Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase

Myosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/β-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients

Оценка буровых растворов применяемых для бурения продуктивных отложений в скважинах Беларуси

The assembly process of a-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson´s disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of a-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of a-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and follwed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substansia nigra of normal and a-synuclein knockout mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed singificant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in a-synuclein knock-out mice injected with accelerator intor the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson´s disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice

Uptake of Aggregating Transthyretin by Fat Body in a Drosophila Model for TTR-Associated Amyloidosis

Background: A functional link has been established between the severe neurodegenerative disorder Familial amyloidotic polyneuropathy and the enhanced propensity of the plasma protein transthyretin (TTR) to form aggregates in patients with single point mutations in the TTR gene. Previous work has led to the establishment of an experimental model based on transgenic expression of normal or mutant forms of human TTR in Drosophila flies. Remarkably, the severity of the phenotype was greater in flies that expressed a single copy than with two copies of the mutated gene. Methodology/Principal Findings: In this study, we analyze the distribution of normal and mutant TTR in transgenic flies, and the ultrastructure of TTR-positive tissues to clarify if aggregates and/or amyloid filaments are formed. We report the formation of intracellular aggregates of 20 nm spherules and amyloid filaments in thoracic adipose tissue and in brain glia, two tissues that do not express the transgene. The formation of aggregates of nanospherules increased with age and was more considerable in flies with two copies of mutated TTR. Treatment of human neuronal cells with protein extracts prepared from TTR flies of different age showed that the extracts from older flies were less toxic than those from younger flies. Conclusions/Significance: These findings suggest that the uptake of TTR from the circulation and its subsequent segregation into cytoplasmic quasi-crystalline arrays of nanospherules is part of a mechanism that neutralizes the toxic effect of TTR.Original Publication:Malgorzata Pokrzywa, Ingrid Dacklin, Monika Vestling, Dan Hultmark, Erik Lundgren and Rafael Cantera, Uptake of Aggregating Transthyretin by Fat Body in a Drosophila Model for TTR-Associated Amyloidosis, 2010, PLOS ONE, (5), 12.http://dx.doi.org/10.1371/journal.pone.0014343Licensee: Public Library of Science (PLoS)http://www.plos.org

A Drosophila Disease-Model for Transthyretin-associated Amyloidosis

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed

Human TTBK1, TTBK2 and MARK1 kinase toxicity in Drosophila melanogaster is exacerbated by co-expression of human Tau

Tau protein is involved in numerous human neurodegenerative diseases, and Tau hyper-phosphorylation has been linked to Tau aggregation and toxicity. Previous studies have addressed toxicity and phospho-biology of human Tau (hTau) in Drosophila melanogaster. However, hTau transgenes have most often been randomly inserted in the genome, thus making it difficult to compare between different hTau isoforms and phospho-mutants. In addition, many studies have expressed hTau also in mitotic cells, causing nonphysiological toxic effects. Here, we overcome these confounds by integrating UAS-hTau isoform transgenes into specific genomic loci, and express hTau post-mitotically in the Drosophila nervous system. Lifespan and locomotor analyses show that all six of the hTau isoforms elicit similar toxicity in flies, although hTau(2N3R) showed somewhat elevated toxicity. To determine if Tau phosphorylation is responsible for toxicity, we analyzed the effects of co-expressing hTau isoforms together with Tau-kinases, focusing on TTBK1, TTBK2 and MARK1. We observed toxicity when expressing each of the three kinases alone, or in combination. Kinase toxicity was enhanced by hTau co-expression, with strongest co-toxicity for TTBK1. Mutagenesis and phosphorylation analysis indicates that hTau-MARK1 combinatorial toxicity may be due to direct phosphorylation of hTau, while hTau-TTBK1/2 combinatorial toxicity may result from independent toxicity mechanisms.Funding Agencies|Konung Gustaf V: s och Drottning Victorias Frimurarestiftelse [700-0557]</p

Учебная программа по учебной дисциплине Высшая математика

Учебная программа "Высшая математика" кафедры "Высшая математика" для дневной формы получения образования: общее количество часов - 570, трудоемкость учебной дисциплины 16 з.е., форма контроля знаний - экзамен, РГР

Systematic A beta Analysis in Drosophila Reveals High Toxicity for the 1-42, 3-42 and 11-42 Peptides, and Emphasizes N- and C-Terminal Residues

