PERK inhibition in zebrafish mimics human Wolcott-Rallison syndrome phenotypes.

Wolcott-Rallison Syndrome (WRS) is the most common cause of permanent neonatal diabetes mellitus among consanguineous families. The diabetes associated with WRS is non-autoimmune, insulin-requiring and associated with skeletal dysplasia and growth retardation. The therapeutic options for WRS patients rely on permanent insulin pumping or on invasive transplants of liver and pancreas. WRS has a well identified genetic cause: loss-of-function mutations in the gene coding for an endoplasmic reticulum kinase named PERK (protein kinase R-like ER kinase). Currently, WRS research is facilitated by cellular and rodent models with PERK ablation. While these models have unique strengths, cellular models incompletely replicate the organ/system-level complexity of WRS, and rodents have limited scalability for efficiently screening potential therapeutics. To address these challenges, we developed a new in vivo model of WRS by pharmacologically inhibiting PERK in zebrafish. This small vertebrate displays high fecundity, rapid development of organ systems and is amenable to highly efficient in vivo drug testing. PERK inhibition in zebrafish produced typical WRS phenotypes such as glucose dysregulation, skeletal defects, and impaired development. PERK inhibition in zebrafish also produced broad-spectrum WRS phenotypes such as impaired neuromuscular function, compromised cardiac function and muscular integrity. These results show that zebrafish holds potential as a versatile model to study WRS mechanisms and contribute to the identification of promising therapeutic options for WRS.

Edaravone counteracts redox and metabolic disruptions in an emerging zebrafish model of sporadic ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which the death of motor neurons leads to loss of muscle function. Additionally, cognitive and circadian disruptions are common in ALS patients, contributing to disease progression and burden. Most ALS cases are sporadic, and environmental exposures contribute to their aetiology. However, animal models of these sporadic ALS cases are scarce. The small vertebrate zebrafish is a leading organism to model neurodegenerative diseases; previous studies have proposed bisphenol A (BPA) or ß-methylamino-l-alanine (BMAA) exposure to model sporadic ALS in zebrafish, damaging motor neurons and altering motor responses. Here we characterise the face and predictive validity of sporadic ALS models, showing their potential for the mechanistic study of ALS drugs. We phenotypically characterise the BPA and BMAA-induced models, going beyond motor activity and motor axon morphology, to include circadian, redox, proteostasis, and metabolomic phenotypes, and assessing their predictive validity for ALS modelling. BPA or BMAA exposure induced concentration-dependent activity impairments. Also, exposure to BPA but not BMAA induced motor axonopathy and circadian alterations in zebrafish larvae. Our further study of the BPA model revealed loss of habituation to repetitive startles, increased oxidative damage, endoplasmic reticulum (ER) stress, and metabolome abnormalities. The BPA-induced model shows predictive validity, since the approved ALS drug edaravone counteracted BPA-induced motor phenotypes, ER stress, and metabolic disruptions. Overall, BPA exposure is a promising model of ALS-related redox and ER imbalances, contributing to fulfil an unmet need for validated sporadic ALS models.

Stress response mechanisms in protein misfolding diseases: Profiling a cellular model of Huntington's disease.

Stress response pathways like the integrated stress response (ISR), the mitochondrial unfolded protein response (UPRmt) and the heat shock response (HSR) have emerged as part of the pathophysiology of neurodegenerative diseases, including Huntington's disease (HD) - a currently incurable disease caused by the production of mutant huntingtin (mut-Htt). Previous data from HD patients suggest that ISR is activated while UPRmt and HSR are impaired in HD. The study of these stress response pathways as potential therapeutic targets in HD requires cellular models that mimic the activation status found in HD patients of such pathways. PC12 cells with inducible expression of the N-terminal fragment of mut-Htt are among the most used cell lines to model HD, however the activation of stress responses remains unclear in this model. The goal of this study is to characterize the activation of ISR, UPRmt and HSR in this HD cell model and evaluate if it mimics the activation status found in HD patients. We show that PC12 HD cell model presents reduced levels of Hsp90 and mitochondrial chaperones, suggesting an impaired activation or function of HSR and UPRmt. This HD model also presents increased levels of phosphorylated eIF2α, the master regulator of the ISR, but overall similar levels of ATF4 and decreased levels of CHOP - transcription factors downstream to eIF2α - in comparison to control, suggesting an initial activation of ISR. These results show that this model mimics the ISR activation and the impaired UPRmt and HSR found in HD patients. This work suggests that the PC12 N-terminal HD model is suitable for studying the role of stress response pathways in the pathophysiology of HD and for exploratory studies investigating the therapeutic potential of drugs targeting stress responses.

