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The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster I. Golomidov a, O. Bolshakova a, A. Komissarov a, V. Sharoyko c, Е. Slepneva a, A. Slobodina a, E. Latypova a, O. Zherebyateva b, T. Tennikova c, S. Sarantseva a, * a b c
Petersburg Nuclear Physics Institute Named By B.P. Konstantinov of National Research Centre, Kurchatov Institute, Gatchina, Russia The Orenburg State Medical University, Orenburg, Russia Institute of Chemistry, Saint-Petersburg State University, Saint-Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2019 Accepted 16 December 2019 Available online xxx
Neuroprotective properties of fullerenols С60(OH)30 and С70(OH)30 have been shown in a Drosophila melanogaster model of Parkinson’s Disease (PD). Fullerenols used in this work demonstrated negligible toxicity even at high concentrations as a result of a specifically developed manufacturing process. It has been shown that the drugs promote restoration of dopamine levels, reduce oxidative stress in transgenic flies expressing the human alpha-synuclein gene, prevent death of dopaminergic neurons in the brain and alleviate aggregation of alpha-synuclein. Thus, the anti-aggregation effect of fullerenols, demonstrated for various forms of amyloid proteins, is also observed for alpha-synuclein, resulting in reduction of formation of insoluble aggregates of this protein. Neuroprotective activity was affected by the Drosophila melanogaster genotype and not by the number of carbon atoms in the fullerenol compounds. We concluded that due to their unique properties, fullerenols might be a promising tool for drug development to treat PD. © 2019 Elsevier Inc. All rights reserved.
Keywords: Fullerenols Drosophila melanogaster Oxidative stress Alpha-synuclein Dopamine Parkinson’s disease
1. Introduction PD is the second most common neurodegenerative disease after Alzheimer’s disease, and affects 1e2% of the population over 60 years of age. The aim of this work was to analyze neuroprotective properties of fullerenols C60(OH)30 and C70(OH)30 employing the Drosophila melanogaster model of PD. The main neuropathological symptom of the disease is dopaminergic neuron death in the substantia nigra. The degeneration of this type of neuron leads to a dopamine level decrease in the striatum resulting in motor disorders such as bradykinesia, akinesia and resting tremor. PD is also characterized by the presence of intracellular inclusions named Levi’s bodies, which are formed as a result of the alpha-synuclein aggregation [1,2]. Alpha-synuclein is a small cytosolic neuronal protein consisting of several parts: N-terminal domain, which adopts an a-helical configuration upon membrane binding, NAC domain e responsible for the formation of B-structures e and charged C-terminal domain with several phosphorylation sites
* Corresponding author. E-mail address: [email protected] (S. Sarantseva).
[3,4]. Alpha-synuclein gene duplication or triplication, as well as mutations A30P, E46K, H50Q, A53T cause familial forms of the disease [5e7]. In addition, the mutant forms of alpha synuclein were shown in cell culture studies to increase toxicity and promote formation of a large number of oligomers and relatively mature fibrils [8,9]. Oxidative stress is one of the factors that mediates the neurotoxic effect of overexpression and/or mutations in the alphasynuclein gene. Alpha-synuclein is partially localized to mitochondria [10,11]. Overexpression of alpha-synuclein can lead to mitochondrial fragmentation [12], whereas mutant forms induce excessive mitophagy resulting in neuronal death [13]. Unfortunately, current treatment for PD cannot be considered effective. The existing treatment strategy is based on prescribing dopamine neurotransmitter agonists, which can be ineffective or even produce negative effects after long-term administration. Therefore, besides understanding the etiology of PD, screening for drugs decelerating or alleviating disease progression is of great importance. In this regard, hydroxylated fullerene derivatives e fullerenols e have considerable potency. Fullerenols are efficient antioxidants due to their ability to absorb and deactivate oxygen-containing free radicals [14], which
https://doi.org/10.1016/j.bbrc.2019.12.075 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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exceeds that of traditional antioxidants [15]. Second, in vitro biochemical assays and in vivo studies in experimental model systems of Alzheimer disease have shown the effectiveness of fullerenols in reduction of amyloid aggregation [16e18]. Since oxidative stress and protein misfolding are the major triggers of neurodegeneration relevant to PD, the properties of fullerenols would suggest positive effects in protection, restoration or regeneration of the nervous system. Due to a well-developed nervous system and complex behavior, Drosophila melanogaster is an excellent model to study neurodegenerative diseases and screen for neuroprotective drugs. Moreover, the binary system UAS-GAL4 allows not only generation of transgenic Drosophila flies but also tight control of expression of genes of interest in various tissues at different stages of the ontogenesis [19]. Finally, a short life cycle and numerous offspring allows working with large number of genetically homogeneous subjects. Therefore, the number of reported studies in Drosophila to test various potential therapeutic compounds is constantly increasing [20,21].
2. Materials and methods 2.1. Fly stocks All strains were obtained from the Drosophila Bloomington Stock Center (USA). Flies were maintained on standard yeast medium in 12-h/12-h light/dark photoperiod at 25 C. Transgene expression was carried out in the UAS-GAL4 system. Transgenic Drosophila strains related to the current study are listed here: w*;P{UAS-Hsap\SNCA.F}5B, expressing human wild-type asynuclein insertion (further SNCA.WT) (RRID: BDSC_8146), w*; w*;P {UAS-Hsap\SNCA.A30P}40.1, expressing human mutant A30P a-synuclein (further SNAC.A30P), w* (RRID:BDSC_8147), w*; P{UASHsap\SNCA.A53T}15.3, expressing human mutant A53T a-synuclein (further SNCA.A53T) (RRID: BDSC_8148); cholinergic neuron driver line e w*;P{ChAT-GAL4.7.4}19B P{UAS-GFP.S65T}Myo31DFT2 (RRID:BDSC_6793) (further, Cha), which promotes expression of genes regulated by UAS in the cholinergic neurons, and w*; P{pleGAL4.F}3 (RRID:BDSC_8848) (further, ple), with expression of targets transgene in dopaminergic neurons. Dopaminergic neurons were visualized using the y1y1w*; P{UAS-mCD8::GFP.L}LL5, P{UASmCD8::GFP.L}2 (further CD8) (RRID: BDSC_8746) strain. P{GawB} elavC155 (RRID: BDSC_458) strain was used for Western blotting and oxidative stress analysis. Flies of both sexes with a populational sex ratio of nearly 1:1 were used for all experiments, except dopaminergic neuron number analysis, where only males were tested.
