Dual topologies of myotomal collagen XV and Tenascin C act in concert to guide and shape developing motor axons.

During development, motor axons are guided toward muscle target by various extrinsic cues including extracellular matrix (ECM) proteins whose identities and cellular source remain poorly characterized. Here, using single-cell RNAseq of sorted GFP+ cells from smyhc1:gfp-injected zebrafish embryos, we unravel the slow muscle progenitors (SMP) pseudotemporal trajectory at the single-cell level and show that differentiating SMPs are a major source of ECM proteins. The SMP core-matrisome was characterized and computationally predicted to form a basement membrane-like structure tailored for motor axon guidance, including basement membrane-associated ECM proteins, as collagen XV-B, one of the earliest core-matrisome gene transcribed in differentiating SMPs and the glycoprotein Tenascin C. To investigate how contact-mediated guidance cues are organized along the motor path to exert their function in vivo, we used microscopy-based methods to analyze and quantify motor axon navigation in tnc and col15a1b knock-out fish. We show that motor axon shape and growth rely on the timely expression of the attractive cue Collagen XV-B that locally provides axons with a permissive soft microenvironment and separately organizes the repulsive cue Tenascin C into a unique functional dual topology. Importantly, bioprinted micropatterns that mimic this in vivo ECM topology were sufficient to drive directional motor axon growth. Our study offers evidence that not only the composition of ECM cues but their topology critically influences motor axon navigation in vertebrates with potential applications in regenerative medicine for peripheral nerve injury as regenerating nerves follow their original path.

Quantitative Image Analysis of Axonal Morphology in In Vivo Model.

Quantifying axonal branching is crucial for understanding neural circuit function, developmental and regeneration processes and disease mechanisms. Factors that regulate patterns of axonal arborization and tune neuronal circuits are investigated for their implication in various disorders in brain connectivity. The lack of a reliable and user-friendly method makes the quantitative analysis of axon morphology difficult. Specifically, methods to visualize and quantify the complex axon arborization are challenging to implement and apply practically. Our study was aimed at developing a robust but simple method of quantification that used ImageJ 2D analysis and compared it with Imaris visualization and analysis of 3D images. We used zebrafish fluorescent transgenic lines to perform in vivo imaging of developing motor neuron axons that adequately reflected the complexity of axonal networks. Our new method, developed on ImageJ, is easy and fast, giving access to new information such as collateral distribution along the axonal shaft. This study describes step-by-step procedures that can be easily applied to a variety of organisms and in vitro systems. Our study provides a basis for further exploration of neural circuits to gain new insights into neuronal disorders and potential therapeutic interventions.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

Superfast excitation-contraction coupling in adult zebrafish skeletal muscle fibers.

The zebrafish has emerged as a very relevant animal model for probing the pathophysiology of human skeletal muscle disorders. This vertebrate animal model displays a startle response characterized by high-frequency swimming activity powered by contraction of fast skeletal muscle fibers excited at extremely high frequencies, critical for escaping predators and capturing prey. Such intense muscle performance requires extremely fast properties of the contractile machinery but also of excitation-contraction coupling, the process by which an action potential spreading along the sarcolemma induces a change in configuration of the dihydropyridine receptors, resulting in intramembrane charge movements, which in turn triggers the release of Ca2+ from the sarcoplasmic reticulum. However, thus far, the fastest Ca2+ transients evoked by vertebrate muscle fibers has been described in muscles used to produce sounds, such as those in the toadfish swim bladder, but not in muscles used for locomotion. By performing intracellular Ca2+ measurements under voltage control in isolated fast skeletal muscle fibers from adult zebrafish and mouse, we demonstrate that fish fast muscle fibers display superfast kinetics of action potentials, intramembrane charge movements, and action potential-evoked Ca2+ transient, allowing fusion and fused sustained Ca2+ transients at frequencies of excitation much higher than in mouse fast skeletal muscle fibers and comparable to those recorded in muscles producing sounds. The present study is the first demonstration of superfast kinetics of excitation-contraction coupling in skeletal muscle allowing superfast locomotor behaviors in a vertebrate.

