Mitochondrial abnormalities contribute to muscle weakness in a Dnajb6 deficient zebrafish model.

Mutations in DNAJB6 are a well-established cause of limb girdle muscular dystrophy type D1 (LGMD D1). Patients with LGMD D1 develop progressive muscle weakness with histology showing fibre damage, autophagic vacuoles, and aggregates. Whilst there are many reports of LGMD D1 patients, the role of DNAJB6 in the muscle is still unclear. In this study, we developed a loss of function zebrafish model in order to investigate the role of Dnajb6. Using a double dnajb6a and dnajb6b mutant model, we show that loss of Dnajb6 leads to a late onset muscle weakness. Interestingly, we find that adult fish lacking Dnajb6 do not have autophagy or myofibril defects, however, they do show mitochondrial changes and damage. This study demonstrates that loss of Dnajb6 causes mitochondrial defects and suggests that this contributes to muscle weakness in LGMD D1. These findings expand our knowledge of the role of Dnajb6 in the muscle and provides a model to screen novel therapies for LGMD D1.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Established and emerging GABAA receptor pharmacotherapy for epilepsy.

Drugs that modulate the GABAA receptor are widely used in clinical practice for both the long-term management of epilepsy and emergency seizure control. In addition to older medications that have well-defined roles for the treatment of epilepsy, recent discoveries into the structure and function of the GABAA receptor have led to the development of newer compounds designed to maximise therapeutic benefit whilst minimising adverse effects, and whose position within the epilepsy pharmacologic armamentarium is still emerging. Drugs that modulate the GABAA receptor will remain a cornerstone of epilepsy management for the foreseeable future and, in this article, we provide an overview of the mechanisms and clinical efficacy of both established and emerging pharmacotherapies.

Manipulation of Proteostasis Networks in Transgenic ZAAT Zebrafish via CRISPR-Cas9 Gene Editing.

The CRISPR-Cas9 genome editing system is used to induce mutations in genes of interest resulting in the loss of functional protein. A transgenic zebrafish α1-antitrypsin deficiency (AATD) model displays an unusual phenotype, in that it lacks the hepatic accumulation of the misfolding Z α1-antitrypsin (ZAAT) evident in human and mouse models. Here we describe the application of the CRISPR-Cas9 system to generate mutant zebrafish with defects in key proteostasis networks likely to be involved in the hepatic processing of ZAAT in this model. We describe the targeting of the atf6a and man1b1 genes as examples.

Genetic model of UBA5 deficiency highlights the involvement of both peripheral and central nervous systems and identifies widespread mitochondrial abnormalities.

Variants in UBA5 have been reported to cause neurological disease with impaired motor function, developmental delay, intellectual disability and brain pathology as recurrent clinical manifestations. UBA5 encodes a ubiquitin-activating-like enzyme that activates ufmylation, a post-translational ubiquitin-like modification pathway, which has been implicated in neurodevelopment and neuronal survival. The reason behind the variation in severity and clinical manifestations in affected individuals and the signal transduction pathways regulated by ufmylation that compromise the nervous system remains unknown. Zebrafish have emerged as a powerful model to study neurodegenerative disease due to its amenability for in vivo analysis of muscle and neuronal tissues, high-throughput examination of motor function and rapid embryonic development allowing an examination of disease progression. Using clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing, we developed and characterized zebrafish mutant models to investigate disease pathophysiology. uba5 mutant zebrafish showed a significantly impaired motor function accompanied by delayed growth and reduced lifespan, reproducing key phenotypes observed in affected individuals. Our study demonstrates the suitability of zebrafish to study the pathophysiology of UBA5-related disease and as a powerful tool to identify pathways that could reduce disease progression. Furthermore, uba5 mutants exhibited widespread mitochondrial damage in both the nervous system and the skeletal muscle, suggesting that a perturbation of mitochondrial function may contribute to disease pathology.

Muscle Pathology in Dystrophic Rats and Zebrafish Is Unresponsive to Taurine Treatment, Compared to the mdx Mouse Model for Duchenne Muscular Dystrophy.

