Autophagy modulates Aß accumulation and formation of aggregates in yeast.

Intracellular accumulation of amyloid-ß protein (Aß) is an early event in Alzheimer's disease (AD). The autophagy-lysosomal pathway is an important pathway for maintaining cellular proteostasis and for the removal of damaged organelles and protein aggregates in all eukaryotes. Despite mounting evidence showing that modulating autophagy promotes clearance of Aß aggregates, the regulatory mechanisms and signalling pathways underlying this process remain poorly understood. In order to gain better insight we used our previously characterised yeast model expressing GFP-Aß42 to identify genes that regulate the removal of Aß42 aggregates by autophagy. We report that GFP-Aß42 is sequestered and is selectively transported to vacuole for degradation and that autophagy is the prominent pathway for clearance of aggregates. Next, to identify genes that selectively promote the removal of Aß42 aggregates, we screened levels of GFP-Aß42 and non-aggregating GFP-Aß42 (19:34) proteins in a panel of 192 autophagy mutants lacking genes involved in regulation and initiation of the pathway, cargo selection and degradation processes. The nutrient and stress signalling genes RRD1, SNF4, GCN4 and SSE1 were identified. Deletion of these genes impaired GFP-Aß42 clearance and their overexpression reduced GFP-Aß42 levels in yeast. Overall, our findings identify a novel role for these nutrient and stress signalling genes in the targeted elimination of Aß42 aggregates, which offer a promising avenue for developing autophagy based therapies to suppress amyloid deposition in AD.

Latrepirdine (dimebon) enhances autophagy and reduces intracellular GFP-Aß42 levels in yeast.

Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-ß (Aß)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aß yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aß42. GFP-Aß42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aß42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aß42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aß42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aß42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aß42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aß42.

Oligomerization and toxicity of Aß fusion proteins.

This study has found that the Maltose binding protein Aß42 fusion protein (MBP-Aß42) forms soluble oligomers while the shorter MBP-Aß16 fusion and control MBP did not. MBP-Aß42, but neither MBP-Aß16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aß42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aß42 characterization.

Alzheimer's amyloid-beta rescues yeast from hydroxide toxicity.

Amyloid-beta(Abeta42), which is known to be toxic to neuronal cells, protects yeast cells from severe sodium hydroxide toxicity. More than 85% cell death was caused by treatment with 1 mM NaOH and approximately 95% was observed at a 2 mM concentration. However, greater than 55% cells survived the treatment in the presence of Abeta42. A strong protective effect of the peptide was also evident from the differential staining of the treated culture with propidium iodide.

Abeta aggregation and possible implications in Alzheimer's disease pathogenesis.

Amyloid beta protein (Abeta) has been associated with Alzheimer's disease (AD) because it is a major component of the extracellular plaque found in AD brains. Increased Abeta levels correlate with the cognitive decline observed in AD. Sporadic AD cases are thought to be chiefly associated with lack of Abeta clearance from the brain, unlike familial AD which shows increased Abeta production. Abeta aggregation leading to deposition is an essential event in AD. However, the factors involved in Abeta aggregation and accumulation in sporadic AD have not been completely characterized. This review summarizes studies that have examined the factors that affect Abeta aggregation and toxicity. By necessity these are studies that are performed with recombinant-derived or chemically synthesized Abeta. The studies therefore are not done in animals but in cell culture, which includes neuronal cells, other mammalian cells and, in some cases, non-mammalian cells that also appear susceptible to Abeta toxicity. An understanding of Abeta oligomerization may lead to better strategies to prevent AD.