[The role of extracellular matrix in the regeneration of motor nerves]. / Le rôle de la matrice extracellulaire dans la régénération des nerfs moteurs.

The motor neurons (MN) form the ultimate route to convey the commands from the central nervous system to muscles. During development, MN extend axons that follow stereotyped trajectories to their muscle targets, guided by various attractive and repulsive molecular cues. Extracellular matrix (ECM) is a major source of guidance cues, but its role in axonal development and regeneration remains poorly documented. Regenerating axons are able to return to their synaptic target following their original trajectory. The same guidance cues could be thus involved in motor nerve regeneration. Zebrafish has become a popular model system in understanding the development of the peripheral nervous system. Thanks to the generation of fluorescent transgenic lines and the optical transparency of embryos and larvae, it allows direct visualization of axonogenesis. Additionally, and contrary to humans, its remarkable capacity to regenerate makes it well suited for the study of nerve regeneration. A laser method to ablate nerves in living zebrafish larvae has been developed in our laboratory that, combined with the use of the fluorescent mnx1:gfp zebrafish transgenic line, allows the follow up of the dynamics of the nerve regeneration process. To study the role of ECM proteins present in the axonal path, mutant lines for different ECM proteins (already available in our laboratory or generated in mnx1:gfp fish using CRISPR-Cas9 method) will be used to analyze their role during the regeneration process. These mutant lines for ECM will be crossed with existing fluorescent transgenic lines to visualize different cell types involved in the nerve regeneration, such as macrophages (mfap4:mcherry), neutrophils (mpx:gfp) or even Schwann cells (sox10:mrfp). Overall, this study will depict the role of ECM in nerve regeneration and will provide essential knowledge for the development of new biomaterials to promote the regeneration of injured motor nerves.

Collagen XV, a multifaceted multiplexin present across tissues and species.

Type XV collagen is a non-fibrillar collagen that is associated with basement membranes and belongs to the multiplexin subset of the collagen superfamily. Collagen XV was initially studied because of its sequence homology with collagen XVIII/endostatin whose anti-angiogenic and anti-tumorigenic properties were subjects of wide interest in the past years. But during the last fifteen years, collagen XV has gained growing attention with increasing number of studies that have attributed new functions to this widely distributed collagen/proteoglycan hybrid molecule. Despite the cumulative evidence of its functional pleiotropy and its evolutionary conserved function, no review compiling the current state of the art about collagen XV is currently available. Here, we thus provide the first comprehensive view of the knowledge gathered so far on the molecular structure, tissue distribution and functions of collagen XV in development, tissue homeostasis and disease with an evolutionary perspective. We hope that our review will open new roads for promising research on collagen XV in the coming years.

Spatio-temporal expression and distribution of collagen VI during zebrafish development.

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

[Unraveling the pathophysiology of Bethlem Myopathy using a unique zebrafish model for the disease]. / Étude physiopathologique de la myopathie de Bethlem à l'aide d'un modèle de poisson zèbre - 16es JSFM : Prix Master 2018.

Bethlem myopathy (BM) is a neuromuscular disease characterized by joint contractures and muscle weakness. BM is caused by mutations in one of the genes encoding one of the three α-chains of collagen VI (COLVI), a component of the skeletal muscle extracellular matrix. Nowadays, an unresolved question is to understand how alteration of COLVI located outside the muscle cells leads to functional modifications in muscle fibers. The zebrafish model col6a1Δex14 is currently the unique animal model of the disease since it is the only model to reproduce a mutation that is the most frequently found in BM patients. In patient and col6a1Δex14 zebrafish muscles, the structure of the sarcoplasmic reticulum has been found to be altered, thus suggesting dysfunction in intracellular Ca2+ handling and/or in ion channels that are known to control Ca2+ homeostasis and to play pivotal roles in muscle function and pathogenesis. Therefore, our project aims at exploring the properties of ion channels and intracellular Ca2+ regulation using electrophysiological approaches and intracellular Ca2+ measurement at rest and during activity in isolated muscle fibers from col6a1Δex14 zebrafish. On one hand, this project should contribute to decipher how alteration in an extracellular matrix component transduces pathogenic signals within muscle fiber and should possibly lead to identify therapeutic targets for this currently incurable disease. On the other hand, because functional studies on zebrafish muscle cells are scarce, this project will provide a sound database on the electrophysiological properties of this cell model.

