Olfactory deficits in an alpha-synuclein fly model of Parkinson's disease

Abstract

Parkinson's disease (PD) is the most common motor neurodegenerative disorder. Olfactory dysfunction is a prevalent feature of PD. It often precedes motor symptoms by several years and is used in assisting PD diagnosis. However, the cellular and molecular bases of olfactory dysfunction in PD are not known. The fruit fly Drosophila melanogaster, expressing human alpha-synuclein protein or its mutant, A30P, captures several hallmarks of PD and has been successfully used to model PD in numerous studies. First, we report olfactory deficits in fly expressing A30P (A30P), showing deficits in two out of three olfactory modalities, tested--olfactory acuity and odor discrimination. The remaining third modality is odor identification/naming. Second, oxidative stress is an important environmental risk factor of PD. We show that oxidative stress exacerbated the two affected olfactory modalities in younger A30P flies. Third, different olfactory receptor neurons are activated differentially by different odors in flies. In a separate experiment, we show that the odor discrimination deficit in A30P flies is general and not restricted to a specific class of chemical structure. Lastly, by restricting A30P expression to dopamine, serotonin or olfactory receptor neurons, we show that A30P expression in dopamine neurons is necessary for development of both acuity and discrimination deficits, while serotonin and olfactory receptor neurons appeared not involved. Our data demonstrate olfactory deficits in a synuclein fly PD model for exploring olfactory pathology and physiology, and for monitoring PD progression and treatment.

Figure 1. A30P flies show climbing deficit.

(A) A30P expression in A30P flies. A30P protein from heads of five days old flies was probed using human-specific αSyn antibody. Pan-actin expression was used as an internal control. (B) Climbing assay. Climbing performance was measured based on negative geotaxis using countercurrent apparatus. By moving the upper five climbing tubes back and forward between each trial per test, flies would distribute between tubes 0 through 5. Each trial is 10 seconds. Details are in materials and methods. (C) Left: Fly distribution based on climbing performance. More CT flies reached the No.5 tube, while more A30P flies stayed in the No.1 tube. The percentage distribution (%) for a tube is [(# of flies in the tube)/(# of flies per test)]×100. The assays were performed with flies of fifteen days old. Fifty to eighty flies were used per trial. Ct vs. A30P: t₀ (8) = 3.588, **P ₀ = 0.007; t₄(8) = 3.770, **P ₄ = 0.0054; t₅(8) = 3.846, **P ₅ = 0.0049. Right: The compounded climbing performance index (PI) from the left. PI = 100%×[Σ⁵ _i = (# of flies)_i×i/(# of flies per test)×5]. t(8) = 4.768, **P = 0.0014; CT is +/+: A30P/+; A30P is Elav/+: A30P/+, hereafter unless noted otherwise. Student t-test.

Figure 2. Fly odor acuity (OA) and odor discrimination (OD) assays.

Fly odor acuity (OA) and odor discrimination (OD) were tested using T-maze apparatus. Flies loaded into T-maze were dropped to a choice point between [BA] and air in the odor acuity assay or between [BA]^e and [MCH]^e+[BA]^e in the odor discrimination assay. To avoid an inherent directional reference in assays, an odor of a pair was delivered from one end of T-maze during the first trial (PI₁) and from the opposite end during the second trial (PI₂). A test comprised two trials. The performance index (PI) of a test is the average of PI₁ and PI₂. When the equilibrium concentrations of BA ([BA]^e) and MCH ([MCH]^e) were delivered from the opposite ends of T-maze, flies would show no preference for either [BA]^e or [MCH]^e by equally distributing between the two end-tubes. Flies with health odor discrimination would detect the presence of [BA]^e, a foreground odor, in the background of [MCH] ^e and run to [MCH]^e tube, avoiding the [MCH]^e+[BA]^e tube. The formula for PI calculation was expressed as a percentage of the absolute number of flies that were differentially distributed between two end-tubes, divided by the total number of flies in a trial. PI₁ or ₂ = 100%×|(# of L)- (# of R)|/(# of total flies in a test).

Figure 3. A30P flies show age-accelerated deficit in odor acuity and odor discrimination.

