Identification of Antiparkinsonian Drugs in the 6-Hydroxydopamine Zebrafish Model 2 Rita L

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1 Identification of antiparkinsonian drugs in the 6-hydroxydopamine zebrafish model 2 Rita L. Vaz 1,2,†, Sara Sousa 1,†,*, Diana Chapela 1,3, Herma C. van der Linde 4, Rob 3 Willemsen 4, Ana D. Correia 3, &, Tiago F. Outeiro 5,6,7,8 and Nuno D. Afonso1* 4

5 1 TechnoPhage, SA, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal 6 2 Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal 7 3 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. 8 Prof. Egas Moniz, 1649-028 Lisboa, Portugal 9 4 Department of Clinical Genetics, Erasmus MC, Rotterdam, the Netherlands. 10 5 Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and 11 Molecular Physiology of the Brain, Center for Biostructural Imaging of 12 Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany 13 6 CEDOC, Chronic Diseases Research Centre, NOVA Medical School | Faculdade de 14 Ciências Médicas, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria, 130, 15 1169-056 Lisboa, Portugal. 16 7 Max Planck Institute for Experimental Medicine, Göttingen, Germany 17 8 Institute of Neuroscience, Medical School, Newcastle University, Framlington Place, 18 Newcastle Upon Tyne, NE2 4HH, UK 19 20 † Equal contribution 21 & Current address: Battelle UK Limited, Chelmsford Business Park, Springfield, 22 Chelmsford CM2 5LB, United Kingdom 23 24 *Co-corresponding authors: 25 Dr. Nuno D. Afonso 26 TechnoPhage, SA, 27 Av. Prof. Egas Moniz 28 1649-028 Lisboa 29 Portugal 30 Telephone: +(351) 217999545 31 Email: [email protected] 32 33 Dr. Sara Sousa

34 TechnoPhage, SA, 35 Av. Prof. Egas Moniz 36 1649-028 Lisboa 37 Portugal 38 Telephone: +(351) 217999545 39 Email: [email protected] 40

41 Abstract

42 Parkinson’s disease (PD) is known as a movement disorder due to characteristic motor 43 features. Existing therapies for PD are only symptomatic, and their efficacy decreases as 44 disease progresses. Zebrafish, a vertebrate in which parkinsonism has been modelled, 45 offers unique features for the identification of molecules with antiparkinsonian properties. 46 Here, we developed a screening assay for the selection of neuroactive agents with 47 antiparkinsonian potential. First, we performed a pharmacological validation of the 48 phenotypes exhibited by the 6-hydroxydopamine zebrafish model, by testing the effects 49 of known antiparkinsonian agents. These drugs were also tested for disease-modifying 50 properties by whole mount immunohistochemistry to TH+ neurons and confocal 51 microscopy in the dopaminergic diencephalic cluster of zebrafish. Next, we optimized a 52 phenotypic screening using the 6-hydroxydopamine zebrafish model and tested 1600 53 FDA-approved bioactive drugs. We found that 6-hydroxydopamine-lesioned zebrafish 54 larvae exhibit bradykinetic and dyskinetic-like behaviours that are rescued by the 55 administration of levodopa, rasagiline, isradipine or amantadine. The rescue of 56 dopaminergic cell loss by isradipine was also verified, through the observation of a higher 57 number of TH+ neurons in 6-OHDA-lesioned zebrafish larvae treated with this compound 58 as compared to untreated lesioned larvae. The phenotypic screening enabled us to identify 59 several compounds previously positioned for PD, as well as, new molecules with potential 60 antiparkinsonian properties. Among these, we selected stavudine, tapentadol and 61 nabumetone as the most promising candidates. Our results demonstrate the functional 62 similarities of the motor impairments exhibited by 6-hydroxydopamine-lesioned 63 zebrafish with mammalian models of PD and with PD patients, and highlights novel 64 molecules with antiparkinsonian potential.

66 Keywords: Drug screening, Parkinson’s disease, repositioning, zebrafish.

68 List of Abbreviations

69 6-OHDA, 6-hydroxydopamine AIMs, abnormal involuntary movements 70 BBB, blood brain barrier BDNF, brain derived neurotrophic factor 71 dpf, days post fertilization EM, embryo medium

72 L-dopa, levodopa PBS, phosphate buffer saline 73 PD, Parkinson’s disease PFA, paraformaldehyde 74 TH, tyrosine hydroxylase TU, Tubingen

76 Introduction

77 Parkinson’s disease (PD) is the most common movement disorder, affecting around 5 78 million people worldwide (Dorsey et al., 2007). The characteristic motor features of this 79 neurodegenerative disorder include bradykinesia, resting tremor, postural instability and 80 muscular rigidity. While the causes of PD are still not completely understood, the 81 degeneration of dopaminergic neurons in the substantia nigra is a major pathological 82 feature of the disease (Hirsch et al., 2013). Current pharmacological therapies, such as 83 levodopa (L-dopa), dopamine agonists (apomorphine, bromocriptine, among others), 84 monoamine oxidase (MAO)-B inhibitors (rasagiline, selegiline and safinamide) and 85 catechol-o-methyl transferase inhibitors (entacapone, tolcapone, nitecapone and 86 opicapone), address the loss of dopamine by either replacing it or controlling its 87 metabolism (Oertel and Schulz, 2016). Although these drugs substantially improve 88 quality of life, they lose efficacy as the disease progresses, and give rise to L-dopa induced 89 motor fluctuations and dyskinesia. Amantadine, an NMDA antagonist, is the most 90 effective anti-dyskinetic drug, but the associated side effects limit its use (Vijayakumar

91 and Jankovic, 2016). Non-dopaminergic agents, including A2A-receptor antagonists and 92 modulators of glutamate receptors, as well as nicotine, caffeine and isradipine are 93 currently under clinical trials (Oertel and Schulz, 2016). Despite the number of new 94 candidate agents that have successfully displayed antiparkinsonian effects in preclinical 95 studies, there is a growing demand for the development of alternative drugs.