TITLE: Étude physiopathologique de la myopathie de Bethlem à l'aide d'un modèle de poisson zèbre - 16es JSFM : Prix Master 2018. ABSTRACT: La myopathie de Bethlem (BM) est une maladie caractérisée par des rétractions et une faiblesse musculaires. Cette pathologie résulte de mutations dans un des gènes codant l'une des trois chaînes α du collagène VI (COLVI), un composant de la matrice extracellulaire musculaire squelettique. Aujourd'hui, une question non résolue est de comprendre comment l'altération de COLVI présent à l'extérieur des cellules musculaires conduit à des modifications fonctionnelles dans les fibres musculaires. Le modèle poisson zèbre col6a1Δex14 est actuellement un modèle animal unique de la BM puisqu'il est le seul à reproduire spécifiquement l'une des mutations la plus fréquemment retrouvée chez les patients. Chez les patients et le poisson col6a1Δex14, la structure du réticulum sarcoplasmique est altérée, suggérant une perturbation de l'homéostasie calcique musculaire et/ou des canaux ioniques qui, en contrôlant cette homéostasie, jouent un rôle crucial dans la fonction et la pathogenèse musculaire. Notre projet vise ainsi à étudier à l'aide de techniques électrophysiologiques et de mesure de Ca2+ les propriétés des canaux ioniques et la régulation du Ca2+ intracellulaire au repos et en activité dans la fibre musculaire du poisson col6a1Δex14. Nos recherches devraient contribuer à mieux comprendre comment la perturbation de la matrice influe sur la fonction musculaire et conduire à terme à identifier des cibles thérapeutiques pour traiter cette maladie actuellement incurable. Enfin, du fait de la rareté des études fonctionnelles sur la cellule musculaire de poisson zèbre, ce projet permettra de constituer une base de données de référence sur les propriétés électrophysiologiques de ce modèle.

Fishing for collagen function: About development, regeneration and disease.

Collagens are the most abundant vertebrate extracellular matrix proteins. They form a superfamily of 28 members that show a remarkable diversity in molecular and supramolecular organization, tissue distribution and function and mutations in collagen genes result in a wide range of inherited connective tissue diseases. In the recent years, unexpected and very diverse regulatory and mechanical collagen functions have been reported. But the structural and functional landscape of the collagen superfamily is still far from being complete. Zebrafish has emerged over the last decades as a powerful model to interrogate gene function and there are numerous advantages of using zebrafish for collagen research, including recent advances in genome editing technologies and the characterization of the zebrafish matrisome. One can confidently predict that zebrafish will rapidly become a popular vertebrate model to investigate the role of collagens in development, disease and regeneration as discussed in this chapter.

Slow Muscle Precursors Lay Down a Collagen XV Matrix Fingerprint to Guide Motor Axon Navigation.

