{"paper_id":"068dfacb-b4f6-4600-83a8-dea0752eaade","body_text":"Early motor deficits, sleep dysfunction and reduction in dopaminergic neurons in a park7 -/- zebrafish larval model of Parkinson’s disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Early motor deficits, sleep dysfunction and reduction in dopaminergic neurons in a park7 -/- zebrafish larval model of Parkinson’s disease Nora Solheim, Brígida R. Pinho, Nuno A. S. Oliveira, Leonor P. Lima, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7848595/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Parkinson's Disease (PD) is the fastest-growing neurological disorder and only symptomatic treatment is available. Zebrafish are ideally suited for high-throughput screening of disease modifying drugs and mechanistic studies. Mutations in park7 are associated with early-onset familial PD. Additionally, altered levels and subcellular location of the park7 protein product (DJ-1) have been found in PD patients without known park7 mutations. Here, we show that larval park7 −/− zebrafish show reduced number of dopaminergic neurons and motor dysfunction, similarly to the 1-methyl-4-phenylpyridinium (MPP + )-induced PD model. Additionally, PD-associated prodromal symptoms, such as reduced sensory function, increased sleep latency and daytime sleepiness, were also observed in the park7 −/− , but not in the MPP + -induced PD model. The park7 −/− is the first stable genetic zebrafish model of Parkinson’s disease which shows both motor and non-motor symptoms, together with a reduction in dopaminergic neurons, at a larval stage. The model should therefore be highly valuable as a tool for PD related drug screening and mechanistic studies. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Neurology Biological sciences/Neuroscience Parkinson’s disease zebrafish model sleep motor non-motor prodromal dopaminergic neurons larva Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Parkinson’s Disease (PD) is the fastest growing neurological disorder ( 1 ) and only symptomatic therapy is available. Although about 10% of PD cases are caused by an inherited genetic variant, the majority of PD cases are sporadic, age-dependent and most possibly the result of a complex combination of/interplay between environmental exposure and genetic risk factors ( 2 ). PD diagnosis is set when motor disabilities occur, although there are prodromal symptoms such as disturbances in REM sleep, changes in smell and vision, reduced sense of touch, mood disorders and others ( 3 , 4 ). PD is characterized by a selective loss of dopaminergic cells in the substantia nigra pars compacta . Cellular changes common to both familiar and spontaneous PD include mitochondrial dysfunction, increased oxidative stress and altered immune-related response (reviewed in ( 5 , 6 )). However, the mechanisms resulting in the selective loss of dopaminergic neurons in PD are still not well understood. Considering the limited success of existing treatments for PD and the increasing prevalence of the disease, there is an evident need for novel disease-modifying treatments and methods to halt disease progression. In this context, in vivo models enabling the understanding of early cellular changes leading to disease and progression are indispensable. Zebrafish serve as an excellent in vivo platform for pharmacological screening as drugs can be added directly to the water. Additionally, zebrafish can be easily genetically modified and in combination with their small and optically transparent larvae they are a unique gateway for elucidating pathological mechanisms. Dopaminergic neurons are detected already at 1 day post fertilization (dpf) in the zebrafish embryo, and at 3 dpf the organization of the CNS is already complete ( 7 , 8 ). Dopaminergic neurons in the zebrafish are also sensitive to oxidative stress induced by mitochondria-directed neurotoxicants ( 9 ). Thus, zebrafish are excellent models for studying pathological mechanisms in PD and directed therapies. Loss of function mutations in park7 are associated with autosomal recessive forms of early-onset PD ( 10 ). Importantly, the park7 protein product, DJ-1 is also linked to spontaneous form of PD, in which increased expression of irreversibly oxidative modified DJ-1 and reduced association to mitochondrial ATP synthase are observed ( 11 , 12 ). DJ-1 is a multifunctional redox sensitive and neuroprotective protein. It has been shown to modulate oxidative stress, inflammatory response, mitochondrial function, synaptic recycling, autophagy, and proteostasis ( 13 ), ( 14 , 15 ), ( 16 ), ( 17 ), ( 18 ) ( 19 ). We have previously established a CRISPR/cas9-based park7 knockout zebrafish line ( 20 ) and demonstrated a progressive increase in motor and non-motor symptoms at the adult stage ( 21 , 22 ). Here, we show that this DJ-1 knockout model, in contrast to other genetic PD-related zebrafish models ( 23 – 25 ), starts to both develop motor deficits and have a reduced number of dopaminergic neurons already at their larval stage. Results Loss of DJ-1 evoked no noticeable effects on general development and early motor activity of zebrafish embryos In wild-type zebrafish DJ-1 is expressed throughout development (Fig. 1 A). To assess whether a loss of DJ-1 affects embryonic development, we measured morphometric parameters (Fig. 1 C-E). As reflected in gross morphology, the loss of DJ-1 in park7 −/− did not hamper general development – at 5 dpf, wild-type and park7 knockout larvae did not differ in body length or eye aspect ratios (Fig. 1 D-E). The earliest motor activity in zebrafish can be observed at 1 dpf as spontaneous tail coiling ( 26 ). No significant difference was observed in tail coiling comparing wild-type and park7 −/− larvae (Fig. 1 B). park7 −/− larvae have a reduced touch-evoked escape response Reduced sense of touch is a common and often prodromal symptom in many PD patients ( 3 ). Mitochondria-directed toxicants affecting dopaminergic neurons have also been shown to impair tactile sensory processing in a mouse model ( 27 ). In view of this, we examined the touch-evoked response in larvae by applying tactile stimulation to the head and tail (Fig. 2 ). Larvae were exposed from 3–5 dpf to 1-methyl-4-phenylpyridinium (MPP + ) which selectively targets dopaminergic neurons. Indeed, at 5 dpf MPP + exposure reduced both head and tail response in wild-type larvae. More importantly, a significant impairment in touch-evoked responses was also observed in park7 −/− compared to wild types, even in the absence of MPP + . Additionally, loss of DJ-1 resulted in an increased sensitivity towards MPP + . park7 −/− larvae have disturbed activity and sleep patterns, but no impairment in motor endurance Larvae behaviour was analysed from 5 to 8 dpf using an infrared-based Locomotor Activity Monitor (LAM) ( 28 ). LAM tracking allows the monitoring of motor function, circadian function and sleep disturbances, the latter being one of the most common prodromal symptoms in PD ( 29 ). All the LAM-monitored zebrafish larvae, independent of genotype or treatment, showed a typical circadian pattern with higher activity in the light phases vs. dark phases (Fig. 3 A). park7 knockout and treatment with MPP + decreased light-phase activity, compared to control larvae (wild type, solvent). There was no further activity reduction (no additive effect) when combining MPP + with park7 knockout (Fig. 3 B-C). Sleep bout durations and sleep latency were analysed in the dark phases. park7 knockout or MPP + treatment had no statistically significant effects on sleep bout duration (Fig. 3 D). Sleep latency, on the other hand, was increased in park7 −/− larvae throughout the experimental period (Fig. 3 E). At 5 dpf, larvae pretreated with MPP + also showed increased sleep latency, but this effect of MPP + fades as the experiment progresses, likely due to larvae recovering after MPP + washout (LAM medium does not contain MPP + ). Interestingly, the MPP + -exposed park7 −/− larvae do not initially show increased sleep latency but approximate the unexposed park7 −/− as the experiment progresses and larvae recover from the MPP + pre-exposure. Moreover, no differences were observed in the sleep ratio (proportion of time spent sleeping) in dark phases, but in light phases, the sleep ratio was increased in park7 −/− zebrafish (Fig. 3 F-G). A similar increase was not observed in MPP + -treated wild types, suggesting a specific effect of park7 on sleep regulation. Motor endurance has been shown to be decreased in mice exposed to the MPP + precursor MPTP ( 30 ). We therefore assessed motor endurance in PD-model zebrafish. While MPP + exposure reduced motor endurance in zebrafish larvae, park7 −/− larvae showed motor endurance similar to wild types (Supplementary Fig. 1A-B). We also did not observe any degradation in neuromuscular junctions in park7 −/− larvae (Supplementary Fig. 1C), a feature that accompanies reduction in motor endurance in zebrafish models of motor neuron disease ( 31 , 32 ). These findings support the hypothesis that park7 −/− induces more selective PD phenotypes than MPP + , regarding motor parameters. park7 −/− larvae exhibit a lower number of DC1 diencephalic dopaminergic neurons compared to wild types Dopaminergic (DA) neurons can be detected already at 18 hours post-fertilization (hpf) and at 3 dpf the distribution of DA neuronal populations is complete ( 7 , 8 ). We studied the number and distribution of DA neurons using an antibody detecting tyrosine hydroxylase (TH), a marker for DA neurons (Fig. 4 ). We measured the number of the diencephalic DA neurons of the posterior tuberculum referred to as DC1 ( 33 ) or 5, 6, 11 population ( 9 )(Fig. 4 A). This is the DA neuronal population comparable to the mammalian substantia nigra, and which is selectively targeted by MPP + ( 9 ). The DC1 population can easily be distinguished from another nearby DA population (DC2) based on morphology and the coronal level (Fig. 4 B-C). Our results showed a significant reduction in the number of DA neurons in the DC1 population in the park7 −/− at 5 dpf, compared to wild type (Fig. 4 D-E). This difference seemed to progress by embryonal/larval aging since at 3 and 4 dpf the difference was not significant (Fig. 4 E). As expected, exposure to MPP + from 3–5 dpf decreased the number of DC1 dopaminergic neurons in the wild type larvae. There was no further reduction in the number of DC1 neurons (no additive effect) when combining MPP + with park7 knockout. In another zebrafish model of PD, the PTEN-induced kinase 1 ( pink1 ) −/− , the larger DC2 dopaminergic neurons show reduced neurogenesis ( 34 ). We determined the number of neurons in the smaller DC2 population but found no differences between genotypes or the effect of MPP + (Supplementary Fig. 2). Thus, these results indicate that loss of DJ-1 selectively targets the DA neuronal population comparable to the mammalian substantia nigra. Discussion No disease-modifying therapeutics exist for Parkinson’s disease, only symptom-relieving drugs. Larval zebrafish offer unique possibilities for high-throughput phenotypic analysis and imaging analysis of a well characterized nervous system. Here we describe the behaviour and dopaminergic cell population at the larval stage of a CRISPR/cas9-based park7 −/− zebrafish. Our results show that park7 −/− larvae not only have sensory and motor deficits but also sleep disturbances and a lower number of the dopaminergic neurons comparable to the mammalian substantia nigra. To our knowledge this combination of PD-relevant behaviour and cell-specific targeting have not previously been shown in other genetic or toxicant based zebrafish models. Mutations in park7 leading to a dysfunction in its protein product DJ-1 are linked to a rare, familial type of early-onset PD ( 10 ). However, even in idiopathic cases of PD, with no known mutations in park7 , post-mortem analyses of brain samples revealed an increase in oxidative cysteine-modified DJ-1 ( 11 , 35 ). Recently, a reduced association of DJ-1 to mitochondrial F1Fo-ATP synthase was shown in sub-cellular compartments (predominantly