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ATP13A2 is the autosomal recessive causative gene for juvenile-onset Parkinson’s disease (PARK9, Parkinson’s disease 9), also known as Kufor-Rakeb syndrome. The disease is characterized by levodopa-responsive Parkinsonism, supranuclear gaze palsy, spasticity, and dementia. Previously, we have reported that Atp13a2 deficient medaka fish showed dopaminergic neurodegeneration and lysosomal dysfunction, indicating that lysosome-autophagy impairment might be one of the key pathogeneses of Parkinson’s disease. Here, we established Atp13a2 deficient zebrafish using CRISPR/Cas9 gene editing. We found that the number of TH + neurons in the posterior tuberculum and the locus coeruleus significantly reduced (dopaminergic neurons, 64 % at 4 months and 37 % at 12 months, p < 0.001 and p < 0.05, respectively; norepinephrine neurons, 52 % at 4 months and 40 % at 12 months, p < 0.001 and p < 0.05, respectively) in Atp13a2 deficient zebrafish, proving the degeneration of dopaminergic neurons. In addition, we found the reduction (60 %, p < 0.05) of cathepsin D protein expression in Atp13a2 deficient zebrafish using immunoblot. Transmission electron microscopy analysis using middle diencephalon samples from Atp13a2 deficient zebrafish showed lysosome-like bodies with vesicle accumulation and fingerprint-like structures, suggesting lysosomal dysfunction. Furthermore, a significant reduction (p < 0.001) in protein expression annotated with vesicle fusion with Golgi apparatus in Atp13a2 deficient zebrafish by liquid-chromatography tandem mass spectrometry suggested intracellular trafficking impairment. Therefore, we concluded that Atp13a2 deficient zebrafish exhibited degeneration of dopaminergic neurons, lysosomal dysfunction and the possibility of intracellular trafficking impairment, which would be the key pathogenic mechanism underlying Parkinson’s disease.

In larviculture of marine finfish, Tisochrysis lutea cultured cells have a potential to be a sustainable enrichment diet for rotifers due to its de novo synthesis of docosahexaenoic acid (DHA). To improve its effectiveness in rotifer enrichment for later larviculture, we determined which growth phase of T. lutea can enhance DHA in polar lipids (PLs) in Brachionus plicatilis sp. complex rotifers without reducing their vigor. Tisochrysis lutea was harvested at logarithmic growth phase, deceleration growth phase, and stationary phase. The ambient concentrations of phosphorus (P) and nitrogen (N), and fatty acid composition of microalgae were measured. P was limited between logarithmic and deceleration growth phases, followed by N starvation in stationary phase. Total lipid DHA was lowest in logarithmic growth phase, whereas DHA levels increased in deceleration growth phase and maintained at high levels even in stationary phase. PL-DHA also increased during deceleration growth phase, although its abundance dropped during stationary phase. In a rotifer enrichment trial, T. lutea harvested in each phase were fed to B. plicatilis sp. complex rotifers for 12 h. The highest PL-DHA value for cells in deceleration growth phase reflected the concentrations of the rotifers. No significant differences were found in rotifer growth rate and egg ratio. Rotifers fed cells in deceleration growth phase exhibited higher swimming speeds than those fed the other cells, potentially linking to increased larval capture success. Therefore, we recommend the deceleration growth phase as an optimal timing for T. lutea harvest for the effective enrichment of B. plicatilis sp. complex rotifers.

Euryhaline rotifer belonging to the genera Brachionus are often used as live feed for first feeding marine finfish larviculture. Two common rotifer enrichments, a single usage of docosahexaenoic acid (DHA)-enriched Chlorella vulgaris and a combination of DHA-enriched C. vulgaris and salmon roe emulsion oil were used to assess: (i) nutritional status of rotifers and, (ii) performance when rotifers were fed to Pagrus major and Paralichthys olivaceus larvae from 2 to 15 days after hatching (DAH). Both rotifer enrichment treatments were found to be nutritionally effective with high unsaturated fatty acid (HUFA) content, especially in the combined treatment with significantly higher eicosapentaenoic acid (EPA). Despite deficient levels of EPA, DHA-enriched C. vulgaris was found to be a suitable enrichment for rotifer and also had positive response in survival, growth and trypsin activity in both P. major and P. olivaceus. DHA-enriched C. vulgaris only treatment had higher rotifer population growth (13% higher), egg bearing capacity (7% higher), almost double the soluble protein content and generally higher free amino acid content. Oxidation of lipids in the combined diet due to its high-saturated fatty acid content could be a possible cause to the inferior performance of the combined treatment. Both P. major (20.1 ± 10.1%) and P. olivaceus (12.3 ± 3.87%) suffered low survival and lower gut content in the combined treatment especially during periods of significant growth in both species (7–11 DAH). Despite high HUFA content in the combined treatment, this study revealed that consideration of free amino acids and soluble protein content as parameters of nutritional indicators for first feeding P. major and P. olivaceus should not be discounted.