(A) A30P flies showed decreased odor acuity (d3: t(6) = 2.916, *P = 0.0268; d5: t(10) = 3.982, **P = 0.0026; d15: t(5) = 4.046, **P = 0.0099). Odors used are 0.05% BA vs. air on day3 and 0.1% BA versus air on day5 and day 10. (B) Aged A30P flies showed decreased performance in odor discrimination (d3: t(8) = 2.094, *P = 0.0695; d5: t(14) = 3.058, **P = 0.0085; d15: t(6) = 5.915, **P = 0.0010). Odor options were 1.5% BA made in 15% MCH background and 15% MCH. (C) A30P flies showed normal climbing on day 5 (t(14) = 0.7625, P = 0.480). (A–C) Olfactory deficits preceded motor deficit. (D) Aging exacerbated both OA and OD deficits in A30P flies. The PIs for OA and OD were presented as ratios of PI_A30P over PI_CT. Odors used in OA assays were 0.1% BA and air. Odors used in OD assays were the same as in Figure 2B. OA ratio: t(14) = 4.563, ***P = 0.0004; OD ratio: t(7) = 2.680, *P = 0.0316, Student t-test.

Figure 4. Oxidative stress exacerbated olfactory deficits in A30P flies.

(A) Odor acuity (OA) and (B) odor discrimination (OD) assays. Five-days old A30P flies showed normal olfactory acuity (0.05% BA vs. air) and mild discrimination deficit. Paraquat (PQ) feeding enhanced OA and OD deficits in A30P (For CT+PQ vs. A30P+PQ comparison: F_OA1,12 = 6.243, *P = 0.0280; F_OD1,23 = 5.5152, *P = 0.0329). In OA assays, the mean differences of CT vs. CT+PQ was 10.04% (n.s.: P>0.05) and of A30P vs. A30P+PQ was 27.96% (***P<0.001). In OD assays, the mean differences of CT vs. CT+PQ was 5.97% (n.s.: P>0.05) and of A30P vs. A30P+PQ was 16.25% (***P<0.001). A30P and PQ-feeding were variables significantly interacting with each other (Interaction: *P _OA = 0.028, *P _OD = 0.033), suggesting an enhancement effect. PQ was 5 mM; wt was an internal control for apparatus assays. Odors used in OA were 0.05%BA and air. Odors used in OD assays were the same as in Figure 3B. Other comparisons: ***P<0.001, **P<0.01, *P<0.05, ns: non significant. Two-way ANOVA followed by Tukey Post-hoc tests among CTs and A30Ps with or without PQ-feeding.

Figure 5. A30P flies showed non-odor-specific discrimination deficits.

Fifteen-days old flies were tested for the ability to discriminate the presence of different odors made in 15% MCH versus 15% MCH alone. Chemical structures of each odor are shown and arranged from aromatics on the left, to esters on the right, based on structure similarity to MCH, the background odor. Behavior-equivalent concentrations used were 1.5% of BA^e, 20% MS^e, 1% of 1-Pro^e, 20% of EA^e, 10% of ButA^e and 5% of EH^e made in 15% of MCH^e. BA: benzaldehyde, MS: methyl salicylate, 1-Pro: 1-propanol, EA: ethyl acetate, ButA: butyl acetate, EH: ethyl hexanoate. For CT vs. A30P comparisons from left to right, t_BA(6) = 5.915,**P = 0.001, t_MS(7) = 2.,*P = , t_1-Pro(6) = 8.986, ***P = 0.0001, t_EA(8) = 3.651,**P = 0.0065, t_ButA(12) = 4.750,***P = 0.0005, and t_EH(11) = 8.404,***P<0.0001; Student t-test.

Figure 6. A30P expression in dopaminergic neurons causes odor acuity and discrimination deficits in aged A30P flies.

Fifteen-days old A30P flies expressing A30P under Elav-gal4 (Elav), TH-gal4 (TH), Cha-gal4 (Cha) or Or83b-gal4 (Or83b) drivers were tested for odor acuity (A) and the performance of odor discrimination (B). Young wild-type flies of 1–2 days old were used as internal assay control. Only A30P expressed in dopamine neurons, A30P_TH, showed olfactory acuity and discrimination deficits as seen in A30PElav. The corresponding controls for each comparison were CT_Elav (Elav/+; +/+; +/+), CT_TH (+/+; +/+; TH/+), CT_Cha (+/+; +/+; Cha/+), and CT_Or83b (Or83b/+; +/+; +/+). OA: t_Elav(14) = 6.388, ***P<0.0001, t_TH(14) = 3.773, **P = 0.0021; OD: t_Elav(14) = 3.338, **P = 0.0049, t_TH(14) = 3.257, **P = 0.0057; Student t-test.