distal neurites) of dopaminergic neurons in substantia nigra of post-mortem brains from sporadic PD patients ( 12 ). In relation to gross morphology and basic body plan, park7 −/− larvae were indistinguishable from wild types (Fig. 1 ). Neither was there any difference in spontaneous motor activity at the embryonal stage (28 hpf). However, at the 5 dpf stage, spontaneous locomotor activity was significantly reduced in park7 −/− larvae compared to wild types (Fig. 3 ). Impaired motor function has also previously been observed in the morpholino-based LRRK2 ( 36 ) and SNCA ( 37 ) knockdown PD models, although LRRK2 knockdown induces developmental defects and conflicting evidence exists for its effect on motor activity ( 38 , 39 ). Mutations in park7 have been associated with signs of amyotrophic lateral sclerosis (ALS) pathology in two independent studies ( 31 , 32 ). However, the park7 −/− larvae showed no reduction in motor endurance as would be expected for ALS-associated phenotypes (Suppl. Figure 1) ( 40 ). Tactile perception is impaired in dopamine-depleted mice ( 27 ). This dopamine-dependent response seems to be a conserved feature as tyrosine hydroxylase-depleted C. elegans had a dysregulation in tactile plasticity which could be rescued by dopamine ( 41 ). In agreement with these reports, we found that park7 −/− had a significantly lower response to both head and tail touch compared to wild type (Fig. 2 ). Thus, reduced touch response in park7 −/− might be coupled to a dysfunctional dopaminergic response and be relevant to PD pathology, which often includes a reduced sense of touch ( 3 ). It is well established that mitochondrial dysfunction is strongly implicated in both familial and spontaneous PD. The mitochondrial toxicant MPTP and its oxidized product MPP + , which target a specific population of dopaminergic neurons, are therefore commonly used to replicate PD pathology in animal models. In this study we used MPP + to compare exposed wild type with unexposed park7 −/− and to evaluate whether loss of DJ-1 renders larvae more sensitive to MPP + . Loss of DJ-1 combined with MPP + exposure induced a greater reduction in the touch-evoked responses than each condition alone (Fig. 2 – 4 ). Sleep disorders are common and evident at the prodromal stage in PD ( 42 ). Rapid eye movement sleep behaviour disorder, excessive daytime sleepiness and increased sleep latency are among these sleep disturbances ( 29 , 43 ). Using the LAM, we were able to monitor both total day-/night-time sleep and sleep latency at the start of the darkness phase. Interestingly, both sleep latency and sleep ratio during daytime were increased in park7 −/− , whilst sleep ratio during the night phase remained unaltered (Fig. 3 E-G). These sleep disturbances persisted throughout the experimental period (5–8 dpf). MPP + -treated wild types did not show a similar pattern of sleep disturbances. Previous reports on exposure of rodents to MPTP have not been able to show a correlation between sleep disruptions and loss of dopaminergic neurons, suggesting that the commonly used MPTP model for PD does not fulfil a comprehensive role as a PD model, specifically in relation to non-motor symptoms ( 44 ). The pronounced PD-related sleep disorder symptoms observed in park7 −/− larvae therefore increases its value as a PD model. The dopaminergic cell population in the DC1 of zebrafish larvae is comparable to the mammalian substantia nigra ( 8 ). In zebrafish the neurotoxicants MPTP and MPP + specifically target these DC1 dopaminergic cells, sparing other dopaminergic cell populations ( 9 ). Focusing on the tyrosine hydroxylase-positive cells in the DC1 region we saw that park7 −/− zebrafish at 5 dpf, even in the absence of MPP + , had less dopaminergic neurons in the DC1 population compared to wild-type zebrafish (Fig. 4 ). Comparing the number of dopaminergic DC1 neurons between park7 −/− and wild type at the 3 and 4 dpf stage did not show a significant difference, thus the reduction in dopaminergic DC1 in park7 −/− seemed to be a progressive effect. In parallel with the behaviour analysis, the loss of DJ-1 did not appear to make park7 −/− more sensitive towards MPP + than wild type. Even though MPP + treatment of park7 −/− further decreased the number of dopaminergic DC1, the number of cells was not significantly different from MPP + -treated wild types. In another stable genetic zebrafish model of PD, the pten-induced putative kinase 1 −/− ( pink1 −/− ), a reduced number of dopaminergic neurons is also evident already at the larval stage ( 24 ). Loss of PINK1 impairs neurogenesis in zebrafish, but also in human PINK1-deficient organoids ( 34 ). As shown in Fig. 4 , both wild type and park7 −/− larvae show a progressive increase in the number of dopaminergic DC1 cells from 3–5 dpf, but this increase is depressed in the park7 −/− . Thus, loss of DJ-1 in the park7 −/− most likely inhibits neurogenesis, as in the pink1 −/− zebrafish. Like DJ-1, PINK1 is also associated with early-onset PD and it has been shown that DJ-1 and PINK1 in complex have a role in oxidative stress protection and promoting unfolded protein degradation ( 45 ). To summarize, loss of DJ-1 in the park7 −/− zebrafish results in reduced touch-evoked response, sleep disturbances and locomotor dysfunction together with a reduced number of dopaminergic neurons. This is the first stable genetic model for Parkinson´s disease presenting motor symptoms, sleep disturbances and reduction of dopaminergic neurons already at the larval stage ( 46 ). This makes it an attractive model for pharmacological screening and mechanistic studies. Materials and methods Zebrafish husbandry Adult zebrafish were maintained at 26–28 °C with a 14/10 light cycle and were fed twice daily. Embryos and larvae were maintained at 28 °C and raised in E3 media (5 mM NaCl, 0.17 mM KCl, and 0.33 mM MgSO4). Ethics declaration and approval for animal experiments Animals used in the experiments were housed either at the Zebrafish Facility located in the Department of Biological Sciences at the University of Bergen, Norway or in the Vivarium of Aquatic Organisms at the University of Porto. The facilities are run according to the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and following the regulation by the European Directive 2010/63/EU. Establishment of the park7 −/− line was approved by the Norwegian National Animal Research Authority at Mattilsynet (FOTS ID8039 and ID14039) (20). Experiments were performed according to the 3Rs principle by reducing the number of animals used and using refined techniques that reduce animal suffering, and are reported according to ARRIVE guidelines. All experimental protocols were approved by National Animal Research Authority at Mattilsynet, Norway and local licensing committee at University of Bergen. All methods were carried out in accordance with relevant guidelines and regulations. Western blot analysis Embryos and larvae were sampled at 24 hpf, 3 dpf and 5 dpf. Deyolking was performed with 24 hpf and 3 dpf embryos/larva. Lysates were prepared by suspending in 150µl homogenization buffer (10 mM K 2 HPO 4 , 10m MKH 2 PO 4 , 1 mM EDTA, 0.6% CHAPS, 0.2 mM Na 3 VO 4 , 50 mM NaF, and protease inhibitor cocktail (Roche Diagnostics GmbH:11836153001)) and sonication (4×5 s) followed by incubation on ice for 20 min. Samples were pelleted at 15,000×g for 15 min at 4 °C and 25µg protein from the supernatant was separated by SDS-PAGE and transferred to PVDF membranes using 14 V overnight at 4 °C. Membranes were blocked in 1% BSA and incubated with anti-DJ-1 (1:3000, Novus Biologicals NB300-270, 1 hr) followed by secondary antibody. Ponceau-S was used as a loading control. Tail Coiling Embryos at 28 1 hpf were transferred to a 96 well-plate containing E3 (6 embryos/well). After a 5 min acclimation period, the embryos were recorded for 3-4 minutes using a Dino-Eye eyepiece camera (AM-423U). The videos were cut to exactly 3 minutes and the number of tail-coilings (random movement) were counted manually. Morphometric analysis of larvae Larvae were fixed o.n. in 4% PFA/PBS and washed in PBS before being mounted in agar molds. Images were taken with a Nikon SMZ800N and morphometric analysis was performed using ImageJ. 1-methyl-4-phenylpyridinium (MPP + ) treatment At 3 dpf, hatched larvae that did not display any abnormalities were randomly selected and placed in 12-well plate (10-15 larvae/1mL/well) and exposed to 500µM MPP + (D048, Merck KGaA, Darmstadt, Germany). The MPP + solution and vehicle were renewed at 4 dpf by replacing half volume with fresh solutions. At 5 dpf, the larvae were transferred to E3 in 6-well plates (1 larva/well) for sensorimotor responses or the LAM system (1 larva/tube) for continuous activity monitoring, without further exposure to MPP + . Sensorimotor responses In 6-well plate (one larva/well) each larva was gently touched with a micropipette tip, alternating between the head and tail. Immediate swimming was registered as a positive response, whereas no movement was registered as a negative response, yielding a binary result (47). Each larva was touched ten times at both head and tail, with a 30-second break in between. A minimum of n = 12 larvae, randomly selected from several breedings, were used for each condition. Locomotor Activity Monitor (LAM) Larvae were individually placed in E3 media-filled LAM tubes (3.65 mL) with a 100 ml air bubble inside. They were evenly dispersed and placed in the LAM for continuous activity monitoring until 8 dpf, while maintaining 28 1°C and a 14h/10h light-dark cycle, as previously described (28). The starting point of the light phase was set to 09.00AM. The LAM data were analysed with the Rtivity software (28). Sleep threshold was set to 10 minutes of inactivity and bout activity threshold to 2 minutes. Larval activity was measured as the number of infrared beam crossings per 30 seconds. Total activity for each day (counts) was analysed. Furthermore, activity (counts), sleep ratio (proportion of time spent sleeping), sleep latency (the mean time between lights turned off and start of the first sleep bout, in minutes) and sleep bout duration (the mean time spent being continuous asleep without interruptions, in minutes) were analysed for each day separated by light/dark phases. Sleep latency and sleep bout duration only contained data from the dark phases. Motor endurance assessment of zebrafish larvae For motor endurance assessment, we used a custom gravity-fed counter-current swimming system, capable of inducing the counter-current swimming reflex in zebrafish larvae without immediately overpowering them (manuscript in preparation). 7 dpf zebrafish larvae were moved to a 90 mm dish with chlorine-free water, 30 minutes prior to the experiment, for acclimation. 5 to 10 larvae were then moved into a 5 mL serological pipette, used as the swimming tunnel, and left to acclimate for 5 minutes. During this period, the larvae were guided to the middle of the swimming tunnel. Afterwards, water flow was initiated. Initial flow speed was set at 3 cm/s, gradually increasing to 5 cm/s over the course of 10 minutes. Endurance times were recorded as the length of time that each fish was able to stay in the swimming tunnel, before being pushed out by the water flow. Whole-mount labelling and image analysis Larvae were fixed in methanol free 4% PFA/PBS 4°C o/n with gentle agitation. Thereafter the larvae were washed in 6x15 min in PBS with 0.05% Triton X (PBS-TX) and stored at 4 ºC until further use. Permeabilization was performed by incubation with acetone at -20 ºC o.n. Thereafter larvae were washed in PBS-TX followed by 2 times wash in PBS. Bleaching was done in 3% H 2 O 2 in 0.89% KOH under bright light (approx. 