Summary: Parkinson’s disease (PD) is a neurodegenerative disease characterized by α-synuclein-positive inclusion bodies and loss of neurons, including dopaminergic neurons. Difficulty in replicating PD phenotypes using animal models partly limits the understanding of PD and the therapy required. Although PD is strongly associated with aging, most experimental animals may not exhibit age-related symptoms. Herein, we demonstrate that Nothobranchius furzeri, a rapidly aging teleost with a short life span, exhibits age-dependent degeneration of dopaminergic and noradrenergic neurons and progression of α-synuclein pathologies. These pathological phenotypes are similar to those observed in human patients with PD. Amelioration of the cell loss by genetic depletion of α-synuclein suggests that α-synuclein is not a bystander but a causative protein of neurodegeneration. N. furzeri can reveal mechanisms underlying PD, especially of the idiopathic form that affects a majority of patients with PD, including α-synuclein-dependent neurodegeneration, age-dependent phenotypes, and progression of α-synuclein pathology. : Matsui et al. find that annual killifish, Nothobranchius furzeri, reveals age-dependent degeneration of dopamine neurons and accumulation of α-synuclein. Genetic depletion of α-synuclein rescues these phenotypes. Keywords: Nothobranchius furzeri, aging, Parkinson’s disease, α-synuclein

Mitochondrial dysfunction and lysosomal dysfunction have been implicated in Parkinson’s disease (PD), but the links between these dysfunctions in PD pathogenesis are still largely unknown. Here we report that cytosolic dsDNA of mitochondrial origin escaping from lysosomal degradation was shown to induce cytotoxicity in cultured cells and PD phenotypes in vivo. The depletion of PINK1, GBA and/or ATP13A2 causes increases in cytosolic dsDNA of mitochondrial origin and induces type I interferon (IFN) responses and cell death in cultured cell lines. These phenotypes are rescued by the overexpression of DNase II, a lysosomal DNase that degrades discarded mitochondrial DNA, or the depletion of IFI16, which acts as a sensor for cytosolic dsDNA of mitochondrial origin. Reducing the abundance of cytosolic dsDNA by overexpressing human DNase II ameliorates movement disorders and dopaminergic cell loss in gba mutant PD model zebrafish. Furthermore, IFI16 and cytosolic dsDNA puncta of mitochondrial origin accumulate in the brain of patients with PD. These results support a common causative role for the cytosolic leakage of mitochondrial DNA in PD pathogenesis., Mitochondrial and lysosomal dysfunction are central to Parkinson’s disease (PD) pathogenesis. Here, the authors show mitochondrial dsDNA in the cytosol in cellular and Zebrafish models of PD induces cytotoxicity and neurodegeneration; knock-down of IFI16, a cytosolic dsDNA sensor, rescues cytotoxicity, as does overexpression of lysosomal DNAse II.