Swimming against ALS: How to model disease in zebrafish for pathophysiological and behavioral studies.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that leads to progressive disability and motor impairment. Existing therapies provide modest improvements in patient survival, raising a need for new treatments for ALS. Zebrafish is a promising model animal for translational and fundamental research in ALS - it is an experimentally tractable vertebrate, with high homology to humans and an ample experimental toolbox. These advantages allow high-throughput study of behavioral and pathophysiological phenotypes. The last decade saw an increased interest in modelling ALS in zebrafish, leading to the current abundance and variety of available methods and models. Additionally, the rise of gene editing techniques and toxin combination studies has created novel opportunities for ALS studies in zebrafish. In this review, we address the relevance of zebrafish as a model animal for ALS studies, the strategies for model induction and key phenotypical evaluation. Furthermore, we discuss established and emerging zebrafish models of ALS, analyzing their validity, including their potential for drug testing, and highlighting research opportunities in this area.

Disruptions of circadian rhythms, sleep, and stress responses in zebrafish: New infrared-based activity monitoring assays for toxicity assessment.

Behavioural disruptions are sensitive indicators of alterations to normal animal physiology and can be used for toxicity assessment. The small vertebrate zebrafish is a leading model organism for toxicological studies. The ability to continuously monitor the toxicity of drugs, pollutants, or environmental changes over several days in zebrafish can have high practical application. Although video-recordings can be used to monitor short-term zebrafish behaviour, it is challenging to videorecord prolonged experiments (e.g. circadian behaviour over several days) because of the darkness periods (nights) and the heavy data storage and image processing requirements. Alternatively, infrared-based activity monitors, widely used in invertebrate models such as drosophila, generate simple and low-storage data and could optimize large-scale prolonged behavioural experiments in zebrafish, thus favouring the implementation of high-throughput testing strategies. Here, we validate the use of a Locomotor Activity Monitor (LAM) to study the behaviour of zebrafish larvae, and we characterize the behavioural phenotypes induced by abnormal light conditions and by the Parkinsonian toxin MPP+. When zebrafish were deprived from daily light-cycle synchronization, the LAM detected various circadian disruptions, such as increased activity period, phase shifts, and decreased inter-daily stability. Zebrafish exposed to MPP+ (10, 100, 500 µM) showed a concentration-dependent decrease in activity, sleep disruptions, impaired habituation to repetitive startles (visual-motor responses), and a slower recovery to normal activity after the startle-associated stress. These phenotypes evidence the feasibility of using infrared-based LAM to assess multi-parameter behavioural disruptions in zebrafish. The procedures in this study have wide applicability and may yield standard methods for toxicity testing.

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Automated analysis of activity, sleep, and rhythmic behaviour in various animal species with the Rtivity software.

Behavioural studies provide insights into normal and disrupted biological mechanisms. In many research areas, a growing spectrum of animal models-particularly small organisms-is used for high-throughput studies with infrared-based activity monitors, generating counts per time data. The freely available software to analyse such data, however, are primarily optimized for drosophila and circadian analysis. Researchers investigating other species or non-circadian behaviour would thus benefit from a more versatile software. Here we report the development of a free and open-source software-Rtivity-allowing customisation of species-specific parameters, and offering a versatile analysis of behavioural patterns, biological rhythms, stimulus responses, and survival. Rtivity is based on the R language and uses Shiny and the recently developed Rethomics package for a user-friendly graphical interface without requiring coding skills. Rtivity automatically assesses survival, computes various activity, sleep, and rhythmicity parameters, and performs fractal analysis of activity fluctuations. Rtivity generates multiple informative graphs, and exports structured data for efficient interoperability with common statistical software. In summary, Rtivity facilitates and enhances the versatility of the behavioural analysis of diverse animal species (e.g. drosophila, zebrafish, daphnia, ants). It is thus suitable for a broad range of researchers from multidisciplinary fields such as ecology, neurobiology, toxicology, and pharmacology.