8 min for 5 dpf larvae). Larvae were then washed 3x5 min in PBS-TX and 2x15 min PBS-TX. Clearing was performed according to Pende et al (48). Larvae were then transferred to blocking solution (5% normal goat serum, 1% BSA and 0.1% Triton X) for 3 hrs at RT and incubated for 3 days with either anti-tyrosine hydroxylase (TH) (1:250, Merck Millipore MAB318) or anti-SV2 (1:1000, DSHB SV2-C) in antibody dilution buffer (5% goat serum, 1% BSA and 0.1% Triton-X in PBS) at 4 ºC with gentle agitation.. It should be noted that available antibodies detecting zebrafish TH only detect TH1 and not TH2, in which TH1 is the isoform predominantly expressed in the brain (49). Samples were then washed 6x15 min in PBS-Tx and incubated for 2 days at 4 ºC with appropriate secondary antibody together with a 1:6000 dilution of DAPI in PBS-Tx. Larvae were then washed 3x15 min in PBS-Tx followed by 3x15 in PBS. Larvae were then transferred 50% refractive index solution (50% sucrose, 12% Antipyrin, 8% nicotinamide and 10% trietanolamine)(50) for at least 2 hrs at RT followed by at least 30 min in 100% refractive index solution. Larvae were then mounted in 2% low melt agarose on glass slides and imaged on Olympus FLUOVIEW FV3000 confocal laser scanning microscope. Confocal images were processed in Image J, using the plugin CLAHE to enhance local contrast(51). The number of DA neurons were calculated by counting tyrosine hydroxylase positive cells in the diencephalic posterior tuberculum DC1 area by moving through the confocal stack from the dorsal side until reaching the easily recognizable larger DA neurons of the DC2 (33). A maximum projection of the described area was generated and the DC1 neurons positioned anterior to the DC2 neurons was counted. The DC1 population of DA neurons includes posterior tuberculi populations 5, 6, and 11 as shown in Sallinen et al (9) known to be specifically targeted by MPP + . Statistical analysis Statistical analysis was done with R, in RStudio. The mean±standard error (SEM) was calculated for each genotype with respective treatment for n larvae from 3-5 different breedings. For each dataset, model choice was based on data distribution and scale. T-test or ANOVA were used for normally distributed data. For non-normally distributed data, generalized linear models (GLM) were created. Analysis was done between genotypes, MPP + exposure, and their interaction. Sensorimotor response was measured as the positive response in percentage from n=10 touches, so the data was modelled with a generalized linear model using a quasibinomial distribution with logit link to account for overdispersion, followed by Tukey-adjusted pairwise comparisons. Total activity, activity separated by light phases, sleep latency and sleep bout duration in the locomotor activity monitor were analysed using a generalised linear model with a negative binomial distribution as the data showed overdispersion (variance > mean). Post hoc comparisons of estimated marginal means were Tukey-adjusted. Sleep ratio separated by light phase were analysed using beta regression, followed by post hoc Tukey-adjusted comparisons on the fitted model. Lastly, differences in the number of cells positively stained for tyrosine hydroxylase were assessed with Type II ANOVA, with post hoc Tukey-adjusted comparisons. Statistical difference was defined as P-value < 0.05. Declarations Conflict of Interest None declared. Funding COST Action ImmuParkNet CA21117 (N.S.) and Advokat Rolf Sandberg Reberg og Ellen Marie Rebergs legat (K.E.F.). Fundação para a Ciência e a Tecnologia (UIDB/04378/2020; UIDP/04378/2020; LA/P/0140/2020) (J.M.A.O). Author Contribution Experimental investigation, N.S., A.B, L.P.L, B.R.P, N.A.S.O; Formal Analysis, N.S., A.B., J.M.A.O, K.E.F.; Original Manuscript Writing, N.S., A.B., N.A.S.O, K.E.F..; All authors have read and agreed to the published version of the manuscript. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Dorsey ER, Sherer T, Okun MS, Bloem BR. The Emerging Evidence of the Parkinson Pandemic. J Parkinsons Dis. 2018;8(s1):S3-S8. Ascherio A, Schwarzschild MA. The epidemiology of Parkinson's disease: risk factors and prevention. Lancet Neurol. 2016;15(12):1257-72. Nolano M, Provitera V, Estraneo A, Selim MM, Caporaso G, Stancanelli A, et al. Sensory deficit in Parkinson's disease: evidence of a cutaneous denervation. Brain. 2008;131(Pt 7):1903-11. Chaudhuri KR, Healy DG, Schapira AH, National Institute for Clinical E. Non-motor symptoms of Parkinson's disease: diagnosis and management. Lancet Neurol. 2006;5(3):235-45. Roodveldt C, Bernardino L, Oztop-Cakmak O, Dragic M, Fladmark KE, Ertan S, et al. The immune system in Parkinson's disease: what we know so far. Brain. 2024;147(10):3306-24. Ye H, Robak LA, Yu M, Cykowski M, Shulman JM. Genetics and Pathogenesis of Parkinson's Syndrome. Annu Rev Pathol. 2023;18:95-121. Holzschuh J, Ryu S, Aberger F, Driever W. Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech Dev. 2001;101(1-2):237-43. Rink E, Wullimann MF. The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res. 2001;889(1-2):316-30. Sallinen V, Torkko V, Sundvik M, Reenila I, Khrustalyov D, Kaslin J, et al. MPTP and MPP+ target specific aminergic cell populations in larval zebrafish. J Neurochem. 2009;108(3):719-31. Bonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. 2003;299(5604):256-9. Choi J, Sullards MC, Olzmann JA, Rees HD, Weintraub ST, Bostwick DE, et al. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J Biol Chem. 2006;281(16):10816-24. Abulimiti A, Bae H, Ali A, Balakrishnan S, Tsujishita M, Gveric D, et al. Reduced DJ-1-F1Fo ATP synthase association correlates with midbrain dopaminergic neuron vulnerability in idiopathic Parkinson's disease. Sci Adv. 2025;11(23):eads3051. Clements CM, McNally RS, Conti BJ, Mak TW, Ting JP. DJ-1, a cancer- and Parkinson's disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci U S A. 2006;103(41):15091-6. Lind-Holm Mogensen F, Sousa C, Ameli C, Badanjak K, Pereira SL, Muller A, et al. PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation. J Neuroinflammation. 2024;21(1):174. Froyset AK, Edson AJ, Gharbi N, Khan EA, Dondorp D, Bai Q, et al. Astroglial DJ-1 over-expression up-regulates proteins involved in redox regulation and is neuroprotective in vivo. Redox Biol. 2018;16:237-47. Franco-Iborra S, Vila M, Perier C. Mitochondrial Quality Control in Neurodegenerative Diseases: Focus on Parkinson's Disease and Huntington's Disease. Front Neurosci. 2018;12:342. Kyung JW, Kim JM, Lee W, Ha TY, Cha SH, Chung KH, et al. DJ-1 deficiency impairs synaptic vesicle endocytosis and reavailability at nerve terminals. Proc Natl Acad Sci U S A. 2018;115(7):1629-34. Imberechts D, Kinnart I, Wauters F, Terbeek J, Manders L, Wierda K, et al. DJ-1 is an essential downstream mediator in PINK1/parkin-dependent mitophagy. Brain. 2022;145(12):4368-84. Moscovitz O, Ben-Nissan G, Fainer I, Pollack D, Mizrachi L, Sharon M. The Parkinson's-associated protein DJ-1 regulates the 20S proteasome. Nat Commun. 2015;6:6609. Edson AJ, Hushagen HA, Froyset AK, Elda I, Khan EA, Di Stefano A, et al. Dysregulation in the Brain Protein Profile of Zebrafish Lacking the Parkinson's Disease-Related Protein DJ-1. Mol Neurobiol. 2019;56(12):8306-22. Chavali LNM, Yddal I, Bifulco E, Mannsaker S, Roise D, Law JO, et al. Progressive Motor and Non-Motor Symptoms in Park7 Knockout Zebrafish. Int J Mol Sci. 2023;24(7). Rostad KO, Trognitz T, Froyset AK, Bifulco E, Fladmark KE. Accelerated Sarcopenia Phenotype in the DJ-1/Park7-Knockout Zebrafish. Antioxidants (Basel). 2024;13(12). Flinn L, Mortiboys H, Volkmann K, Koster RW, Ingham PW, Bandmann O. Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio). Brain. 2009;132(Pt 6):1613-23. Flinn LJ, Keatinge M, Bretaud S, Mortiboys H, Matsui H, De Felice E, et al. TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency. Ann Neurol. 2013;74(6):837-47. Fett ME, Pilsl A, Paquet D, van Bebber F, Haass C, Tatzelt J, et al. Parkin is protective against proteotoxic stress in a transgenic zebrafish model. PLoS One. 2010;5(7):e11783. Saint-Amant L, Drapeau P. Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol. 1998;37(4):622-32. Ketzef M, Spigolon G, Johansson Y, Bonito-Oliva A, Fisone G, Silberberg G. Dopamine Depletion Impairs Bilateral Sensory Processing in the Striatum in a Pathway-Dependent Manner. Neuron. 2017;94(4):855-65 e5. Silva RFO, Pinho BR, Santos MM, Oliveira JMA. Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment. Chemosphere. 2022;305:135449. Breen DP, Vuono R, Nawarathna U, Fisher K, Shneerson JM, Reddy AB, et al. Sleep and circadian rhythm regulation in early Parkinson disease. JAMA Neurol. 2014;71(5):589-95. Li H, Zhang J, Shen Y, Ye Y, Jiang Q, Chen L, et al. Targeting Mitochondrial Complex I Deficiency in MPP(+)/MPTP-induced Parkinson's Disease Cell Culture and Mouse Models by Transducing Yeast NDI1 Gene. Biol Proced Online. 2024;26(1):9. Annesi G, Savettieri G, Pugliese P, D'Amelio M, Tarantino P, Ragonese P, et al. DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex. Ann Neurol. 2005;58(5):803-7. Hanagasi HA, Giri A, Kartal E, Guven G, Bilgic B, Hauser AK, et al. A novel homozygous DJ1 mutation causes parkinsonism and ALS in a Turkish family. Parkinsonism Relat Disord. 2016;29:117-20. Rink E, Wullimann MF. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Brain Res Dev Brain Res. 2002;137(1):89-100. Brown SJ, Boussaad I, Jarazo J, Fitzgerald JC, Antony P, Keatinge M, et al. PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons. Sci Rep. 2021;11(1):6617. Bandopadhyay R, Kingsbury AE, Cookson MR, Reid AR, Evans IM, Hope AD, et al. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson's disease. Brain. 2004;127(Pt 2):420-30. Sheng D, Qu D, Kwok KH, Ng SS, Lim AY, Aw SS, et al. Deletion of the WD40 domain of LRRK2 in Zebrafish causes Parkinsonism-like loss of neurons and locomotive defect. PLoS Genet. 2010;6(4):e1000914. Milanese C, Sager JJ, Bai Q, Farrell TC, Cannon JR, Greenamyre JT, et al. Hypokinesia and reduced dopamine levels in zebrafish lacking beta- and gamma1-synucleins. J Biol Chem. 2012;287(5):2971-83. Ren G, Xin S, Li S, Zhong H, Lin S. Disruption of LRRK2 does not cause specific loss of dopaminergic neurons in zebrafish. PLoS One. 2011;6(6):e20630. Prabhudesai S, Bensabeur FZ, Abdullah R, Basak I, Baez S, Alves G, et al. LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation. J Neurosci Res. 2016;94(8):717-35. Ramesh T, Lyon AN, Pineda RH, Wang C, Janssen PM, Canan BD, et al. A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease. Dis Model Mech. 2010;3(9-10):652-62. Sanyal S, Wintle RF, Kindt KS, Nuttley WM, Arvan R, Fitzmaurice P, et al. Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans. EMBO J. 2004;23(2):473-82. Dodet P, Houot M, Leu-Semenescu S, Corvol JC, Lehericy S, Mangone G, et al. Sleep disorders in Parkinson's disease, an early and multiple problem. NPJ Parkinsons Dis. 2024;10(1):46. Kalia LV, Lang AE. Parkinson's disease. Lancet. 2015;386(9996):896-912. Hunt J, Coulson EJ, Rajnarayanan R, Oster H, Videnovic A, Rawashdeh O. Sleep and circadian rhythms in Parkinson's disease and preclinical models. Mol Neurodegener. 2022;17(1):2. Xiong H, Wang D, Chen L, Choo YS, Ma H, Tang C, et al. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J Clin Invest. 2009;119(3):650-60. Doyle JM, Croll RP. A Critical Review of Zebrafish Models of Parkinson's Disease. Front Pharmacol. 2022;13:835827. Pinho BR, Reis SD, Guedes-Dias P, Leitao-Rocha A, Quintas C, Valentao P, et al. Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease. Pharmacol Res. 2016;103:328-39. Pende M, Vadiwala K, Schmidbaur H, Stockinger AW, Murawala P, Saghafi S, et al. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity. Sci Adv. 2020;6(22):eaba0365. Chen YC, Priyadarshini M, Panula P. Complementary developmental expression of the two tyrosine hydroxylase transcripts in zebrafish. Histochem Cell Biol. 2009;132(4):375-81. Lempereur S, Machado E, Licata F, Simion M, Buzer L, Robineau I, et al. ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 days post-fertilization zebrafish following simultaneous visualization with identical orientations. Dev Biol. 2022;490:86-99. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82. Additional Declarations No competing interests reported. 