Prion-like proteins sequester and suppress the toxicity of huntingtin exon 1.

Expansions of preexisting polyglutamine (polyQ) tracts in at least nine different proteins cause devastating neurodegenerative diseases. There are many unique features to these pathologies, but there must also be unifying mechanisms underlying polyQ toxicity. Using a polyQ-expanded fragment of huntingtin exon-1 (Htt103Q), the causal protein in Huntington disease, we and others have created tractable models for investigating polyQ toxicity in yeast cells. These models recapitulate key pathological features of human diseases and provide access to an unrivalled genetic toolbox. To identify toxicity modifiers, we performed an unbiased overexpression screen of virtually every protein encoded by the yeast genome. Surprisingly, there was no overlap between our modifiers and those from a conceptually identical screen reported recently, a discrepancy we attribute to an artifact of their overexpression plasmid. The suppressors of Htt103Q toxicity recovered in our screen were strongly enriched for glutamine- and asparagine-rich prion-like proteins. Separated from the rest of the protein, the prion-like sequences of these proteins were themselves potent suppressors of polyQ-expanded huntingtin exon-1 toxicity, in both yeast and human cells. Replacing the glutamines in these sequences with asparagines abolished suppression and converted them to enhancers of toxicity. Replacing asparagines with glutamines created stronger suppressors. The suppressors (but not the enhancers) coaggregated with Htt103Q, forming large foci at the insoluble protein deposit in which proteins were highly immobile. Cells possessing foci had fewer (if any) small diffusible oligomers of Htt103Q. Until such foci were lost, cells were protected from death. We discuss the therapeutic implications of these findings.

Clioquinol promotes the degradation of metal-dependent amyloid-ß (Aß) oligomers to restore endocytosis and ameliorate Aß toxicity.

Alzheimer's disease (AD) is a common, progressive neurodegenerative disorder without effective disease-modifying therapies. The accumulation of amyloid-ß peptide (Aß) is associated with AD. However, identifying new compounds that antagonize the underlying cellular pathologies caused by Aß 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 Aß toxicity where the Aß peptide transits through the secretory and endocytic compartments as it does in neurons. The pathogenic Aß 1-42 peptide forms more oligomers and is more toxic than Aß 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 Aß toxicity. Of ∼30 hits, several were 8-hydroxyquinolines (8-OHQs). Clioquinol (CQ), an 8-OHQ previously reported to reduce Aß burden, restore metal homeostasis, and improve cognition in mouse AD models, was also effective and rescued the toxicity of Aß secreted from glutamatergic neurons in Caenorhabditis elegans. In yeast, CQ dramatically reduced Aß 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 Aß oligomer formation contributes to Aß toxicity within the secretory/endosomal pathways where it can be targeted with selective metal binding compounds. Establishing the ability of the Aß yeast model to identify disease-relevant compounds supports its further exploitation as a validated early discovery platform.

Functional links between Aß toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast.

Aß (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aß toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aß was previously unknown. The factors identified in yeast modified Aß toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aß impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aß, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.

Membrane-protein integration and the role of the translocation channel.

Most eukaryotic membrane proteins are integrated into the lipid bilayer during their synthesis at the endoplasmic reticulum (ER). Their integration occurs with the help of a protein-conducting channel formed by the heterotrimeric Sec61 membrane-protein complex. The crystal structure of an archaeal homolog of the complex suggests mechanisms that enable the channel to open across the membrane and to release laterally hydrophobic transmembrane segments of nascent membrane proteins into lipid. Many aspects of membrane-protein integration remain controversial and poorly understood, but new structural data provide testable hypotheses. We propose a model of how the channel recognizes transmembrane segments, orients them properly with respect to the plane of the membrane and releases them into lipid. We also discuss how the channel would prevent small molecules from crossing the lipid bilayer while it is integrating proteins.