96 Zebrafish is particularly suitable for large-scale drugs screening due to its small size, 97 transparency and high permeability to compounds diluted in the surrounding media. In 98 addition, studies have explored the organization and function of the zebrafish 99 dopaminergic system and suggest its overall conserved when compared to mammalian 100 vertebrates (Godoy et al., 2015; Rico et al., 2011; Rink and Wullimann, 2002, 2001), 101 rendering zebrafish as a practical and economic alternative vertebrate model for testing 102 the effect of neuroactive compounds (Parng et al., 2006; Sun et al., 2012). The sensitivity 103 of zebrafish to specific neurotoxins known to induce dopaminergic cell loss in rodent 104 models of PD has also been well validated (Anichtchik et al., 2004; Babu et al., 2016; 105 Wang et al., 2017). Specifically, the exposure of zebrafish larvae to 6-hydroxydopamine 106 (6-OHDA) induces the typical phenotypic features of PD, namely death of dopaminergic 107 neurons and bradykinesia (Feng et al., 2014). Furthermore, 6-OHDA-lesioned zebrafish 108 respond to antiparkinsonian compounds, such as levodopa or rasagiline (Cronin and

109 Grealy, 2017; Feng et al., 2014). Therefore, this model constitutes an excellent tool for 110 evaluating novel therapeutic options, not only as an alternative but also to complement 111 studies in mammalian models (Chong et al., 2013; Flinn et al., 2008; Vaz et al., 2018; 112 Wang et al., 2011; Xi et al., 2011; Zhang et al., 2012, 2011).

113 Using the 6-OHDA zebrafish model, we developed a platform for the selection of novel 114 potential antiparkinsonian molecules. First, we evaluated the effects of established anti- 115 parkinsonian agents, such as L-dopa, rasagiline and isradipine on two specific measures 116 of motor performance, total distance moved and burst swimming. These three compounds 117 were also tested for their disease-modifying properties as determined by dopaminergic 118 neuronal loss. We then evaluated a new parameter, immobile events, as a surrogate 119 marker for dyskinetic-like behaviour (Babu et al., 2016), using the previously described 120 drugs and amantadine. Finally, we performed a phenotypic screen of 1600 FDA approved 121 drugs and selected the most promising candidates based on their ability to rescue motor 122 impairments in 6-OHDA-lesioned zebrafish larvae and evaluated their potential for 123 repositioning.

124

125 Material and methods

126 Chemicals and reagents

127 6-hydroxydopamine hydrobromide (6-OHDA) stock solution was prepared in 0.2% (w/v) 128 ascorbic acid and tested at 250, 500, 600, 750 and 800 µM, (Supplementary fig. S1A and 129 B). Tapentadol hydrochloride was purchased from Thonson Technology Ltd. (Shanghai, 130 China). The library of bioactive, FDA approved drugs was purchased from Microsource 131 (Gaylordsville, CT, USA). Nabumetone (Cat# N6142), stavudine (Cat# Y0000408), 6- 132 OHDA (Cat# 162957), levodopa methyl ester (L-dopa, Cat# D9628), rasagiline (Cat# 133 SML0124), isradipine (Cat# I6658), paraformaldehyde (Cat# P6148), bovine serum 134 albumin (Cat# A2153) and triton X-100 (Cat# X100) were purchased from Sigma- 135 Aldrich (St Louis, MO, USA). Amantadine was used directly from the library of drugs. 136 DABCO (Cat# 803456) was obtained from Millipore (Darmstadt, Germany). Mouse anti- 137 tyrosine hydroxylase (TH, Cat# 22941) antibody was purchased from ImmunoStar 138 (Hudson, WI, USA) and AlexaFluor 568 (Cat# A11004) secondary antibody was 139 obtained from ThermoFisher Scientific (Waltham, MA, USA). All other reagents were 140 purchased from AppliChem (Darmstadt, Germany).

141

142 Zebrafish maintenance

143 Animal procedures were performed in accordance with the European Community 144 guidelines (Directive 2010/63/EU), Portuguese law on animal care (DL 113/2013), and 145 approved by the Instituto de Medicina Molecular Internal Committee and the Portuguese 146 Animal Ethics Committee (Direcção Geral de Alimentação e Veterinária). Tubingen (TU) 147 wild-type zebrafish were obtained from ZIRC (University of Oregon, Eugene, USA), 148 maintained and bred in constant conditions, by following standard guidelines for fish care 149 and maintenance protocols (Westerfield, 2000). 150

151 Treatment protocol

152 The protocol optimized for exposure of zebrafish larvae to the compounds was the same 153 for testing therapeutic controls, screening the library and determining dose-response 154 curves (Fig. 1). All experimental procedures were conducted between 8AM and 8PM.

155 Investigators were blind to the experimental groups during the screening protocol. 156 Briefly, zebrafish larvae were allowed to develop in embryo medium (EM [5 mM NaCl,

157 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, pH 7.4]) with 1µM methylene blue 158 until 4 days post fertilization (dpf). At this developmental stage, the zebrafish 159 catecholaminergic system is fully developed and the blood brain barrier (BBB) is already 160 functional (Du et al., 2016; Jeong et al., 2008; Kastenhuber et al., 2010). On the other 161 hand, 6-OHDA induces dopaminergic lesion in zebrafish larvae with up to 5 dpf, 162 suggesting the BBB permeability to this neurotoxin (Feng et al., 2014). 4 dpf zebrafish 163 larvae were arbitrarily distributed into 24-well plate (8 larvae/well) containing EM + 10 164 mM HEPES and treated with 750 µM of 6-OHDA, for 24h. The next day, compounds 165 were added to the medium at the established concentration and larvae (n= 8-16 larvae) 166 were incubated for 24h. To counteract zebrafish larvae neuroregenerative capability, 167 further 6-OHDA was added to each well simultaneously. For each experiment, healthy 168 larvae treated with 0.2% ascorbic acid (vehicle, n= 8-16 larvae) and untreated 6-OHDA- 169 lesioned larvae (6-OHDA, n= 8-16 larvae) were used as controls. The sample size was 170 predetermined by non-statistical methods. Particularly, the exposure to 6-OHDA resulted 171 in a variable rate of survival dependent on zebrafish larvae fitness. For suitable statistical 172 analysis and in compliance with the 3Rs, this study was designed to ensure a minimum 173 of 5 zebrafish larvae per group and, given only experiments with more than 65% of 174 surviving animals were considered, minimum 8 zebrafish larvae were assigned to each 175 group. Behavioural assessment was performed at the end of the incubation with the 176 compounds.

177

178 Figure 1 – Schematic representation of the platform developed for the selection of 179 novel compounds with antiparkinsonian potential. The protocol of the phenotypic 180 screening starts with dopaminergic lesion by exposure of the zebrafish larvae to 6-OHDA. 181 The next day, the compounds are added to the medium and zebrafish larvae are incubated 182 for 24h. Finally, the behavioural analysis is conducted with a video tracking system and 183 drugs are selected for their capability to improve the motor performance of 6-OHDA- 184 lesioned zebrafish larvae.