Allosteric activation of Hsp70 reduces mutant huntingtin levels, the clustering of N-terminal fragments, and their nuclear accumulation.

AIMS: Huntington's disease (HD) is caused by a mutant huntingtin protein that misfolds, yields toxic N-terminal fragments, aggregates, and disrupts proteostasis. The Hsp70 chaperone is a potential therapeutic target as it prevents proteotoxicity by favouring protein folding, disaggregation, or degradation. We tested the hypothesis that allosteric Hsp70 activation with a pharmacological mimetic of the Hsp70 co-chaperone Hip, YM-1, could modulate huntingtin proteostasis. MAIN METHODS: We used HD cell models expressing either N-terminal or full-length huntingtin. Using single-cell analysis we studied huntingtin aggregation in different cellular compartments by fluorescence microscopy. Protein interaction was evaluated by immunoprecipitation, while protein levels were quantified by immunofluorescence and western-blot. KEY FINDINGS: N-terminal huntingtin interacted with Hsp70 and increased its levels. Treatment with YM-1 reduced N-terminal huntingtin clustering and nuclear aggregation. Full-length mutant huntingtin also interacted with Hsp70, and treatment with YM-1 reduced huntingtin levels when combined with Hsp70 induction by heat shock. Mechanistically, YM-1 increases the Hsp70 affinity for substrates, promoting their proteasomal degradation. Consistently, YM-1 reduced the levels of ubiquitinated proteins. Interestingly, YM-1 accumulated in mitochondria, interfered with its Hsp70 isoform involved in protein import, and increased NRF1 levels, a regulator of proteasome genes. We thus suggest that YM-1 may trigger the coordination of mitochondrial and cytosolic proteostasis, enhancing protein degradation. SIGNIFICANCE: Our findings show that the strategy of allosteric Hsp70 activation holds potential for HD. While drug efficacy may be limited to tissues with elevated Hsp70, combined therapies with Hsp70 elevating strategies could harness the full potential of allosteric Hsp70 activators for HD.

The interplay between redox signalling and proteostasis in neurodegeneration: In vivo effects of a mitochondria-targeted antioxidant in Huntington's disease mice.

Abnormal protein homeostasis (proteostasis), dysfunctional mitochondria, and aberrant redox signalling are often associated in neurodegenerative disorders, such as Huntington's (HD), Alzheimer's and Parkinson's diseases. It remains incompletely understood, however, how changes in redox signalling affect proteostasis mechanisms, including protein degradation pathways and unfolded protein responses (UPR). Here we address this open question by investigating the interplay between redox signalling and proteostasis in a mouse model of HD, and by examining the in vivo effects of the mitochondria-targeted antioxidant MitoQ. We performed behavioural tests in wild-type and R6/2 HD mice, examined markers of oxidative stress, UPR activation, and the status of key protein degradation pathways in brain and peripheral tissues. We show that R6/2 mice present widespread markers of oxidative stress, with tissue-specific changes in proteostasis that were more pronounced in the brain and muscle than in the liver. R6/2 mice presented increased levels of cytosolic and mitochondrial chaperones, particularly in muscle, indicating UPR activation. Treatment with MitoQ significantly ameliorated fine motor control of R6/2 mice, and reduced markers of oxidative damage in muscle. Additionally, MitoQ attenuated overactive autophagy induction in the R6/2 muscle, which has been associated with muscle wasting. Treatment with MitoQ did not alter autophagy markers in the brain, in agreement with its low brain bioavailability, which limits the risk of impairing neuronal protein clearance mechanisms. This study supports the hypotheses that abnormal redox signalling in muscle contributes to altered proteostasis and motor impairment in HD, and that redox interventions can improve muscle performance, highlighting the importance of peripheral therapeutics in HD.

Mitochondrial superoxide generation induces a parkinsonian phenotype in zebrafish and huntingtin aggregation in human cells.

