Familial amyloidotic polyneuropathy is an autosomal dominant neurodegenerative disorder caused by accumulation of mutated transthyretin (TTR) amyloid fibrils in different organs and prevalently around peripheral nerves. We have constructed transgenic flies, expressing the clinical amyloidogenic variant TTRL55P and the engineered variant TTR-A (TTRV14N/V16E) as well as the wild-type protein, all in secreted form. Within a few weeks, both mutants but not the wild-type TTR demonstrated a time-dependent aggregation of misfolded molecules. 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 wild-type TTR had no discernible effect on either longevity or behavior. These results suggest that Drosophila can be used as a disease-model to study TTR amyloid formation, and to screen for pharmacological agents and modifying genes.

Brain amyloid plaques are a hallmark of Alzheimer's disease (AD), and primarily consist of aggregated Aβ peptides. While Aβ 1-40 and Aβ 1-42 are the most abundant, a number of other Aβ peptides have also been identified. Studies have indicated differential toxicity for these various Aβ peptides, but in vivo toxicity has not been systematically tested. To address this issue, we generated improved transgenic Drosophila UAS strains expressing 11 pertinent Aβ 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 Aβ 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 Aβ (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 Aβ accumulation and toxicity. Histological and biochemical analysis revealed strong correlation between in vivo toxicity and brain Aβ aggregate load, as well as amount of insoluble Aβ. This systematic Drosophila in vivo and in vitro analysis reveals crucial N- and C-terminal specificity for Aβ neurotoxicity and aggregation, and underscores the importance of residues 1-10 and E11, as well as a pivotal role of A42.

Transthyretin (TTR) is a homotetrameric plasma protein with amyloidogenic properties that has been linked to the development of familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and senile systemic amyloidosis. The in vivo role of TTR is associated with transport of thyroxine hormone T4 and retinol-binding protein. Loss of the tetrameric integrity of TTR is a rate-limiting step in the process of TTR amyloid formation, and ligands with the ability to bind within the thyroxin binding site (TBS) can stabilize the tetramer, a feature that is currently used as a therapeutic approach for FAP. Several different flavonoids have recently been identified that impair amyloid formation. The flavonoid luteolin shows therapeutic potential with low incidence of unwanted side effects. In this work, we show that luteolin effectively attenuates the cytotoxic response to TTR in cultured neuronal cells and rescues the phenotype of a Drosophila melanogaster model of FAP. The plant-derived luteolin analogue cynaroside has a glucoside group in position 7 of the flavone A-ring and as opposed to luteolin is unable to stabilize TTR tetramers and thus prevents a cytotoxic effect. We generated high-resolution crystal-structures of both TTR wild type and the amyloidogenic mutant V30M in complex with luteolin. The results show that the A-ring of luteolin, in contrast to what was previously suggested, is buried within the TBS, consequently explaining the lack of activity from cynaroside. The flavonoids represent an interesting group of drug candidates for TTR amyloidosis. The present investigation shows the potential of luteolin as a stabilizer of TTR in vivo. We also show an alternative orientation of luteolin within the TBS which could represent a general mode of binding of flavonoids to TTR and is of importance concerning the future design of tetramer stabilizing drugs.

Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in encoding slow/β-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the α-helical rod domain of are mainly associated with Laing distal myopathy. To investigate the mechanisms underlying the pathology of the recurrent causative mutation (K1729del), we have developed a model of Laing distal myopathy by genomic engineering of the locus. Homozygous animals die during larval/pupal stages, and both homozygous and heterozygous larvae display reduced muscle function. Flies expressing only in indirect flight and jump muscles, and heterozygous animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous larvae also demonstrated structural and functional impairments in heart muscle, which were not observed in heterozygous animals, indicating a dose-dependent effect of the mutated allele. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. Our observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.

The assembly process of α-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 α-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of α-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and followed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substantia nigra of normal and α-synuclein knock-out 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 significant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in α-synuclein knock-out mice injected with accelerator into 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.