185

186 Behavioural analysis

187 Spontaneous locomotion was recorded using the DanioVision™ (Noldus Information 188 Technology, the Netherlands) automated tracking system for zebrafish larvae. Larvae 189 were allowed to swim freely in a 96-well plate (1 larva/well) with 1200 µl of EM + 10 190 mM HEPES and their swimming activity was tracked for 90 minutes, under 10 min light- 191 dark cycles. 30 min acclimatization period was done followed by 60 min test. The 192 acquired track data was analysed using the Ethovision X.T. 10 software (Noldus,

193 Wageningen, Netherlands). Only swimming activity obtained in the dark periods (i.e. 194 under infrared light) were analysed (Esch et al., 2012). The parameters automatically 195 measured were total distance moved (mm), burst swimming (number of times larvae 196 reached velocities higher than 25 mm/s) and immobile events (mean time spent moving 197 less than 2 mm/s divided by number of events; protocol adapted from noldus technical 198 specifications (http://www.noldus.com/EthoVision-XT/Gathering-data; 199 http://www.noldus.com/animal-behavior-research/solutions/research-small-lab- 200 animals/open-field-set)). To filter system noise, 0.2 mm was defined as the minimum 201 distance of movement. All values were normalized as a percentage of the mean of the 202 healthy control.

203

204 Dose-response curves

205 Dose-response curves were determined by adding to 6-OHDA-lesioned larvae different 206 doses of the compound under test (n= 8 larvae per condition). Dopaminergic lesion 207 induction, exposure to the compounds and behavioural analysis were conducted as 208 described above. Exceptionally, to depict L-dopa curve, larvae were treated 30 min prior 209 to the behavioural evaluation. The parameters were normalized as a percentage of the 210 mean of healthy control. Anti-TH immunostaining was performed after behavioural 211 evaluation to test the viability of dopaminergic neurons in zebrafish larvae treated with 212 an effective dose of the compound.

213

214 Screening of the library of FDA-approved compounds

215 To screen for compounds that can rescue 6-OHDA-induced motor impairments in 216 zebrafish, the treatment protocol and behavioural analysis were performed as described 217 above. 6-OHDA-lesioned larvae treated with 1250 µM of L-dopa were concomitantly 218 tested as positive control. L-dopa treated larvae were exposed to freshly prepared L-dopa, 219 30 min prior to behavioural evaluation. All experimental conditions (n= 8 larvae per 220 condition) were performed at 1% DMSO and with compounds at 25 µM, labelled with a 221 predefined code by an investigator not involved in the analysis, to ensure the blinding 222 procedure. Healthy larvae treated with vehicle and untreated 6-OHDA-lesioned larvae 223 were also exposed to 1% DMSO, a non-toxic concentration to zebrafish (Hallare et al.,

224 2006). Compounds were considered as a positive hit, when there was a statistically 225 significant recovery of motor performance (total distance moved and burst swimming) in 226 the 3 independent experiments performed. 227 228 Evaluation of repositioning potential 229 After the first round of screenings, the feasibility of repositioning of the compounds that 230 rescued motor performance of 6-OHDA-lesioned zebrafish larvae was evaluated by an 231 investigator not involved in the screening experiments. Relevant data about the selected 232 molecules was searched in publicly available scientific databases and revised. All 233 compounds with active intellectual property, prior art for PD, prone to off-label use (e.g. 234 vitamins) and with safety concerns were excluded from further screenings. The 235 compounds that rescued motor performance in the three independent rounds of screenings 236 and feasible for repositioning were further investigated. Previous medical indications, 237 targets and BBBpermeability were evaluated. 238

239 Whole mount immunostaining and confocal microscopy

240 Whole mount immunostaining in zebrafish was performed as previously described (Wang 241 et al., 2011) with modifications. Zebrafish larvae were fixed overnight at 4 °C in 4% 242 paraformaldehyde (PFA). Larvae were then gradually dehydrated to methanol (Cat# 243 A3493) 100% and stored at −20 °C. For whole mount immunostaining, larvae were 244 gradually rehydrated to phosphate buffer saline (PBS). The tissue was then permeated in 245 100% acetone (Cat# 211007) for 15 min at -20°C, washed with 0.5% PBS-Triton X-100 246 and blocked in blocking solution (1% bovine serum albumin in PBS with 1% DMSO and 247 0.05% Triton X-100) for 2 hours at room temperature. Whole mount tissues were 248 incubated overnight at 4 ºC with anti-TH primary antibody (1:200 in blocking solution; 249 ImmunoStar Cat# 22941), washed in 0.1% PBS-Triton X-100, PBS and re-incubated 250 overnight at 4 ºC with AlexaFluor 568 secondary antibody (1:1000 in blocking solution; 251 ThermoFisher Scientific Cat# A-11004). After staining, larvae were washed in PBS and 252 flat-mounted on a fluorescent mounting medium with DABCO, under a stereoscope. Z- 253 stack compositions of the dopaminergic diencephalic cluster were acquired in a confocal 254 microscope (Zeiss LSM 510 META, Carl Zeiss MicroImaging, Göttingen, Germany) 255 with 40x magnification. Dopaminergic cell content was assessed by counting the number 256 of TH+ cells from average intensity projections with Image J software (Schneider et al.,

257 2012), as described by (Wang et al., 2011). The zebrafish dopaminergic diencephalic 258 cluster was outlined according to (Kastenhuber et al., 2010; Tay et al., 2011) (Figure 3D). 259 The number of TH+ neurons ranged between X and X in the 6-OHDA-lesioned zebrafish 260 larvae as compared to Y and Y in the healthy larvae. Results are expressed as a percentage 261 of the mean of TH+ cells in healthy controls.

262

263 Statistical analysis

264 Data analysis and graphical representation were performed using Prism 5 software 265 (GraphPad Software, Inc., San Diego, CA, USA). All values were normalized as 266 percentage of the mean of healthy control, because substantial variability was evidenced 267 in the behaviour of zebrafish larvae (previously described at this developmental stage 268 (Farrell et al., 2011). This variability was also observed in the number of TH+ neurons in 269 the dopaminergic diencephalic cluster, as well as, in the extent of the lesion induced by 270 different lots of 6-OHDA. This variability probably resulted from the instability of 6- 271 OHDA, which is highly sensitive to light and easily oxidized. Values presented are mean 272 ± s.e.m. of n animals. All statistical tests used were two-tailed and chosen according to 273 the distribution of the data. Mean comparisons between the different groups and 6- 274 OHDA-lesioned untreated larvae were performed using one-way ANOVA with 275 Dunnett’s post-hoc test for experiments independently replicated or Kruskal-Wallis 276 ANOVA with Dunn’s post-hoc test for experiments performed once. Difference was 277 considered significant when P value < 0.05.

