Clioquinol promotes the degradation of metal-dependent amyloid-β (Aβ) oligomers to restore endocytosis and ameliorate Aβ toxicity

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.Alzheimer’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 conservation of protein homeostasis mechanisms—such as protein trafficking and chaperone networks—among all eukaryotes makes yeast a powerful discovery platform for modeling the cellular toxicities caused by neurodegenerative disease proteins (6). In neurons, the cleavage of APP to generate the Aβ peptide occurs within the secretory and endosomal pathways (7). APP is trafficked through the secretory pathway to the plasma membrane and subsequently internalized and recycled through endosomal vesicles and the trans-Golgi network back to the plasma membrane (7). During this recycling, the Aβ peptide is liberated from APP by β/γ-secretases, thus enabling the peptide to interact with multiple membranous compartments within the cell.We have taken advantage of the great conservation of the secretory and endocytic pathways between yeast and neurons to study Aβ in a simple, highly tractable model organism—budding yeast. By expressing Aβ as a fusion to an endoplasmic reticulum (ER) targeting signal, we have mimicked in yeast the multicompartmental distribution of Aβ (8). This approach bypasses the need to recapitulate the entire APP processing pathway and generates an Aβ peptide with exactly the same sequence as is found in the human brain. The ER targeting signal directs cotranslational transport of the primary translation product into the ER, where the signal sequence is removed by signal peptidase. The peptide then transits through the secretory pathway and is secreted from the cell. In yeast, the cell wall prevents secreted Αβ from diffusing away, allowing it to interact with the plasma membrane and undergo endocytosis. As in the human nervous system (2), the aggregation-prone 42-aa peptide is more prone to forming oligomeric species than the 40-aa peptide (9) and is more toxic (8).This model allowed us to take advantage of yeast genetics to perform a completely unbiased screen of the yeast genome for suppressers or enhancers of Aβ toxicity. Of the ∼6,000 genes we tested, we recovered only a handful of modifiers. There are ∼25,000 genes in the human genome and less than 20 (10) have been shown to confer risk for AD. However, several of the yeast genes that alter Aβ toxicity are either direct homologs or interacting partners of human risk factors (8, 10). For example, YAP1802, is the yeast homolog of PICALM, one the most highly validated risk factors for AD (10, 11); INP52, is homologous to SYNJ1, which interacts with the risk factor BIN1 (12, 13); and SLA1 is homologous to SH3KBP1, which interacts with the risk factor CD2AP (14–16). All of these proteins are involved in clathrin-mediated endocytosis in yeast and humans. In addition to ameliorating the toxicity of Aβ in yeast, these proteins reduced Aβ toxicity in both Caenorhabditis elegans and rat cortical neuronal models (8). The recovery of genes that promote clathrin-mediated endocytosis in unbiased genome-wide screens suggested that Aβ poisons this process (8). Indeed, the peptide compromised endocytosis of a transmembrane receptor (Ste3), an activity crucial for neuronal function. Importantly, the mechanism of action of these risk factors had not previously been linked to Aβ. Thus, the yeast model has already provided key insights on the nature of Aβ’s cellular toxicity in the human brain.In this study, we used our yeast Aβ model to identify small molecules that ameliorate toxicity. In an extremely stringent and unbiased screen of 140,000 compounds, we identified a small number of cytoprotective compounds, including 8-hydroxyquinolines (8-OHQs). Members of this family bind metals and are among the few compounds that have been shown to alleviate Aβ toxicity in mouse models of AD (17, 18), and to show early potential as an AD therapeutic (19). Here, we investigate the mechanism of action for the most efficacious member of this family, clioquinol (CQ).     

Global transcriptome response to ionic liquid by a tropical rain forest soil bacterium, Enterobacter lignolyticus

To process plant-based renewable biofuels, pretreatment of plant feedstock with ionic liquids has significant advantages over current methods for deconstruction of lignocellulosic feedstocks. However, ionic liquids are often toxic to the microorganisms used subsequently for biomass saccharification and fermentation. We previously isolated Enterobacter lignolyticus strain SCF1, a lignocellulolytic bacterium from tropical rain forest soil, and report here that it can grow in the presence of 0.5 M 1-ethyl-3-methylimidazolium chloride, a commonly used ionic liquid. We investigated molecular mechanisms of SCF1 ionic liquid tolerance using a combination of phenotypic growth assays, phospholipid fatty acid analysis, and RNA sequencing technologies. Potential modes of resistance to 1-ethyl-3-methylimidazolium chloride include an increase in cyclopropane fatty acids in the cell membrane, scavenging of compatible solutes, up-regulation of osmoprotectant transporters and drug efflux pumps, and down-regulation of membrane porins. These findings represent an important first step in understanding mechanisms of ionic liquid resistance in bacteria and provide a basis for engineering microbial tolerance.     

