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, however also idiopathic PD patients without known mutations in PARK7 show altered subcellular location and levels of its protein product DJ-1 in pathological tissues. Here, we show that PARK7-/- zebrafish already at their larval stage show a correlation between reduced number of dopaminergic neurons and motor dysfunction. Additionally, PD associated prodromal symptoms as reduced sensory function, increased sleep latency and light phase sleepiness were also observed. The 1-methyl-4-phenylpyridinium (MPP+) exposed PD model did not reproduce similar sleep disturbances. This is the first stable genetic larval zebrafish model of Parkinson’s disease which shows both motor and non-motor symptoms together with a reduction in dopaminergic neurons. The model should therefore be highly valuable as a tool for PD related drug screening and mechanistic studies.

Graphene, a material composed of a two-dimensional lattice of carbon atoms, has due to its many unique properties a wide array of potential applications in the biomedical field. One of the most common production methods is exfoliation through sonication, which is simple but has low yields. Another approach, using microfluidization, has shown promise through its scalability for commercial production. Regardless of their production method, materials made for biomedical applications need to be tested for biocompatibility. Here, we investigated the differences in toxicity, macrophage response, and complement activation of similar-sized graphene flakes produced through sonication and microfluidization, using in vitro cell assays and in vivo assays on zebrafish larvae. In vitro toxicity testing showed that sonicated graphene had a high toxicity, with an EC50 of 100 μg mL-1 for endothelial cells and 60 μg mL-1 for carcinoma cells. In contrast, microfluidized graphene did not reach EC50 at any of the tested concentrations. The potency to activate the complement system in whole blood was 10-fold higher for sonicated than for microfluidized graphene. In zebrafish larvae, graphene of either production method was found to mainly agglomerate in the caudal hematopoietic tissue; however, no acute toxic effects were found. Sonicated graphene led to an increase in macrophage count and a macrophage migration to the ventral tail area, while microfluidized graphene led to a transient reduction in macrophage count and fewer cells in the ventral trail area. The observed reduction in macrophages and change in macrophage distribution following exposure to microfluidized graphene was less pronounced compared to sonicated graphene and contributed to masking of the fluorescent signal rather than cytotoxic effects. Summarized, we observed higher toxicity, macrophage response, and complement activation with graphene produced through sonication, which could be due to oxygen-containing functional groups introduced to the edge of the carbon lattice by this production method. These findings indicate that microfluidization produces graphene more suitable for biomedical applications.

Parkinson's disease is characterized neuropathologically by the degeneration of dopaminergic neurons in the ventral midbrain, the accumulation of α-synuclein (α-syn) aggregates in neurons and chronic neuroinflammation. In the past two decades, in vitro, ex vivo and in vivo studies have consistently shown the involvement of inflammatory responses mediated by microglia and astrocytes, which may be elicited by pathological α-syn or signals from affected neurons and other cell types, and are directly linked to neurodegeneration and disease development. Apart from the prominent immune alterations seen in the CNS, including the infiltration of T cells into the brain, more recent studies have demonstrated important changes in the peripheral immune profile within both the innate and adaptive compartments, particularly involving monocytes, CD4+ and CD8+ T cells. This review aims to integrate the consolidated understanding of immune-related processes underlying the pathogenesis of Parkinson's disease, focusing on both central and peripheral immune cells, neuron-glia crosstalk as well as the central-peripheral immune interaction during the development of Parkinson's disease. Our analysis seeks to provide a comprehensive view of the emerging knowledge of the mechanisms of immunity in Parkinson's disease and the implications of this for better understanding the overall pathogenesis of this disease.

Age-dependent loss of muscle mass and function is associated with oxidative stress. DJ-1/Park7 acts as an antioxidant through multiple signalling pathways. DJ-1-knockout zebrafish show a decline in swimming performance and loss of weight gain between 6 and 9 months of age. Here, we address the degree to which this is associated with muscle degeneration and identify molecular changes preceding dysregulation of muscle performance. Loss of DJ-1 reduced the skeletal muscle fibre cross-section area. The highly mitochondrial-dependent red slow muscle was more affected than the white muscle, and degeneration of sub-sarcolemma red muscle mitochondria was observed. Using TandemMassTag-based quantitative proteomics, we identified a total of 3721 proteins in the multiplex sample of 4 and 12-month-old muscles. A total of 68 proteins, mainly associated with inflammation and mitochondrial function, were dysregulated in the young DJ-1-null adults, with Annexin A3, Sphingomyelin phosphodiesterase acid-like 3B, Complement C3a, and 2,4-dienoyl CoA reductase 1 being the most affected. The loss of DJ-1 also accelerated molecular features associated with sarcopenia, such as a decrease in the NAD+/NADH ratio and a reduction in Prostaglandin reductase 2 and Cytosolic glycerol-3-phosphate dehydrogenase levels. In view of the experimental power of zebrafish, the DJ-1-null zebrafish makes a valuable model for understanding the connection between oxidative stress and age-dependent muscle loss and function.