Superoxide generation by mitochondria respiratory complexes is a major source of reactive oxygen species (ROS) which are capable of initiating redox signaling and oxidative damage. Current understanding of the role of mitochondrial ROS in health and disease has been limited by the lack of experimental strategies to selectively induce mitochondrial superoxide production. The recently-developed mitochondria-targeted redox cycler MitoParaquat (MitoPQ) overcomes this limitation, and has proven effective in vitro and in Drosophila. Here we present an in vivo study of MitoPQ in the vertebrate zebrafish model in the context of Parkinson's disease (PD), and in a human cell model of Huntington's disease (HD). We show that MitoPQ is 100-fold more potent than non-targeted paraquat in both cells and in zebrafish in vivo. Treatment with MitoPQ induced a parkinsonian phenotype in zebrafish larvae, with decreased sensorimotor reflexes, spontaneous movement and brain tyrosine hydroxylase (TH) levels, without detectable effects on heart rate or atrioventricular coordination. Motor phenotypes and TH levels were partly rescued with antioxidant or monoaminergic potentiation strategies. In a HD cell model, MitoPQ promoted mutant huntingtin aggregation without increasing cell death, contrasting with the complex I inhibitor rotenone that increased death in cells expressing either wild-type or mutant huntingtin. These results show that MitoPQ is a valuable tool for cellular and in vivo studies of the role of mitochondrial superoxide generation in redox biology, and as a trigger or co-stressor to model metabolic and neurodegenerative disease phenotypes.

Targeting the proteostasis network in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion mutation in the huntingtin protein. Expansions above 40 polyglutamine repeats are invariably fatal, following a symptomatic period characterised by choreiform movements, behavioural abnormalities, and cognitive decline. While mutant huntingtin (mHtt) is widely expressed from early life, most patients with HD present in mid-adulthood, highlighting the role of ageing in disease pathogenesis. mHtt undergoes proteolytic cleavage, misfolding, accumulation, and aggregation into inclusion bodies. The emerging model of HD pathogenesis proposes that the chronic production of misfolded mHtt overwhelms the chaperone machinery, diverting other misfolded clients to the proteasome and the autophagy pathways, ultimately leading to a global collapse of the proteostasis network. Multiple converging hypotheses also implicate ageing and its impact in the dysfunction of organelles as additional contributing factors to the collapse of proteostasis in HD. In particular, mitochondrial function is required to sustain the activity of ATP-dependent chaperones and proteolytic machinery. Recent studies elucidating mitochondria-endoplasmic reticulum interactions and uncovering a dedicated proteostasis machinery in mitochondria, suggest that mitochondria play a more active role in the maintenance of cellular proteostasis than previously thought. The enhancement of cytosolic proteostasis pathways shows promise for HD treatment, protecting cells from the detrimental effects of mHtt accumulation. In this review, we consider how mHtt and its post translational modifications interfere with protein quality control pathways, and how the pharmacological and genetic modulation of components of the proteostasis network impact disease phenotypes in cellular and in vivo HD models.

Modulation of Molecular Chaperones in Huntington's Disease and Other Polyglutamine Disorders.

Polyglutamine expansion mutations in specific proteins underlie the pathogenesis of a group of progressive neurodegenerative disorders, including Huntington's disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and several spinocerebellar ataxias. The different mutant proteins share ubiquitous expression and abnormal proteostasis, with misfolding and aggregation, but nevertheless evoke distinct patterns of neurodegeneration. This highlights the relevance of the full protein context where the polyglutamine expansion occurs and suggests different interactions with the cellular proteostasis machinery. Molecular chaperones are key elements of the proteostasis machinery and therapeutic targets for neurodegeneration. Here, we provide a focused review on Hsp90, Hsp70, and their co-chaperones, and how their genetic or pharmacological modulation affects the proteostasis and disease phenotypes in cellular and animal models of polyglutamine disorders. The emerging picture is that, in principle, Hsp70 modulation may be more amenable for long-term treatment by promoting a more selective clearance of mutant proteins than Hsp90 modulation, which may further decrease the necessary wild-type counterparts. It seems, nevertheless, unlikely that a single Hsp70 modulator will benefit all polyglutamine diseases. Indeed, available data, together with insights from effects on tau and alpha-synuclein in models of Alzheimer's and Parkinson's diseases, indicates that Hsp70 modulators may lead to different effects on the proteostasis of different mutant and wild-type client proteins. Future studies should include the further development of isoform selective inhibitors, namely to avoid off-target effects on Hsp in the mitochondria, and their characterization in distinct polyglutamine disease models to account for client protein-specific differences.