The euryhaline copepod Pseudodiaptomus inopinus play important roles in coastal waters as vectors of docosahexanoic (DHA) and eicosapentaenoic (EPA) acids for larval fish. While DHA and EPA in polar lipids (PLs) are more effective for fish larval development than non-polar lipid forms (NLs), there is little knowledge how much these lipids are accumulated in copepods from microalgae and are effective for early development of fish larvae. We report PLs fatty acid profiles of P. inopinus fed DHA-poor microalgae and evaluate its significance as a food source for larvae development of Pagrus major, compared with DHA-enriched rotifers. Copepods and rotifers were fed a mixed diet of three algal species (Phaeodactylum tricornutum, Tisochrysis lutea, and Pavlova lutheri), in addition of DHA-supplemented Super Fresh Chlorella (SFC) for rotifers. Compared with SFC, the algal mixture had higher EPA but lower DHA. Copepods had higher DHA and EPA in total lipids than rotifers fed each diet. Copepod PLs were specifically enriched with DHA and their contents were higher than both rotifers. On the other hand, PLs EPA contents were comparable between preys, indicating that copepods selectively fortified the PLs. Fish culture experiment showed that larvae fed copepods had higher growth than those fed SFC-enriched rotifers. Principal component analysis for each organism fatty acid composition emphasized trophic modification of DHA by copepods toward larval fish. This study highlighted that P. inopinus contribute to enhanced growth of coastal larval fish by efficiently transferring DHA via copepod fatty acid metabolism.

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p

Parkinson's disease is a neurodegenerative disorder that involves movement discloses, degeneration of dopaminergic neurons, and presence of cytoplasmic inclusion bodies. Various animal models have been developed and small fish including zebrafish and medaka fish have recently been employed as a new model for Parkinson disease. In this review, we summarize fish models of Parkinson's disease mainly using our own findings and explain two major hypotheses of PD: lysosome dysfunction theory and mitochondrial dysfunction theory. Finally, we discuss the potential for future application of small fish model.

Small teleost fish including zebrafish and medaka have been used as animal models for research because of their small body size, vast amounts of eggs produced, their rapid development, low husbandry costs, and transparency during embryogenesis. Although the body size and appearance seem different, fish and mammals including human still possess anatomical and functional similarities in their brains. This review summarizes the similarities of brain structures and functions between teleost fish and mammalian brains, focusing on the dopamine system, functional regionalization of the cerebellum, and presence of the nucleus ruber.

The effective use of distillery by-products of the shochu sweet potato has long been considered as a rich source of protein, fiber, amino acids, fatty acids, probiotics with a great potential as a functional feed for animals. This study applied fermented by-product of Shochu distillery (FBPSD) and salmon roe emulsion oil for rotifer enrichment to evaluate its effects on the: (i) nutritional composition of rotifers and (ii) feeding responses of Paralichthys olivaceus larvae when fed with enriched rotifers. The salmon roe oil enrichment was set as a control diet and FBPSD was supplemented at different doses (1, 5 and 10 % based on the salmon roe oil amount). The results showed that the rotifers supplemented with FBPSD at 5 and 10 % significantly enhanced protein, carbohydrate, and HUFA content. Similarly, the activity and expression profiles of targeted enzymes including trypsin, chymotrypsin, lipase and amylase effectively responded at early and middle-metamorphosis of the larvae to 5 and 10 % enrichment regiment. These two treatments were also found to be better supported in energy metabolism evidenced by Cytochrome-C-Oxidase (cox) and ATPase-pk (atp) gene expression in the larvae. Moreover, the expression levels of immune-related genes (IL-10, IL-1β, TNF-α, P2Y6) and antioxidant-related genes (SOD, CAT) were significantly improved at 5 and 10 % of FBPSD. Cessation of feeding and delay in growth during the metamorphic process was also molecularly demonstrated. Larvae suffered low survival (24.5–25.3 %) in control and 1 % supplementation of FBPSD, but significantly improved at 5 and 10 % (36.4–38.7%). However, the growth performance of larval fish was not remarkable after 40 days of culture. The results of this study revealed that FBPSD could be considered as a potential supplementary enrichment for live feed in fish larviculture, especially at 5 and 10 % rotifer enrichment dosage.