Parkinson’s disease (PD) is a chronic, age-related, progressive multisystem disease associated with neuroinflammation and immune dysfunction. This review discusses the methodological approaches used to study the changes in central and peripheral immunity in PD, the advantages and limitations of the techniques, and their applicability to humans. Although a single animal model cannot replicate all pathological features of the human disease, neuroinflammation is present in most animal models of PD and plays a critical role in understanding the involvement of the immune system (IS) in the pathogenesis of PD. The IS and its interactions with different cell types in the central nervous system (CNS) play an important role in the pathogenesis of PD. Even though culture models do not fully reflect the complexity of disease progression, they are limited in their ability to mimic long-term effects and need validation through in vivo studies. They are an indispensable tool for understanding the interplay between the IS and the pathogenesis of this disease. Understanding the immune-mediated mechanisms may lead to potential therapeutic targets for the treatment of PD. We believe that the development of methodological guidelines for experiments with animal models and PD patients is crucial to ensure the validity and consistency of the results.

DJ-1 is a redox sensitive protein with a wide range of functions related to oxidative stress protection. Mutations in the park7 gene, which codes for DJ-1 are associated with early onset familial Parkinson's disease and increased astrocytic DJ-1 levels are found in pathologic tissues from idiopathic Parkinson's disease. We have previously established a DJ-1 knockout zebrafish line that developed normally, but with aging the DJ-1 null fish had a lowered level of tyrosine hydroxylase, respiratory mitochondrial failure and a lower body mass. Here we have examined the DJ-1 knockout from the early adult stage and show that loss of DJ-1 results in a progressive, age-dependent increase in both motoric and non-motoric symptoms associated to Parkinson's disease. These changes coincide with changes in mitochondrial and mitochondrial associated proteins. Recent studies have suggested that a decline in NAD+ can contribute to Parkinson's disease and that supplementation of NAD+ precursors may delay disease progression. We found that the brain NAD+/NADH ratio decreased in aging zebrafish but did not correlate with DJ-1 induced altered behavior. Differences were first observed at the late adult stage in which NAD+ and NADPH levels were decreased in DJ-1 knockouts. Considering the experimental power of zebrafish and the development of Parkinson's disease-related symptoms in the DJ-1 null fish, this model can serve as a useful tool both to understand the progression of the disease and the effect of suggested treatments.

Acute myeloid leukemia and myelodysplastic syndromes are cancers of the bone marrow with poor prognosis in frail and older patients. To investigate cancer pathophysiology and therapies, confocal imaging of fluorescent cancer cells and their response to treatments in zebrafish larvae yields valuable information. While zebrafish larvae are well suited for confocal imaging, the lack of efficient processing of large datasets remains a severe bottleneck. To alleviate this problem, we present a software tool that segments cells from confocal images and track characteristics such as volume, location in the larva and fluorescent intensity on a single-cell basis. Using this software tool, we were able to characterise the responses of the cancer cell lines Molm-13 and MDS-L to established treatments. By utilizing the computer-assisted processing of confocal images as presented here, more information can be obtained while being less time-consuming and reducing the demand of manual data handling, when compared to a manual approach, thereby accelerating the pursuit of novel anti-cancer treatments. The presented software tool is available as an ImageJ java-plugin at https:/zenodo.org/10.5281/zenodo. 7383160 and the source code at https:/github.com/Jfo004/ConfocalCellSegmentation.

The eye is continuously under oxidative stress due to high metabolic activity and reactive oxygen species generated by daily light exposure. The redox-sensitive protein DJ-1 has proven to be essential in order to protect retina and retinal pigment epithelium (RPE) from oxidative-stress-induced degeneration. Here, we analyzed the specific role of Müller cell DJ-1 in the adult zebrafish retina by re-establishing Müller-cell-specific DJ-1 expression in a DJ-1 knockout retina. Loss of DJ-1 resulted in an age-dependent retinal degeneration, including loss of cells in the ganglion cell layer, retinal thinning, photoreceptor disorganization and RPE cell dysfunction. The degenerative phenotype induced by the absence of DJ-1 was inhibited by solely expressing DJ-1 in Müller cells. The protective effect was dependent upon the cysteine-106 residue of DJ-1, which has been shown to be an oxidative sensor of DJ-1. In a label-free proteomics analysis of isolated retinas, we identified proteins differentially expressed after DJ-1 knockout, but with restored levels after Müller cell DJ-1 re-insertion. Our data show that Müller cell DJ-1 has a major role in protecting the retina from age-dependent oxidative stress.

