Suppression of Aß toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity.

The accumulation of ß-amyloid (Aß) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aß toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aß levels by clearing the Aß plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aß are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aß toxicity is indirect was supported by the finding that Aß is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aß toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aß toxicity.

Expression in drosophila of tandem amyloid ß peptides provides insights into links between aggregation and neurotoxicity.

The generation and subsequent aggregation of amyloid ß (Aß) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aß(40)) or 42 residues (Aß(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aß peptides to probe the manner in which changes in the aggregation kinetics of Aß affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aß(40) and Aß(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aß(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aß(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aß(40) rather than simply to its higher rate of aggregation.

Testing the therapeutic potential of doxycycline in a Drosophila melanogaster model of Alzheimer disease.

Therapies for Alzheimer disease that reduce the production of pathogenic amyloid ß (Aß) peptides have been associated with a range of unwanted effects. For this reason, alternative strategies that promote the clearance of the peptide by preventing its aggregation and deposition in the brain have been favored. In this context we have studied doxycycline, a member of the tetracycline family of antibiotics that has shown neuroprotective effects in a number of models of neurodegenerative disease. We investigated the neuroprotective potential of doxycycline in a Drosophila model of Aß toxicity and sought to correlate any effects with the aggregation state of the peptide. We found that administration of doxycycline to Aß42-expressing flies did not improve their lifespan but was able to slow the progression of their locomotor deficits. We also measured the rough eye phenotype of transgenic flies expressing the E22G variant of Aß42 and showed that doxycycline administration partially rescued the toxicity of Aß in the developing eye. We correlated these in vivo effects with in vitro observations using transmission electron microscopy, dynamic light scattering, and thioflavin T binding. We found that doxycycline prevents Aß fibrillization and favors the generation of smaller, non-amyloid structures that were non-toxic as determined by the lack of caspase 3 activation in a neuroblastoma cell line. Our confirmation that doxycycline can prevent amyloid ß toxicity both in vitro and in vivo supports its therapeutic potential in AD.

The DNA polymerases of Drosophila melanogaster.

DNA synthesis during replication or repair is a fundamental cellular process that is catalyzed by a set of evolutionary conserved polymerases. Despite a large body of research, the DNA polymerases of Drosophila melanogaster have not yet been systematically reviewed, leading to inconsistencies in their nomenclature, shortcomings in their functional (Gene Ontology, GO) annotations and an under-appreciation of the extent of their characterization. Here, we describe the complete set of DNA polymerases in D. melanogaster, applying nomenclature already in widespread use in other species, and improving their functional annotation. A total of 19 genes encode the proteins comprising three replicative polymerases (alpha-primase, delta, epsilon), five translesion/repair polymerases (zeta, eta, iota, Rev1, theta) and the mitochondrial polymerase (gamma). We also provide an overview of the biochemical and genetic characterization of these factors in D. melanogaster. This work, together with the incorporation of the improved nomenclature and GO annotation into key biological databases, including FlyBase and UniProtKB, will greatly facilitate access to information about these important proteins.

Methods and models in neurodegenerative and systemic protein aggregation diseases.

Protein misfolding and aggregation are implicated in a wide range of increasingly prevalent human diseases ranging from dementia to diabetes. In this review we discuss the current experimental strategies that are being employed in the investigation of the pathogenesis of three important protein misfolding disorders. The first, Alzheimer's disease (AD), is the most prevalent neurodegenerative disease and is thought to be initiated by the aggregation of a natively unstructured peptide called amyloid beta (Abeta). We discuss methods for the characterization of the aggregation properties of Abeta in vitro and how the results of such experiments can be correlated with data from animal models of disease. We then consider another form of amyloidosis, where a systemic distribution of amyloid deposit is caused by aggregation and deposition of mutational variants of lysozyme. We describe how experiments in vitro, and more recently in vivo, have provided insights into the origins of this disease. Finally we outline the varied paradigms that have been employed in the study of the serpinopathies, and in particular, a dementia caused by neuroserpin polymerization.