Kawakawa tuna (Euthynnus affinis) is a novel target species for aquaculture in Japan and currently there is very limited knowledge on its early ontogenetic development in captivity. The ontogeny of E. affinis larvae from hatching to 20 days after hatching (DAH) was illustrated in the present study through an integrated approach of histology, biochemistry and molecular approaches. Larvae were fed enriched L-type rotifer Brachionus plicatilis species complex from 2 to 10 DAH, followed by enriched Artemia nauplii. Target digestive enzymes; trypsin, pepsin, lipase and amylase were assessed in order to evaluate nutritional condition. The larvae began exogenous feeding from 2 DAH, coinciding with the opening of the oral capacity and anus. The yolk sac was rapidly consumed and completely resorbed at 3 DAH, while the oil globule disappeared by 5 DAH. Enzymatic activity and expression of the target enzymes were detectable at hatching, except pepsin. With the development of the gastric glands, pepsin activity was then detected at 10 DAH, which was followed by rapid increase in pepsin activity at 15 DAH. The larvae showed rapid growth, however, high mortality also occurred at 6–10 DAH which has been highlighted as a critical stage in the larviculture of E. affinis. From 15 to 20 DAH, the histological structures of the digestive tract were completely segmented with the digestive tract resembling that of its juvenile stages. The activity of trypsin, pepsin, and lipase tended to increase sharply, while amylase dramatically dropped, possibly indicating a change in dietary preference. Findings from the present study provides a preliminary insight into the early development of the digestive system and associated digestive enzymes of E. affinis larvae and establishes an initial step towards the rearing and feeding protocol optimization of this novel target species.

The microalga Nannochloropsis oculata is used as a diet for rotifers and can enrich the highly unsaturated fatty acid (HUFA) proportion of rotifer polar lipids (PL-HUFA). For efficient enrichment of PL-HUFAs in rotifers, we evaluated nutrient–sufficient or –deficient cell populations to clarify when the alga should be harvested and fed to rotifers. The nutrient conditions for N. oculata were spectrophotometrically monitored during cultivation and cells with nutrient sufficiency (NS) and phosphorous deficiency (PD) were harvested. The PD cells exhibited reduced chlorophyll a content, photosynthetic activity, and growth rate and accumulated total lipids as non-polar lipids (NLs). A decrease in the proportion of HUFA to total lipids between two nutrient states corresponded to a similar phenomenon observed in NLs. On the other hand, PLs, including glycolipids and phospholipids, showed different trends. Glycolipids did not change fatty acid composition between two states. Phospholipids, however, showed significantly higher contents of arachidonic acid (ArA) and eicosapentaenoic acid (EPA) only in the early periods of PD culture. It was concluded that phosphorous deficiency stimulated cells to increase the fatty acid desaturation rate of non-plastidial membranes. Mitochondria in the PD cells decreased in number but increased in size and mass. Such adaptation of mitochondria can be associated with high desaturation of non-plastidial membranes. When the early P–limited phase culture was used for the enrichment of rotifers, PL-ArA and EPA of the rotifers were enhanced by twice as much as those fed on NS cells. This study provides evidence that the beginning of phosphorous deficiency is the optimum time to harvest N. oculata cells for enhancing rotifer PL-HUFAs.

BACKGROUND Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

A modified method of cerebrospinal fluid injection was developed for the efficient and reliable administration of substances to the zebrafish central nervous system. The accuracy of this modified method was evaluated using Alexa Fluor dye injection. A high survival ratio was achieved due to the simplicity of the procedure and ice-tricaine combined anaesthesia. To validate this new method, we injected ammonium chloride, which successfully blocked lysosome function resulting in elevated LC3-II and the accumulation of ubiquitinated proteins. Injection of human α-synuclein fibrils initiated a prion-like propagation of α-synuclein pathology in zebrafish. This method can be used to investigate the effects of various substances and the propagation of α-synuclein in the central nervous system.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output. Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon's start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Teleost fish have recently been employed as a model for human neurodegenerative diseases. We used toxin exposure and genetic engineering to develop models of Parkinson's disease (PD) in the teleost fish, medaka. Among the toxins examined, 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors, and tunicamycin all induced important features of PD in medaka. Specifically, these agents induced dopaminergic cell loss and reduced spontaneous movement, and the latter three toxins produced inclusion bodies that were ubiquitously distributed in the medaka brain. Despite the extensive distribution of these inclusion bodies, the middle diencephalic dopaminergic neurons were particularly susceptible to the effect of the toxins, suggesting that this cluster of dopaminergic neurons is analogous to the human substantia nigra. We have also created a variety of different genetic models using the Targeting Induced Local Lesions in Genomes (TILLING) method, and found that neither PTEN-induced putative kinase 1 (PINK1) mutants nor Parkin mutants disclosed significant dopaminergic cell loss. Surprisingly however, PINK1 and Parkin double mutants exhibited selective dopamine cell loss, as well as aggregation and deficit of mitochondrial activity. Another mutant, the ATP13A2 mutant, also expressed a PD phenotype, exhibiting marked cathepsin D reduction and fingerprint-like structures that are generally found in lysosome storage diseases. Taken together, these data indicate that medaka fish can serve as a new animal model for PD. In this review, we summarize our data and discuss the potential for future application of this animal model.