278

279 Results

280 L-dopa, rasagiline and isradipine rescue bradykinesia in zebrafish larvae

281 6-OHDA-lesioned zebrafish present bradykinetic-like behaviour that can be depicted 282 from quantification of the total distance moved (Feng et al., 2014). To further characterize 283 the motor impairments in this model, we calculated the number of times that larvae 284 reached velocities corresponding to escaping behaviour (>25 mm/s, burst swimming). 285 This parameter allows the evaluation of motor fitness. After 2 days of exposure to 6- 286 OHDA, lesioned larvae exhibit a decrease of the burst swimming when compared to 287 healthy larvae, as well as, a reduction of the total distance moved (Fig. 2). We then 288 assessed whether, in this model, the motor impairments could be rescued by L-dopa (the 289 most effective treatment for PD to date, (Oertel and Schulz, 2016)), rasagiline (showed 290 disease-modifying properties in preclinical models of PD and induces motor 291 improvement in patients with PD (Oertel and Schulz, 2016)) and isradipine (in phase III 292 clinical trials as a disease-modifying agent for PD (Oertel and Schulz, 2016)). To 293 determine the effective doses, 6-OHDA-lesioned larvae were incubated with different 294 concentrations of each compound and the dose-response curves were outlined. First, we 295 tested a larger range of concentrations and determined the LD50 of each drug. The LD50 296 of L-dopa, rasagiline and isradipine on 6-OHDA-lesioned zebrafish larvae was above 297 5000, 150 and 40 µM, respectively. Then, we tested concentrations around the optimal 298 dose. As shown by the dose-response curve, L-dopa rescued the total distance moved at 299 125 and 1250 µM (Supplementary fig. S2A), but only at 125 µM there was rescue of the 300 burst swimming (Supplementary fig. S2B). Rasagiline was effective at lower 301 concentrations, 0.8 and 0.9 µM rescued the total distance moved and burst swimming 302 (Supplementary fig. S2C and D). Isradipine showed a wider range of effective doses, 0.04 303 µM of this drug rescued the total distance moved, but did not affect the burst swimming 304 (Supplementary fig. S2E). Additionally, several concentrations between 0.08 and 0.8 µM 305 rescued both parameters, with a peak effective dose at 0.5 µM (Supplementary fig. S2E 306 and F). To confirm the effective doses obtained from this data, three independent 307 experiments were performed with each of the compounds at two different concentrations, 308 125 and 1250 µM for L-dopa, 0.8 and 0.9 µM for rasagiline and 0.04 and 0.5 µM of 309 isradipine. 0.8 µM and 0.5 µM were confirmed to be the effective doses of rasagiline (Fig. 310 2C and D) and isradipine (Fig. 2E and F), respectively, as highlighted by the dose- 311 response curves of each compound. In turn, while the dose of 125 µM was highlighted

312 by the dose-response curves of L-dopa, the dose of 1250 µM revealed more consistent 313 rescue of motor performance of 6-OHDA-lesioned zebrafish larvae in the three 314 independent experiments performed. The specificity of the motor effects induced by L- 315 dopa, rasagiline and isradipine was then confirmed, as none of the compounds induced 316 behavioural changes in healthy zebrafish larvae (Supplementary fig. S3). This data shows 317 that the bradykinetic-like behaviour in 6-OHDA-lesioned zebrafish larvae can be 318 recovered by antiparkinsonian compounds, namely L-dopa, rasagiline and isradipine, and 319 that the effects observed are specific.

320

321

322 Figure 2 – Rescue of bradykinetic-like behaviour by L-dopa, rasagiline and 323 isradipine in 6-OHDA-lesioned zebrafish larvae. Motor performance depicted from 324 (A, C and E) total distance moved and (B, D and F) burst swimming in 6-OHDA-lesioned 325 zebrafish larvae treated with 1250 µM of L-dopa (n= 24 larvae), 0.8 µM of rasagiline (n= 326 36 larvae) and 0.5 µM of isradipine (n= 24 larvae), as compared to untreated larvae (n= 327 35-44 larvae). Percentage relative to the mean of vehicle treated larvae (healthy control, 328 n= 24-32 larvae). Mean ± s.e.m. of three independent experiments is presented. 329 ***p<0.001, one-way ANOVA with Dunnett’s post-hoc test.

330

331

332 Isradipine rescues dopaminergic cell loss in zebrafish larvae

333 Previous studies demonstrated neuronal loss in the dopaminergic diencephalic cluster of 334 larvae lesioned with 6-OHDA (Feng et al., 2014). Therefore, we explored disease- 335 modifying properties of L-dopa, rasagiline and isradipine in this model. Larvae were 336 treated with each compound at the effective dose and, after behavioural analysis, the 337 cellular content in the dopaminergic diencephalic cluster was determined by 338 immunohistochemistry against TH. 6-OHDA-lesioned larvae presented a reduction in the 339 number of dopaminergic cells, that was partially recovered by 0.5 µM of isradipine (Fig. 340 3C and D). On the contrary, neither L-dopa (Fig. 3A), nor rasagiline showed disease- 341 modifying properties in this zebrafish model, at the optimal dose. Considering that, in 342 rats, rasagiline shows disease-modifying properties (Blandini et al., 2004), in addition to 343 the optimal dose, we also tested this compound at the concentration used in the phenotypic 344 screening, 25 µM, but no statistically significant cell recovery was observed (Fig. 3B). 345 This result indicates that the 6-OHDA-lesioned zebrafish larvae has a limited predictive 346 value to test disease-modifying agents for PD.

347

348

349 Figure 3 – Rescue of dopaminergic cell loss by isradipine, but not rasagiline and L- 350 dopa in 6-OHDA-lesioned zebrafish larvae. Neuronal death depicted from percentage 351 of TH+ neurons in 6-OHDA-lesioned zebrafish larvae treated with (A) 1250 µM of L- 352 dopa (n= 18 larvae), (B) 25 µM of rasagiline (n= 16 larvae) and (C) 0.5 µM of isradipine 353 (n= 18 larvae), as compared to untreated larvae (n= 19-32 larvae), determined by whole 354 mount immunohistochemistry for TH. Percentage relative to the mean of vehicle treated 355 larvae (healthy control, n= 18-21 larvae). Mean ± s.e.m. of three independent experiments 356 is presented. Ns – not significant, *p<0.05 and ***p<0.001, one-way ANOVA with 357 Dunnett’s post-hoc test. Representative Z-projections (D) of confocal stacks of 358 wholemount anti-TH immunohistochemistry in the dopaminergic diencephalic cluster of 359 6-OHDA-lesioned zebrafish larvae treated with isradipine (isradipine) as compared to 6- 360 OHDA-lesioned untreated larvae (6-OHDA) and healthy larvae (vehicle). Ventral views, 361 anterior to the top and posterior to the bottom. Scale bar is 20 µM.

