Arabidopsis KCBP interacts with AIR9 but stays in the cortical division zone throughout mitosis via its MyTH4-FERM domain.

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 calmodulin-binding 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.

Aß43 is neurotoxic and primes aggregation of Aß40 in vivo.

The involvement of Amyloid-ß (Aß) in the pathogenesis of Alzheimer's disease (AD) is well established. However, it is becoming clear that the amyloid load in AD brains consists of a heterogeneous mixture of Aß peptides, implying that a thorough understanding of their respective role and toxicity is crucial for the development of efficient treatments. Besides the well-studied Aß40 and Aß42 species, recent data have raised the possibility that Aß43 peptides might be instrumental in AD pathogenesis, because they are frequently observed in both dense and diffuse amyloid plaques from human AD brains and are highly amyloidogenic in vitro. However, whether Aß43 is toxic in vivo is currently unclear. Using Drosophila transgenic models of amyloid pathology, we show that Aß43 peptides are mainly insoluble and highly toxic in vivo, leading to the progressive loss of photoreceptor neurons, altered locomotion and decreased lifespan when expressed in the adult fly nervous system. In addition, we demonstrate that Aß43 species are able to trigger the aggregation of the typically soluble and non-toxic Aß40, leading to synergistic toxic effects on fly lifespan and climbing ability, further suggesting that Aß43 peptides could act as a nucleating factor in AD brains. Altogether, our study demonstrates high pathogenicity of Aß43 species in vivo and supports the idea that Aß43 contributes to the pathological events leading to neurodegeneration in AD.

Pseudo-acetylation of multiple sites on human Tau proteins alters Tau phosphorylation and microtubule binding, and ameliorates amyloid beta toxicity.

Tau is a microtubule-associated protein that is highly soluble and natively unfolded. Its dysfunction is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer's disease (AD), where it aggregates within neurons. Deciphering the physiological and pathogenic roles of human Tau (hTau) is crucial to further understand the mechanisms leading to its dysfunction in vivo. We have used a knock-out/knock-in strategy in Drosophila to generate a strain with hTau inserted into the endogenous fly tau locus and expressed under the control of the endogenous fly tau promoter, thus avoiding potential toxicity due to genetic over-expression. hTau knock-in (KI) proteins were expressed at normal, endogenous levels, bound to fly microtubules and were post-translationally modified, hence displaying physiological properties. We used this new model to investigate the effects of acetylation on hTau toxicity in vivo. The simultaneous pseudo-acetylation of hTau at lysines 163, 280, 281 and 369 drastically decreased hTau phosphorylation and significantly reduced its binding to microtubules in vivo. These molecular alterations were associated with ameliorated amyloid beta toxicity. Our results indicate acetylation of hTau on multiple sites regulates its biology and ameliorates amyloid beta toxicity in vivo.

Acetylation mimic of lysine 280 exacerbates human Tau neurotoxicity in vivo.

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.

Deletion of endogenous Tau proteins is not detrimental in Drosophila.

Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer's disease (AD), constituting, together with accumulated ß-amyloid (Aß) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, we aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. We observed that the specific removal of endogenous dTau proteins did not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function were unchanged in tau KO flies. In addition, we did not find any overt positive or negative effect of dTau removal on human Aß-induced toxicity. Altogether, our results indicate that the absence of dTau proteins has no major functional impact on flies, and suggests that our tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions.

Increased Glucose Transport into Neurons Rescues Aß Toxicity in Drosophila.

Glucose hypometabolism is a prominent feature of the brains of patients with Alzheimer's disease (AD). Disease progression is associated with a reduction in glucose transporters in both neurons and endothelial cells of the blood-brain barrier. However, whether increasing glucose transport into either of these cell types offers therapeutic potential remains unknown. Using an adult-onset Drosophila model of Aß (amyloid beta) toxicity, we show that genetic overexpression of a glucose transporter, specifically in neurons, rescues lifespan, behavioral phenotypes, and neuronal morphology. This amelioration of Aß toxicity is associated with a reduction in the protein levels of the unfolded protein response (UPR) negative master regulator Grp78 and an increase in the UPR. We further demonstrate that genetic downregulation of Grp78 activity also protects against Aß toxicity, confirming a causal effect of its alteration on AD-related pathology. Metformin, a drug that stimulates glucose uptake in cells, mimicked these effects, with a concomitant reduction in Grp78 levels and rescue of the shortened lifespan and climbing defects of Aß-expressing flies. Our findings demonstrate a protective effect of increased neuronal uptake of glucose against Aß toxicity and highlight Grp78 as a novel therapeutic target for the treatment of AD.

Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies.

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.

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins.

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, but not stop codon-interrupted "RNA-only" repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) 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.