This study aimed to determine whether enteric neurons are involved in multiple system atrophy (MSA). Four-μm-thick slices of small intestine were prepared from 10%-formalin-fixed and paraffin-embedded materials obtained from autopsied cases. Enteric neurons were stained using an anti-peripherin antibody. Immunostaining of phosphorylated α-synuclein was also performed. Areas of the cytoplasm and nucleus that showed nucleoli were measured using computer software. Both areas of myenteric neurons were significantly smaller in MSA cases (n = 3) than in control subjects (n = 3) (P 0.0001); however, no deposits of phosphorylated α-synuclein were observed. These findings suggest that myenteric neurons in MSA are affected independent of α-synuclein accumulation.

Small teleost fish including zebrafish and medaka have been used as animal models in basic science research due to the relative ease of handling and transparency during embryogenesis. Current advances in genetic engineering and progress in disease genetics allowed utilization of these fish to study neurological diseases and psychiatric disorders. This review summarizes the advantages and disadvantages of using fish for neuropsychiatric research using primarily our own studies as examples. We discuss how fish belong to a class of vertebrates, are feasible for imaging, and include diverse species with multiple research possibilities yet to be discovered.

The teleost fish has been widely used in creating neurodegenerative models. Here we describe the teleost medaka fish Parkinson's disease (PD) models we developed using toxin treatment and genetic engineering. 1-Methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), proteasome inhibitors, lysosome inhibitors and tunicamycin treatment in our model fish replicated some salient features of PD: selective dopamine cell loss and reduced spontaneous movement with the last three toxins producing inclusion bodies ubiquitously in the brain. Despite the ubiquitous distribution of the inclusion bodies, the middle diencephalic dopaminergic neurons were particularly vulnerable to these toxins, supporting the idea that this dopamine cluster is similar to the human substantia nigra. PTEN-induced putative kinase 1 (PINK1) homozygous mutants also showed reduced spontaneous swimming movements. These data indicate that medaka fish can serve as a new model animal of PD. In this review we summarize our previous data and discuss future prospects.

Perturbations in protein folding and degradation are key pathological mechanisms in neurodegenerative diseases, including Parkinson's disease (PD). Recent evidence suggests that mishandling of proteins may play an important role in the pathogenesis of PD. We have utilized medaka fish to monitor the effects of injecting neurotoxins into the CSF space. In this study, ammonium chloride, tunicamycin, and lactacystin were tested for their ability to disturb lysosomal proteolysis, N-glycosylation in the endoplasmic reticulum, and proteasomal degradation, respectively. All of the substances tested induced selective loss of dopaminergic neurons, movement disorders and inclusion bodies. Among them, the features of the inclusion bodies that developed after ammonium chloride injection mimicked those of PD: co-localization of ubiquitin and phosphorylated α-synuclein, as well as the presence of LC3 protein in the inclusion bodies. Our study demonstrated that medaka fish are useful for examining the effects of environmental toxins and lysosome inhibition, and lysosome inhibitors may be factors in the development of PD.

Recent findings suggest that a defect in the ubiquitin-proteasome system plays an important role in the pathogenesis of Parkinson's disease (PD). A previous report (McNaught et al. 2004) demonstrated that rats systemically injected with proteasome inhibitors exhibited PD-like clinical symptoms and pathology. However, because these findings have not been consistently replicated, this model is not commonly used to study PD. We used medaka fish to test the effect of systemic administration of proteasome inhibitors because of the high level of accessibility of the cerebrospinal fluid in fish. We injected lactacystin or epoxomicin into the CSF of medaka. With proteasome inhibition in the medaka brain, selective dopaminergic and noradrenergic cell loss was observed. Furthermore, treated fish exhibited reduced spontaneous movement. Treatment with proteasome inhibitors also induced the formation of inclusion bodies resembling Lewy bodies, which are characteristic of PD. Treatment with 6-OHDA also induced dopaminergic cell loss but did not produce inclusion bodies. These findings in medaka are consistent with previous results reporting that non-selective proteasome inhibition replicates the cardinal features of PD: locomotor dysfunction, selective dopaminergic cell loss, and inclusion body formation.