2.2. Fullerenols Fullerenols C60(OH)30 and C70(OH)30 were produced at the Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre«Kurchatov Institute»d[22,23]. The concentration of stock solutions was 20 mg/ml. Fullerenols were added to inactivated by heating to 65 C yeast solution and thoroughly mixed. In preliminary studies on lifespan and negative geotaxis, various concentrations of fullerenols have been tested. The optimal concentration to perform the described experiments was determined as 0.2 mg/ml 80 ml of this preparation was spread on the surface of the agar, which does not contain a nutrient medium for flies. Every 2 days flies were exposed to the fresh agar with the drug. Thus, the animals were receiving only yeast (Saccharomyces cerevisiae) with fullerenols as a source of food through their entire lifespan.
2.3. Sample preparation for confocal microscopy Fly heads were separated from their bodies and transferred to a fresh Phosphate Buffered Saline solution (PBS). Heads were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 7 min at room temperature. After fixation, heads were washed with PBS and the brain was isolated. The prepared brain was immersed in a Vectaschield solution (Vector).
2.4. Image analysis Confocal microscopy was performed using a Leica TCS SP5 microscope (Leica, Germany) with 35-мВт integrated argon laser. All samples were analyzed with the same settings. Brain preparations were analyzed at a wavelength of l ¼ 488 nm. Dopaminergic neuron analysis was performed on offspring of flies obtained from crossing: ple and SNCA.WT, SNAC.A30P and SNCA.A53T on the 5th and 30th day of life. During scanning, the thickness of optical sections was 2 mm. Cholinergic neuron analysis was performed on offspring of flies obtained from crossing cha and SNCA.WT, SNAC.A30P and SNCA.A53T on the 5th and 30th day of life. During scanning, the thickness of optical sections was 1 mm. Neuron counting was performed using confocal micrographs of brain slices. The number of intermediate neuron cells (cholinergic neurons) was counted on 3D projections (n ¼ 3) in the ImageJ software (version 1.38a for Windows). Dopaminergic neurons were counted manually, according to the technique described elsewhere [24]. At least six brain samples of three or more replicates for each age group in each genetic cross were analyzed.
2.5. Western blot For each experiment, 50 Drosophila heads were frozen in liquid nitrogen and homogenized. Lysis buffer N1 [1% (w/v) SDS in PBS with Complete Protease Inhibitor Cocktail tablets (Roche, Penzberg, Upper Bavaria, Germany)] was added to the homogenized heads to extract proteins. After centrifugation at 14 000g for 1 min, the supernatant was collected and used for the detection of soluble asynuclein. Lysis buffer N2 [9 M urea, 1% (w/v) SDS, 25 mM Tris (hydroxymethyl) aminomethane, 1 mM EDTA] was added to the pellets and the mixture was incubated at 55 C for 1 h. After centrifugation at 14 000g for 1 min, the supernatant was collected and used for the detection of insoluble a-synuclein. The protein concentration was determined by the Bradford method using Sarstedt reagents and IMPLENP 330 nanophotometer. Equivalent amounts of total protein were separated in a 12% SDS- polyacrylamide gels and transferred to PVDF membranes (Thermo Fisher Scientific, Prod # 88520, USA). The membranes were incubated overnight at 4 C with primary Rabbit anti-a-synuclein antibody [MJFR1] (Abcam Cat # ab138501, RRID: AB_2537217) or mouse monoclonal anti-b-tubulin antibody (Santa Cruz Biotechnology Cat # sc-365791, RRID: AB_10841919) followed by secondary goat antibody to rabbit IgG - H&L (HRP) (Abcam Cat # ab6721, RRID: AB_955447) or goat antibody to mouse IgG - H&L (HRP) (Abcam Cat # ab97023, RRID: AB_10679675). All blots were developed using an ECL Pierce Fast Western Blot Kit (Thermo Fisher Scientific). Bands were quantified using ImageJ (NIH, Bethesda, MD, USA). The density of the background staining was subtracted from all signals and the densities of the 20 kDa asynuclein bands were normalized by the densities of the tubulin bands (55 kDa).