Inflammation and oxidative stress are strongly implicated in the pathology of Duchenne muscular dystrophy (DMD), and the sulphur-containing amino acid taurine ameliorates both and decreases dystropathology in the mdx mouse model for DMD. We therefore further tested taurine as a therapy using dystrophic DMDmdx rats and dmd zebrafish models for DMD that have a more severe dystropathology. However, taurine treatment had little effect on the indices of dystropathology in both these models. While we and others have previously observed a deficiency in taurine in mdx mice, in the current study we show that the rat and zebrafish models had increased taurine content compared with wild-type, and taurine treatment did not increase muscle taurine levels. We therefore hypothesised that endogenous levels of taurine are a key determinate in potential taurine treatment efficacy. Because of this, we felt it important to measure taurine levels in DMD patient plasma samples and showed that in non-ambulant patients (but not in younger patients) there was a deficiency of taurine. These data suggest that taurine homeostasis varies greatly between species and may be influenced by age and disease progression. The potential for taurine to be an effective therapy may depend on such variables.

Expression of the Z Variant of α1-Antitrypsin Suppresses Hepatic Cholesterol Biosynthesis in Transgenic Zebrafish.

Individuals homozygous for the Pi*Z allele of SERPINA1 (ZAAT) are susceptible to lung disease due to insufficient α1-antitrypsin secretion into the circulation and may develop liver disease due to compromised protein folding that leads to inclusion body formation in the endoplasmic reticulum (ER) of hepatocytes. Transgenic zebrafish expressing human ZAAT show no signs of hepatic accumulation despite displaying serum insufficiency, suggesting the defect in ZAAT secretion occurs independently of its tendency to form inclusion bodies. In this study, proteomic, transcriptomic, and biochemical analysis provided evidence of suppressed Srebp2-mediated cholesterol biosynthesis in the liver of ZAAT-expressing zebrafish. To investigate the basis for this perturbation, CRISPR/Cas9 gene editing was used to manipulate ER protein quality control factors. Mutation of erlec1 resulted in a further suppression in the cholesterol biosynthesis pathway, confirming a role for this ER lectin in targeting misfolded ZAAT for ER-associated degradation (ERAD). Mutation of the two ER mannosidase homologs enhanced ZAAT secretion without inducing hepatic accumulation. These insights into hepatic ZAAT processing suggest potential therapeutic targets to improve secretion and alleviate serum insufficiency in this form of the α1-antitrypsin disease.

Novel preclinical model for CDKL5 deficiency disorder.

Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for in vivo imaging and genetic manipulation, allows for the detailed assessment of disease pathogenesis and therapeutic discovery. We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies. This article has an associated First Person interview with the first author of the paper.

Metformin rescues muscle function in BAG3 myofibrillar myopathy models.

Dominant de novo mutations in the co-chaperone BAG3 cause a severe form of myofibrillar myopathy, exhibiting progressive muscle weakness, muscle structural failure, and protein aggregation. To elucidate the mechanism of disease in, and identify therapies for, BAG3 myofibrillar myopathy, we generated two zebrafish models, one conditionally expressing BAG3P209L and one with a nonsense mutation in bag3. While transgenic BAG3P209L-expressing fish display protein aggregation, modeling the early phase of the disease, bag3-/- fish exhibit exercise dependent fiber disintegration, and reduced swimming activity, consistent with later stages of the disease. Detailed characterization of the bag3-/- fish, revealed an impairment in macroautophagic/autophagic activity, a defect we confirmed in BAG3 patient samples. Taken together, our data highlights that while BAG3P209L expression is sufficient to promote protein aggregation, it is the loss of BAG3 due to its sequestration within aggregates, which results in impaired autophagic activity, and subsequent muscle weakness. We therefore screened autophagy-promoting compounds for their effectiveness at removing protein aggregates, identifying nine including metformin. Further evaluation demonstrated metformin is not only able to bring about the removal of protein aggregates in zebrafish and human myoblasts but is also able to rescue the fiber disintegration and swimming deficit observed in the bag3-/- fish. Therefore, repurposing metformin provides a promising therapy for BAG3 myopathy.Abbreviations:ACTN: actinin, alpha; BAG3: BAG cochaperone 3; CRYAB: crystallin alpha B; DES: desmin; DMSO: dimethyl sulfoxide; DNAJB6: DnaJ heat shock protein family (Hsp40) member B6; dpf: days post fertilization; eGFP: enhanced green fluorescent protein; FDA: Food and Drug Administration; FHL1: four and a half LIM domains 1; FLNC: filamin C; hpf: hours post-fertilization; HSPB8: heat shock protein family B [small] member 8; LDB3/ZASP: LIM domain binding 3; MYOT: myotilin; TTN: titin; WT: wild-type.

Functional validation of CHMP7 as an ADHD risk gene.