The biological relevance of extracellular vesicles (EV) released in an ischemia/reperfusion setting is still unclear. We hypothesized that the inflammatory microenvironment prevents cardioprotection mediated by endothelial cell (EC)-derived extracellular vesicles. The effects of naïve EC-derived EV (eEV) or eEV released in response to interleukin-3 (IL-3) (eEV-IL-3) were evaluated in cardiomyoblasts (H9c2) and rat hearts. In transwell assay, eEV protected the H9c2 exposed to hypoxia/reoxygenation (H/R) more efficiently than eEV-IL-3. Conversely, only eEV directly protected H9c2 cells to H/R-induced damage. Consistent with this latter observation, eEV, but not eEV-IL-3, exerted beneficial effects in the whole heart. Protein profiles of eEV and eEV-IL-3, established using label-free mass spectrometry, demonstrated that IL-3 drives changes in eEV-IL-3 protein cargo. Gene ontology analysis revealed that both eEV and eEV-IL-3 were equipped with full cardioprotective machinery, including the Nitric Oxide Signaling in the Cardiovascular System. eEV-IL-3 were also enriched in the endothelial-nitric oxide-synthase (eNOS)-antagonist caveolin-1 and proteins related to the inflammatory response. In vitro and ex vivo experiments demonstrated that a functional Mitogen-Activated Protein Kinase Kinase (MEK1/2)/eNOS/guanylyl-cyclase (GC) pathway is required for eEV-mediated cardioprotection. Consistently, eEV were found enriched in MEK1/2 and able to induce the expression of B-cell-lymphoma-2 (Bcl-2) and the phosphorylation of eNOS in vitro. We conclude that an inflammatory microenvironment containing IL-3 changes the eEV cargo and impairs eEV cardioprotective action.

Astrocytes are vital for maintaining and regulating redox environments in the brain. Astrocytic protection extends in a transcellular action toward the neurons, which are highly sensitive to oxidative stress. The complete mechanism of astrocytic neuroprotection has yet to be elucidated; however, astrocytic DJ-1 appears to play a vital role in this neuroprotective effect. High levels of DJ-1 in astrocytes surrounding pathological areas observed in a number of neurodegenerative diseases support a key role of astrocytic DJ-1 in oxidative stress response. Astrocytic DJ-1 can act as an intracellular sensor of oxidative stress from reactive oxygen/nitrogen species. In animal models, increased astrocytic DJ-1 levels have been shown to be neuroprotective to oxidative stressors. The mechanisms of this neuroprotection appear to be multifaceted including: suppression of proapoptotic signaling, activating the Nrf2/Keap1 pathway, controlling mitochondrial turnover, and regulating astrogliosis and inflammatory pathways.

DJ-1 is a protein with a wide range of functions importantly related to redox regulation in the cell. In humans, dysfunction of the PARK7 gene is associated with neurodegeneration and Parkinson’s disease. Our objective was to establish a novel DJ-1 knockout zebrafish line and to identify early brain proteome changes, which could be linked to later pathology. The CRISPR-Cas9 method was used to target exon 1 of the park7-/- gene to produce a transgenic DJ-1-deficient zebrafish model of Parkinson’s disease. Label-free mass spectrometry was employed to identify altered protein expression in the DJ-1 null brain of early adult animals. The park7−/− line appears to develop normally at young adult and larval stages. With aging however, DJ-1 null fish exhibit lower tyrosine hydroxylase levels, respiratory failure in skeletal muscle, and lower body mass which is especially prevalent among male fish. By proteomic analysis of early adult brains, we determined that less than 5% of the 4091 identified proteins were influenced by the lack of DJ-1. The dysregulated proteins were mainly proteins known to be involved in mitochondrial metabolism, mitophagy, stress response, redox regulation, and inflammation. This dysregulation in protein networks of our novel DJ-1-deficient zebrafish model occurs in the early adult stage preceding a Parkinson’s disease-related phenotype and the reduction of tyrosine hydroxylase level. The identified protein changes provide new mechanistic background for DJ-1 function. The experimental power of zebrafish makes this model a highly valuable tool to understand and modulate cellular signaling leading to neurodegeneration.

