-LacZ/+; ok107-Gal4/+). Flies co-expressing the cytosolic Hsp70 and Aβ42 (UAS-Aβ42/+; UAS-cytHsp70/+; ok107-Gal4/+) performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (UAS-Aβ42/+; UAS-LacZ/+; ok107-Gal4/+, p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p > 0.9999; UAS-Aβ42/+; UAS-LacZ/+, p > 0.9999; UAS-Aβ42/+; UAS-cytHsp70/+, p = 0.9871). (d) Five-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0001) than control flies expressing LacZ alone. Flies co-expressing cytosolic Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p = 0.9963; UAS-Aβ42/+; UAS-LacZ/+, p > 0.9999; UAS-Aβ42/+; UAS-cytHsp70/+, p = 0.7362). (e) Fifteen-day-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0001) than control flies expressing LacZ alone. Flies co-expressing the cytosolic form of Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p < 0.0001) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p = 0.4423; UAS-Aβ42/+; UAS-LacZ/+, p = 0.9863; UAS-Aβ42/+; UAS-cytHsp70/+, p = 0.9997). (f) Thirty-days-old flies expressing Aβ42 and LacZ in the MB neurons display a significantly lower memory performance (p < 0.0001) than control flies expressing LacZ alone. Flies co-expressing the cytosolic Hsp70 and Aβ42 performed at a significantly higher level than flies co-expressing Aβ42 and LacZ (p = 0.0002) and their memory performance was statistically undistinguishable from control flies (UAS-LacZ/+; ok107-Gal4/+, p > 0.9999; UAS-Aβ42/+; UAS-LacZ/+, p = 0.3980; UAS-Aβ42/+; UAS-cytHsp70/+, p > 0.9999). Error bars indicate SEM; n = 10 per group; Tukey’s comparison test: *p < 0.05, ***p < 0.001.

Figure 5

Cytosolic and secreted forms of Hsp70 do not enhance memory performance in young or elder flies. All genotypes were raised at 27 °C. Flies were trained at days 1 (a), 5 (b), 15 (c) or 30 (d) post-eclosion using olfactory classical conditioning and then tested immediately after training. Memory performance index (P.I.) is shown for control flies (ok107-Gal4/+; UAS-LacZ/+) and flies expressing either a secreted (ok107-Gal4/+; UAS-secHsp70/+) or a cytosolic (ok107-Gal4/+; UAS-cytHsp70/+) form of the Hsp70 chaperone. Memory performance of flies expressing the secreted (ok107-Gal4/+; UAS-secHsp70/+; day 1, p = 0.0550; day 5, p = 0.2960; day 15, p = 0.4265; day 30, p = 0.5554) or the cytosolic (ok107-Gal4/+; UAS-cytHsp70/+; day 1, p = 0.1339; day 5, p = 0.7947; day 15, p = 0.6182; day 30, p = 0.8622) form of Hsp70 is statistically undistinguishable from the performance of control flies (ok107-Gal4/+; UAS-LacZ/+) at all ages tested. Error bars indicate SEM; n = 14 per group; Tukey’s comparison test: no significant.