The extracellular matrix (ECM) provides local positional information to guide motoneuron axons toward their muscle target. Collagen XV is a basement membrane component mainly expressed in skeletal muscle. We have identified two zebrafish paralogs of the human COL15A1 gene, col15a1a and col15a1b, which display distinct expression patterns. Here we show that col15a1b is expressed and deposited in the motor path ECM by slow muscle precursors also called adaxial cells. We further demonstrate that collagen XV-B deposition is both temporally and spatially regulated before motor axon extension from the spinal cord in such a way that it remains in this region after the adaxial cells have migrated toward the periphery of the myotome. Loss- and gain-of-function experiments in zebrafish embryos demonstrate that col15a1b expression and subsequent collagen XV-B deposition and organization in the motor path ECM depend on a previously undescribed two-step mechanism involving Hedgehog/Gli and unplugged/MuSK signaling pathways. In silico analysis predicts a putative Gli binding site in the col15a1b proximal promoter. Using col15a1b promoter-reporter constructs, we demonstrate that col15a1b participates in the slow muscle genetic program as a direct target of Hedgehog/Gli signaling. Loss and gain of col15a1b function provoke pathfinding errors in primary and secondary motoneuron axons both at and beyond the choice point where axon pathway selection takes place. These defects result in muscle atrophy and compromised swimming behavior, a phenotype partially rescued by injection of a smyhc1:col15a1b construct. These reveal an unexpected and novel role for collagen XV in motor axon pathfinding and neuromuscular development. SIGNIFICANCE STATEMENT: In addition to the archetypal axon guidance cues, the extracellular matrix provides local information that guides motor axons from the spinal cord to their muscle targets. Many of the proteins involved are unknown. Using the zebrafish model, we identified an unexpected role of the extracellular matrix collagen XV in motor axon pathfinding. We show that the synthesis of collagen XV-B by slow muscle precursors and its deposition in the common motor path are dependent on a novel two-step mechanism that determines axon decisions at a choice point during motor axonogenesis. Zebrafish and humans use common molecular cues and regulatory mechanisms for the neuromuscular system development. And as such, our study reveals COL15A1 as a candidate gene for orphan neuromuscular disorders.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

Knockdown of col22a1 gene in zebrafish induces a muscular dystrophy by disruption of the myotendinous junction.

The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of sub-phenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7ß1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

TigarB causes mitochondrial dysfunction and neuronal loss in PINK1 deficiency.

OBJECTIVE: Loss of function mutations in PINK1 typically lead to early onset Parkinson disease (PD). Zebrafish (Danio rerio) are emerging as a powerful new vertebrate model to study neurodegenerative diseases. We used a pink1 mutant (pink(-/-) ) zebrafish line with a premature stop mutation (Y431*) in the PINK1 kinase domain to identify molecular mechanisms leading to mitochondrial dysfunction and loss of dopaminergic neurons in PINK1 deficiency. METHODS: The effect of PINK1 deficiency on the number of dopaminergic neurons, mitochondrial function, and morphology was assessed in both zebrafish embryos and adults. Genome-wide gene expression studies were undertaken to identify novel pathogenic mechanisms. Functional experiments were carried out to further investigate the effect of PINK1 deficiency on early neurodevelopmental mechanisms and microglial activation. RESULTS: PINK1 deficiency results in loss of dopaminergic neurons as well as early impairment of mitochondrial function and morphology in Danio rerio. Expression of TigarB, the zebrafish orthologue of the human, TP53-induced glycolysis and apoptosis regulator TIGAR, was markedly increased in pink(-/-) larvae. Antisense-mediated inactivation of TigarB gave rise to complete normalization of mitochondrial function, with resulting rescue of dopaminergic neurons in pink(-/-) larvae. There was also marked microglial activation in pink(-/-) larvae, but depletion of microglia failed to rescue the dopaminergic neuron loss, arguing against microglial activation being a key factor in the pathogenesis. INTERPRETATION: Pink1(-/-) zebrafish are the first vertebrate model of PINK1 deficiency with loss of dopaminergic neurons. Our study also identifies TIGAR as a promising novel target for disease-modifying therapy in PINK1-related PD.

The influence of the zebrafish genetic background on Parkinson's disease-related aspects.