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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2.6. Measurement of dopamine level The level of dopamine was measured using dopamine research ImmuSmol Dopamine ELISA kit according to the manufacturer’s instructions. Reaction was terminated by adding stop solution; optical density was detected by spectrophotometer Multiskan FC (Thermo Scientific) at a wavelength of 450 nm. 2.7. Oxidative stress analysis Oxidative stress was measured using the H2DCF-DA probe. 20 flies heads were used for the lysate preparation. The heads were homogenized in a mixture containing 100 ml of 10 mM Tris and 3 ml of protease inhibitor Cocktail tablets (Roche, Penzberg, Upper Bavaria, Germany) (pH ¼ 7.4). The homogenate was centrifuged for 10 min at 10,000 rpm. 5 ml of lysate and 60 ml of 5 mM H2DCF-DA (Invintrogen) were dropped into a well of a 96-well plate and were incubated for 60 min at 37 C. The signal was measured on a flatbed analyser with lwt ¼ 490 nm, lem ¼ 530 nm, and h ¼ 4.7 mm. The signal was normalized to protein concentration measured by the Bradford method. 2.8. Statistical analysis Significance of the differences between the control and the samples was determined using the one-way analysis of variance (ANOVA) method and the Tukey-Kramer test for multiple comparisons with KyPlot software. The differences were considered statistically significant at p < 0.05. 3. Results 3.1. Analyses of alpha-synuclein level The level of insoluble alpha-synuclein protein in brains was significantly reduced in flies receiving food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose for 30 days relative to control flies receiving regular food (Fig. 1). 3.2. Analyses of dopamine level It was reported that alpha-synuclein modulates the membrane turnover of a number of neurospecific transporters, which are involved in the neurotransmitter reuptake from synaptic clefts, in particular dopamine [25]. In addition, an important role was suggested for alpha-synuclein in regulation of dopamine biosynthesis and release. We measured the level of dopamine in Drosophila brain lysates and found a significant decrease in dopamine level by day 30, compared to the dopamine level at day 5 (control) in flies carrying any of the three genetic constructs (Fig. 2). If flies received food containing fullerenols during the 30 days, the level of dopamine significantly increased in the strains with mutations, unlike the strain expressing wild-type protein. This increase was observed for both fullerenols, C60(OH)30 and C70(OH)30. At the same time, the dopamine concentration reached wild-type level, suggesting that fullerenols contributed to the normalization of dopamine biosynthesis regulation and release. 3.3. Oxidative stress analyses Oxidative stress is considered to be a general mechanism leading to cellular dysfunction and even cell death. Oxidative stress is a result of an imbalance between the production of reactive oxygen species (ROS) and cellular antioxidant activity. Dopamine can be
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oxidized to reactive species of dopamine quinones, which contribute to elevation of ROS level. Dopamine-induced toxicity has been observed in several in vitro and in vivo models [26]. We analyzed oxidative stress in 5 and 30 day-old flies and demonstrated that oxidative stress was increased in all studied strains by day 30. In strains with the expression of the wild-type or A30P mutant form of the protein, the ROS level increased almost 4 times on day 30, whereas a similar increase was observed on day 5 in the strain expressing alpha-synuclein with the A53T mutation. We also evaluated the effectiveness of fullerenols as antioxidants. The maintenance of flies on a medium containing fullerenols С60(OH)30d and С70(OH)30d resulted in a statistically significant decrease of ROS level in Drosophila strains harboring A53T mutation. Fullerenol С70(OH)30d, but not С60(OH)30d, demonstrated similar effect on wild-type strain (Fig. 3). 3.4. Analysis of neurodegeneration in distinct groups of neurons Excessive levels of oxidative stress can cause lipid peroxidation, DNA damage, and protein oxidation. The accumulation of free radicals causes mitochondrial impairment and, in turn, may lead to neuronal dysfunction and even death. We performed analysis of the dopaminergic neuron numbers by counting GFP signals in flies with expression of wild-type and mutant forms of human alphasynuclein in dopaminergic neurons (Fig. 4 and S1). The numbers of dopaminergic neurons (GFP signals) were shown to be significantly reduced in all strains on day 30. Fullerenols contributed to the preservation of a greater number of dopaminergic neurons (GFP signals) only in flies with the expression of A53T mutant protein. Acetylcholine is one of the major synaptic transmitters in the Drosophila’s central nervous system. Consistently, we detected a very high density of fluorescent signals throughout the brain. Intermediate neuronal cells (cholinergic neurons) were chosen for analysis. Their number did not change with age in the examined strains, therefore they cannot be used as a models for studying fullerenols effects on acetylcholinergic neurons (Fig. S2d). 4. Discussion In the current view, the potential of alpha-synuclein to form aggregates is considered to be a key pathological factor of PD. This has been confirmed by the lack of the toxic effect of beta synuclein and non-aggregating alpha synuclein with truncated NAC domain [27]. At the same time, dopamine and its metabolites act as inhibitors of the conversion of protofibrils into mature fibrils by forming dopamine/alpha-synuclein adducts [28]. Overexpression, as well as the presence of mutations contributes to a more rapid formation of fibrils leading to a loss of synaptic functions of alpha synuclein. This results in increase of dopamine synthesis and impairment of turnover of the synaptic vesicles [29e31]. Dopamine in cytosol is oxidized to form toxic adducts - dopamine-quinone and ROS [32]. This, in turn, can modify alpha-synuclein and help stabilize toxic protofibrils [28]. Alpha-synuclein overexpression further increases susceptibility of the cells to oxidative conditions induced by oxidized dopamine [33]. Some features of PD can be modeled in Drosophila. As described above, we observed an elevation of oxidative stress, as well as a decrease in dopamine levels and in number of GFP signals in dopaminergic neurons, in all used strains on the 30th day of life, which can be interpreted as neuronal death or ultimately lethal functional disorder [34]. We also observed locomotor activity dysfunction (data not shown). Recently, Danielle E. Mor et al. [35] has demonstrated that, normally, dopamine can reduce the level of aggregated alpha synuclein, but oxidation of dopamine may lead to the formation of toxic oligomers and high level of ROS. Based on this, it can be concluded
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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Fig. 1. Fullerenols affects the level of a-synuclein insoluble fractions. (A) Representative western blots showing levels of insoluble a-synuclein in whole brain lysates of SNCA.WT, SNCA.A30P and SNCA.A53T D. melanogaster fed with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose for 30 days. (B) The bar graphs showing the decrease in the relative level of insoluble a-synuclein fractions. Data are expressed as mean ± S.E.M., **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 4 separate experiments, one-way ANOVA.
