An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycinearginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are adult-onset, neurodegenerative diseases associated with personality change, language dysfunction and † Corresponding authors. a.isaacs@prion.ucl.ac.uk; l.partridge@ucl.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts progressive muscle weakness. These syndromes overlap genetically and pathologically, and can also co-occur in individuals, and within families (1). An intronic GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of both FTD and ALS (C9FTD/ALS) (2-4), and can be found in patients diagnosed with all common neurodegenerative diseases (5). Healthy individuals carry fewer than 33 hexanucleotide repeats, with 2 repeats being the most common, but C9FTD/ALS cases carry between 400 and 4400 repeats (2, 5, 6).The repeat expansion could cause disease by three possible mechanisms: i) toxic sense and/or antisense repeat RNA species that sequester key RNA-binding proteins, ii) toxic dipeptide repeat (DPR) proteins, generated by repeat-associated, non-ATG (RAN) translation, or iii) reduced expression of C9orf72. The absence of a severe phenotype in a homozygous C9orf72 mutation case (7), and the lack of C9orf72 coding mutations (8) argue against loss-of-function as a primary mechanism. Neuronal aggregates of RNA, termed RNA foci, generated from both sense and antisense repeat transcripts are frequent in C9FTD/ALS patient brain (9-13). The GGGGCC repeat can be translated in all sense and antisense frames, two of which encode the same DPR, resulting in five DPR proteins, all of which form inclusions in widespread brain regions (10,12,(14)(15)(16)(17)(18). It is therefore of fundamental importance to understand the contributions of repeat RNA and DPR proteins to C9orf72-mediated neurodegeneration.A major obstacle in the investigation of large expanded repeats is that they are inherently unstable. We used recombination-deficient E. coli and a cloning strategy termed recursive directional ligati...

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease.

Dysfunction and accumulation of the microtubule-associated human Tau (hTau) protein into intraneuronal aggregates is observed in many neurodegenerative disorders including Alzheimer’s disease (AD). Reversible lysine acetylation has recently emerged as a post-translational modification that may play an important role in the modulation of hTau pathology. Acetylated hTau species have been observed within hTau aggregates in human AD brains and multi-acetylation of hTau in vitro regulates its propensity to aggregate. However, whether lysine acetylation at position 280 (K280) modulates hTau-induced toxicity in vivo is unknown. We generated new Drosophila transgenic models of hTau pathology to evaluate the contribution of K280 acetylation to hTau toxicity, by analysing the respective toxicity of pseudo-acetylated (K280Q) and pseudo-de-acetylated (K280R) mutant forms of hTau. We observed that mis-expression of pseudo-acetylated K280Q-hTau in the adult fly nervous system potently exacerbated fly locomotion defects and photoreceptor neurodegeneration. In addition, modulation of K280 influenced total hTau levels and phosphorylation without changing hTau solubility. Altogether, our results indicate that pseudo-acetylation of the single K280 residue is sufficient to exacerbate hTau neurotoxicity in vivo, suggesting that acetylated K280-hTau species contribute to the pathological events leading to neurodegeneration in AD.

The preprophase band of microtubules performs the crucial function of marking the plane of cell division. Although the preprophase band depolymerises at the onset of mitosis, the division plane is 'memorized' by a cortical division zone to which the phragmoplast is attracted during cytokinesis. Proteins have been discovered that are part of the molecular memory but little is known about how they contribute to phragmoplast guidance. Previously, we found that the microtubule-associated protein AIR9 is found in the cortical division zone at preprophase and returns during cell plate insertion but is absent from the cortex during the intervening mitosis. To identify new components of the preprophase memory, we searched for proteins that interact with AIR9. We detected the kinesin-like calmodulinbinding protein, KCBP, which can be visualized at the predicted cortical site throughout division. A truncation study of KCBP indicates that its MyTH4-FERM domain is required for linking the motor domain to the cortex. These results suggest a mechanism by which minus-end-directed KCBP helps guide the centrifugally expanding phragmoplast to the cortical division site.