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11:11:04\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":470436,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLarval gross morphology and embryonic spontaneous coilings are not altered in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003epark7\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cstrong\\u003e-/-\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e. (A)\\u003c/strong\\u003e Western blot shows expression of DJ-1 at 1, 3 and 5 dpf in total lysates from wild type and the lack of DJ-1 in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/-\\u003c/sup\\u003e. Arrow points to DJ-1 band. Pon-S is used as loading control. \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Spontaneous coilings of the tail were recorded for 3 min at 28 hpf. T-test of genotypes showed no significant difference (\\u003cem\\u003ep\\u003c/em\\u003e \\u0026gt; 0.05). Graph shows the mean ± SEM;\\u003cem\\u003e n \\u003c/em\\u003e= 63-66 embryos per condition, from at least five independent breedings. \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Images of 5 dpf wild type and \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/-\\u003c/sup\\u003e larvae indicating eye aspect ratio (white, \\u003cstrong\\u003eD\\u003c/strong\\u003e) and body length (red, \\u003cstrong\\u003eE\\u003c/strong\\u003e) measurement.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/a893d14d72c8f5e05fa6a0e4.png\"},{\"id\":95914049,\"identity\":\"9550162a-2240-4797-9967-eba9100023e7\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 11:11:04\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":84468,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003epark7\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cstrong\\u003e-/-\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003elarvae have lower sensorimotor responsiveness compared to wild types. \\u003c/strong\\u003eTouch-evoked response of both head \\u003cstrong\\u003e(A) \\u003c/strong\\u003eand tail \\u003cstrong\\u003e(B)\\u003c/strong\\u003e show a significant decrease (**\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001; generalised linear model (GLM) with a quasibinomial distribution followed by Tukey-adjusted pairwise comparisons of model-adjusted means to assess the effects of genotype, drug and their interaction) in sensorimotor responses for \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/-\\u003c/sup\\u003e.\\u003cem\\u003e \\u003c/em\\u003eMPP\\u003csup\\u003e+\\u003c/sup\\u003e further decreased responsiveness for both wild type and \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/- \\u003c/sup\\u003elarvae.\\u003cem\\u003e n\\u003c/em\\u003e = 48-84 per condition, from 4-7 independent breedings.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/9090575e33e813a538006ac5.png\"},{\"id\":95914052,\"identity\":\"d22b448f-eeac-4e98-a3c0-eeb196c1ee75\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 11:11:04\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":479558,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eActivity and sleep in \\u003c/strong\\u003e\\u003cem\\u003e\\u003cstrong\\u003epark7\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cstrong\\u003e-/-\\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003e larvae are altered\\u003c/strong\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/em\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003ecompared to wild types. \\u003c/strong\\u003e\\u003cem\\u003epark\\u003c/em\\u003e7\\u003csup\\u003e-/-\\u003c/sup\\u003e and wild type zebrafish larvae were exposed to MPP\\u003csup\\u003e+\\u003c/sup\\u003e or vehicle from 3-5 dpf and thereafter washed and put into LAM tubes for continuous behaviour analysis under light-dark cycles from 5-8 dpf. \\u003cstrong\\u003e(A)\\u003c/strong\\u003e Representative behaviour tracing of activity per 60 min. Y-axis shows number of beam crossings +/- SEM (n=16). \\u003cstrong\\u003e(B-G) \\u003c/strong\\u003eQuantification of activity and sleep parameters from three individual experiments. Values are mean ± SEM; \\u003cem\\u003en\\u003c/em\\u003e = 48 from three independent breedings. *\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.05, **\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.01, ***\\u003cem\\u003ep\\u003c/em\\u003e \\u0026lt; 0.001, different GLMs were created depending on the data distribution and scale followed by Tukey-adjusted comparisons of the model-adjusted means to assess the effects of genotype, drug and their interaction. \\u003cstrong\\u003e(B-C) \\u003c/strong\\u003eActivity in light\\u003cstrong\\u003e (B) \\u003c/strong\\u003edark\\u003cstrong\\u003e (C) \\u003c/strong\\u003ephases.\\u003cstrong\\u003e (D-G)\\u003c/strong\\u003e Sleep analysis of LAM data: sleep bout duration \\u003cstrong\\u003e(D)\\u003c/strong\\u003e; sleep latency \\u003cstrong\\u003e(E)\\u003c/strong\\u003e; sleep ratio in light phases \\u003cstrong\\u003e(F)\\u003c/strong\\u003e; sleep ratio in dark phases \\u003cstrong\\u003e(G)\\u003c/strong\\u003e.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/dfd65cc037f24c83b602970d.png\"},{\"id\":95914056,\"identity\":\"8e96925e-bdd0-4f90-824a-ad05010fd771\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 11:11:04\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":3638427,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003e\\u003cstrong\\u003epark7\\u003c/strong\\u003e\\u003c/em\\u003e\\u003csup\\u003e\\u003cstrong\\u003e-/- \\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003elarvae\\u003c/strong\\u003e\\u003csup\\u003e\\u003cstrong\\u003e \\u003c/strong\\u003e\\u003c/sup\\u003e\\u003cstrong\\u003ehave a lower number of dopaminergic neurons compared to wild types. \\u003c/strong\\u003eZebrafish were exposed to 500 µM MPP\\u003csup\\u003e+\\u003c/sup\\u003e or vehicle from 3-5 dpf thereafter fixed, bleached, whole-mount immune-stained using anti-tyrosine hydroxylase and mounted in clearing solution. Larvae were imaged by confocal microscopy. \\u003cstrong\\u003eA: \\u003c/strong\\u003eDopaminergic cell populations at 5 dpf.\\u003cstrong\\u003e \\u003c/strong\\u003eCells of interest are encircled with a dotted line.\\u003cstrong\\u003e \\u003c/strong\\u003eColours refer to the stack level.\\u003cstrong\\u003e B: \\u003c/strong\\u003eCircled cell population from (A) after selecting stack images of interest corresponding to DC1 and DC2 population.\\u003cstrong\\u003e C: \\u003c/strong\\u003eSchematic sagittal section of the 5 dpf brain showing the location of the DC1 and DC2 dopaminergic cell population from a lateral view. \\u003cstrong\\u003eD:\\u003c/strong\\u003e Projections of\\u003cstrong\\u003e \\u003c/strong\\u003edopaminergic neurons in the selected DC1 and part of DC2 population from wild type, wild type plus MPP\\u003csup\\u003e+\\u003c/sup\\u003e, \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/-\\u003c/sup\\u003e and \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e-/-\\u003c/sup\\u003e + MPP\\u003csup\\u003e+\\u003c/sup\\u003e at 5 dpf. Arrows point to the larger pear-shaped DC2 dopaminergic neurons. \\u003cstrong\\u003eE: \\u003c/strong\\u003eThe number of dopaminergic cells in the DC1 population at 3, 4, and 5 dpf. Numbers are the mean ± SEM; \\u003cem\\u003en \\u003c/em\\u003e= 3-6 larvae. **\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt;0.01, ***\\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt;0.001 using Type II ANOVA and Tukey-adjusted pairwise comparisons. Bars, 50µm. Dopaminergic cell populations are named according to Rink and Wulliman(33), which corresponds to MPP\\u003csup\\u003e+\\u003c/sup\\u003e responsive cell population (5, 6, and 11) as shown in Sallinen et al(9).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/2c1095ce4fd686fbc172aa96.png\"},{\"id\":103251151,\"identity\":\"a9919eeb-f191-44ba-b1ff-2d145f2f8583\",\"added_by\":\"auto\",\"created_at\":\"2026-02-23 16:05:19\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4851426,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/821124c3-9e8e-4ce1-8a65-3d597c7bb9be.pdf\"},{\"id\":95914068,\"identity\":\"715db6bd-03b4-445f-856a-58e222b6fccb\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 11:11:04\",\"extension\":\"docx\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":6736548,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplementaryfiguresfile.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/9cd9acc3036ec9447b556d67.docx\"},{\"id\":95914051,\"identity\":\"ff5d8910-6691-4e69-bb45-6d1d8b85d6d4\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 11:11:04\",\"extension\":\"pdf\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":36407,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"UncroppedWesternblotforFig.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7848595/v1/6f6a414cace18705da5cb60b.pdf\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"Early motor deficits, sleep dysfunction and reduction in dopaminergic neurons in a park7 -/- zebrafish larval model of Parkinson’s disease\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eParkinson\\u0026rsquo;s Disease (PD) is the fastest growing neurological disorder (\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e) and only symptomatic therapy is available. Although about 10% of PD cases are caused by an inherited genetic variant, the majority of PD cases are sporadic, age-dependent and most possibly the result of a complex combination of/interplay between environmental exposure and genetic risk factors (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003ePD diagnosis is set when motor disabilities occur, although there are prodromal symptoms such as disturbances in REM sleep, changes in smell and vision, reduced sense of touch, mood disorders and others (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e). PD is characterized by a selective loss of dopaminergic cells in the \\u003cem\\u003esubstantia nigra pars compacta\\u003c/em\\u003e. Cellular changes common to both familiar and spontaneous PD include mitochondrial dysfunction, increased oxidative stress and altered immune-related response (reviewed in (\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e)). However, the mechanisms resulting in the selective loss of dopaminergic neurons in PD are still not well understood. Considering the limited success of existing treatments for PD and the increasing prevalence of the disease, there is an evident need for novel disease-modifying treatments and methods to halt disease progression. In this context, \\u003cem\\u003ein vivo\\u003c/em\\u003e models enabling the understanding of early cellular changes leading to disease and progression are indispensable.\\u003c/p\\u003e\\u003cp\\u003eZebrafish serve as an excellent \\u003cem\\u003ein vivo\\u003c/em\\u003e platform for pharmacological screening as drugs can be added directly to the water. Additionally, zebrafish can be easily genetically modified and in combination with their small and optically transparent larvae they are a unique gateway for elucidating pathological mechanisms. Dopaminergic neurons are detected already at 1 day post fertilization (dpf) in the zebrafish embryo, and at 3 dpf the organization of the CNS is already complete (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). Dopaminergic neurons in the zebrafish are also sensitive to oxidative stress induced by mitochondria-directed neurotoxicants (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). Thus, zebrafish are excellent models for studying pathological mechanisms in PD and directed therapies.