Fig. 2. Dopamine levels in Drosophila melanogaster strains SNCA.WT, SNCA.A30P and SNCA.A53T obtaining food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose on 5 and 30 days. Data are expressed as mean ± S.E.M., *р < 0.05, **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 3 separate experiments, one-way ANOVA.
that the main factors in preventing neuronal death would be minimization of dopamine oxidation, reduction of ROS levels, and maintenance of alpha-synuclein in a functionally active state, which prevents formation of the oligomers and alpha fibrils. It raises a question: “Is it essential to have all factors in place or only some of them could do the job”? C60(OH)30 and C70(OH)30 significantly reduced the levels of the insoluble (fibrillar) form of alphasynuclein in all strains. Anti-aggregation activity of fullerenes and their derivatives has already been demonstrated in several studies [13,15]. Interaction between nanoparticles and alpha-synuclein is assumed to inhibit formation of beta-structure by weakening hydrogen bonds between the peptide groups. Alpha-synuclein belongs to the family of amyloid proteins, and its aggregation is also accompanied by the formation of beta-fold structures. Based on this, fullerenes are suspected to interact with alpha synucleins
by similar mechanisms. The use of fullerenols demonstrated different efficacy in different PD models. Despite the fact that fullerenols are considered to be effective antioxidants, our study indicates that drugs reduce the levels of ROS only in the A35T strain. This strain differed from the others in the presence of high levels of ROS on the 5th day of life and persisting through the 30th day of life. In this strain, we observed the effectiveness of fullerenols in relation to the dopamine level that increased significantly by day 30. Normalization of these parameters contributed to the increase of the functional dopaminergic neuron numbers in this fly strain, determined by the GFP signal. In the strain with expression of another mutant protein, fullerenols contributed to the maintenance of dopamine level, but oxidative stress and the number of the dopaminergic neurons did not change. In the strain with wild-type protein expression, we
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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Fig. 3. ROS levels in Drosophila melanogaster strains SNCA.WT, SNCA.A30P and SNCA.A53T obtaining food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose on 5 and 30 days. Data are expressed as mean ± S.E.M., *р < 0.05, **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 3 separate experiments, one-way ANOVA.
Fig. 4. Effect of C60(OH)30 and C70(OH)30 fullerenols on dopaminergic neurons of SNCA.WT, SNCA.A30P and SNCA.A53T D. melanogaster. The bar graphs show the total number of dopaminergic neuron cells in the brain of flies on 5 and 30 days. Data are expressed as mean ± S.E.M., **р < 0.005, ***р < 0.001; 8 heads/genotype, n ¼ 3 separate experiments, oneway ANOVA.
observed a slight decrease in oxidative stress, but the dopamine levels and the numbers of the dopaminergic neurons did not change. Currently, there are a number of studies showing the neuroprotective properties of various fullerene derivatives on PD models [36e38]. According to our data, fullerenols indeed demonstrate neuroprotective activity. They reduce oxidative stress and the level of insoluble alpha-synuclein, restore the level of dopamine and the number of the dopaminergic neurons. However, the result is significantly affected by the genetic background. The positive effect on the preservation of the dopaminergic neurons was only observed when fullerenols corrected all three parameters examined. The mechanism of this phenomenon is unclear, and Wang B et al. also demonstrated dependence of the effect on fly genotype, sex, and drug dose [39]. In our preliminary studies, we also observed the dependence of the effect on the dose of fullerenols, notwithstanding a large dose (2 mg/ml) was less effective. We did not observe the difference between the effects of fullerenols C60(OH)30 and C70(OH)30, that differ from each other in the number of carbon atoms, but contain the same number of hydroxyl groups. C70(OH)30 had a lower tendency to aggregate and formed smaller clusters in solution [23]d, but this did not affect its neuroprotective activity. It should be noted that both fullerenols passed the BBB Drosophila, which was also shown in preliminary experiments. Thus, both studied fullerenols, regardless of the number of carbon atoms in the molecule, displayed similar neuroprotective activity in Drosophila PD models. In one model, fullerenols were effective in all experiments, while in others the effects were only observed in individual tests. This favors a personalized approach in drug treatment. Interestingly, fullerenols reduced the level of
insoluble alpha synuclein in all fly models. These data additionally confirm anti-aggregation properties of fullerenols with respect to alpha-synuclein protein. Acknowledgements We thank Dr. Alina Borisenkova for fyllerenols, and Dr Andrew Oleinikov for critical reading of the manuscript. This work was supported by Russian Foundation for Basic Research 15-04-99647 А. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.075. References [1] K.A. Jellinger, Neuropathology of sporadic Parkinson’s disease: evaluation and changes of concepts, Mov. Disord. 27 (1) (2012) 8e30, https://doi.org/ 10.1002/mds.23795. [2] H. Braak, K. Del Tredici, Neuropathological staging of brain pathology in sporadic Parkinson’s disease: separating the wheat from the chaff, J. Parkinson’s Dis. 7 (s1) (2017) 73e87, https://doi.org/10.3233/JPD-179001. [3] J.T. Bendor, T.P. Logan, R.H. Edwards, The function of a-synuclein, Neuron 79 (2013) 1044e1066, https://doi.org/10.1016/j.neuron.2013.09.004. [4] D. Snead, D. Eliezer, Alpha-synuclein function and dysfunction on cellular membranes, Exp. Neurobiol. 23 (2014) 292e313, https://doi.org/10.5607/ en.2014.23.4.292. [5] D.P. Karpinar, M.B. Balija, S. Kügler, et al., Pre-fibrillar a-synuclein variants with impaired b-structure increase neurotoxicity in Parkinson’s disease models, EMBO J. 28 (2009) 3256e3268, https://doi.org/10.1038/ emboj.2009.257. [6] B. Winner, R. Jappelli, S.K. Maji, et al., In vivo demonstration that alpha-
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The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster I. Golomidov a, O. Bolshakova a, A. Komissarov a, V. Sharoyko c, Е. Slepneva a, A. Slobodina a, E. Latypova a, O. Zherebyateva b, T. Tennikova c, S. Sarantseva a, * a b c
Petersburg Nuclear Physics Institute Named By B.P. Konstantinov of National Research Centre, Kurchatov Institute, Gatchina, Russia The Orenburg State Medical University, Orenburg, Russia Institute of Chemistry, Saint-Petersburg State University, Saint-Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 December 2019 Accepted 16 December 2019 Available online xxx
Neuroprotective properties of fullerenols С60(OH)30 and С70(OH)30 have been shown in a Drosophila melanogaster model of Parkinson’s Disease (PD). Fullerenols used in this work demonstrated negligible toxicity even at high concentrations as a result of a specifically developed manufacturing process. It has been shown that the drugs promote restoration of dopamine levels, reduce oxidative stress in transgenic flies expressing the human alpha-synuclein gene, prevent death of dopaminergic neurons in the brain and alleviate aggregation of alpha-synuclein. Thus, the anti-aggregation effect of fullerenols, demonstrated for various forms of amyloid proteins, is also observed for alpha-synuclein, resulting in reduction of formation of insoluble aggregates of this protein. Neuroprotective activity was affected by the Drosophila melanogaster genotype and not by the number of carbon atoms in the fullerenol compounds. We concluded that due to their unique properties, fullerenols might be a promising tool for drug development to treat PD. © 2019 Elsevier Inc. All rights reserved.