Zebrafish are increasingly used to study neurodegenerative conditions such as Parkinson's disease (PD). In rodents, the influence of the genetic background on important experimental parameters in PD research such as susceptibility to toxin exposure or motor behavior is well established. In contrast, little is known about the impact of the genetic background in commonly used zebrafish wild-type strains on these important experimental parameters. We determined the effect of the genetic background in five commonly used zebrafish wild-type strains on crucial, PD-related aspects, in particular the number of ascending dopaminergic neurons, their susceptibility to PD-related neurotoxins, and the expression levels of five genes involved in oxidative stress defense, protein degradation, cell death, and apoptosis. We also investigated whether the susceptibility to morpholino-mediated knockdown of the PD gene DJ-1 may have a varying effect on neuronal cell loss depending on the genetic background. Finally, we determined the influence of the genetic background on spontaneous motor behavior. There was remarkably little variation between the different wild-type strains for most parameters investigated. However, the susceptibility to the neurotoxin 1-methyl-4-phenylpyridinium differed between the five investigated strains and so did their spontaneous motor behavior.

Characterization of spatial and temporal expression pattern of Col15a1b during zebrafish development.

In mammals, collagen XV is primarily expressed in skeletal and cardiac muscles, and loss of its expression in mice results in a mild skeletal myopathy. We recently identified Col15a1a, a zebrafish ortholog of the human collagen XV gene which expression was restricted to notochord in embryos. Col15a1a knockdown led to defects in muscle maintenance via Shh signaling. Here we report that zebrafish express a second ortholog Col15a1b. The identification of its complete primary sequence showed that the overall structure of collagen XV is well conserved between vertebrates. Whole mount in situ hybridization and RT-PCR analysis revealed that at 12hpf Col15a1b is mainly expressed in slow muscle cell lineage and in nervous tissues, and, at later stages transcripts are detected in eyes, otic placodes and aortic arches. Based on the expression pattern of col15a1b, sequence alignments and synteny comparisons, we conclude that, contrary to collagen XVa, the zebrafish collagen XVb likely displays the same or similar function than the mammalian orthologs.

Ethanol-modulated camouflage response screen in zebrafish uncovers a novel role for cAMP and extracellular signal-regulated kinase signaling in behavioral sensitivity to ethanol.

Ethanol, a widely abused substance, elicits evolutionarily conserved behavioral responses in a concentration-dependent manner in vivo. The molecular mechanisms underlying such behavioral sensitivity to ethanol are poorly understood. While locomotor-based behavioral genetic screening is successful in identifying genes in invertebrate models, such complex behavior-based screening has proven difficult for recovering genes in vertebrates. Here we report a novel and tractable ethanol response in zebrafish. Using this ethanol-modulated camouflage response as a screening assay, we have identified a zebrafish mutant named fantasma (fan), which displays reduced behavioral sensitivity to ethanol. Positional cloning reveals that fan encodes type 5 adenylyl cyclase (AC5). fan/ac5 is required to maintain the phosphorylation of extracellular signal-regulated kinase (ERK) in the forebrain structures, including the telencephalon and hypothalamus. Partial inhibition of phosphorylation of ERK in wild-type zebrafish mimics the reduction in sensitivity to stimulatory effects of ethanol observed in the fan mutant, whereas, strikingly, strong inhibition of phosphorylation of ERK renders a stimulatory dose of ethanol sedating. Since previous studies in Drosophila and mice show a role of cAMP signaling in suppressing behavioral sensitivity to ethanol, our findings reveal a novel, isoform-specific role of AC signaling in promoting ethanol sensitivity, and suggest that the phosphorylation level of the downstream effector ERK is a critical "gatekeeper" of behavioral sensitivity to ethanol.

Zebrafish as a new animal model for movement disorders.

