Presynaptic accumulation of α-synuclein causes synaptopathy and progressive neurodegeneration in Drosophila

Abstract Alpha-synuclein (α-syn) mislocalization and accumulation in intracellular inclusions is the major pathological hallmark of degenerative synucleinopathies, including Parkinson’s disease, Parkinson’s disease with dementia and dementia with Lewy bodies. Typical symptoms are behavioural abnormalities including motor deficits that mark disease progression, while non-motor symptoms and synaptic deficits are already apparent during the early stages of disease. Synucleinopathies have therefore been considered synaptopathies that exhibit synaptic dysfunction prior to neurodegeneration. However, the mechanisms and events underlying synaptopathy are largely unknown. Here we investigated the cascade of pathological events underlying α-syn accumulation and toxicity in a Drosophila model of synucleinopathy by employing a combination of histological, biochemical, behavioural and electrophysiological assays. Our findings demonstrate that targeted expression of human α-syn leads to its accumulation in presynaptic terminals that caused downregulation of synaptic proteins, cysteine string protein, synapsin, and syntaxin 1A, and a reduction in the number of Bruchpilot puncta, the core component of the presynaptic active zone essential for its structural integrity and function. These α-syn-mediated presynaptic alterations resulted in impaired neuronal function, which triggered behavioural deficits in ageing Drosophila that occurred prior to progressive degeneration of dopaminergic neurons. Comparable alterations in presynaptic active zone protein were found in patient brain samples of dementia with Lewy bodies. Together, these findings demonstrate that presynaptic accumulation of α-syn impairs the active zone and neuronal function, which together cause synaptopathy that results in behavioural deficits and the progressive loss of dopaminergic neurons. This sequence of events resembles the cytological and behavioural phenotypes that characterise the onset and progression of synucleinopathies, suggesting that α-syn-mediated synaptopathy is an initiating cause of age-related neurodegeneration.


Fig. 1. Presynaptic accumulation of α-Syn causes specific presynaptic deficits.
Representative confocal images of NMJ immunolabeled with anti-Synaptotagmin (A), anti-SNAP-25 (B), anti-nSynaptobrevin (C) and anti-GFP at the NMJ shows they are unaffected. Quantification of fluorescence intensity showed that WT-α-Syn expression under control of the pan-neuronal driver nSyb-Gal4 caused no alterations in these proteins compared to control group expressing GFP only; ns -not significant p>0.05; n=5-10 NMJ from 5/7 flies (A), 10 flies (B), and 8 flies (C)/genotype. Mean ± SEM are shown; statistical analyses were performed using unpaired t test. Scale bars: 10 µm.            Membrane images were acquired Odyssey CLx Imaging System and quantified with ImageJ.

Analysis of neuronal function
The Steady State Visual Evoked Potential (SSVEP) assay measured the output of the photoreceptors and second-order lamina neurons. On the day of eclosion, flies were placed in the dark at 29°C, 3 day-old-flies were prepared for SSVEP measurements as described 11,12 .
The same protocol was used, except that stimuli were generated, and responses recorded by an Arduino Due system instead of a PC. Data was analysed in Matlab and R. Full code at https://github.com/wadelab/flyCode.

Behavioural Analyses
Drosophila ARousal Tracking (DART). DART was used to perform single fly tracking of age-matched mated females 13,14 . During each experiment, a total of 80 flies including controls and experimental genotypes were recorded. Briefly, flies were quickly anesthetised on ice and individually placed into glass tubes. The flies were allowed to recover for 30 min at 25°C prior to the beginning of the experiment. The recording was continuously performed at 5 frames per second for 2 hours using a USB-webcam (Logitech). The x/y position of every fly was tracked and analysed using DART software in order to evaluate the relative speed and activity during the recording.

Startle-induced negative geotaxis (SING).
SING was used to assess the locomotor ability of flies following a startle stimulus to which flies display a negative geotaxis response (modified from Ruan et al 15 . A group of ten mated age-matched female flies, per genotype, were selected by a mouth aspirator and transferred into the experimental tubes containing 1 cm of fresh ageing food at room temperature. After the tubes of all genotypes tested being placed in custom-made apparatus (see Fig. 7A), flies were allowed to acclimatise for 20 min prior the beginning of the assay. Control and experimental groups were always assayed together by tapping all the flies to the bottom of the tubes and allowing them to climb as a negative geotaxis response. After 10 seconds, the number of flies that successfully climbed above the 7 cm line was recorded. This assay was repeated and recorded, at 30 frames per second; 5 trials were performed for each cohort and the flies were allowed to rest during 1 min between trials. The averaged data were represented as percentage. A minimum of 9 cohorts, each consisting of 10 flies (= 90 flies in total) were tested per genotype.

Proboscis extension response (PER) -Akinesia assay.
The PER assay was recorded from 5-8-day-old flies. Flies were restrained as previously described 16   Lite, respectively.

Statistical analysis
GraphPad Prism 8 was used to perform the statistical analyses. Comparison of means from 2 experimental conditions was performed using unpaired parametric two-tailed Student's t-test.
Comparison of means from multiple experimental conditions was performed using ANOVA, followed by Dunnett's multiple comparison post-hoc test, when comparing the experimental groups to control only. Alternatively, when comparing all groups among each other, Tukey's multiple comparison post-hoc test was used. Samples not normally distributed were assessed by Kruskal-Wallis test followed by Dunn's multiple comparison post-hoc test. The significance was defined as p<0.05, error bars are shown as SEM.