Keywords: Fullerenols Drosophila melanogaster Oxidative stress Alpha-synuclein Dopamine Parkinson’s disease
1. Introduction PD is the second most common neurodegenerative disease after Alzheimer’s disease, and affects 1e2% of the population over 60 years of age. The aim of this work was to analyze neuroprotective properties of fullerenols C60(OH)30 and C70(OH)30 employing the Drosophila melanogaster model of PD. The main neuropathological symptom of the disease is dopaminergic neuron death in the substantia nigra. The degeneration of this type of neuron leads to a dopamine level decrease in the striatum resulting in motor disorders such as bradykinesia, akinesia and resting tremor. PD is also characterized by the presence of intracellular inclusions named Levi’s bodies, which are formed as a result of the alpha-synuclein aggregation [1,2]. Alpha-synuclein is a small cytosolic neuronal protein consisting of several parts: N-terminal domain, which adopts an a-helical configuration upon membrane binding, NAC domain e responsible for the formation of B-structures e and charged C-terminal domain with several phosphorylation sites
* Corresponding author. E-mail address: [email protected] (S. Sarantseva).
[3,4]. Alpha-synuclein gene duplication or triplication, as well as mutations A30P, E46K, H50Q, A53T cause familial forms of the disease [5e7]. In addition, the mutant forms of alpha synuclein were shown in cell culture studies to increase toxicity and promote formation of a large number of oligomers and relatively mature fibrils [8,9]. Oxidative stress is one of the factors that mediates the neurotoxic effect of overexpression and/or mutations in the alphasynuclein gene. Alpha-synuclein is partially localized to mitochondria [10,11]. Overexpression of alpha-synuclein can lead to mitochondrial fragmentation [12], whereas mutant forms induce excessive mitophagy resulting in neuronal death [13]. Unfortunately, current treatment for PD cannot be considered effective. The existing treatment strategy is based on prescribing dopamine neurotransmitter agonists, which can be ineffective or even produce negative effects after long-term administration. Therefore, besides understanding the etiology of PD, screening for drugs decelerating or alleviating disease progression is of great importance. In this regard, hydroxylated fullerene derivatives e fullerenols e have considerable potency. Fullerenols are efficient antioxidants due to their ability to absorb and deactivate oxygen-containing free radicals [14], which
https://doi.org/10.1016/j.bbrc.2019.12.075 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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exceeds that of traditional antioxidants [15]. Second, in vitro biochemical assays and in vivo studies in experimental model systems of Alzheimer disease have shown the effectiveness of fullerenols in reduction of amyloid aggregation [16e18]. Since oxidative stress and protein misfolding are the major triggers of neurodegeneration relevant to PD, the properties of fullerenols would suggest positive effects in protection, restoration or regeneration of the nervous system. Due to a well-developed nervous system and complex behavior, Drosophila melanogaster is an excellent model to study neurodegenerative diseases and screen for neuroprotective drugs. Moreover, the binary system UAS-GAL4 allows not only generation of transgenic Drosophila flies but also tight control of expression of genes of interest in various tissues at different stages of the ontogenesis [19]. Finally, a short life cycle and numerous offspring allows working with large number of genetically homogeneous subjects. Therefore, the number of reported studies in Drosophila to test various potential therapeutic compounds is constantly increasing [20,21].
2. Materials and methods 2.1. Fly stocks All strains were obtained from the Drosophila Bloomington Stock Center (USA). Flies were maintained on standard yeast medium in 12-h/12-h light/dark photoperiod at 25 C. Transgene expression was carried out in the UAS-GAL4 system. Transgenic Drosophila strains related to the current study are listed here: w*;P{UAS-Hsap\SNCA.F}5B, expressing human wild-type asynuclein insertion (further SNCA.WT) (RRID: BDSC_8146), w*; w*;P {UAS-Hsap\SNCA.A30P}40.1, expressing human mutant A30P a-synuclein (further SNAC.A30P), w* (RRID:BDSC_8147), w*; P{UASHsap\SNCA.A53T}15.3, expressing human mutant A53T a-synuclein (further SNCA.A53T) (RRID: BDSC_8148); cholinergic neuron driver line e w*;P{ChAT-GAL4.7.4}19B P{UAS-GFP.S65T}Myo31DFT2 (RRID:BDSC_6793) (further, Cha), which promotes expression of genes regulated by UAS in the cholinergic neurons, and w*; P{pleGAL4.F}3 (RRID:BDSC_8848) (further, ple), with expression of targets transgene in dopaminergic neurons. Dopaminergic neurons were visualized using the y1y1w*; P{UAS-mCD8::GFP.L}LL5, P{UASmCD8::GFP.L}2 (further CD8) (RRID: BDSC_8746) strain. P{GawB} elavC155 (RRID: BDSC_458) strain was used for Western blotting and oxidative stress analysis. Flies of both sexes with a populational sex ratio of nearly 1:1 were used for all experiments, except dopaminergic neuron number analysis, where only males were tested.