DJ-1, a Parkinson's disease-associated protein, is strongly up-regulated in reactive astrocytes in Parkinson's disease. This is proposed to represent a neuronal protective response, although the mechanism has not yet been identified. We have generated a transgenic zebrafish line with increased astroglial DJ-1 expression driven by regulatory elements from the zebrafish GFAP gene. Larvae from this transgenic line are protected from oxidative stress-induced injuries as caused by MPP+, a mitochondrial complex I inhibitor shown to induce dopaminergic cells death. In a global label-free proteomics analysis of wild type and transgenic larvae exposed to MPP+, 3418 proteins were identified, in which 366 proteins were differentially regulated. In particular, we identified enzymes belonging to primary metabolism to be among proteins affected by MPP+ in wild type animals, but not affected in the transgenic line. Moreover, by performing protein profiling on isolated astrocytes we showed that an increase in astrocytic DJ-1 expression up-regulated a large group of proteins associated with redox regulation, inflammation and mitochondrial respiration. The majority of these proteins have also been shown to be regulated by Nrf2. These findings provide a mechanistic insight into the protective role of astroglial up-regulation of DJ-1 and show that our transgenic zebrafish line with astrocytic DJ-1 over-expression can serve as a useful animal model to understand astrocyte-regulated neuroprotection associated with oxidative stress-related neurodegenerative disease.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.

The non-protein amino acid β-methylamino-L-alanine (BMAA) is a neurotoxin present in microalgae and shown to accumulate in the food web. BMAA has been linked to the complex neurodegenerative disorder of Guam and to increased incidents sporadic ALS. Two main neurotoxic routes are suggested; an excitotoxic by acting as an agonist towards glutamate receptors and a metabolic by misincorporating into cellular proteins. We have used zebrafish, an increasingly used model for neurodegenerative diseases, to further identify signaling components involved in BMAA-induced toxicity. Zebrafish embryos were exposed to sub-lethal dosages of BMAA and a label-free proteomics analysis was conducted on larvae 4 days post fertilization. The exposed larvae showed no developmental abnormalities, but a reduced heart rate and increased expression of GSK3 isoforms. Search towards a reviewed database containing 2968 entries identified 480 proteins. Only 17 of these were regulated 2-fold or more in the exposed larvae. Seven of these proteins could be associated to glutamate receptor signaling and recycling. The remaining nine have all been linked to disturbance in protein homeostasis, reactive oxygen species (ROS) development or neuronal cell death. We also found that BMAA influenced the endocannabinoid system by up-regulation of fatty acid amide hydrolase (FAAH) and that FAAH inhibitor URB597 reduced the BMAA effect on heart rate and GSK3 expression.

N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish.

Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies.

Azaspiracid-1 is an algal toxin that accumulates in edible mussels, and ingestion may result in human illness as manifested by vomiting and diarrhoea. When injected into mice, it causes neurotoxicological symptoms and death. Although it is well known that azaspiracid-1 is toxic to most cells and cell lines, little is known about its biological target(s). A rat PC12 cell line, commonly used as a model for the peripheral nervous system, was used to study the neurotoxicological effects of azaspiracid-1. Azaspiracid-1 induced differentiation-related morphological changes followed by a latter cell death. The differentiated phenotype showed peripherin-labelled neurite-like processes simultaneously as a specific isoform of peripherin was down-regulated. The precise mechanism behind this down-regulation remains uncertain. However, this study provides new insights into the neurological effects of azaspiracid-1 and into the biological significance of specific isoforms of peripherin.

In rainbow trout (Oncorhynchus mykiss) it has been shown that high affinity IgM antibodies have a higher degree of disulfide polymerization and a longer half life time. In the present study, distinct IgM sub-variants related to ancestral tetraploidy in salmonid fish were analyzed to reveal possible characteristic differences between these. A monoclonal antibody (MAb4C10) which distinguishes between IgM-A and IgM-B in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta) was further characterized. It was shown that substitution of a proline located in the loop between the B and C beta strands of the third constant domain (μ3) of salmon μA eliminated MAb4C10 reactivity. Accordingly, the reverse substitution in salmon μB restored MAb4C10 reactivity. Molecular cloning of μ cDNA from arctic char (Salvelinus alpinus) revealed two sub-variants (μA-1 and μA-2), i.e. a similar situation as in Atlantic salmon and brown trout. However, arctic char IgM eluted in one peak by anion exchange chromatography, in contrast to salmon and brown trout IgM that are eluted in two peaks. The only characteristic residue of salmon and brown trout μB is an additional cysteine in the C-terminal part of μ4. Most likely, this cysteine is involved in inter-chain disulfide bonding and influences the elution profiles of IgM-A and IgM-B on anion exchange chromatography. Neither of the μ sub-variants in arctic char have the additional cysteine, and char IgM, as well as salmon and brown trout IgM-A, showed a lower degree of inter-chain disulfide bonding than IgM-B when subjected to denaturation and gel electrophoresis under non-reducing conditions. Hybrids of char/salmon expressed μA-1, μA-2, μA and μB, indicating that there are two paralogous Ig heavy chain gene complexes in the haploid genome of char, like in Atlantic salmon. A comparison of salmonid μ sequences is presented, including representatives of Salmoninae (trout, salmon and char), Thymallinae (grayling) and Coregoninae (whitefish).