Brain amyloid plaques are a hallmark of Alzheimers disease (AD), and primarily consist of aggregated A beta peptides. While A beta 1-40 and A beta 1-42 are the most abundant, a number of other A beta peptides have also been identified. Studies have indicated differential toxicity for these various A beta peptides, but in vivo toxicity has not been systematically tested. To address this issue, we generated improved transgenic Drosophila UAS strains expressing 11 pertinent A beta peptides. UAS transgenic flies were generated by identical chromosomal insertion, hence removing any transgenic position effects, and crossed to a novel and robust Gal4 driver line. Using this improved Gal4/UAS set-up, survival and activity assays revealed that A beta 1-42 severely shortens lifespan and reduces activity. N-terminal truncated peptides were quite toxic, with 3-42 similar to 1-42, while 11-42 showed a pronounced but less severe phenotype. N-terminal mutations in 3-42 (E3A) or 11-42 (E11A) resulted in reduced toxicity for 11-42, and reduced aggregation for both variants. Strikingly, C-terminal truncation of A beta (1-41, -40, -39, -38, -37) were non-toxic. In contrast, C-terminal extension to 1-43 resulted in reduced lifespan and activity, but not to the same extent as 1-42. Mutating residue 42 in 1-42 (A42D, A42R and A42W) greatly reduced A beta accumulation and toxicity. Histological and biochemical analysis revealed strong correlation between in vivo toxicity and brain A beta aggregate load, as well as amount of insoluble A beta. This systematic Drosophila in vivo and in vitro analysis reveals crucial N- and C-terminal specificity for A beta neurotoxicity and aggregation, and underscores the importance of residues 1-10 and E11, as well as a pivotal role of A42.Funding Agencies|Swedish VINNOVA; King Gustaf Vs and Queen Victorias Freemasons Foundation; AstraZeneca, Sodertalje; Swedish Research Council; VINNOVA grant, "Innovations for future health"</p

Myosin Storage Myopathy in C. elegans and Human Cultured Muscle Cells.

Myosin storage myopathy is a protein aggregate myopathy associated with the characteristic subsarcolemmal accumulation of myosin heavy chain in muscle fibers. Despite similar histological findings, the clinical severity and age of onset are highly variable, ranging from no weakness to severe impairment of ambulation, and usually childhood-onset to onset later in life. Mutations located in the distal end of the tail of slow/ß-cardiac myosin heavy chain are associated with myosin storage myopathy. Four missense mutations (L1793P, R1845W, E1883K and H1901L), two of which have been reported in several unrelated families, are located within or closed to the assembly competence domain. This location is critical for the proper assembly of sarcomeric myosin rod filaments. To assess the mechanisms leading to protein aggregation in myosin storage myopathy and to evaluate the impact of these mutations on myosin assembly and muscle function, we expressed mutated myosin proteins in cultured human muscle cells and in the nematode Caenorhabditis elegans. While L1793P mutant myosin protein efficiently incorporated into the sarcomeric thick filaments, R1845W and H1901L mutants were prone to formation of myosin aggregates without assembly into striated sarcomeric thick filaments in cultured muscle cells. In C. elegans, mutant alleles of the myosin heavy chain gene unc-54 corresponding to R1845W, E1883K and H1901L, were as effective as the wild-type myosin gene in rescuing the null mutant worms, indicating that they retain functionality. Taken together, our results suggest that the basis for the pathogenic effect of the R1845W and H1901L mutations are primarily structural rather than functional. Further analyses are needed to identify the primary trigger for the histological changes seen in muscle biopsies of patients with L1793P and E1883K mutations

Effects of SAP on amyloidogenic aggregates.

<p><i>(A)</i> The effect of SAP on TTR-induced toxicity. IMR-32 cells were incubated with the indicated concentrations of either TTR-A (▴) or TTR-D (•) for 12 h. Solid lines represent the toxic response when cells were incubated with the respective proteins, and dashed lines represent experiments with the addition of 3 µM SAP. One-way ANOVA with sequential Bonferroni <i>post-hoc</i> test revealed significant protective effects of SAP on cells in the presence of either TTR-A or TTR-D (<i>P = </i>0.004 and <i>P = </i>0.003, respectively) <i>(B)</i> The effect of SAP on H₂O₂-induced cytotoxicity. IMR-32 cells were treated with different concentrations of H₂O₂ (in the range 0–5 mM) without addition of (▪) or in the presence of 1,000 U/ml catalase (▴) or 3 µM SAP (•). Oxidative stress-induced toxicity in IMR-32 cells was significantly reduced by catalase treatment (<i>P</i><0.001; one-way ANOVA, sequential Bonferroni <i>post-hoc</i> test) but not by SAP treatment (<i>P = </i>0.4). Error bars indicate SD.</p