\\u003c/p\\u003e\\u003cp\\u003eLoss of function mutations in \\u003cem\\u003epark7\\u003c/em\\u003e are associated with autosomal recessive forms of early-onset PD (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). Importantly, the \\u003cem\\u003epark7\\u003c/em\\u003e protein product, DJ-1 is also linked to spontaneous form of PD, in which increased expression of irreversibly oxidative modified DJ-1 and reduced association to mitochondrial ATP synthase are observed (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e). DJ-1 is a multifunctional redox sensitive and neuroprotective protein. It has been shown to modulate oxidative stress, inflammatory response, mitochondrial function, synaptic recycling, autophagy, and proteostasis (\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e), (\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e), (\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e), (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e), (\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e) (\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eWe have previously established a CRISPR/cas9-based \\u003cem\\u003epark7\\u003c/em\\u003e knockout zebrafish line (\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e) and demonstrated a progressive increase in motor and non-motor symptoms at the adult stage (\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Here, we show that this DJ-1 knockout model, in contrast to other genetic PD-related zebrafish models (\\u003cspan additionalcitationids=\\\"CR24\\\" citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e), starts to both develop motor deficits and have a reduced number of dopaminergic neurons already at their larval stage.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003eLoss of DJ-1 evoked no noticeable effects on general development and early motor activity of zebrafish embryos\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn wild-type zebrafish DJ-1 is expressed throughout development (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA). To assess whether a loss of DJ-1 affects embryonic development, we measured morphometric parameters (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC-E). As reflected in gross morphology, the loss of DJ-1 in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e did not hamper general development \\u0026ndash; at 5 dpf, wild-type and \\u003cem\\u003epark7\\u003c/em\\u003e knockout larvae did not differ in body length or eye aspect ratios (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD-E). The earliest motor activity in zebrafish can be observed at 1 dpf as spontaneous tail coiling (\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). No significant difference was observed in tail coiling comparing wild-type and \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003epark7\\u003c/span\\u003e\\u003csup\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026minus;/\\u0026minus;\\u003c/span\\u003e\\u003c/sup\\u003e \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003elarvae have a reduced touch-evoked escape response\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003eReduced sense of touch is a common and often prodromal symptom in many PD patients (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e). Mitochondria-directed toxicants affecting dopaminergic neurons have also been shown to impair tactile sensory processing in a mouse model (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). In view of this, we examined the touch-evoked response in larvae by applying tactile stimulation to the head and tail (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Larvae were exposed from 3\\u0026ndash;5 dpf to 1-methyl-4-phenylpyridinium (MPP\\u003csup\\u003e+\\u003c/sup\\u003e) which selectively targets dopaminergic neurons. Indeed, at 5 dpf MPP\\u003csup\\u003e+\\u003c/sup\\u003e exposure reduced both head and tail response in wild-type larvae. More importantly, a significant impairment in touch-evoked responses was also observed in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e compared to wild types, even in the absence of MPP\\u003csup\\u003e+\\u003c/sup\\u003e. Additionally, loss of DJ-1 resulted in an increased sensitivity towards MPP\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003epark7\\u003c/span\\u003e\\u003csup\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026minus;/\\u0026minus;\\u003c/span\\u003e\\u003c/sup\\u003e \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003elarvae have disturbed activity and sleep patterns, but no impairment in motor endurance\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003eLarvae behaviour was analysed from 5 to 8 dpf using an infrared-based Locomotor Activity Monitor (LAM) (\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). LAM tracking allows the monitoring of motor function, circadian function and sleep disturbances, the latter being one of the most common prodromal symptoms in PD (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eAll the LAM-monitored zebrafish larvae, independent of genotype or treatment, showed a typical circadian pattern with higher activity in the light phases vs. dark phases (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). \\u003cem\\u003epark7\\u003c/em\\u003e knockout and treatment with MPP\\u003csup\\u003e+\\u003c/sup\\u003e decreased light-phase activity, compared to control larvae (wild type, solvent). There was no further activity reduction (no additive effect) when combining MPP\\u003csup\\u003e+\\u003c/sup\\u003e with \\u003cem\\u003epark7\\u003c/em\\u003e knockout (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB-C).\\u003c/p\\u003e\\u003cp\\u003eSleep bout durations and sleep latency were analysed in the dark phases. \\u003cem\\u003epark7\\u003c/em\\u003e knockout or MPP\\u003csup\\u003e+\\u003c/sup\\u003e treatment had no statistically significant effects on sleep bout duration (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). Sleep latency, on the other hand, was increased in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae throughout the experimental period (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE). At 5 dpf, larvae pretreated with MPP\\u003csup\\u003e+\\u003c/sup\\u003e also showed increased sleep latency, but this effect of MPP\\u003csup\\u003e+\\u003c/sup\\u003e fades as the experiment progresses, likely due to larvae recovering after MPP\\u003csup\\u003e+\\u003c/sup\\u003e washout (LAM medium does not contain MPP\\u003csup\\u003e+\\u003c/sup\\u003e). Interestingly, the MPP\\u003csup\\u003e+\\u003c/sup\\u003e-exposed \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae do not initially show increased sleep latency but approximate the unexposed \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e as the experiment progresses and larvae recover from the MPP\\u003csup\\u003e+\\u003c/sup\\u003e pre-exposure. Moreover, no differences were observed in the sleep ratio (proportion of time spent sleeping) in dark phases, but in light phases, the sleep ratio was increased in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF-G). A similar increase was not observed in MPP\\u003csup\\u003e+\\u003c/sup\\u003e-treated wild types, suggesting a specific effect of \\u003cem\\u003epark7\\u003c/em\\u003e on sleep regulation.\\u003c/p\\u003e\\u003cp\\u003eMotor endurance has been shown to be decreased in mice exposed to the MPP\\u003csup\\u003e+\\u003c/sup\\u003e precursor MPTP (\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e). We therefore assessed motor endurance in PD-model zebrafish. While MPP\\u003csup\\u003e+\\u003c/sup\\u003e exposure reduced motor endurance in zebrafish larvae, \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae showed motor endurance similar to wild types (Supplementary Fig.\\u0026nbsp;1A-B). We also did not observe any degradation in neuromuscular junctions in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae (Supplementary Fig.\\u0026nbsp;1C), a feature that accompanies reduction in motor endurance in zebrafish models of motor neuron disease (\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). These findings support the hypothesis that \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e induces more selective PD phenotypes than MPP\\u003csup\\u003e+\\u003c/sup\\u003e, regarding motor parameters.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cspan type=\\\"ItalicUnderline\\\" class=\\\"ItalicUnderline\\\" name=\\\"Emphasis\\\"\\u003epark7\\u003c/span\\u003e\\u003csup\\u003e\\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003e\\u0026minus;/\\u0026minus;\\u003c/span\\u003e\\u003c/sup\\u003e \\u003cspan type=\\\"Underline\\\" class=\\\"Underline\\\" name=\\\"Emphasis\\\"\\u003elarvae exhibit a lower number of DC1 diencephalic dopaminergic neurons compared to wild types\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003eDopaminergic (DA) neurons can be detected already at 18 hours post-fertilization (hpf) and at 3 dpf the distribution of DA neuronal populations is complete (\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). We studied the number and distribution of DA neurons using an antibody detecting tyrosine hydroxylase (TH), a marker for DA neurons (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eWe measured the number of the diencephalic DA neurons of the posterior tuberculum referred to as DC1 (\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e) or 5, 6, 11 population (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e)(Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). This is the DA neuronal population comparable to the mammalian substantia nigra, and which is selectively targeted by MPP\\u003csup\\u003e+\\u003c/sup\\u003e (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). The DC1 population can easily be distinguished from another nearby DA population (DC2) based on morphology and the coronal level (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB-C). Our results showed a significant reduction in the number of DA neurons in the DC1 population in the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e at 5 dpf, compared to wild type (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD-E). This difference seemed to progress by embryonal/larval aging since at 3 and 4 dpf the difference was not significant (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE). As expected, exposure to MPP\\u003csup\\u003e+\\u003c/sup\\u003e from 3\\u0026ndash;5 dpf decreased the number of DC1 dopaminergic neurons in the wild type larvae. There was no further reduction in the number of DC1 neurons (no additive effect) when combining MPP\\u003csup\\u003e+\\u003c/sup\\u003e with \\u003cem\\u003epark7\\u003c/em\\u003e knockout.\\u003c/p\\u003e\\u003cp\\u003eIn another zebrafish model of PD, the PTEN-induced kinase 1 (\\u003cem\\u003epink1\\u003c/em\\u003e)\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e, the larger DC2 dopaminergic neurons show reduced neurogenesis (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). We determined the number of neurons in the smaller DC2 population but found no differences between genotypes or the effect of MPP\\u003csup\\u003e+\\u003c/sup\\u003e (Supplementary Fig.\\u0026nbsp;2). Thus, these results indicate that loss of DJ-1 selectively targets the DA neuronal population comparable to the mammalian substantia nigra.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eNo disease-modifying therapeutics exist for Parkinson\\u0026rsquo;s disease, only symptom-relieving drugs. Larval zebrafish offer unique possibilities for high-throughput phenotypic analysis and imaging analysis of a well characterized nervous system. Here we describe the behaviour and dopaminergic cell population at the larval stage of a CRISPR/cas9-based \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish. Our results show that \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae not only have sensory and motor deficits but also sleep disturbances and a lower number of the dopaminergic neurons comparable to the mammalian substantia nigra. To our knowledge this combination of PD-relevant behaviour and cell-specific targeting have not previously been shown in other genetic or toxicant based zebrafish models.