Altered lipid metabolism in Drosophila model of Huntington's disease

Abstract

Huntington's disease (HD) is late-onset, progressive neurodegenerative disorder caused by expansion of polyglutamine (polyQ) repeat within Huntingtin (Htt) protein. In HD patients, energy-related manifestations such as modulation of weight during entire course of disease with energy deficit at terminal stage have been reported, however, underlying reason remains elusive till date. Lipids, carbohydrate and protein constitute a predominant fraction of body's energy reservoir and perturbation in their homeostasis may influence weight. To discern role of these energy molecules in weight alteration, we quantified them in an in vivo transgenic Drosophila model of HD. We document that diseased flies exhibit change in weight due to an altered lipid metabolism, as evident from considerably high lipid levels at the time of disease onset followed by a pathologic decline at end-stage. An alteration in intracellular lipid droplet size suggested altered cellular lipid turnover. Furthermore, diseased flies displayed substantial changes in carbohydrate and protein content. Interestingly, alteration in weight and lipid levels are independent of the feeding pattern in diseased condition and exhibit weak correlation with insulin-like peptide or adipokinetic hormone producing cells. We propose that therapeutic intervention aimed at restoring lipid levels and associated metabolic pathways may improve longevity and quality of patient's life.

Figure 1. Modulation of body size, weight, dry mass and fluid content in diseased flies.

(a) The flies on the right are elav>Httex1p Q93 (diseased) and those on the left are control. The diseased flies are heavy at an initial stage (day 5, 7 and 9) and lean at later stage (day 11 and 13). (b) Diseased flies (grey bars) show significantly low fresh weight at day 0 followed by an increase in weight from day 3–9 that declined significantly at day 13 as compared to the age-matched controls (black bars). Diseased flies do not survive beyond day 13 post-eclosion. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (c) Diseased flies (grey bars) display significantly low dry mass at the time of emergence and gain from 3–9 days as compared to the age-matched control flies (black bars). The dry weight is comparable to controls by day 11 and 13. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (d) Diseased flies (grey bars) show significantly low water content upon hatching with increase from day 3–9. The fluid level subsequently declined at day 13 as compared to the age-matched controls (black bars). P-value: ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 2. Arrhythmic feeding behavior of HD flies.

(a) Normal (black bars) and diseased (grey bars) third instar larvae show comparable food intake. (b) Diseased flies (grey bars) show differential food intake as compared to age-matched controls (black bars and pattern bars) monitored from day 0–12. Black bars represent non-expressing UAS-Httex1p Q93 flies and pattern bars represent unexpanded elav>Httex1p Q20 flies that serves as control. M, D & N denotes morning, day and night, respectively. # represents comparison between diseased and control flies; *represents comparison between elav>Httex1p Q93 (diseased) and unexpanded flies. P-value: ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 3. Altered trehalose, glycogen and protein levels in HD flies.

(a) elav>Httex1p Q93 flies (diseased, grey bars) have significantly high circulating hemolymph sugar at an initial stage of disease (day 3–5) that becomes comparable to age-matched controls (black bars) at later stages (day 7–13). P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (b) Diseased flies (grey bars) exhibit significantly high glycogen levels at 3–5 days of age, however, at later stage it is comparable to age-matched controls. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (c) Diseased flies (grey bars) display significantly high protein content upon disease progression (day 7–9) with significant decline at day 13 as compared to age-matched control flies (black bars). P-value: ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 4. Dysregulated lipid levels and lipid droplets in HD flies.

(a) elav>Httex1p Q93 flies (diseased, grey bars) have significantly low lipid content at the time of emergence that shoots up upon disease progression (day 3–7). The lipid level becomes significantly low at terminal stage of the disease (day 11–13) as compared to the age-matched control flies (black bars). P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (b) Lipid droplets (LD) in control flies did not undergo much variation in their size whereas diseased flies had bigger lipid droplets in abdominal fat cells at day 7. At the later stage of disease (11–13 days) the size and intensity of LD is significantly reduced as compared to their age-matched controls. Arrowheads mark large LD at day 7 and smaller LD at day 11–13. Scale bar: 10 μm. (c) Quantification of total surface area of LD representing significant increase in area occupied by lipid droplets at day 3 followed by decline at day 11 and 13 in diseased condition (grey bars). Error bars represent ± SEM. P-value: *P < 0.05.

Figure 5. Modulation of fresh weight and metabolic activity by mutant Htt expression in insulin-like peptide 2 producing cells.

(a) Flies expressing Httex1p Q93 exclusively in Ilp2 producing cells (pattern bars) show significantly lower fresh weight at day 0, 7 and 13 as compared to the age-matched control flies (black bars). P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (b) Significantly high lipid content at day 5 and 9 as compared to age-matched controls. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (c) Significantly low glycogen content at 0, 3 and 7 days which becomes significantly high at day 5 and 11. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (d) High circulating trehalose levels at day 0 and 11 and significantly low levels at day 3 and 5. P-value: ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 6. Altered body weight, lipid content and carbohydrate in flies expressing mutant Htt in Akh specific neurons.

(a) Flies expressing Httex1p Q93 under the control of Akh driver (pattern bars) have significantly low fresh weight from day 5 to 13 as compared to age-matched control flies (black bars). P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (b) Significantly low lipid content at day 0, followed by increase in lipid content at day 3, 5, 7 and 11. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (c) Significantly low glycogen content at day 3 which increases at day 5, 7, 11 and 13 in comparison to age-matched controls. P-value: ***P < 0.001; **P < 0.01; *P < 0.05. (d) Significantly high trehalose levels at day 0, 3 and 5 that declines at day 11. P-value: ***P < 0.001; **P < 0.01; *P < 0.05.