2.2. Fullerenols Fullerenols C60(OH)30 and C70(OH)30 were produced at the Petersburg Nuclear Physics Institute named by B.P. Konstantinov of National Research Centre«Kurchatov Institute»d[22,23]. The concentration of stock solutions was 20 mg/ml. Fullerenols were added to inactivated by heating to 65 C yeast solution and thoroughly mixed. In preliminary studies on lifespan and negative geotaxis, various concentrations of fullerenols have been tested. The optimal concentration to perform the described experiments was determined as 0.2 mg/ml 80 ml of this preparation was spread on the surface of the agar, which does not contain a nutrient medium for flies. Every 2 days flies were exposed to the fresh agar with the drug. Thus, the animals were receiving only yeast (Saccharomyces cerevisiae) with fullerenols as a source of food through their entire lifespan.
2.3. Sample preparation for confocal microscopy Fly heads were separated from their bodies and transferred to a fresh Phosphate Buffered Saline solution (PBS). Heads were fixed in 4% paraformaldehyde (Sigma-Aldrich, USA) for 7 min at room temperature. After fixation, heads were washed with PBS and the brain was isolated. The prepared brain was immersed in a Vectaschield solution (Vector).
2.4. Image analysis Confocal microscopy was performed using a Leica TCS SP5 microscope (Leica, Germany) with 35-мВт integrated argon laser. All samples were analyzed with the same settings. Brain preparations were analyzed at a wavelength of l ¼ 488 nm. Dopaminergic neuron analysis was performed on offspring of flies obtained from crossing: ple and SNCA.WT, SNAC.A30P and SNCA.A53T on the 5th and 30th day of life. During scanning, the thickness of optical sections was 2 mm. Cholinergic neuron analysis was performed on offspring of flies obtained from crossing cha and SNCA.WT, SNAC.A30P and SNCA.A53T on the 5th and 30th day of life. During scanning, the thickness of optical sections was 1 mm. Neuron counting was performed using confocal micrographs of brain slices. The number of intermediate neuron cells (cholinergic neurons) was counted on 3D projections (n ¼ 3) in the ImageJ software (version 1.38a for Windows). Dopaminergic neurons were counted manually, according to the technique described elsewhere [24]. At least six brain samples of three or more replicates for each age group in each genetic cross were analyzed.
2.5. Western blot For each experiment, 50 Drosophila heads were frozen in liquid nitrogen and homogenized. Lysis buffer N1 [1% (w/v) SDS in PBS with Complete Protease Inhibitor Cocktail tablets (Roche, Penzberg, Upper Bavaria, Germany)] was added to the homogenized heads to extract proteins. After centrifugation at 14 000g for 1 min, the supernatant was collected and used for the detection of soluble asynuclein. Lysis buffer N2 [9 M urea, 1% (w/v) SDS, 25 mM Tris (hydroxymethyl) aminomethane, 1 mM EDTA] was added to the pellets and the mixture was incubated at 55 C for 1 h. After centrifugation at 14 000g for 1 min, the supernatant was collected and used for the detection of insoluble a-synuclein. The protein concentration was determined by the Bradford method using Sarstedt reagents and IMPLENP 330 nanophotometer. Equivalent amounts of total protein were separated in a 12% SDS- polyacrylamide gels and transferred to PVDF membranes (Thermo Fisher Scientific, Prod # 88520, USA). The membranes were incubated overnight at 4 C with primary Rabbit anti-a-synuclein antibody [MJFR1] (Abcam Cat # ab138501, RRID: AB_2537217) or mouse monoclonal anti-b-tubulin antibody (Santa Cruz Biotechnology Cat # sc-365791, RRID: AB_10841919) followed by secondary goat antibody to rabbit IgG - H&L (HRP) (Abcam Cat # ab6721, RRID: AB_955447) or goat antibody to mouse IgG - H&L (HRP) (Abcam Cat # ab97023, RRID: AB_10679675). All blots were developed using an ECL Pierce Fast Western Blot Kit (Thermo Fisher Scientific). Bands were quantified using ImageJ (NIH, Bethesda, MD, USA). The density of the background staining was subtracted from all signals and the densities of the 20 kDa asynuclein bands were normalized by the densities of the tubulin bands (55 kDa).