The zebrafish, long recognized as a model organism for the analysis of basic developmental processes, is now also emerging as an alternative animal model for human diseases. This review will first provide an overview of the particular characteristics of zebrafish in general and their dopaminergic nervous system in particular. We will then summarize all work undertaken so far to establish zebrafish as a new animal model for movement disorders and will finally emphasize its particular strength - amenability to high throughput in vivo drug screening.

p53-dependent neuronal cell death in a DJ-1-deficient zebrafish model of Parkinson's disease.

Mutations in DJ-1 lead to early onset Parkinson's disease (PD). The aim of this study was to elucidate further the underlying mechanisms leading to neuronal cell death in DJ-1 deficiency in vivo and determine whether the observed cell loss could be prevented pharmacologically. Inactivation of DJ-1 in zebrafish, Danio rerio, resulted in loss of dopaminergic neurons after exposure to hydrogen peroxide and the proteasome inhibitor MG132. DJ-1 knockdown by itself already resulted in increased p53 and Bax expression levels prior to toxin exposure without marked neuronal cell death, suggesting subthreshold activation of cell death pathways in DJ-1 deficiency. Proteasome inhibition led to a further increase of p53 and Bax expression with widespread neuronal cell death. Pharmacological p53 inhibition either before or during MG132 exposure in vivo prevented dopaminergic neuronal cell death in both cases. Simultaneous knockdown of DJ-1 and the negative p53 regulator mdm2 led to dopaminergic neuronal cell death even without toxin exposure, further implicating involvement of p53 in DJ-1 deficiency-mediated neuronal cell loss. Our study demonstrates the utility of zebrafish as a new animal model to study PD gene defects and suggests that modulation of downstream mechanisms, such as p53 inhibition, may be of therapeutic benefit.

Sensitivity of zebrafish to environmental toxins implicated in Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra and movement defects, including bradykinesia, tremor, and postural imbalance. Whereas the etiology and pathogenesis of PD is still poorly understood, studies in animal models are providing important insights. One valuable type of animal model for PD is established by treating animals with PD-inducing neurotoxins, including 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, and paraquat. These neurotoxins are thought to inhibit mitochondrial complex I activity leading to oxidative stress, impaired energy metabolism, proteasomal dysfunction, and, eventually, dopamine neuronal loss. However, the genes and pathways that underlie the neurotoxicity of these agents are not known. In this study, we explored the effect of MPTP, rotenone, and paraquat in both adult and larval zebrafish, which are highly amenable to genetic analysis that can lead to the identification of the underlying genes and pathways. Here, we report that adult zebrafish display behavioral alterations, including decreased locomotor activity in response to MPTP, whereas larval zebrafish exhibited developmental, behavioral, and DA sensitivity to these agents. Taken together, these findings suggest that zebrafish could be a valuable model for genetically dissecting the molecular mechanisms underlying the neurotoxicity of PD-inducing agents.

Biochemical and behavioral effects of carbofuran in goldfish (Carassius auratus).

The effects of concentration (5, 50, and 500 microg/L) and duration (24, 48 h) of exposure to carbofuran, a carbamate insecticide, were assessed on brain catecholamine (norepinephrine [NE] and dopamine), plasma glucose, and hepatic glycogen contents and behavioral activities of goldfish (Carassius auratus). After 24 h of exposure to 50 and 500 microg/L, the level of NE was increased in the olfactory bulbs. The same effect was observed after a 48-h exposure to 500 and 50 microg/L in the telencephalic hemispheres and in the hypothalamus, respectively. An increase in the level of dopamine was also found in hypothalamus after 48 h of exposure to 500 microg/L carbofuran. Plasma glucose increased in concentration after both periods of exposure to carbofuran at 50 and 500 microg/L. Hepatic glycogen concentration decreased after a 48-h exposure to the highest concentration. Behavioral endpoints related to swimming pattern and social interactions were affected after a 24-h exposure to the lowest concentration tested (5 microg/L). The relative sensitivities of these different types of responses to exposure to carbofuran are discussed in light of data on the neurotoxic effects of carbamate and organophosphate insecticides in fish.