Attention deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder of childhood with a strong genetic component. Despite the success of mapping ADHD risk loci, little work has been done to experimentally verify the contribution of these loci to ADHD phenotypes. Meta-analysis of four genome-wide association studies in ADHD suggested CHMP7 as a predisposing gene for ADHD. A DNA variant (rs2294123) mapped to CHMP7 has been shown (via bioinformatic analysis) to have a high likelihood for functionality and correlate with reduced transcript levels. We used CRISPR-Cas9 genome editing to generate a chmp7 zebrafish model for ADHD. chmp7+/- fish showed comparable reductions in mRNA levels to individuals homozygous for the CHMP7 ADHD risk allele. These fish displayed significant hyperactivity over a 24-h period at 6 days post-fertilisation compared to chmp7+/+, but this effect did not persist into juvenile and adulthood stages. In addition, chmp7+/- fish had significantly smaller total brain volumes than chmp7+/+ fish. Finally, the hyperactivity at 6 days post-fertilisation was significantly reduced through the application of methylphenidate, a mainstay pharmacological treatment for ADHD. Overall, this study highlights an important role for CHMP7 in the neurodevelopment of ADHD, and demonstrates the utility of zebrafish for modelling the functional effects of genes conferring risk to ADHD.

Creating Predictive Weed Emergence Models Using Repeat Photography and Image Analysis.

Weed emergence models have the potential to be important tools for automating weed control actions; however, producing the necessary data (e.g., seedling counts) is time consuming and tedious. If similar weed emergence models could be created by deriving emergence data from images rather than physical counts, the amount of generated data could be increased to create more robust models. In this research, repeat RGB images taken throughout the emergence period of Raphanus raphanistrum L. and Senna obtusifolia (L.) Irwin and Barneby underwent pixel-based spectral classification. Relative cumulative pixels generated by the weed of interest over time were used to model emergence patterns. The models that were derived from cumulative pixel data were validated with the relative emergence of true seedling counts. The cumulative pixel model for R. raphanistrum and S. obtusifolia accounted for 92% of the variation in relative emergence of true counts. The results demonstrate that a simple image analysis approach based on time-dependent changes in weed cover can be used to generate weed emergence predictive models equivalent to those produced based on seedling counts. This process will help researchers working on weed emergence models, providing a new low-cost and technologically simple tool for data collection.

BAG3P215L/KO Mice as a Model of BAG3P209L Myofibrillar Myopathy.

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.

KBTBD13 is an actin-binding protein that modulates muscle kinetics.

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.

Biomass of the Cyanobacterium Lyngbya wollei Alters Copper Algaecide Exposure and Risks to a Non-target Organism.

Nuisance algal infestations are increasing globally in distribution and frequency. Copper-based algaecides are routinely applied to control these infestations, though there is an ever-present concern of risks to non-target species. This research evaluated risks associated with a commonly applied chelated copper algaecide (Captain® XTR; SePRO Corporation) to a sentinel non-target species (Daphnia magna) and further assessed alteration of the exposure and toxicity when a nuisance mat-forming cyanobacterium, Lyngbya wollei, was present in exposures. Aqueous copper concentrations in treatments with algae significantly decreased within 1 h after treatment and averaged 57.5% of nominal amended Cu through the experiment duration. The 48 h LC50 values were 371 µg Cu/L with no algae present in exposures and increased significantly to 531 µg Cu/L when L. wollei was simultaneously exposed. This research provides information on the short-term fate of copper and hazard assessment by incorporating targeted binding ligands, as present in operational treatments.

Integration of remote-weed mapping and an autonomous spraying unmanned aerial vehicle for site-specific weed management.

BACKGROUND: Unmanned aerial vehicles (UAVs) have been used in agriculture to collect imagery for crop and pest monitoring, and for decision-making purposes. Spraying-capable UAVs are now commercially available worldwide for agricultural applications. Combining UAV weed mapping and UAV sprayers into an UAV integrated system (UAV-IS) can offer a new alternative to implement site-specific pest management. RESULTS: The UAV-IS was 0.3- to 3-fold more efficient at identifying and treating target weedy areas, while minimizing treatment on non-weedy areas, than ground-based broadcast applications. The UAV-IS treated 20-60% less area than ground-based broadcast applications, but also missed up to 26% of the target weedy area, while broadcast applications covered almost the entire experimental area and only missed 2-3% of the target weeds. The efficiency of UAV-IS management practices increased as weed spatial aggregation increased (patchiness). CONCLUSION: Integrating UAV imagery for pest mapping and UAV sprayers can provide a new strategy for integrated pest management programs to improve efficiency and efficacy while reducing the amount of pesticide being applied. The UAV-IS has the potential to improve the detection and control of weed escapes to reduce/delay herbicide resistance evolution. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency.