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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2.6. Measurement of dopamine level The level of dopamine was measured using dopamine research ImmuSmol Dopamine ELISA kit according to the manufacturer’s instructions. Reaction was terminated by adding stop solution; optical density was detected by spectrophotometer Multiskan FC (Thermo Scientific) at a wavelength of 450 nm. 2.7. Oxidative stress analysis Oxidative stress was measured using the H2DCF-DA probe. 20 flies heads were used for the lysate preparation. The heads were homogenized in a mixture containing 100 ml of 10 mM Tris and 3 ml of protease inhibitor Cocktail tablets (Roche, Penzberg, Upper Bavaria, Germany) (pH ¼ 7.4). The homogenate was centrifuged for 10 min at 10,000 rpm. 5 ml of lysate and 60 ml of 5 mM H2DCF-DA (Invintrogen) were dropped into a well of a 96-well plate and were incubated for 60 min at 37 C. The signal was measured on a flatbed analyser with lwt ¼ 490 nm, lem ¼ 530 nm, and h ¼ 4.7 mm. The signal was normalized to protein concentration measured by the Bradford method. 2.8. Statistical analysis Significance of the differences between the control and the samples was determined using the one-way analysis of variance (ANOVA) method and the Tukey-Kramer test for multiple comparisons with KyPlot software. The differences were considered statistically significant at p < 0.05. 3. Results 3.1. Analyses of alpha-synuclein level The level of insoluble alpha-synuclein protein in brains was significantly reduced in flies receiving food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose for 30 days relative to control flies receiving regular food (Fig. 1). 3.2. Analyses of dopamine level It was reported that alpha-synuclein modulates the membrane turnover of a number of neurospecific transporters, which are involved in the neurotransmitter reuptake from synaptic clefts, in particular dopamine [25]. In addition, an important role was suggested for alpha-synuclein in regulation of dopamine biosynthesis and release. We measured the level of dopamine in Drosophila brain lysates and found a significant decrease in dopamine level by day 30, compared to the dopamine level at day 5 (control) in flies carrying any of the three genetic constructs (Fig. 2). If flies received food containing fullerenols during the 30 days, the level of dopamine significantly increased in the strains with mutations, unlike the strain expressing wild-type protein. This increase was observed for both fullerenols, C60(OH)30 and C70(OH)30. At the same time, the dopamine concentration reached wild-type level, suggesting that fullerenols contributed to the normalization of dopamine biosynthesis regulation and release. 3.3. Oxidative stress analyses Oxidative stress is considered to be a general mechanism leading to cellular dysfunction and even cell death. Oxidative stress is a result of an imbalance between the production of reactive oxygen species (ROS) and cellular antioxidant activity. Dopamine can be
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oxidized to reactive species of dopamine quinones, which contribute to elevation of ROS level. Dopamine-induced toxicity has been observed in several in vitro and in vivo models [26]. We analyzed oxidative stress in 5 and 30 day-old flies and demonstrated that oxidative stress was increased in all studied strains by day 30. In strains with the expression of the wild-type or A30P mutant form of the protein, the ROS level increased almost 4 times on day 30, whereas a similar increase was observed on day 5 in the strain expressing alpha-synuclein with the A53T mutation. We also evaluated the effectiveness of fullerenols as antioxidants. The maintenance of flies on a medium containing fullerenols С60(OH)30d and С70(OH)30d resulted in a statistically significant decrease of ROS level in Drosophila strains harboring A53T mutation. Fullerenol С70(OH)30d, but not С60(OH)30d, demonstrated similar effect on wild-type strain (Fig. 3). 3.4. Analysis of neurodegeneration in distinct groups of neurons Excessive levels of oxidative stress can cause lipid peroxidation, DNA damage, and protein oxidation. The accumulation of free radicals causes mitochondrial impairment and, in turn, may lead to neuronal dysfunction and even death. We performed analysis of the dopaminergic neuron numbers by counting GFP signals in flies with expression of wild-type and mutant forms of human alphasynuclein in dopaminergic neurons (Fig. 4 and S1). The numbers of dopaminergic neurons (GFP signals) were shown to be significantly reduced in all strains on day 30. Fullerenols contributed to the preservation of a greater number of dopaminergic neurons (GFP signals) only in flies with the expression of A53T mutant protein. Acetylcholine is one of the major synaptic transmitters in the Drosophila’s central nervous system. Consistently, we detected a very high density of fluorescent signals throughout the brain. Intermediate neuronal cells (cholinergic neurons) were chosen for analysis. Their number did not change with age in the examined strains, therefore they cannot be used as a models for studying fullerenols effects on acetylcholinergic neurons (Fig. S2d). 4. Discussion In the current view, the potential of alpha-synuclein to form aggregates is considered to be a key pathological factor of PD. This has been confirmed by the lack of the toxic effect of beta synuclein and non-aggregating alpha synuclein with truncated NAC domain [27]. At the same time, dopamine and its metabolites act as inhibitors of the conversion of protofibrils into mature fibrils by forming dopamine/alpha-synuclein adducts [28]. Overexpression, as well as the presence of mutations contributes to a more rapid formation of fibrils leading to a loss of synaptic functions of alpha synuclein. This results in increase of dopamine synthesis and impairment of turnover of the synaptic vesicles [29e31]. Dopamine in cytosol is oxidized to form toxic adducts - dopamine-quinone and ROS [32]. This, in turn, can modify alpha-synuclein and help stabilize toxic protofibrils [28]. Alpha-synuclein overexpression further increases susceptibility of the cells to oxidative conditions induced by oxidized dopamine [33]. Some features of PD can be modeled in Drosophila. As described above, we observed an elevation of oxidative stress, as well as a decrease in dopamine levels and in number of GFP signals in dopaminergic neurons, in all used strains on the 30th day of life, which can be interpreted as neuronal death or ultimately lethal functional disorder [34]. We also observed locomotor activity dysfunction (data not shown). Recently, Danielle E. Mor et al. [35] has demonstrated that, normally, dopamine can reduce the level of aggregated alpha synuclein, but oxidation of dopamine may lead to the formation of toxic oligomers and high level of ROS. Based on this, it can be concluded
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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Fig. 1. Fullerenols affects the level of a-synuclein insoluble fractions. (A) Representative western blots showing levels of insoluble a-synuclein in whole brain lysates of SNCA.WT, SNCA.A30P and SNCA.A53T D. melanogaster fed with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose for 30 days. (B) The bar graphs showing the decrease in the relative level of insoluble a-synuclein fractions. Data are expressed as mean ± S.E.M., **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 4 separate experiments, one-way ANOVA.