Parkinson's disease is a neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. The PTEN-induced kinase 1 gene (PINK1) is responsible for recessive inherited familial Parkinson's disease (PARK6). Neither the function of PINK1 nor its role in the prevention of Parkinson's disease is fully understood. Gene disruption of PINK1 causes remarkably different phenotypes in animal models such as Drosophila melanogaster, zebrafish, and mouse, none of which recapitulate Parkinson's-disease-like symptoms. We established PINK1-gene-disrupted medaka fish. These mutant fish grew normally at first, then developed significant decrease in the frequency of spontaneous swimming movements in the late-adult stage. Although the mutants did not show any dopaminergic cell loss, the amount of 3,4-dihydroxyphenylacetic acid, a major metabolite of dopamine, decreased. Thus, PINK1 contributes to the maintenance of dopamine metabolism, even before the selective death of dopaminergic neurons. Our animal model is therefore a valuable tool to detect pathogenesis in Parkinson's patients in the early stages.

The red nucleus is located in the rostral midbrain of the vertebrate brain and controls motor coordination during locomotion. It receives input from the cerebellum and sends its output to the spinal cord. The presence of the red nucleus is well established in tetrapods, and its existence has also been suggested in teleosts but its presence and position has still been under discussion. By using wheat germ agglutinin (WGA) as a genetically encoded anterograde tracer, we recently identified contralateral projections from the cerebellum to a putative red nucleus in the zebrafish midbrain tegmentum. In this report we further revealed red nucleus derived from this contralateral afferent from the cerebellum using WGA and contralateral projections to the hindbrain-spinal cord junction site using DiI-mediated retrograde tracing. Thus the structure that we have identified by anterograde and retrograde tracing fulfills the anatomical demands for the red nucleus: the location in the midbrain tegmentum, contralateral afferent from the cerebellum (cerebello- ruber projection) and contralateral efferent to the spinal cord (rubro-spinal projection).

In recent years, considerable effort has been devoted to the development of a fish model for Parkinson’s disease (PD) to examine the pathological mechanisms of neurodegeneration. To effectively evaluate PD pathology, the ability to accurately and reliably count dopaminergic neurons is important. However, there is currently no such standardized method. Due to the relatively small number of dopaminergic neurons in fish, stereological estimation would not be suitable. In addition, serial sectioning requires proficiency to not lose any sections, and it permits double counting due to the large size of some of the dopaminergic neurons. In this study, we report an optimized protocol for staining dopaminergic neurons in zebrafish and provide a reliable counting method. Finally, using our optimized protocol, we confirmed that administration of 6-hydroxydopamine (a neurotoxin) or the deletion of the PINK1 gene (one of the causative genes of familiar PD) in zebrafish caused significant reduction in the number of dopaminergic and noradrenergic neurons. In summary, this method will serve as an important tool for the appropriate evaluation and establishment of fish PD models.

There has been accumulating evidence for a regionalized organization of the cerebellum, which was mostly deduced from anatomical mapping of axonal projections of cerebellar afferents. A likewise regionalization of the cerebellar output has been suggested from lesion studies and dye-tracer experiments, but its physiological targets as well as the functional relevance of such an output regionalization are less clear. Ideally, such functional regionalization should be proven noninvasively in vivo. We here provide evidence for such a regionalization of the output from the cerebellar cortex by genetically encoded transneuronal mapping of efferent circuits of zebrafish Purkinje neurons. These identified circuits correspond to distinct regionalized Purkinje cell activity patterns in freely behaving zebrafish larvae during the performance of cerebellar-dependent behaviors. Furthermore, optogenetic interrogation of selected Purkinje cell regions during animal behavior confirms the functional regionalization of Purkinje cell efferents and reveals their contribution to behavior control as well as their function in controlling lateralized behavioral output. Our findings reveal how brain compartments serve to fulfill a multitude of functions by dedicating specialized efferent circuits to distinct behavioral tasks.