Conserved features of intermediates in amyloid assembly determine their benign or toxic states

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.     

Tegillarca granosa extract Haishengsu (HSS) suppresses expression of mdr1, BCR/ABL and sorcin in drug-resistant K562/ADM tumors in mice

PurposeTo evaluate the effect of Haishengsu (HSS), a protein extract from Tegillarca granosa, on multidrug-resistance genes mdr1, BCR/ABL and sorcin in transplanted tumors.Material/MethodsMice were inoculated subcutaneously with a drug resistant leukemia cell line K562/ADM. Tumor-bearing animals were divided into control, adriamycin, HSS and combination therapy (adriamycin plus HSS) groups. Flow cytometry was used to detect apoptosis of tumor cells, and RT-PCR was used to evaluate the expression of mdr1, BCR/ABL and sorcin.ResultsThe apoptosis rate in the high (71.8%), medium (72.3%) and low doses HSS group (72.4%) was higher than in control (1.2%, p<0.01), adriamycin (34.4%, p<0.05) or combination therapy group (46.4%, p<0.05). The mean optical density of mdr1, BCR/ABL and sorcin in HSS groups was lower than in control, adriamycin and combination therapy group (p<0.01). The optical density of the three genes in high HSS group was lower than in medium and low HSS group (p<0.01).ConclusionsHaishengsu promotes apoptosis of drug-resistant K562/ADM tumors in mice in a dose-dependent manner. The pro-apoptotic effect of Haishengsu may be related to a reduced expression of multidrug-resistance genes mdr1, BCR/ABL and sorcin.     

Experimental bacterial meningitis in the rabbit: cerebrospinal fluid changes and its relation to leukocyte response

This study was focused on cerebrospinal fluid (CSF) manifestations in experimental Streptococcus pneumoniae and Escherichia coli meningitis in rabbits. An increased (p less than 0.001) CSF lactate concentration was found in infected animals, mostly not accompanied by a decrease in CSF glucose concentrations. Despite a marked difference in CSF cellular response between the 2 etiological groups no significant difference in CSF lactate levels was found. Neither did CSF lactate levels correlate to CSF polymorphonuclear cell counts. CSF concentrations of albumin were with large variations above control levels in all infected animals. Also a small or moderate increase in CSF albumin levels was generally associated with a marked increase in CSF lactate concentration. The concentration of total amino acids in the CSF was above control values (mean + 2 SD) in 9/21 infected animals. Halothane/N2O anesthesia for 25 min increased (p less than 0.05) CSF levels of glucose, partly independent of alterations in plasma glucose concentrations, in both infected rabbits and in controls.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:In vivo functions of the Saccharomyces cerevisiae Hsp90 chaperone

In the highly concentrated environment of the cell, polypeptide chains are prone to aggregation during synthesis (as nascent chains await the emergence of the remainder of their folding domain), translocation, assembly, and exposure to stresses that cause previously folded proteins to unfold. A large and diverse group of proteins, known as chaperones, transiently associate with such folding intermediates to prevent aggregation, but in many cases the specific functions of individual chaperones are still not clear. In vivo, Hsp90 (heat shock protein 90) plays a role in the maturation of components of signal transduction pathways but also exhibits chaperone activity with diverse proteins in vitro, suggesting a more general function. We used a unique temperature-sensitive mutant of Hsp90 in Saccharomyces cerevisiae, which rapidly and completely loses activity on shift to high temperatures, to examine the breadth of Hsp90 functions in vivo. The data suggest that Hsp90 is not required for the de novo folding of most proteins, but it is required for a specific subset of proteins that have greater difficulty reaching their native conformations. Under conditions of stress, Hsp90 does not generally protect proteins from thermal inactivation but does enhance the rate at which a heat-damaged protein is reactivated. Thus, although Hsp90 is one of the most abundant chaperones in the cell, its in vivo functions are highly restricted.