\\u003c/p\\u003e\\u003cp\\u003eMutations in \\u003cem\\u003epark7\\u003c/em\\u003e leading to a dysfunction in its protein product DJ-1 are linked to a rare, familial type of early-onset PD (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). However, even in idiopathic cases of PD, with no known mutations in \\u003cem\\u003epark7\\u003c/em\\u003e, post-mortem analyses of brain samples revealed an increase in oxidative cysteine-modified DJ-1 (\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e). Recently, a reduced association of DJ-1 to mitochondrial F1Fo-ATP synthase was shown in sub-cellular compartments (predominantly distal neurites) of dopaminergic neurons in substantia nigra of post-mortem brains from sporadic PD patients (\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eIn relation to gross morphology and basic body plan, \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae were indistinguishable from wild types (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). Neither was there any difference in spontaneous motor activity at the embryonal stage (28 hpf). However, at the 5 dpf stage, spontaneous locomotor activity was significantly reduced in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae compared to wild types (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). Impaired motor function has also previously been observed in the morpholino-based \\u003cem\\u003eLRRK2\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e) and \\u003cem\\u003eSNCA\\u003c/em\\u003e (\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e) knockdown PD models, although \\u003cem\\u003eLRRK2\\u003c/em\\u003e knockdown induces developmental defects and conflicting evidence exists for its effect on motor activity (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e). Mutations in \\u003cem\\u003epark7\\u003c/em\\u003e have been associated with signs of amyotrophic lateral sclerosis (ALS) pathology in two independent studies (\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). However, the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae showed no reduction in motor endurance as would be expected for ALS-associated phenotypes (Suppl. Figure\\u0026nbsp;1) (\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTactile perception is impaired in dopamine-depleted mice (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). This dopamine-dependent response seems to be a conserved feature as tyrosine hydroxylase-depleted \\u003cem\\u003eC. elegans\\u003c/em\\u003e had a dysregulation in tactile plasticity which could be rescued by dopamine (\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e). In agreement with these reports, we found that \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e had a significantly lower response to both head and tail touch compared to wild type (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Thus, reduced touch response in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e might be coupled to a dysfunctional dopaminergic response and be relevant to PD pathology, which often includes a reduced sense of touch (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eIt is well established that mitochondrial dysfunction is strongly implicated in both familial and spontaneous PD. The mitochondrial toxicant MPTP and its oxidized product MPP\\u003csup\\u003e+\\u003c/sup\\u003e, which target a specific population of dopaminergic neurons, are therefore commonly used to replicate PD pathology in animal models. In this study we used MPP\\u003csup\\u003e+\\u003c/sup\\u003e to compare exposed wild type with unexposed \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u003cem\\u003e\\u0026minus;/\\u0026minus;\\u003c/em\\u003e\\u003c/sup\\u003e and to evaluate whether loss of DJ-1 renders larvae more sensitive to MPP\\u003csup\\u003e+\\u003c/sup\\u003e. Loss of DJ-1 combined with MPP\\u003csup\\u003e+\\u003c/sup\\u003e exposure induced a greater reduction in the touch-evoked responses than each condition alone (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e\\u0026ndash;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eSleep disorders are common and evident at the prodromal stage in PD (\\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e). Rapid eye movement sleep behaviour disorder, excessive daytime sleepiness and increased sleep latency are among these sleep disturbances (\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e). Using the LAM, we were able to monitor both total day-/night-time sleep and sleep latency at the start of the darkness phase. Interestingly, both sleep latency and sleep ratio during daytime were increased in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e, whilst sleep ratio during the night phase remained unaltered (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE-G). These sleep disturbances persisted throughout the experimental period (5\\u0026ndash;8 dpf). MPP\\u003csup\\u003e+\\u003c/sup\\u003e-treated wild types did not show a similar pattern of sleep disturbances. Previous reports on exposure of rodents to MPTP have not been able to show a correlation between sleep disruptions and loss of dopaminergic neurons, suggesting that the commonly used MPTP model for PD does not fulfil a comprehensive role as a PD model, specifically in relation to non-motor symptoms (\\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e44\\u003c/span\\u003e). The pronounced PD-related sleep disorder symptoms observed in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae therefore increases its value as a PD model.\\u003c/p\\u003e\\u003cp\\u003eThe dopaminergic cell population in the DC1 of zebrafish larvae is comparable to the mammalian substantia nigra (\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e). In zebrafish the neurotoxicants MPTP and MPP\\u003csup\\u003e+\\u003c/sup\\u003e specifically target these DC1 dopaminergic cells, sparing other dopaminergic cell populations (\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). Focusing on the tyrosine hydroxylase-positive cells in the DC1 region we saw that \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish at 5 dpf, even in the absence of MPP\\u003csup\\u003e+\\u003c/sup\\u003e, had less dopaminergic neurons in the DC1 population compared to wild-type zebrafish (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Comparing the number of dopaminergic DC1 neurons between \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e and wild type at the 3 and 4 dpf stage did not show a significant difference, thus the reduction in dopaminergic DC1 in \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e seemed to be a progressive effect. In parallel with the behaviour analysis, the loss of DJ-1 did not appear to make \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e more sensitive towards MPP\\u003csup\\u003e+\\u003c/sup\\u003e than wild type. Even though MPP\\u003csup\\u003e+\\u003c/sup\\u003e treatment of \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e further decreased the number of dopaminergic DC1, the number of cells was not significantly different from MPP\\u003csup\\u003e+\\u003c/sup\\u003e-treated wild types.\\u003c/p\\u003e\\u003cp\\u003eIn another stable genetic zebrafish model of PD, the pten-induced putative kinase 1\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e (\\u003cem\\u003epink1\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e), a reduced number of dopaminergic neurons is also evident already at the larval stage (\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). Loss of PINK1 impairs neurogenesis in zebrafish, but also in human PINK1-deficient organoids (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e). As shown in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e, both wild type and \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e larvae show a progressive increase in the number of dopaminergic DC1 cells from 3\\u0026ndash;5 dpf, but this increase is depressed in the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e. Thus, loss of DJ-1 in the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e most likely inhibits neurogenesis, as in the \\u003cem\\u003epink1\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish. Like DJ-1, PINK1 is also associated with early-onset PD and it has been shown that DJ-1 and PINK1 in complex have a role in oxidative stress protection and promoting unfolded protein degradation (\\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e45\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTo summarize, loss of DJ-1 in the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish results in reduced touch-evoked response, sleep disturbances and locomotor dysfunction together with a reduced number of dopaminergic neurons. This is the first stable genetic model for Parkinson\\u0026acute;s disease presenting motor symptoms, sleep disturbances and reduction of dopaminergic neurons already at the larval stage (\\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e46\\u003c/span\\u003e). This makes it an attractive model for pharmacological screening and mechanistic studies.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cp\\u003eZebrafish husbandry\\u003c/p\\u003e\\n\\u003cp\\u003eAdult zebrafish were maintained at 26\\u0026ndash;28 \\u0026deg;C with a 14/10 light cycle and were fed twice daily. Embryos and larvae were maintained at 28 \\u0026deg;C and raised in E3 media (5 mM NaCl, 0.17 mM KCl, and 0.33 mM MgSO4).\\u003c/p\\u003e\\n\\u003cp\\u003eEthics declaration and approval for animal experiments\\u003c/p\\u003e\\n\\u003cp\\u003eAnimals used in the experiments were housed either at the Zebrafish Facility located in the Department of Biological Sciences at the University of Bergen, Norway or in the Vivarium of\\u0026nbsp;Aquatic Organisms\\u0026nbsp;at the University of Porto. The facilities are run according to the guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes, and following the regulation by the European Directive 2010/63/EU. Establishment of the park7\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e line was approved by the Norwegian National Animal Research Authority at Mattilsynet (FOTS ID8039 and ID14039) (20).\\u003c/p\\u003e\\n\\u003cp\\u003eExperiments were performed according to the 3Rs principle by reducing the number of animals used and using refined techniques that reduce animal suffering, and are reported according to ARRIVE guidelines. All experimental protocols were approved by National Animal Research Authority at Mattilsynet, Norway and local licensing committee at University of Bergen. All methods were carried out in accordance with relevant guidelines and regulations.\\u003c/p\\u003e\\n\\u003cp\\u003eWestern blot analysis\\u003c/p\\u003e\\n\\u003cp\\u003eEmbryos and larvae were sampled at 24 hpf, 3 dpf and 5 dpf. Deyolking was performed with 24 hpf and 3 dpf embryos/larva. Lysates were prepared by suspending in 150\\u0026micro;l homogenization buffer (10 mM K\\u003csub\\u003e2\\u003c/sub\\u003eHPO\\u003csub\\u003e4\\u003c/sub\\u003e, 10m MKH\\u003csub\\u003e2\\u003c/sub\\u003ePO\\u003csub\\u003e4\\u003c/sub\\u003e, 1 mM EDTA, 0.6% CHAPS, 0.2 mM Na\\u003csub\\u003e3\\u003c/sub\\u003eVO\\u003csub\\u003e4\\u003c/sub\\u003e, 50 mM NaF, and protease inhibitor cocktail (Roche Diagnostics GmbH:11836153001)) and sonication (4\\u0026times;5 s) followed by incubation on ice for 20 min. Samples were pelleted at 15,000\\u0026times;g for 15 min at 4 \\u0026deg;C and 25\\u0026micro;g protein from the supernatant was separated by SDS-PAGE and transferred to PVDF membranes using 14 V overnight at 4 \\u0026deg;C.\\u003c/p\\u003e\\n\\u003cp\\u003eMembranes were blocked in 1% BSA and incubated with anti-DJ-1 (1:3000, Novus Biologicals NB300-270, 1 hr) followed by secondary antibody. Ponceau-S was used as a loading control.\\u003c/p\\u003e\\n\\u003cp\\u003eTail Coiling\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eEmbryos at 28 \\u0026nbsp; 1 hpf were transferred to a 96 well-plate containing E3 (6 embryos/well). After a 5 min acclimation period, the embryos were recorded for 3-4 minutes using a Dino-Eye eyepiece camera (AM-423U). The videos were cut to exactly 3 minutes and the number of tail-coilings (random movement) were counted manually.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMorphometric analysis of larvae\\u003c/p\\u003e\\n\\u003cp\\u003eLarvae were fixed o.n. in 4% PFA/PBS and washed in PBS before being mounted in agar molds. Images were taken with a Nikon SMZ800N and morphometric analysis was performed using ImageJ.