Mitochondrial dynamics and quality control in Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion mutations in the huntingtin protein. Despite its ubiquitous distribution, expression of mutant huntingtin (mHtt) is particularly detrimental to medium spiny neurons within the striatum. Mitochondrial dysfunction has been associated with HD pathogenesis. Here we review the current evidence for mHtt-induced abnormalities in mitochondrial dynamics and quality control, with a particular focus on brain and neuronal data pertaining to striatal vulnerability. We address mHtt effects on mitochondrial biogenesis, protein import, complex assembly, fission and fusion, mitochondrial transport, and on the degradation of damaged mitochondria via autophagy (mitophagy). For an integrated perspective on potentially converging pathogenic mechanisms, we also address impaired autophagosomal transport and abnormal mHtt proteostasis in HD.

Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease.

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson's Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.

HDAC6 inhibition induces mitochondrial fusion, autophagic flux and reduces diffuse mutant huntingtin in striatal neurons.

Striatal neurons are vulnerable to Huntington's disease (HD). Decreased levels of acetylated alpha-tubulin and impaired mitochondrial dynamics, such as reduced motility and excessive fission, are associated with HD; however, it remains unclear whether and how these factors might contribute to the preferential degeneration of striatal neurons. Inhibition of the alpha-tubulin deacetylase HDAC6 has been proposed as a therapeutic strategy for HD, but remains controversial - studies in neurons show improved intracellular transport, whereas studies in cell-lines suggest it may impair autophagosome-lysosome fusion, and reduce clearance of mutant huntingtin (mHtt) and damaged mitochondria (mitophagy). Using primary cultures of rat striatal and cortical neurons, we show that mitochondria are intrinsically less motile and more balanced towards fission in striatal than in cortical neurons. Pharmacological inhibition of the HDAC6 deacetylase activity with tubastatin A (TBA) increased acetylated alpha-tubulin levels, and induced mitochondrial motility and fusion in striatal neurons to levels observed in cortical neurons. Importantly, TBA did not block neuronal autophagosome-lysosome fusion, and did not change mitochondrial DNA levels, suggesting no impairment in autophagy or mitochondrial clearance. Instead, TBA increased autophagic flux and reduced diffuse mHtt in striatal neurons, possibly by promoting transport of initiation factors to sites of autophagosomal biogenesis. This study identifies the pharmacological inhibition of HDAC6 deacetylase activity as a potential strategy to reduce the vulnerability of striatal neurons to HD.

Trends in Mitochondrial Therapeutics for Neurological Disease.

Neuronal homeostasis is critically dependent on healthy mitochondria. Mutations in mitochondrial DNA (mtDNA), in nuclear-encoded mitochondrial components, and age-dependent mitochondrial damage, have all been connected with neurological disorders. These include not only typical mitochondrial syndromes with neurological features such as encephalomyopathy, myoclonic epilepsy, neuropathy and ataxia; but also secondary mitochondrial involvement in neurodegenerative disorders such as Alzheimer's, Parkinson's and Huntington's disease. Unravelling the molecular aetiology of mitochondrial dysfunction opens new therapeutic prospects for diseases thus far lacking effective treatments. In this review we address recent advances on preventive strategies, such as pronuclear, spindle-chromosome complex, or polar body genome transfer to replace mtDNA and avoid disease transmission to newborns; we also address experimental mitochondrial therapeutics aiming to benefit symptomatic patients and prevent disease manifestation in those at risk. Specifically, we focus on: (1) gene therapy to reduce mutant mtDNA, such as anti-replicative therapies and mitochondriatargeted nucleases allowing favourable heteroplasmic shifts; (2) allotopic expression of recoded wild-type mitochondrial genes, including targeted tRNAs and xenotopic expression of cognate genes to compensate for pathogenic mutations; (3) mitochondria targeted-peptides and lipophilic cations for in vivo delivery of antioxidants or other putative therapeutics; and (4) modulation of mitochondrial dynamics at the level of biogenesis, fission, fusion, movement and mitophagy. Further advances in therapeutic development are hindered by scarce in vivo models for mitochondrial disease, with the bulk of available data coming from cellular models. Nevertheless, wherever available, we also address data from in vivo experiments and clinical trials, focusing on neurological disease models.

Effects of Echium plantagineum L. bee pollen on basophil degranulation: relationship with metabolic profile.