Fig. 2. Dopamine levels in Drosophila melanogaster strains SNCA.WT, SNCA.A30P and SNCA.A53T obtaining food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose on 5 and 30 days. Data are expressed as mean ± S.E.M., *р < 0.05, **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 3 separate experiments, one-way ANOVA.
that the main factors in preventing neuronal death would be minimization of dopamine oxidation, reduction of ROS levels, and maintenance of alpha-synuclein in a functionally active state, which prevents formation of the oligomers and alpha fibrils. It raises a question: “Is it essential to have all factors in place or only some of them could do the job”? C60(OH)30 and C70(OH)30 significantly reduced the levels of the insoluble (fibrillar) form of alphasynuclein in all strains. Anti-aggregation activity of fullerenes and their derivatives has already been demonstrated in several studies [13,15]. Interaction between nanoparticles and alpha-synuclein is assumed to inhibit formation of beta-structure by weakening hydrogen bonds between the peptide groups. Alpha-synuclein belongs to the family of amyloid proteins, and its aggregation is also accompanied by the formation of beta-fold structures. Based on this, fullerenes are suspected to interact with alpha synucleins
by similar mechanisms. The use of fullerenols demonstrated different efficacy in different PD models. Despite the fact that fullerenols are considered to be effective antioxidants, our study indicates that drugs reduce the levels of ROS only in the A35T strain. This strain differed from the others in the presence of high levels of ROS on the 5th day of life and persisting through the 30th day of life. In this strain, we observed the effectiveness of fullerenols in relation to the dopamine level that increased significantly by day 30. Normalization of these parameters contributed to the increase of the functional dopaminergic neuron numbers in this fly strain, determined by the GFP signal. In the strain with expression of another mutant protein, fullerenols contributed to the maintenance of dopamine level, but oxidative stress and the number of the dopaminergic neurons did not change. In the strain with wild-type protein expression, we
Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075
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Fig. 3. ROS levels in Drosophila melanogaster strains SNCA.WT, SNCA.A30P and SNCA.A53T obtaining food with fullerenols C60(OH)30 or C70(OH)30 at 0.2 mg/ml dose on 5 and 30 days. Data are expressed as mean ± S.E.M., *р < 0.05, **р < 0.005, ***р < 0.001; 50 heads/genotype, n ¼ 3 separate experiments, one-way ANOVA.
Fig. 4. Effect of C60(OH)30 and C70(OH)30 fullerenols on dopaminergic neurons of SNCA.WT, SNCA.A30P and SNCA.A53T D. melanogaster. The bar graphs show the total number of dopaminergic neuron cells in the brain of flies on 5 and 30 days. Data are expressed as mean ± S.E.M., **р < 0.005, ***р < 0.001; 8 heads/genotype, n ¼ 3 separate experiments, oneway ANOVA.
observed a slight decrease in oxidative stress, but the dopamine levels and the numbers of the dopaminergic neurons did not change. Currently, there are a number of studies showing the neuroprotective properties of various fullerene derivatives on PD models [36e38]. According to our data, fullerenols indeed demonstrate neuroprotective activity. They reduce oxidative stress and the level of insoluble alpha-synuclein, restore the level of dopamine and the number of the dopaminergic neurons. However, the result is significantly affected by the genetic background. The positive effect on the preservation of the dopaminergic neurons was only observed when fullerenols corrected all three parameters examined. The mechanism of this phenomenon is unclear, and Wang B et al. also demonstrated dependence of the effect on fly genotype, sex, and drug dose [39]. In our preliminary studies, we also observed the dependence of the effect on the dose of fullerenols, notwithstanding a large dose (2 mg/ml) was less effective. We did not observe the difference between the effects of fullerenols C60(OH)30 and C70(OH)30, that differ from each other in the number of carbon atoms, but contain the same number of hydroxyl groups. C70(OH)30 had a lower tendency to aggregate and formed smaller clusters in solution [23]d, but this did not affect its neuroprotective activity. It should be noted that both fullerenols passed the BBB Drosophila, which was also shown in preliminary experiments. Thus, both studied fullerenols, regardless of the number of carbon atoms in the molecule, displayed similar neuroprotective activity in Drosophila PD models. In one model, fullerenols were effective in all experiments, while in others the effects were only observed in individual tests. This favors a personalized approach in drug treatment. Interestingly, fullerenols reduced the level of
insoluble alpha synuclein in all fly models. These data additionally confirm anti-aggregation properties of fullerenols with respect to alpha-synuclein protein. Acknowledgements We thank Dr. Alina Borisenkova for fyllerenols, and Dr Andrew Oleinikov for critical reading of the manuscript. This work was supported by Russian Foundation for Basic Research 15-04-99647 А. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.075. References [1] K.A. Jellinger, Neuropathology of sporadic Parkinson’s disease: evaluation and changes of concepts, Mov. Disord. 27 (1) (2012) 8e30, https://doi.org/ 10.1002/mds.23795. [2] H. Braak, K. Del Tredici, Neuropathological staging of brain pathology in sporadic Parkinson’s disease: separating the wheat from the chaff, J. Parkinson’s Dis. 7 (s1) (2017) 73e87, https://doi.org/10.3233/JPD-179001. [3] J.T. Bendor, T.P. Logan, R.H. Edwards, The function of a-synuclein, Neuron 79 (2013) 1044e1066, https://doi.org/10.1016/j.neuron.2013.09.004. [4] D. Snead, D. Eliezer, Alpha-synuclein function and dysfunction on cellular membranes, Exp. Neurobiol. 23 (2014) 292e313, https://doi.org/10.5607/ en.2014.23.4.292. [5] D.P. Karpinar, M.B. Balija, S. Kügler, et al., Pre-fibrillar a-synuclein variants with impaired b-structure increase neurotoxicity in Parkinson’s disease models, EMBO J. 28 (2009) 3256e3268, https://doi.org/10.1038/ emboj.2009.257. [6] B. Winner, R. Jappelli, S.K. Maji, et al., In vivo demonstration that alpha-
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Please cite this article as: I. Golomidov et al., The neuroprotective effect of fullerenols on a model of Parkinson’s disease in Drosophila melanogaster, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.075