Nicotine-Mediated Rescue of α-Synuclein Toxicity Requires Synaptic Vesicle Glycoprotein 2 in Drosophila

Abstract

Background: Parkinson's disease (PD) is characterized by α-synuclein aggregation and loss of dopamine neurons. Risk of PD arises due to a combination of genetic and environmental factors, which may interact, termed gene-environment (G×E) interactions. An inverse association between smoking and the risk of PD is well established, and a previous genome-wide G×E interaction study identified genetic variation in the synaptic-vesicle glycoprotein 2C (SV2C) locus as an important mediator of the degree to which smoking is inversely associated with PD.

Objective: We sought to determine the mechanism of the smoking-SV2C interaction in a Drosophila model of PD.

Methods: Flies expressing human α-synuclein in all neurons develop the hallmarks of PD, including motor dysfunction, loss of dopaminergic (DA) neurons, and formation of α-synuclein inclusions. We assessed the effects of increasing doses of nicotine on these parameters of neurodegeneration, in the presence or absence of knockdown of two Drosophila orthologues of SV2, hereafter referred to as SV2L1 and SV2L2.

Results: The α-synuclein-expressing flies treated with nicotine had improved locomotion, DA neuron counts, and α-synuclein aggregation. However, in α-synuclein-expressing flies in which SV2L1 and SV2L2 were knocked down, nicotine failed to rescue neurodegeneration.

Conclusions: This work confirms a G×E interaction between nicotine and SV2, defines a role for this interaction in α-synuclein proteostasis, and suggests that future clinical trials on nicotine should consider genetic variation in SV2C. Furthermore, this provides proof of concept that our model can be used for the mechanistic study of G×E, paving the way for the investigation of additional G×E interactions or the identification of novel G×E. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords: Drosophila; Gene-environment; Nicotine; Parkinson's disease; Synaptic; vesicle glycoprotein.

Figure 1:. Rescue of α-synuclein toxicity by nicotine requires SV2.

A. α-synuclein or control flies were fed media with 0.1–0.4 mg/ml nicotine or vehicle control from the time of eclosion up to 10 days of age. Locomotion was measured at 4, 7, and 10 days. The day 7 timepoint is shown; results at 4 and 10 days were similar. Nicotine improved locomotion in α-synuclein expressing flies (far left) in a dose-dependent manner. However, nicotine failed to rescue the locomotor deficit when the SV2 orthologs SV2L1 and SV2L2 were knocked down and in fact further worsened locomotion in this condition in a dose-dependent manner. N = minimum 6 biological replicates of 9–14 flies each per genotype. *p <0.05, **p < 0.01, *** p < 0.005, **** p < 0.001. Within each genotype, statistical significance was determined using two-way ANOVA with Dunnet’s multiple comparison test, comparing each dose of nicotine to vehicle control. B. Quantification of total neurons from hematoxylin-stained slides of anterior medulla, n = 6 replicates per genotype. * = p <0.05, ** = p <0.01, determined with one-way ANOVA with Dunnet’s test for multiple comparisons. C. Quantification of dopaminergic neurons. n = 6 replicates per genotype. * = p <0.05, ** = p <0.01, determined with one-way ANOVA with Dunnet’s test for multiple comparisons. D. Representative sections of the anterior medulla. Scale = 10 μm. E. Anterior medulla sections were stained with tyrosine hydroxylase (red) and mounted with media containing DAPI (blue). Scale = 5 μm.

Figure 2.. High dose nicotine decreases α-synuclein aggregation and oligomerization.

A. Slides were stained with anti-α-synuclein (clone 5G4, mouse, 1:50,000) and DAPI. Representative images are shown. B. Representative immunoblot demonstrating high molecular oligomers as well as monomeric α-synuclein. C. Quantification of α-synuclein aggregates. D. Quantification from 4 independent experiments. Each nicotine condition was normalized to its vehicle control, and statistical significance was determined using a 1 sample t-test.

Figure 3:. High dose nicotine reduces markers of pathologic autophagy.

Immunohistochemistry was performed for ref(2)P (p62), and Atg8a (LC3). Images were thresholded in ImageJ, and puncta >0.25 μm in area were counted using the Analyze Particles function. n = 6 flies per genotype. A. Quantification of Atg8a (LC3) puncta. B. Quantification of ref(2)P (p62) puncta. C. Representative images of Atg8a (LC3) puncta. D. Representative images of ref(2)P (p62) puncta. Scale = 10 μm. Knockdown of SV2 increases Atg8a puncta even in the absence of α-synuclein but has no effect of ref(2)P puncta. Statistical analysis was performed with a one-way ANOVA with Dunnet’s multiple comparison test. Nicotine and/or SV2 knockdown conditions were compared to α-synuclein or control flies treated with vehicle and with no RNAi. * = p <0.05, ** = p <0.01, *** = p <0.005, **** = p <0.001.

Figure 4:. SV2 knockdown increases physiologic autophagy.

A. UAS-GFP-mCherry-Atg8a was expressed in control or α-synuclein flies, with or without SV2L2 knockdown, treated with nicotine or vehicle control. Brains were dissected in PBS and imaged immediately and sequentially in the red and green channels. A-E. Representative images. In control flies, numerous mCherry+ puncta are seen, with exceedingly rare dual positive puncta (see inset). Scale = 5 μm. F-G. Fluorescence intensity of mCherry:GFP was averaged across all puncta. n = 3 flies per condition. Genotype is UAS-GFP-mCherry-Atg8a/+; nSybQF2, Snyb-GAL4/UAS-CG14691 RNAi.

Figure 5:. Proposed model.

1. α-synuclein expressing flies have impaired autophagic flux. 2. Nicotine can restore autophagic flux in α-synuclein flies in an SV2-dependent manner. 3. SV2 knockdown increases basal autophagy, resulting in more autophagosomes but no delay in maturation. 4. In the absence of SV2C nicotine is unable to rescue autophagic flux, perhaps because the increase in basal autophagy resulting in more autophagosomes overwhelms the autophagy-lysosomal system.