\\u003c/p\\u003e\\n\\u003cp\\u003e1-methyl-4-phenylpyridinium (MPP\\u003csup\\u003e+\\u003c/sup\\u003e) treatment\\u003c/p\\u003e\\n\\u003cp\\u003eAt 3 dpf, hatched larvae that did not display any abnormalities were randomly selected and placed in 12-well plate (10-15 larvae/1mL/well) and exposed to 500\\u0026micro;M MPP\\u003csup\\u003e+\\u003c/sup\\u003e (D048, Merck KGaA, Darmstadt, Germany). The MPP\\u003csup\\u003e+\\u003c/sup\\u003e solution and vehicle were renewed at 4 dpf by replacing half volume with fresh solutions. At 5 dpf, the larvae were transferred to E3 in 6-well plates (1 larva/well) for sensorimotor responses or the LAM system (1 larva/tube) for continuous activity monitoring, without further exposure to MPP\\u003csup\\u003e+\\u003c/sup\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003eSensorimotor responses\\u003c/p\\u003e\\n\\u003cp\\u003eIn 6-well plate (one larva/well) each larva was gently touched with a micropipette tip, alternating between the head and tail. Immediate swimming was registered as a positive response, whereas no movement was registered as a negative response, yielding a binary result (47). Each larva was touched ten times at both head and tail, with a 30-second break in between. A minimum of \\u003cem\\u003en\\u003c/em\\u003e = 12 larvae, randomly selected from several breedings, were used for each condition.\\u003c/p\\u003e\\n\\u003cp id=\\\"_Toc180508021\\\"\\u003eLocomotor Activity Monitor (LAM)\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eLarvae were individually placed in E3 media-filled LAM tubes (3.65 mL) with a 100 ml air bubble inside. They were evenly dispersed and placed in the LAM for continuous activity monitoring until 8 dpf, while maintaining 28 1\\u0026deg;C and a 14h/10h light-dark cycle, as previously described (28). The starting point of the light phase was set to 09.00AM. The LAM data were analysed with the \\u003cem\\u003eRtivity\\u003c/em\\u003e software (28). Sleep threshold was set to 10 minutes of inactivity and bout activity threshold to 2 minutes. Larval activity was measured as the number of infrared beam crossings per 30 seconds. Total activity for each day (counts) was analysed. Furthermore, activity (counts), sleep ratio (proportion of time spent sleeping), sleep latency (the mean time between lights turned off and start of the first sleep bout, in minutes) and sleep bout duration (the mean time spent being continuous asleep without interruptions, in minutes) were analysed for each day separated by light/dark phases. Sleep latency and sleep bout duration only contained data from the dark phases.\\u003c/p\\u003e\\n\\u003cp\\u003eMotor endurance\\u0026nbsp;assessment of zebrafish larvae\\u003c/p\\u003e\\n\\u003cp\\u003eFor motor endurance assessment, we used a custom gravity-fed counter-current swimming system, capable of inducing the counter-current swimming reflex in zebrafish larvae without immediately overpowering them (manuscript in preparation). 7 dpf zebrafish larvae were moved to a 90 mm dish with chlorine-free water, 30 minutes prior to the experiment, for acclimation. 5 to 10 larvae were then moved into a 5 mL serological pipette, used as the swimming tunnel, and left to acclimate for 5 minutes. During this period, the larvae were guided to the middle of the swimming tunnel. Afterwards, water flow was initiated. Initial flow speed was set at 3 cm/s, gradually increasing to 5 cm/s over the course of 10 minutes. Endurance times were recorded as the length of time that each fish was able to stay in the swimming tunnel, before being pushed out by the water flow.\\u003c/p\\u003e\\n\\u003cp id=\\\"_Toc180508022\\\"\\u003eWhole-mount labelling and image analysis\\u003c/p\\u003e\\n\\u003cp\\u003eLarvae were fixed in methanol free 4% PFA/PBS 4\\u0026deg;C o/n with gentle agitation. Thereafter the larvae were washed in 6x15 min in PBS with 0.05% Triton X (PBS-TX) and stored at 4 \\u0026ordm;C until further use. Permeabilization was performed by incubation with acetone at -20 \\u0026ordm;C o.n. Thereafter larvae were washed in PBS-TX followed by 2 times wash in PBS. Bleaching was done in 3% H\\u003csub\\u003e2\\u003c/sub\\u003eO\\u003csub\\u003e2\\u003c/sub\\u003e in 0.89% KOH under bright light (approx. 8 min for 5 dpf larvae). Larvae were then washed 3x5 min in PBS-TX and 2x15 min PBS-TX. Clearing was performed according to Pende et al (48). Larvae were then transferred to blocking solution (5% normal goat serum, 1% BSA and 0.1% Triton X) for 3 hrs at RT and incubated for 3 days with either anti-tyrosine hydroxylase (TH) (1:250, Merck Millipore MAB318) or anti-SV2 (1:1000, DSHB SV2-C) in antibody dilution buffer (5% goat serum, 1% BSA and 0.1% Triton-X in PBS) at 4 \\u0026ordm;C with gentle agitation.. It should be noted that available antibodies detecting zebrafish TH only detect TH1 and not TH2, in which TH1 is the isoform predominantly expressed in the brain (49). Samples were then washed 6x15 min in PBS-Tx and incubated for 2 days at 4 \\u0026ordm;C with appropriate secondary antibody together with a 1:6000 dilution of DAPI in PBS-Tx. Larvae were then washed 3x15 min in PBS-Tx followed by 3x15 in PBS. Larvae were then transferred 50% refractive index solution (50% sucrose, 12% Antipyrin, 8% nicotinamide and 10% trietanolamine)(50) for at least 2 hrs at RT followed by at least 30 min in 100% refractive index solution. Larvae were then mounted in 2% low melt agarose on glass slides and imaged on Olympus FLUOVIEW FV3000 confocal laser scanning microscope.\\u003c/p\\u003e\\n\\u003cp\\u003eConfocal images were processed in Image J, using the plugin CLAHE to enhance local contrast(51). The number of DA neurons were calculated by counting tyrosine hydroxylase positive cells in the diencephalic posterior tuberculum DC1 area by moving through the confocal stack from the dorsal side until reaching the easily recognizable larger DA neurons of the DC2 (33). A maximum projection of the described area was generated and the DC1 neurons positioned anterior to the DC2 neurons was counted. The DC1 population of DA neurons includes \\u003cem\\u003eposterior tuberculi\\u003c/em\\u003e populations 5, 6, and 11 as shown in Sallinen et al (9) known to be specifically targeted by MPP\\u003csup\\u003e+\\u003c/sup\\u003e.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eStatistical analysis\\u003c/p\\u003e\\n\\u003cp\\u003eStatistical analysis was done with R, in RStudio. The mean\\u0026plusmn;standard error (SEM) was calculated for each genotype with respective treatment for \\u003cem\\u003en\\u003c/em\\u003e larvae from 3-5 different breedings. For each dataset, model choice was based on data distribution and scale. T-test or ANOVA were used for normally distributed data. For non-normally distributed data, generalized linear models (GLM) were created. Analysis was done between genotypes, MPP\\u003csup\\u003e+\\u003c/sup\\u003e exposure, and their interaction.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eSensorimotor response was measured as the positive response in percentage from n=10 touches, so the data was modelled with a generalized linear model using a quasibinomial distribution with logit link to account for overdispersion, followed by Tukey-adjusted pairwise comparisons. Total activity, activity separated by light phases, sleep latency and sleep bout duration in the locomotor activity monitor were analysed using a generalised linear model with a negative binomial distribution as the data showed overdispersion (variance \\u0026gt; mean). Post hoc comparisons of estimated marginal means were Tukey-adjusted. Sleep ratio separated by light phase were analysed using beta regression, followed by post hoc Tukey-adjusted comparisons on the fitted model. Lastly, differences in the number of cells positively stained for tyrosine hydroxylase were assessed with Type II ANOVA, with post hoc Tukey-adjusted comparisons. Statistical difference was defined as P-value \\u0026lt; 0.05. \\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003ch2\\u003eConflict of Interest\\u003c/h2\\u003e\\u003cp\\u003eNone declared.\\u003c/p\\u003e\\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e\\u003cp\\u003eCOST Action ImmuParkNet CA21117 (N.S.) and Advokat Rolf Sandberg Reberg og Ellen Marie Rebergs legat (K.E.F.). Funda\\u0026ccedil;\\u0026atilde;o para a Ci\\u0026ecirc;ncia e a Tecnologia (UIDB/04378/2020; UIDP/04378/2020; LA/P/0140/2020) (J.M.A.O).\\u003c/p\\u003e\\u003ch2\\u003eAuthor Contribution\\u003c/h2\\u003e\\u003cp\\u003eExperimental investigation, N.S., A.B, L.P.L, B.R.P, N.A.S.O; Formal Analysis, N.S., A.B., J.M.A.O, K.E.F.; Original Manuscript Writing, N.S., A.B., N.A.S.O, K.E.F..; All authors have read and agreed to the published version of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eData Availability\\u003c/h2\\u003e\\u003cp\\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eDorsey ER, Sherer T, Okun MS, Bloem BR. The Emerging Evidence of the Parkinson Pandemic. J Parkinsons Dis. 2018;8(s1):S3-S8.\\u003c/li\\u003e\\n\\u003cli\\u003eAscherio A, Schwarzschild MA. The epidemiology of Parkinson\\u0026apos;s disease: risk factors and prevention. Lancet Neurol. 2016;15(12):1257-72.\\u003c/li\\u003e\\n\\u003cli\\u003eNolano M, Provitera V, Estraneo A, Selim MM, Caporaso G, Stancanelli A, et al. Sensory deficit in Parkinson\\u0026apos;s disease: evidence of a cutaneous denervation. Brain. 2008;131(Pt 7):1903-11.\\u003c/li\\u003e\\n\\u003cli\\u003eChaudhuri KR, Healy DG, Schapira AH, National Institute for Clinical E. Non-motor symptoms of Parkinson\\u0026apos;s disease: diagnosis and management. Lancet Neurol. 2006;5(3):235-45.\\u003c/li\\u003e\\n\\u003cli\\u003eRoodveldt C, Bernardino L, Oztop-Cakmak O, Dragic M, Fladmark KE, Ertan S, et al. The immune system in Parkinson\\u0026apos;s disease: what we know so far. Brain. 2024;147(10):3306-24.\\u003c/li\\u003e\\n\\u003cli\\u003eYe H, Robak LA, Yu M, Cykowski M, Shulman JM. Genetics and Pathogenesis of Parkinson\\u0026apos;s Syndrome. Annu Rev Pathol. 2023;18:95-121.\\u003c/li\\u003e\\n\\u003cli\\u003eHolzschuh J, Ryu S, Aberger F, Driever W. Dopamine transporter expression distinguishes dopaminergic neurons from other catecholaminergic neurons in the developing zebrafish embryo. Mech Dev. 2001;101(1-2):237-43.\\u003c/li\\u003e\\n\\u003cli\\u003eRink E, Wullimann MF. The teleostean (zebrafish) dopaminergic system ascending to the subpallium (striatum) is located in the basal diencephalon (posterior tuberculum). Brain Res. 2001;889(1-2):316-30.\\u003c/li\\u003e\\n\\u003cli\\u003eSallinen V, Torkko V, Sundvik M, Reenila I, Khrustalyov D, Kaslin J, et al. MPTP and MPP+ target specific aminergic cell populations in larval zebrafish. J Neurochem. 2009;108(3):719-31.\\u003c/li\\u003e\\n\\u003cli\\u003eBonifati V, Rizzu P, van Baren MJ, Schaap O, Breedveld GJ, Krieger E, et al. Mutations in the DJ-1 gene associated with autosomal recessive early-onset parkinsonism. Science. 2003;299(5604):256-9.\\u003c/li\\u003e\\n\\u003cli\\u003eChoi J, Sullards MC, Olzmann JA, Rees HD, Weintraub ST, Bostwick DE, et al. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J Biol Chem. 2006;281(16):10816-24.\\u003c/li\\u003e\\n\\u003cli\\u003eAbulimiti A, Bae H, Ali A, Balakrishnan S, Tsujishita M, Gveric D, et al. Reduced DJ-1-F1Fo ATP synthase association correlates with midbrain dopaminergic neuron vulnerability in idiopathic Parkinson\\u0026apos;s disease. Sci Adv. 2025;11(23):eads3051.\\u003c/li\\u003e\\n\\u003cli\\u003eClements CM, McNally RS, Conti BJ, Mak TW, Ting JP. DJ-1, a cancer- and Parkinson\\u0026apos;s disease-associated protein, stabilizes the antioxidant transcriptional master regulator Nrf2. Proc Natl Acad Sci U S A. 2006;103(41):15091-6.\\u003c/li\\u003e\\n\\u003cli\\u003eLind-Holm Mogensen F, Sousa C, Ameli C, Badanjak K, Pereira SL, Muller A, et al. PARK7/DJ-1 deficiency impairs microglial activation in response to LPS-induced inflammation. J Neuroinflammation. 2024;21(1):174.\\u003c/li\\u003e\\n\\u003cli\\u003eFroyset AK, Edson AJ, Gharbi N, Khan EA, Dondorp D, Bai Q, et al. Astroglial DJ-1 over-expression up-regulates proteins involved in redox regulation and is neuroprotective in vivo. Redox Biol. 2018;16:237-47.