In recent genome-wide association studies, the extracellular chaperone protein, clusterin, has been identified as a novel risk factor in Alzheimer's disease (AD). We have examined the interactions between clusterin and the AD-associated amyloid-β 1-40 peptide (Aβ 1-40 ) which is prone to aggregate into an ensemble of oligomeric intermediates implicated in both the proliferation of amyloid fibrils and in neuronal toxicity. Using highly sensitive single molecule fluorescence methods, we have found that Aβ 1-40 forms a heterogeneous distribution of small oligomers (from dimers to 50mers), all of which interact with clusterin to form long-lived, stable complexes. Consequently, clusterin is able to influence strongly both the aggregation and disaggregation of Aβ 1-40 by sequestration of the Aβ oligomers. These results not only elucidate the protective role of clusterin but also provide a molecular basis for the genetic link between clusterin and AD.
Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-β peptide (Aβ) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Aβ has been hindered by a lack of cellular models amenable to high-throughput chemical screening. To address this gap, we use a robust and scalable yeast model of Aβ toxicity where the Aβ peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aβ 1-42 peptide forms more oligomers and is more toxic than Aβ 1-40 and genome-wide genetic screens identified genes that are known risk factors for AD. Here, we report an unbiased screen of ∼140,000 compounds for rescue of Aβ toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aβ burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aβ secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aβ peptide levels in a copper-dependent manner by increasing degradation, ultimately restoring endocytic function. This mirrored its effects on copper-dependent oligomer formation in vitro, which was also reversed by CQ. This unbiased screen indicates that copper-dependent Aβ oligomer formation contributes to Aβ toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aβ yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.phenotypic small-molecule screen | metal chelation A lzheimer's disease (AD) is a common and devastating neurodegenerative disorder that is projected to increase in frequency as our population ages. The lack of disease-modifying therapies requires new approaches to address the underlying mechanisms of cellular dysfunction and identify potential therapeutics.The amyloid-β peptide (Aβ) plays a major role in AD and ultimately leads to neuronal death and cognitive impairment (1). Aβ peptides of ∼40 aa are generated by the successive cleavage of the amyloid precursor protein (APP) by β-and γ-secretases. Mutations in APP or γ-secretase cause familial AD and bias APP cleavage toward a 42-aa Aβ peptide that predominates in Aβ plaques, is more aggregation prone, and is toxic to neurons (2, 3). Although Aβ plaques are a common, conspicuous feature of pathology in diseased brains, increasing evidence suggests that small oligomers of Aβ are the most toxic species (4, 5).
The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer’s disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)–derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4. Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele.
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