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by selective dopaminergic cell loss in the substantia nigra, but its pathogenesis remains unclear. The recessively inherited familial PD genes PARK2 and PARK6 have been attributed to mutations in the Parkin and PTEN-induced kinase 1 (PINK1) genes, respectively. Recent reports suggest that PINK1 works upstream of Parkin in the same pathway to regulate mitochondrial dynamics and/or conduct autophagic clearance of damaged mitochondria. This phenomenon is preserved from Drosophila to human cell lines but has not been demonstrated in a vertebrate animal model in vivo. Here, we developed a medaka fish (Oryzias latipes) model that is deficient in Pink1 and Parkin. We found that despite the lack of a conspicuous phenotype in single mutants for Pink1 or Parkin, medaka that are deficient in both genes developed phenotypes similar to that of human PD: late-onset locomotor dysfunction, a decrease in dopamine levels and a selective degeneration of dopaminergic neurons. Further analysis also revealed defects in mitochondrial enzymatic activity as well as cell death. Consistently, PINK1 and Parkin double-deficient MEF showed a further decrease in mitochondrial membrane potential and mitochondrial complex I activity as well as apoptosis compared with single-deficient MEF. Interestingly, these mitochondrial abnormalities in Parkin-deficient MEF were compensated by exogenous PINK1, but not by disease-related mutants. These results suggest that PINK1 and Parkin work in a complementary way to protect dopaminergic neurons by maintaining mitochondrial function in vertebrates.

Kufor-Rakeb syndrome (KRS) was originally described as an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia. ATP13A2 was identified as the causative gene in KRS. ATP13A2 encodes the ATP13A2 protein, which is a lysosomal type5 P-type ATPase, and ATP13A2 mutations are linked to autosomal recessive familial parkinsonism.Here, we report that normal ATP13A2 localizes in the lysosome, whereas disease-associated variants remain in the endoplasmic reticulum. Cathepsin D activity was decreased in ATP13A2-knockdown cells that displayed lysosome-like bodies characterized by fingerprint-like structures. Furthermore, an atp13a2 mutation in medaka fish resulted in dopaminergic neuronal death, decreased cathepsin D activity, and fingerprint-like structures in the brain. Based on these results, lysosome abnormality is very likely to be the primary cause of KRS/PARK9.

Autosomal recessively inherited, loss of function mutations in PTEN‐induced kinase 1 (PINK1) typically lead to early onset Parkinson disease (EOPD).1 The PINK1 protein is expressed ubiquitously throughout the human brain.2 Impaired mitochondrial function and morphology have been described in both human PINK1 mutant patient tissue and different PINK1‐deficient in vitro or in vivo model systems.3 PINK1 has also been implicated in oxidative stress defense, mitophagy, and the regulation of mitochondrial calcium homeostasis.3 However, the precise mechanisms leading to neuronal cell death remain unclear. Pink1 knockout mice do not develop loss of dopamine (DA) neurons in the substantia nigra and can therefore only be of limited use in investigating the mechanisms leading to neuronal cell death in human Parkinson disease (PD).6 Zebrafish are increasingly being used to model neurodegenerative diseases.7 As vertebrates, they are closer to humans than other genetically tractable model organisms such as Drosophila or Caenorhabditis elegans. Zebrafish embryos develop externally, are transparent, and have a well‐characterized DA nervous system.8 To date, investigations of the functional consequences of PD gene dysfunction in zebrafish have relied on the injection of morpholino antisense oligonucleotides (MOs).7 A major limitation of this approach is that MOs injected into the fertilized egg lose their effect within 3 to 5 days postfertilization, thus precluding investigation of the morphological, biochemical, or behavioral effects of gene dysfunction at larval and adult stages. In addition, morpholinos are frequently associated with nonspecific, off‐target effects.9 Previous studies using the MO strategy to investigate the effects of PINK1 deficiency in zebrafish have led to conflicting results.10 Using the targeting induced local lesions in genomes (TILLING) approach, we have now established a stable line carrying a premature stop mutation in the kinase‐encoding domain of pink1 (Y431*), the zebrafish orthologue of human PINK1. We provide confirmation that this mutation leads to inactivation of PINK1 catalytic activity and decreased mRNA stability. We further demonstrate that PINK1 deficiency in Danio rerio results in highly specific abnormalities in early development, which closely match the biochemical and morphological manifestations of the human disease, with persisting loss of dopaminergic neurons in adulthood and also persisting mitochondrial impairment. Genome‐wide gene expression studies identified upregulation of TigarB, the zebrafish homologue of the TP53‐induced glycolysis and apoptosis regulator (TIGAR).13 Remarkably, TigarB knockdown resulted in normalization of mitochondrial function and complete rescue of ascending dopaminergic neurons. Modulation of TIGAR‐related mechanisms may therefore be a promising novel strategy to develop disease‐modifying therapy for PINK1‐related PD.