362

363 Zebrafish larvae exhibit dyskinetic-like behaviour that is reduced by isradipine and 364 amantadine

365 A dyskinetic-like behaviour has been previously described in MPTP-lesioned adult 366 zebrafish as diminished voluntary movements (Babu et al., 2016). Here, we investigated 367 whether 6-OHDA-lesioned larvae present a similar phenotype, observed as immobile 368 events, and tested the effects of L-dopa, rasagiline and isradipine. In addition, we tested 369 amantadine, the only anti-dyskinetic agent clinically available for PD (Vijayakumar and 370 Jankovic, 2016). 6-OHDA-lesioned larvae were exposed to each compound and their 371 behaviour was analysed. This zebrafish model showed an increase of the duration of the 372 immobile events, that was rescued by isradipine, at 0.06 µM (Fig. 4C), and amantadine, 373 at 25 µM (Fig. 4D). L-dopa was tested at 125, 250 (Fig. 4A) and 1250 µM, and rasagiline 374 at 0.95 (Fig. 4B) and 25 µM, but both compounds failed to alter the parameter, at the 375 doses tested. This result reveals that 6-OHDA-lesioned zebrafish larvae exhibit a 376 behaviour that resembles dyskinesia, sensitive to isradipine and amantadine.

377

378

379 Figure 4 – Rescue of dyskinetic-like behaviour by isradipine and amantadine, but 380 not L-dopa and rasagiline in 6-OHDA-lesioned zebrafish larvae. Dyskinetic-like 381 behaviour depicted from immobile events in 6-OHDA-lesioned zebrafish larvae treated 382 with (A) 250 µM of L-dopa (n= 30 larvae), (B) 0.95 µM of rasagiline (n= 31 larvae), (C) 383 0.06 µM of isradipine (n= 26 larvae) and (D) 25 µM of amantadine (n= 23 larvae), as 384 compared to untreated larvae (n= 44-49 larvae). Percentage relative to the mean of vehicle 385 treated larvae (healthy control, n= 23-31 larvae). Mean ± s.e.m. of three independent 386 experiments is presented. Ns – not significant, **p<0.01 and ***p<0.001, one-way 387 ANOVA with Dunnett’s post-hoc test.

388

389 A phenotypic-based screen identifies compounds with antiparkinsonian potential

390 To select candidate compounds capable of rescuing motor impairments in zebrafish 391 larvae lesioned with 6-OHDA, we developed a phenotypic assay that enabled the 392 screening of a library of 1600 FDA approved drugs (Fig. 1). The assay was three days 393 long and encompassed dopaminergic lesion with 6-OHDA (0h-24h), incubation with the 394 screening compounds (24h-48h) and behavioural evaluation (48h-72h). The locomotor 395 behaviour was tested by automatic measurement of total distance moved and burst 396 swimming, as previously described. From the 1600 compounds screened (Fig. 5), 258 397 (16%) rescued motor impairments in 6-OHDA-lesioned zebrafish larvae during the first 398 round of experiments performed (Table 1). These compounds were then evaluated for 399 repositioning and 83 (32%) were excluded from further analysis, based on pre-determined 400 exclusion criteria. From these 83 compounds, 26 (31%) had prior art for PD, 25 (30%) 401 had an active patent for other indications or were prone to off-label use, and 7 (8%) raised 402 safety concerns (Table 2). Nevertheless, these compounds were useful for validation of 403 the screen. This was the case, for example, of caffeine and carbinoxamine maleate, which 404 were blindly selected during the screen for rescuing motor impairments in 6-OHDA- 405 lesioned zebrafish larvae. In contrast, bromocriptine mesylate and apomorphine 406 hydrochloride, two dopamine agonists used in the clinic, were also screened, but showed 407 no effect in the 6-OHDA-lesioned zebrafish larvae at the concentration tested. In the 408 second and third rounds of screenings, 78 (30%) and 23 (29%) compounds, respectively, 409 rescued motor impairments in 6-OHDA-lesioned zebrafish larvae (Table 1). After further 410 analysis, which included the evaluation of previous medical indications and targets and

411 BBB permeability, 3 drugs (13%), stavudine, tapentadol and nabumetone, showed to be 412 particularly promising for further experiments (Table 3). These three compounds had 413 minimally explored targets for the treatment of PD, high permeability to the BBB and no 414 further concerns from previous indications. The other 20 compounds had either no known 415 targets, low or non-described BBB permeability or concerns regarding safety, and were 416 discarded. Overall, the screening protocol developed was suitable for a quick selection of 417 neuroactive drugs with antiparkinsonian potential, but also presented limitations 418 concerning the activity of dopamine agonists. 419

Round I Round II Round III

420

-3 Intelectual property issues

-2 Toxic to larvae

-1 No rescue of motor performance

0 Rescue of burst swimming (BS)

1 Rescue of total distance moved (TDM)

2 Rescue of TDM + BS 421 Not Applicable

422 Figure 5 – Hit map representation of the screening of 1600 bioactive drugs. Each 423 square corresponds to a compound, identified as rescuing none of the parameters 424 measured (burst swimming, BS, and total distance moved, TDM), rescuing one of the two 425 parameters measured or rescuing both parameters measured in 6-OHDA-lesioned 426 zebrafish larvae. Only compounds that rescued both, BS and TDM, are represented in the 427 subsequent round of screenings. Compounds that presented toxic effects on zebrafish 428 larvae and intellectual property concerns, and therefore excluded from further screenings 429 are also represented.