In human α1-antitrypsin deficiency, homozygous carriers of the Z (E324K) mutation in the gene SERPINA1 have insufficient circulating α1-antitrypsin and are predisposed to emphysema. Misfolding and accumulation of the mutant protein in hepatocytes also causes endoplasmic reticulum stress and underpins long-term liver damage. Here, we describe transgenic zebrafish (Danio rerio) expressing the wildtype or the Z mutant form of human α1-antitrypsin in hepatocytes. As observed in afflicted humans, and in rodent models, about 80% less α1-antitrypsin is evident in the circulation of zebrafish expressing the Z mutant. Although these zebrafish also show signs of liver stress, they do not accumulate α1-antitrypsin in hepatocytes. This new zebrafish model will provide useful insights into understanding and treatment of α1-antitrypsin deficiency.

Linking life-history theory and metabolic theory explains the offspring size-temperature relationship.

Temperature often affects maternal investment in offspring. Across and within species, mothers in colder environments generally produce larger offspring than mothers in warmer environments, but the underlying drivers of this relationship remain unresolved. We formally evaluated the ubiquity of the temperature-offspring size relationship and found strong support for a negative relationship across a wide variety of ectotherms. We then tested an explanation for this relationship that formally links life-history and metabolic theories. We estimated the costs of development across temperatures using a series of laboratory experiments on model organisms, and a meta-analysis across 72 species of ectotherms spanning five phyla. We found that both metabolic and developmental rates increase with temperature, but developmental rate is more temperature sensitive than metabolic rate, such that the overall costs of development decrease with temperature. Hence, within a species' natural temperature range, development at relatively cooler temperatures requires mothers to produce larger, better provisioned offspring.

Selective NaV1.1 activation rescues Dravet syndrome mice from seizures and premature death.

Dravet syndrome is a catastrophic, pharmacoresistant epileptic encephalopathy. Disease onset occurs in the first year of life, followed by developmental delay with cognitive and behavioral dysfunction and substantially elevated risk of premature death. The majority of affected individuals harbor a loss-of-function mutation in one allele of SCN1A, which encodes the voltage-gated sodium channel NaV1.1. Brain NaV1.1 is primarily localized to fast-spiking inhibitory interneurons; thus the mechanism of epileptogenesis in Dravet syndrome is hypothesized to be reduced inhibitory neurotransmission leading to brain hyperexcitability. We show that selective activation of NaV1.1 by venom peptide Hm1a restores the function of inhibitory interneurons from Dravet syndrome mice without affecting the firing of excitatory neurons. Intracerebroventricular infusion of Hm1a rescues Dravet syndrome mice from seizures and premature death. This precision medicine approach, which specifically targets the molecular deficit in Dravet syndrome, presents an opportunity for treatment of this intractable epilepsy.

L-tyrosine supplementation does not ameliorate skeletal muscle dysfunction in zebrafish and mouse models of dominant skeletal muscle α-actin nemaline myopathy.

L-tyrosine supplementation may provide benefit to nemaline myopathy (NM) patients, however previous studies are inconclusive, with no elevation of L-tyrosine levels in blood or tissue reported. We evaluated the ability of L-tyrosine treatments to improve skeletal muscle function in all three published animal models of NM caused by dominant skeletal muscle α-actin (ACTA1) mutations. Highest safe L-tyrosine concentrations were determined for dosing water and feed of wildtype zebrafish and mice respectively. NM TgACTA1D286G-eGFP zebrafish treated with 10 µM L-tyrosine from 24 hours to 6 days post fertilization displayed no improvement in swimming distance. NM TgACTA1D286G mice consuming 2% L-tyrosine supplemented feed from preconception had significant elevations in free L-tyrosine levels in sera (57%) and quadriceps muscle (45%) when examined at 6-7 weeks old. However indicators of skeletal muscle integrity (voluntary exercise, bodyweight, rotarod performance) were not improved. Additionally no benefit on the mechanical properties, energy metabolism, or atrophy of skeletal muscles of 6-7 month old TgACTA1D286G and KIActa1H40Y mice eventuated from consuming a 2% L-tyrosine supplemented diet for 4 weeks. Therefore this study yields important information on aspects of the clinical utility of L-tyrosine for ACTA1 NM.