\\u003c/li\\u003e\\n\\u003cli\\u003eFranco-Iborra S, Vila M, Perier C. Mitochondrial Quality Control in Neurodegenerative Diseases: Focus on Parkinson\\u0026apos;s Disease and Huntington\\u0026apos;s Disease. Front Neurosci. 2018;12:342.\\u003c/li\\u003e\\n\\u003cli\\u003eKyung JW, Kim JM, Lee W, Ha TY, Cha SH, Chung KH, et al. DJ-1 deficiency impairs synaptic vesicle endocytosis and reavailability at nerve terminals. Proc Natl Acad Sci U S A. 2018;115(7):1629-34.\\u003c/li\\u003e\\n\\u003cli\\u003eImberechts D, Kinnart I, Wauters F, Terbeek J, Manders L, Wierda K, et al. DJ-1 is an essential downstream mediator in PINK1/parkin-dependent mitophagy. Brain. 2022;145(12):4368-84.\\u003c/li\\u003e\\n\\u003cli\\u003eMoscovitz O, Ben-Nissan G, Fainer I, Pollack D, Mizrachi L, Sharon M. The Parkinson\\u0026apos;s-associated protein DJ-1 regulates the 20S proteasome. Nat Commun. 2015;6:6609.\\u003c/li\\u003e\\n\\u003cli\\u003eEdson AJ, Hushagen HA, Froyset AK, Elda I, Khan EA, Di Stefano A, et al. Dysregulation in the Brain Protein Profile of Zebrafish Lacking the Parkinson\\u0026apos;s Disease-Related Protein DJ-1. Mol Neurobiol. 2019;56(12):8306-22.\\u003c/li\\u003e\\n\\u003cli\\u003eChavali LNM, Yddal I, Bifulco E, Mannsaker S, Roise D, Law JO, et al. Progressive Motor and Non-Motor Symptoms in Park7 Knockout Zebrafish. Int J Mol Sci. 2023;24(7).\\u003c/li\\u003e\\n\\u003cli\\u003eRostad KO, Trognitz T, Froyset AK, Bifulco E, Fladmark KE. Accelerated Sarcopenia Phenotype in the DJ-1/Park7-Knockout Zebrafish. Antioxidants (Basel). 2024;13(12).\\u003c/li\\u003e\\n\\u003cli\\u003eFlinn L, Mortiboys H, Volkmann K, Koster RW, Ingham PW, Bandmann O. Complex I deficiency and dopaminergic neuronal cell loss in parkin-deficient zebrafish (Danio rerio). Brain. 2009;132(Pt 6):1613-23.\\u003c/li\\u003e\\n\\u003cli\\u003eFlinn LJ, Keatinge M, Bretaud S, Mortiboys H, Matsui H, De Felice E, et al. TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency. Ann Neurol. 2013;74(6):837-47.\\u003c/li\\u003e\\n\\u003cli\\u003eFett ME, Pilsl A, Paquet D, van Bebber F, Haass C, Tatzelt J, et al. Parkin is protective against proteotoxic stress in a transgenic zebrafish model. PLoS One. 2010;5(7):e11783.\\u003c/li\\u003e\\n\\u003cli\\u003eSaint-Amant L, Drapeau P. Time course of the development of motor behaviors in the zebrafish embryo. J Neurobiol. 1998;37(4):622-32.\\u003c/li\\u003e\\n\\u003cli\\u003eKetzef M, Spigolon G, Johansson Y, Bonito-Oliva A, Fisone G, Silberberg G. Dopamine Depletion Impairs Bilateral Sensory Processing in the Striatum in a Pathway-Dependent Manner. Neuron. 2017;94(4):855-65 e5.\\u003c/li\\u003e\\n\\u003cli\\u003eSilva RFO, Pinho BR, Santos MM, Oliveira JMA. Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment. Chemosphere. 2022;305:135449.\\u003c/li\\u003e\\n\\u003cli\\u003eBreen DP, Vuono R, Nawarathna U, Fisher K, Shneerson JM, Reddy AB, et al. Sleep and circadian rhythm regulation in early Parkinson disease. JAMA Neurol. 2014;71(5):589-95.\\u003c/li\\u003e\\n\\u003cli\\u003eLi H, Zhang J, Shen Y, Ye Y, Jiang Q, Chen L, et al. Targeting Mitochondrial Complex I Deficiency in MPP(+)/MPTP-induced Parkinson\\u0026apos;s Disease Cell Culture and Mouse Models by Transducing Yeast NDI1 Gene. Biol Proced Online. 2024;26(1):9.\\u003c/li\\u003e\\n\\u003cli\\u003eAnnesi G, Savettieri G, Pugliese P, D\\u0026apos;Amelio M, Tarantino P, Ragonese P, et al. DJ-1 mutations and parkinsonism-dementia-amyotrophic lateral sclerosis complex. Ann Neurol. 2005;58(5):803-7.\\u003c/li\\u003e\\n\\u003cli\\u003eHanagasi HA, Giri A, Kartal E, Guven G, Bilgic B, Hauser AK, et al. A novel homozygous DJ1 mutation causes parkinsonism and ALS in a Turkish family. Parkinsonism Relat Disord. 2016;29:117-20.\\u003c/li\\u003e\\n\\u003cli\\u003eRink E, Wullimann MF. Development of the catecholaminergic system in the early zebrafish brain: an immunohistochemical study. Brain Res Dev Brain Res. 2002;137(1):89-100.\\u003c/li\\u003e\\n\\u003cli\\u003eBrown SJ, Boussaad I, Jarazo J, Fitzgerald JC, Antony P, Keatinge M, et al. PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons. Sci Rep. 2021;11(1):6617.\\u003c/li\\u003e\\n\\u003cli\\u003eBandopadhyay R, Kingsbury AE, Cookson MR, Reid AR, Evans IM, Hope AD, et al. The expression of DJ-1 (PARK7) in normal human CNS and idiopathic Parkinson\\u0026apos;s disease. Brain. 2004;127(Pt 2):420-30.\\u003c/li\\u003e\\n\\u003cli\\u003eSheng D, Qu D, Kwok KH, Ng SS, Lim AY, Aw SS, et al. Deletion of the WD40 domain of LRRK2 in Zebrafish causes Parkinsonism-like loss of neurons and locomotive defect. PLoS Genet. 2010;6(4):e1000914.\\u003c/li\\u003e\\n\\u003cli\\u003eMilanese C, Sager JJ, Bai Q, Farrell TC, Cannon JR, Greenamyre JT, et al. Hypokinesia and reduced dopamine levels in zebrafish lacking beta- and gamma1-synucleins. J Biol Chem. 2012;287(5):2971-83.\\u003c/li\\u003e\\n\\u003cli\\u003eRen G, Xin S, Li S, Zhong H, Lin S. Disruption of LRRK2 does not cause specific loss of dopaminergic neurons in zebrafish. PLoS One. 2011;6(6):e20630.\\u003c/li\\u003e\\n\\u003cli\\u003ePrabhudesai S, Bensabeur FZ, Abdullah R, Basak I, Baez S, Alves G, et al. LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation. J Neurosci Res. 2016;94(8):717-35.\\u003c/li\\u003e\\n\\u003cli\\u003eRamesh T, Lyon AN, Pineda RH, Wang C, Janssen PM, Canan BD, et al. A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease. Dis Model Mech. 2010;3(9-10):652-62.\\u003c/li\\u003e\\n\\u003cli\\u003eSanyal S, Wintle RF, Kindt KS, Nuttley WM, Arvan R, Fitzmaurice P, et al. Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans. EMBO J. 2004;23(2):473-82.\\u003c/li\\u003e\\n\\u003cli\\u003eDodet P, Houot M, Leu-Semenescu S, Corvol JC, Lehericy S, Mangone G, et al. Sleep disorders in Parkinson\\u0026apos;s disease, an early and multiple problem. NPJ Parkinsons Dis. 2024;10(1):46.\\u003c/li\\u003e\\n\\u003cli\\u003eKalia LV, Lang AE. Parkinson\\u0026apos;s disease. Lancet. 2015;386(9996):896-912.\\u003c/li\\u003e\\n\\u003cli\\u003eHunt J, Coulson EJ, Rajnarayanan R, Oster H, Videnovic A, Rawashdeh O. Sleep and circadian rhythms in Parkinson\\u0026apos;s disease and preclinical models. Mol Neurodegener. 2022;17(1):2.\\u003c/li\\u003e\\n\\u003cli\\u003eXiong H, Wang D, Chen L, Choo YS, Ma H, Tang C, et al. Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation. J Clin Invest. 2009;119(3):650-60.\\u003c/li\\u003e\\n\\u003cli\\u003eDoyle JM, Croll RP. A Critical Review of Zebrafish Models of Parkinson\\u0026apos;s Disease. Front Pharmacol. 2022;13:835827.\\u003c/li\\u003e\\n\\u003cli\\u003ePinho BR, Reis SD, Guedes-Dias P, Leitao-Rocha A, Quintas C, Valentao P, et al. Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson\\u0026apos;s disease. Pharmacol Res. 2016;103:328-39.\\u003c/li\\u003e\\n\\u003cli\\u003ePende M, Vadiwala K, Schmidbaur H, Stockinger AW, Murawala P, Saghafi S, et al. A versatile depigmentation, clearing, and labeling method for exploring nervous system diversity. Sci Adv. 2020;6(22):eaba0365.\\u003c/li\\u003e\\n\\u003cli\\u003eChen YC, Priyadarshini M, Panula P. Complementary developmental expression of the two tyrosine hydroxylase transcripts in zebrafish. Histochem Cell Biol. 2009;132(4):375-81.\\u003c/li\\u003e\\n\\u003cli\\u003eLempereur S, Machado E, Licata F, Simion M, Buzer L, Robineau I, et al. ZeBraInspector, a platform for the automated segmentation and analysis of body and brain volumes in whole 5 days post-fertilization zebrafish following simultaneous visualization with identical orientations. Dev Biol. 2022;490:86-99.\\u003c/li\\u003e\\n\\u003cli\\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Parkinson’s disease, zebrafish model, sleep, motor, non-motor, prodromal, dopaminergic neurons, larva\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7848595/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7848595/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eParkinson's Disease (PD) is the fastest-growing neurological disorder and only symptomatic treatment is available. Zebrafish are ideally suited for high-throughput screening of disease modifying drugs and mechanistic studies. Mutations in \\u003cem\\u003epark7\\u003c/em\\u003e are associated with early-onset familial PD. Additionally, altered levels and subcellular location of the \\u003cem\\u003epark7\\u003c/em\\u003e protein product (DJ-1) have been found in PD patients without known \\u003cem\\u003epark7\\u003c/em\\u003e mutations. Here, we show that larval \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e zebrafish show reduced number of dopaminergic neurons and motor dysfunction, similarly to the 1-methyl-4-phenylpyridinium (MPP\\u003csup\\u003e+\\u003c/sup\\u003e)-induced PD model. Additionally, PD-associated prodromal symptoms, such as reduced sensory function, increased sleep latency and daytime sleepiness, were also observed in the \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e, but not in the MPP\\u003csup\\u003e+\\u003c/sup\\u003e-induced PD model. The \\u003cem\\u003epark7\\u003c/em\\u003e\\u003csup\\u003e\\u0026minus;/\\u0026minus;\\u003c/sup\\u003e is the first stable genetic zebrafish model of Parkinson\\u0026rsquo;s disease which shows both motor and non-motor symptoms, together with a reduction in dopaminergic neurons, at a larval stage. The model should therefore be highly valuable as a tool for PD related drug screening and mechanistic studies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Early motor deficits, sleep dysfunction and reduction in dopaminergic neurons in a park7 -/- zebrafish larval model of Parkinson’s disease\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-14 11:10:59\",\"doi\":\"10.21203/rs.3.rs-7848595/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-12-29T08:34:12+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-12-23T19:51:11+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"316860798451877162828626952425387481053\",\"date\":\"2025-12-03T14:58:03+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-12-02T13:50:07+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"57905877311213699266014068535930099850\",\"date\":\"2025-11-06T09:09:38+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-11-04T08:54:32+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-10-27T10:07:49+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-10-20T08:42:11+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-10-17T15:04:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Scientific Reports\",\"date\":\"2025-10-17T15:01:13+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"scientific-reports\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"scirep\",\"sideBox\":\"Learn more about [Scientific Reports](http://www.nature.com/srep/)\",\"snPcode\":\"\",\"submissionUrl\":\"\",\"title\":\"Scientific Reports\",\"twitterHandle\":\"\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"stoa\",\"reportingPortfolio\":\"Scientific Reports\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"44d40a75-8560-4b83-a02b-6beba8be5d0f\",\"owner\":[],\"postedDate\":\"November 14th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[{\"id\":57614501,\"name\":\"Health sciences/Diseases\"},{\"id\":57614502,\"name\":\"Biological sciences/Drug discovery\"},{\"id\":57614503,\"name\":\"Health sciences/Neurology\"},{\"id\":57614504,\"name\":\"Biological sciences/Neuroscience\"}],\"tags\":[],\"updatedAt\":\"2026-02-23T16:02:02+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7848595\",\"link\":\"https://doi.org/10.1038/s41598-026-39692-0\",\"journal\":{\"identity\":\"scientific-reports\",\"isVorOnly\":false,\"title\":\"Scientific Reports\"},\"publishedOn\":\"2026-02-18 15:58:25\",\"publishedOnDateReadable\":\"February 18th, 2026\"},\"versionCreatedAt\":\"2025-11-14 11:10:59\",\"video\":\"\",\"vorDoi\":\"10.1038/s41598-026-39692-0\",\"vorDoiUrl\":\"https://doi.org/10.1038/s41598-026-39692-0\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7848595\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7848595\",\"identity\":\"rs-7848595\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}