430

431 432 Table 1 – Overview results of the screening of 1600 bioactive drugs. Number and 433 percentage of compounds with no effects, and with effects on one or both parameters 434 (burst swimming, BS, and total distance moved, TDM) measured during each round of 435 the screening assay, to depict motor performance in 6-OHDA-lesioned zebrafish larvae. 436 Number and percentage of compounds with toxic effects on zebrafish larvae and 437 intellectual property concerns also indicated. Percentage relative to the total of 438 compounds screened (1600) for round I and to the number of positive hits in the previous 439 round, for rounds II and III. 440

441 442 Table 2 – Intellectual property issues considered for exclusion of positive hits from 443 round I of phenotypic screenings. Number and percentage of compounds with prior art

444 for PD, active patent, prone to off-label use and safety concerns. Percentage relative to 445 the total of compounds selected for rescuing motor performance in 6-OHDA-lesioned 446 zebrafish larvae during round I of screenings. 447

Hits Targets BBB permeability 1 Human immunodeficiency virus type 1 protease inhibitor Low Nuclear receptor subfamily 1 group I member 2 activator

2 Tubulin beta-1 chain High Apoptosis regulator Bcl-2 Microtubule-associated protein 2 and 4 Microtubule-associated protein tau Nuclear receptor subfamily 1 group I member 2

3 Not found High

4 Sodium channel protein type 5 subunit alpha antagonist High

5 Not found Not found

6 cAMP and cGMP-specific 3',5'-cyclic phosphodiesterases inhibitor Low Adenosine deaminase inhibitor Calcipressin-1 Alpha-1-acid glycoprotein 1

7 Not found Not found

8 5-hydroxytryptamine receptors agonist High Alpha-2A adrenergic receptor agonist

9 Cross-linking/alkylation of DNA High Nuclear receptor subfamily 1 group I member 2

10 Arachidonate 5-lipoxygenase inhibitor High Prostaglandin G/H synthase 1 inhibitor

11 Prostaglandin G/H synthase 1 and 2 inhibitor High

12 Not found Not found

13 Reverse transcriptase/RNaseH inhibitor High

14 Mu-type opioid receptor agonist High Sodium-dependent noradrenaline transporter inhibitor Kappa-type opioid receptor Delta-type opioid receptor 5-hydroxytryptamine receptor 3A Sodium-dependent serotonin transporter inhibitor

15 Alpha-1A, 1D and 1B adrenergic receptor antagonist Low

16 3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 1 and 2 inhibitor Low Estrogen receptor alpha and beta allosteric modulator

17 Not found Not found

18 Not found Not found

19 Beta-1 and 2 adrenergic receptor antagonist Low Alpha-1A, 1B, 1D, 2A-2C adrenergic receptor antagonist NADH dehydrogenase [ubiquinone] 1 subunit C2 inhibitor Vascular endothelial growth factor A Natriuretic peptides B Gap junction alpha-1 protein Potassium voltage-gated channel subfamily H member 2 inhibitor Vascular cell adhesion protein 1 inhibitor E-selectin inhibitor Hypoxia-inducible factor 1-alpha modulator Inward rectifier potassium channel 4

20 Penicillin binding protein 2a, 1A and 1B inhibitor High Peptidoglycan synthase FtsI inhibitor D-alanyl-D-alanine carboxypeptidase DacA and DacC inhibitor

21 Muscarinic acetylcholine receptor M1 antagonist High

22 Not found Not found

23 Potassium voltage-gated channel subfamily H member 2 Not found 448 24

449 Table 3 – Overview of the 23 lead compounds of the screening assay. Targets and 450 BBB permeability of the 23 compounds selected for rescuing motor performance in 6- 451 OHDA-lesioned zebrafish larvae during the screening assay. Data obtained from an initial 452 search at https://www.drugbank.ca/. 453 454 455 Three new compounds display antiparkinsonian activity in 6-OHDA-lesioned 456 zebrafish larvae

457 Stavudine is a nucleoside reverse transcriptase inhibitor, that stimulates the production of 458 brain derived neurotrophic factor (BDNF) (Renn et al., 2011), tapentadol is a µ-opioid 459 receptor agonist and norepinephrine reuptake inhibitor (Tzschentke et al., 2007) and 460 nabumetone is a cyclooxygenase enzyme inhibitor (Davies, 1997). These three 461 compounds were selected from the screening assay, for their antiparkinsonian properties 462 in 6-OHDA-lesioned zebrafish larvae (Supplementary fig. S4). Next, we determined the 463 effective dose of each of the purified compounds and evaluated their effect on dyskinetic- 464 like behaviour. 6-OHDA-lesioned larvae were treated with different concentrations of 465 each compound and behavioural analysis was conducted. For both parameters, total 466 distance moved and burst swimming, we observed a significant recovery of larvae treated 467 with 50 µM of stavudine (Fig. 6A and B), 49 µM of tapentadol (Fig. 6D and E) and 0.9 468 µM of nabumetone (Fig. 6G and H). Stavudine and tapentadol also rescued dyskinetic- 469 like behaviour at these doses, as opposed to nabumetone, in 6-OHDA-lesioned zebrafish 470 larvae (Fig. 6C, F and I). This data confirms that stavudine, tapentadol and nabumetone 471 are potential antiparkinsonian agents and supports further studies in mammalian models 472 of PD. 473

474 475 Figure 6 – Rescue of motor impairments by stavudine, tapentadol and nabumetone 476 in 6-OHDA-lesioned zebrafish larvae. Motor performance and dyskinetic-like 477 behaviour depicted from (A, D and G) total distance moved, (B, E and H) burst swimming 478 and (C, F and I) immobile events in 6-OHDA-lesioned zebrafish larvae treated with 50 479 µM of stavudine (n= 38-40 larvae), 49 µM of tapentadol (n= 32-46 larvae) and 0.9 µM 480 of nabumetone (n= 38-49 larvae), as compared to untreated larvae (n= 30-66 larvae). 481 Percentage relative to the mean of vehicle treated larvae (healthy control, n= 23-31 482 larvae). Mean ± s.e.m. of three independent experiments is presented. Ns – not significant, 483 and ***p<0.001, one-way ANOVA with Dunnett’s post-hoc test. 484

485 Discussion

486 Here, we developed a phenotypic screening assay for the selection of new drugs with 487 antiparkinsonian potential, using 6-OHDA-lesioned zebrafish larvae. This model exhibits 488 death of dopaminergic neurons, accompanied by a decrease of dopamine levels and by 489 motor impairments (Anichtchik et al., 2004; Feng et al., 2014; Vijayanathan et al., 2017). 490 These phenotypes can be rescued by antiparkinsonian compounds (L-dopa and rasagiline) 491 (Cronin and Grealy, 2017; Feng et al., 2014). After deeper characterization of this model, 492 we used it for screening of 1600 FDA approved drugs and identified 23 drugs with 493 antiparkinsonian potential. We selected stavudine, tapentadol and nabumetone due to 494 their effects on relevant targets for the treatment of PD.