This study aimed to evaluate the anti-allergic potential of Echium plantagineum L. bee pollen and to characterize its primary metabolites. The activity of E. plantagineum hydromethanolic extract, devoid of alkaloids, was tested against ß-hexosaminidase release in rat basophilic leukemic cells (RBL-2H3). Two different stimuli were used: calcium ionophore A23187 and IgE/antigen. Lipoxygenase inhibitory activity was evaluated in a cell-free system using soybean lipoxygenase. Additionally, the extract was analysed by HPLC-UV for organic acids and by GC-IT/MS for fatty acids. In RBL-2H3 cells stimulated either with calcium ionophore or IgE/antigen, the hydromethanolic extract significantly decreased ß-hexosaminidase release until the concentration of 2.08 mg/mL, without compromising cellular viability. No effect was found on lipoxygenase. Concerning extract composition, eight organic acids and five fatty acids were determined for the first time. Malonic acid (80%) and α-linolenic acid (27%) were the main compounds in each class. Overall, this study shows promising results, substantiating for the first time the utility of intake of E. plantagineum bee pollen to prevent allergy and ameliorate allergy symptoms, although a potentiation of an allergic response can occur, depending on the dose used.

Modulation of basophils' degranulation and allergy-related enzymes by monomeric and dimeric naphthoquinones.

Allergic disorders are characterized by an abnormal immune response towards non-infectious substances, being associated with life quality reduction and potential life-threatening reactions. The increasing prevalence of allergic disorders demands for new and effective anti-allergic treatments. Here we test the anti-allergic potential of monomeric (juglone, menadione, naphthazarin, plumbagin) and dimeric (diospyrin and diosquinone) naphthoquinones. Inhibition of RBL-2H3 rat basophils' degranulation by naphthoquinones was assessed using two complementary stimuli: IgE/antigen and calcium ionophore A23187. Additionally, we tested for the inhibition of leukotrienes production in IgE/antigen-stimulated cells, and studied hyaluronidase and lipoxidase inhibition by naphthoquinones in cell-free assays. Naphthazarin (0.1 µM) decreased degranulation induced by IgE/antigen but not A23187, suggesting a mechanism upstream of the calcium increase, unlike diospyrin (10 µM) that reduced degranulation in A23187-stimulated cells. Naphthoquinones were weak hyaluronidase inhibitors, but all inhibited soybean lipoxidase with the most lipophilic diospyrin, diosquinone and menadione being the most potent, thus suggesting a mechanism of competition with natural lipophilic substrates. Menadione was the only naphthoquinone reducing leukotriene C4 production, with a maximal effect at 5 µM. This work expands the current knowledge on the biological properties of naphthoquinones, highlighting naphthazarin, diospyrin and menadione as potential lead compounds for structural modification in the process of improving and developing novel anti-allergic drugs.

Nature as a source of metabolites with cholinesterase-inhibitory activity: an approach to Alzheimer's disease treatment.

OBJECTIVES: Alzheimer's disease (AD) is the most common cause of dementia, being responsible for high healthcare costs and familial hardships. Despite the efforts of researchers, no treatment able to delay or stop AD progress exists. Currently, the available treatments are only symptomatic, cholinesterase inhibitors being the most widely used drugs. Here we describe several natural compounds with anticholinesterase (acetylcholinesterase and butyrylcholinesterase) activity and also some synthetic compounds whose structures are based on those of natural compounds. KEY FINDINGS: Galantamine and rivastigmine are two cholinesterase inhibitors used in therapeutics: galantamine is a natural alkaloid that was extracted for the first time from Galanthus nivalis L., while rivastigmine is a synthetic alkaloid, the structure of which is modelled on that of natural physostigmine. Alkaloids include a high number of compounds with anticholinesterases activity at the submicromolar range. Quinones and stilbenes are less well studied regarding cholinesterase inhibition, although some of them, such as sargaquinoic acid or (+)-α-viniferin, show promising activity. Among flavonoids, flavones and isoflavones are the most potent compounds. Xanthones and monoterpenes are generally weak cholinesterase inhibitors. SUMMARY: Nature is an almost endless source of bioactive compounds. Several natural compounds have anticholinesterase activity and others can be used as leader compounds for the synthesis of new drugs.

How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs.

BACKGROUND AND PURPOSE: Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. EXPERIMENTAL APPROACH: Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. KEY RESULTS: Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. CONCLUSION AND IMPLICATIONS: This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further, it evidences zebrafish's potential for in vivo efficacy or toxicity screening of ubiquinone analogues or antiparasitic mitochondria-targeted drugs.