Parkinson's disease (PD) is the second most common neurodegenerative disease associated with the degeneration of dopaminergic neurons in the substantia nigra. To create a new model of PD, we used medaka (Oryzias latipes), a small teleost that has been used in genetics and environmental biology. We identified tyrosine hydroxylase (TH) immunopositive dopaminergic and noradrenergic fibers and neurons in the medaka brain. Following establishment of a method for counting the number of dopaminergic neurons and an assay for the evaluation of the medaka behavior, we exposed medaka to 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP). The treatment of medaka at the larval stage, but not at adult stage, decreased the number of dopaminergic cells in the diencephalon and reduced spontaneous movement, which is reminiscent of human PD patients and other MPTP-induced animal PD models. Among TH(+) neurons in the medaka brain, only a specific cluster in the paraventricular area of the middle diencephalon was vulnerable to MPTP toxicity. Detailed examinations of medaka transiently exposed to MPTP at the larval stage revealed that the number of dopaminergic cells was not fully recovered at their adult stage. Moreover, the amounts of dopamine persistently decreased in the brain of these MPTP-treated fish. MPTP-treated medaka is valuable for modeling human PD.

Homozygous mutations in the glucocerebrosidase (GBA) gene result in Gaucher disease (GD), the most common lysosomal storage disease. Recent genetic studies have revealed that GBA mutations confer a strong risk for sporadic Parkinson’s disease (PD). To investigate how GBA mutations cause PD, we generated GBA nonsense mutant (GBA-/-) medaka that are completely deficient in glucocerebrosidase (GCase) activity. In contrast to the perinatal death in humans and mice lacking GCase activity, GBA-/- medaka survived for months, enabling analysis of the pathological progression. GBA-/- medaka displayed the pathological phenotypes resembling human neuronopathic GD including infiltration of Gaucher cell-like cells into the brains, progressive neuronal loss, and microgliosis. Detailed pathological findings represented lysosomal abnormalities in neurons and alpha-synuclein (α-syn) accumulation in axonal swellings containing autophagosomes. Unexpectedly, disruption of α-syn did not improve the life span, formation of axonal swellings, neuronal loss, or neuroinflammation in GBA-/- medaka. Taken together, the present study revealed GBA-/- medaka as a novel neuronopathic GD model, the pahological mechanisms of α-syn accumulation caused by GCase deficiency, and the minimal contribution of α-syn to the pathogenesis of neuronopathic GD., Author Summary Parkinson’s disease (PD) is a neurodegenerative disease characterized by intraneuronal accumulation of alpha-synuclein (α-syn) called Lewy bodies and Lewy neurites. Recent genetic studies have revealed that mutations in glucocerebrosidase (GBA), a causative gene of Gaucher disease (GD), are a strong risk for PD. However, its pathological mechanisms leading to PD remain largely unknown. Here, we generated GBA nonsense mutant (GBA -/-) medaka which survive long enough for pathological analysis of disease progression. These mutant medaka display not only the phenotypes resembling human neuronopathic GD but also axonal accumulation of α-syn accompanied by impairment of the autophagy-lysosome pathway. Furthermore, the present study demonstrates this α-syn accumulation has negligible contribution to the pathogenesis of neuronopathic GD in medaka. GBA -/- medaka represent a valuable model for exploring the pathological mechanisms of PD with GBA mutations as well as neuronopathic GD, and our findings have important implications for the association of GBA mutations with PD.