495 In agreement with previous studies (Anichtchik et al., 2004; Cronin and Grealy, 2017; 496 Feng et al., 2014), we observed that zebrafish larvae exposed to 6-OHDA display 497 bradykinetic-like behaviour and loss of dopaminergic neurons. We also introduce the 498 burst swimming as a suitable parameter to assess motor performance. This parameter 499 measures the number of times that larvae reach velocities correspondent to escaping 500 behaviour (>25 mm/s). Burst swimming has been catalogued as a swimming behaviour 501 of zebrafish larvae, which exhibit locomotion at slow speed, sometimes followed by a 502 faster and more vigorous swimming, that is typically above 20 mm/s (Budick and 503 O’Malley, 2000; Kalueff et al., 2013). Importantly, we showed that burst swimming can 504 be rescued by L-dopa and rasagiline, as previously demonstrated for total distance moved 505 (Cronin and Grealy, 2017; Feng et al., 2014).

506 Since isradipine has been described as a disease-modifying agent for PD (Chan et al., 507 2007; Ilijic et al., 2011), we also explored the antiparkinsonian potential of this drug. We 508 report, for the first time, that isradipine reduces the loss of dopaminergic neurons and 509 rescues bradykinetic-like behaviour in 6-OHDA-lesioned zebrafish larvae. It has been 510 suggested that isradipine (calcium channel blocker) prevents the death of dopaminergic 511 cells through the reduction of calcium influx in neurons and consequently the decrease of 512 mitochondrial activity (Chan et al., 2007). Therefore, isradipine could have induced 513 changes in the mitochondrial activity of zebrafish larvae which decreased the oxidative 514 stress and cytotoxicity induced by 6-OHDA over dopaminergic neurons. In contrast, we 515 did not detect disease-modifying effect of rasagiline. This finding differs from previous 516 reports in zebrafish and mice (Blandini et al., 2004; Cronin and Grealy, 2017), likely due

517 to differences in the treatment protocols adopted. The time between dopaminergic lesion 518 and the exposure to rasagiline is a key factor that may dramatically influence the outcome 519 in the zebrafish model, where the regeneration capacity is greater in comparison to 520 mammals (Zupanc, 2008). In fact, rasagiline failed to demonstrate disease-modifying 521 properties in humans and isradipine is still under clinical evaluation (Oertel and Schulz, 522 2016; Olanow et al., 2009).

523 Previously, dyskinetic-like behaviour in MPTP-lesioned adult zebrafish was reported 524 (Babu et al., 2016). Now, we observed that 6-OHDA-lesioned zebrafish larvae exhibit a 525 similar behaviour, as measured by the average duration of immobile events (velocity <2 526 mm/s). While the evaluation of an hyperkinetic disease through complete lack of 527 movement might appear counter-intuitive, there is already evidence supporting this 528 approach in a zebrafish model of dystonia (Friedrich et al., 2012). On the other hand, 529 dyskinesia only occurs in PD patients under L-dopa or dopamine-agonist treatment. 530 Therefore, although the parameter we measured cannot be directly related with L-dopa- 531 induced dyskinesia, it reports on non-induced dyskinesias, indicative of abnormal 532 plasticity of the motor circuitry. This could result from the dysregulation of different 533 neurotransmitters, sensitive to the impairment of the dopaminergic system, that are 534 essential for generating a normal swimming behaviour. Consistently, L-dopa and 535 rasagiline, two drugs that rely on the dopaminergic system, did not alter the average 536 duration of immobile events in 6-OHDA-lesioned zebrafish larvae. Accordingly, these 537 drugs do not have an anti-dyskinetic effect in humans (Pistacchi et al., 2014). In turn, one 538 would expect this parameter to worsen in larvae treated with L-dopa. However, this may 539 only be observed with chronic treatment, so it could not be assessed. Additionally, we 540 found that the drugs with non-dopaminergic targets, isradipine and amantadine, reduced 541 the average duration of immobile events. Amantadine is widely used in the clinic as an 542 anti-dyskinetic agent for PD (Vijayakumar and Jankovic, 2016). In the case of isradipine, 543 the pre-clinical evidence for anti-dyskinetic properties is limited (Rylander et al., 2009; 544 Schuster et al., 2009) and reports of this therapeutic indication in humans do not exist to 545 date. Whereas, amantadine is an NMDA antagonist and could have balanced the 546 dysregulation induced by the impaired dopaminergic system in zebrafish, isradipine does 547 not act in a specific neuronal circuitry. Therefore, the changes observed in the duration 548 of immobile events of zebrafish larvae treated with isradipine could have resulted from 549 the rescue of dopaminergic neurons and, consequently, from a lower dysregulation of this

550 and other neuronal systems. Additional studies will be necessary to further investigate the 551 predictive value of this parameter. As such, the phenotypic screening assay we developed 552 relied on the total distance moved and burst swimming parameters for the selection of 553 compounds with antiparkinsonian potential.

554 The phenotypic screening of 1600 FDA approved drugs, resulted in the identification of 555 26 drugs (31% of the drugs selected during round I) with prior art for PD, revealing the 556 sensitivity of the assay. In contrast, the screening set up failed to identify some dopamine 557 agonists used as monotherapy or in combination with L-dopa (Oertel and Schulz, 2016). 558 In zebrafish, eight proteins homologous to the human dopamine receptors have been 559 identified (Panula et al., 2010), but their affinity for dopamine agonists has not been 560 explored. Therefore, one possibility is that the dose tested during the screening was not 561 effective. Previously, it was reported that the non-selective dopamine agonist, 562 apomorphine, has a biphasic effect on the locomotor activity of zebrafish, that depends 563 on the dose applied (Irons et al., 2013). This dose-dependent effect has also been 564 evidenced in mice treated with dopamine receptor agonists (Lundblad et al., 2005). All 565 drugs were screened at a concentration of 25 µM, the highest concentration more 566 commonly used for drug screenings in zebrafish (Rennekamp and Peterson, 2015). 567 Importantly, only 16% of the screened drugs showed toxic effects in these conditions, 568 which did not compromise the screening.

569 Overall, the phenotypic screening identified 23 drugs (1.4% of the total drugs screened) 570 with antiparkinsonian potential in 6-OHDA-lesioned zebrafish larvae. Previous studies 571 allowed the analysis of targets, BBB permeability and clinical indications, and selection 572 of three drugs that were considered particularly interesting for further studies. Stavudine 573 is a reverse transcriptase inhibitor and previous reports have described the stimulation of 574 BDNF expression by this drug (Renn et al., 2011). Neurotrophic factors have proven their 575 efficacy in animal models of PD, but their inability to cross the BBB has limited their 576 application (Aron and Klein, 2011). In turn, tapentadol is a µ-opioid receptor agonist and 577 norepinephrine reuptake inhibitor (Tzschentke et al., 2007), two pathways long 578 implicated in PD (Espay et al., 2014; Samadi et al., 2006). Lastly, nabumetone is a 579 prostaglandin synthase inhibitor with anti-inflammatory properties (Davies, 1997). 580 Neuroinflammation is a key pathogenic mechanism of PD and anti-inflammatory agents 581 have also shown promising results in preclinical studies, although convincing clinical data 582 is still missing (Athauda and Foltynie, 2015). Interestingly, the three drugs rescued

583 bradykinetic-like behaviour, but nabumetone failed to alter the duration of immobile 584 events in 6-OHDA-lesioned zebrafish larvae. As discussed above, the target described for 585 nabumetone is not specific of a neuronal circuitry, while stavudine induces the expression 586 of BDNF and tapentadol acts on the opioid and noradrenergic systems. Further studies 587 need to be conducted to determine the exact mechanism of action behind the therapeutic 588 potential of these three compounds and to validate their antiparkinsonian efficacy in other 589 vertebrate models. Since the effects of stavudine, tapentadol and nabumetone in the motor 590 performance of healthy zebrafish larvae were not assessed, further studies should 591 determine the specificity of the therapeutic effects to dopaminergic disfunction. In 592 contrast to other screenings previously reported (Buckley et al., 2010; Parng et al., 2006; 593 Robertson et al., 2016; Sun et al., 2012), our selection of drugs was completely agnostic 594 to target or mechanism of action, which is a key distinction of the phenotypic screening, 595 as it can result in the identification of drugs with new mechanisms of action.

596

597 Conclusions

598 In general, our study provides further demonstration that the 6-OHDA-lesioned zebrafish 599 larvae exhibit bradykinetic-like behaviours that are sensitive to the motor improvement 600 of antiparkinsonian compounds. A dyskinetic-like behaviour was also observed, but 601 further investigations will be necessary in order to fully demonstrate the predictive value 602 of this parameter in zebrafish. Although we did not detect disease-modifying properties 603 for rasagiline or anti-bradykinetic properties for dopamine agonists, this might be due to 604 the screening protocol, and will require further investigation. Nevertheless, the 605 phenotypic screening we describe is a valid strategy for rapid selection of potential 606 antiparkinsonian agents. Importantly, it is simple, is based in objective and automated 607 parameters, and does not require invasive or stressful manipulation of the animals. The 608 screening led to the identification of three neuroactive drugs, stavudine, tapentadol and 609 nabumetone, and further studies regarding their mechanism of action could lead to the 610 discovery of targets or pathways relevant for PD mechanisms.

611 Acknowledgements

612 Funding: This study was sponsored by TechnoPhage S.A. and Eurostars program 613 (ES#5553) from EUREKA (a program run by the European Commission). Rita L. Vaz 614 was supported by a grant (SFRH/BD/78077/2011) from Fundação para a Ciência e 615 Tecnologia. Tiago F. Outeiro was supported by the DFG Center for Nanoscale 616 Microscopy and Molecular Physiology of the Brain (CNMPB).

617

618 Conflict of interest: S.S., D.C. and N.D.A. were employees of Technophage SA, at the 619 time of the study. The other authors declare no conflicts of interest.

620

621 Author contributions: S.S., R.W., T.F.O. and N.D.A. did study conception and design. 622 R.L.V., D.C. and S.S. performed experiments, did data acquisition and analysed the data. 623 All authors interpreted the data. R.L.V. drafted the paper. S.S. and N.D.A. did critical 624 revision.

625

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824 Supplementary material

825

826 Supplementary figure S1 – Sensitivity of zebrafish larvae to different concentrations 827 of 6-OHDA. Motor performance depicted from (A) total distance moved and (B) burst 828 swimming in zebrafish larvae lesioned with different concentrations of 6-OHDA (n= 8 829 larvae per concentration) as compared to vehicle treated larvae (n= 8 larvae). Percentage 830 relative to the mean of vehicle treated larvae. Mean ± s.e.m. of one experiment is 831 presented. *p<0.05, **p<0.01, ***p<0.001, δp<0.05 500 µM vs 750 µM, Kruskal-Wallis 832 ANOVA with Dunn’s post-hoc test.

833

834

835 Supplementary figure S2 – Dose-response curves of motor performance of 6-OHDA- 836 lesioned zebrafish larvae treated with L-dopa, rasagiline and isradipine. Motor 837 performance depicted from (A, C and E) total distance moved and (B, D and F) burst 838 swimming in 6-OHDA-lesioned zebrafish larvae treated with different doses of L-dopa 839 (n= 7-16 larvae), rasagiline (n= 7-8 larvae) and isradipine (n= 6-8 larvae) as compared to 840 untreated larvae (n= 13-14 larvae). Percentage relative to the mean of vehicle treated

841 larvae (healthy control, n= 8 larvae). Dashed lines signalize healthy and disease state, on 842 the top and at the bottom, respectively. Peak effective doses are highlighted by arrows.

843

844

845 Supplementary figure S3 – Motor performance of healthy zebrafish larvae treated 846 with L-dopa, rasagiline and isradipine. Motor performance depicted from (A) total 847 distance moved and (B) burst swimming in healthy zebrafish larvae treated with 1250 848 µM of L-dopa (n= 16 larvae), 0.8 µM of rasagiline (n= 16 larvae) and 0.5 µM of isradipine 849 (n= 15 larvae), as compared to untreated healthy zebrafish larvae (n= 16 larvae). 850 Percentage relative to the mean of healthy larvae. Mean ± s.e.m. of one experiment is 851 presented. Ns – not significant, Kruskal-Wallis ANOVA with Dunn’s post-hoc test.

852

853

854 Supplementary figure S4 - Rescue of motor impairments by stavudine, tapentadol 855 and nabumetone in 6-OHDA-lesioned zebrafish larvae, during the phenotypic 856 screening. Motor performance depicted from (A, C and E) total distance moved and (B, 857 D and F) burst swimming in 6-OHDA-lesioned zebrafish larvae treated with 25 µM of

858 stavudine (n= 19 larvae), tapentadol (n= 24 larvae) and nabumetone (n= 20 larvae), 859 picked from a library of FDA approved drugs, as compared to untreated larvae (n= 38-45 860 larvae). Percentage relative to the mean of vehicle treated larvae (healthy control, n= 23- 861 24 larvae). Mean ± s.e.m. of three independent experiments is presented. ***p<0.001, 862 one-way ANOVA with Dunnett’s post-hoc test.

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