A yeast model for amyloid-β aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity

SUMMARY Alzheimer’s disease is the most common neurodegenerative disease, associated with aggregation of amyloid-β (Aβ) peptides. The exact mechanism of neuronal cell dysfunction in Alzheimer’s disease is poorly understood and numerous models have been used to decipher the mechanisms leading to cellular death. Yeast cells might be a good model to understand the intracellular toxicity triggered by Aβ peptides. Indeed, yeast has been used as a model to examine protein functions or cellular pathways that mediate the secretion, aggregation and subsequent toxicity of proteins associated with human neurodegenerative disorders. In the present study, we use the yeast Saccharomyces cerevisiae as a model system to study the effects of intracellular Aβ in fusion with green fluorescent protein. We sent this fusion protein into the secretory pathway and showed that intracellular traffic pathways are necessary for the generation of toxic species. Yeast PICALM orthologs are involved in cellular toxicity, indicating conservation of the mechanisms of toxicity from mammals to yeast. Finally, our model demonstrates the capacity for intracellular Aβ to cross intracellular membranes and target mitochondrial organelles

ERAD defects and the HFE-H63D variant are associated with increased risk of liver damages in Alpha 1-Antitrypsin Deficiency.

The most common and severe disease causing allele of Alpha 1-Antitrypsin Deficiency (1ATD) is Z-1AT. This protein aggregates in the endoplasmic reticulum, which is the main cause of liver disease in childhood. Based on recent evidences and on the frequency of liver disease occurrence in Z-1AT patients, it seems that liver disease progression is linked to still unknown genetic factors.We used an innovative approach combining yeast genetic screens with next generation exome sequencing to identify and functionally characterize the genes involved in 1ATD associated liver disease.Using yeast genetic screens, we identified HRD1, an Endoplasmic Reticulum Associated Degradation (ERAD) associated protein, as an inducer of Z-mediated toxicity. Whole exome sequencing of 1ATD patients resulted in the identification of two variants associated with liver damages in Z-1AT homozygous cases: HFE H63D and HERPUD1 R50H. Functional characterization in Z-1AT model cell lines demonstrated that impairment of the ERAD machinery combined with the HFE H63D variant expression decreased both cell proliferation and cell viability, while Unfolded Protein Response (UPR)-mediated cell death was hyperstimulated.This powerful experimental pipeline allowed us to identify and functionally validate two genes involved in Z-1AT-mediated severe liver toxicity. This pilot study moves forward our understanding on genetic modifiers involved in 1ATD and highlights the UPR pathway as a target for the treatment of liver diseases associated with 1ATD. Finally, these findings support a larger scale screening for HERPUD1 R50H and HFE H63D variants in the sub-group of 1ATD patients developing significant chronic hepatic injuries (hepatomegaly, chronic cholestasis, elevated liver enzymes) and at risk developing liver cirrhosis

Identification of HRD1 as an important actor in Z-1AT-mediated toxicity in a toxicity assay set up in <i>S</i>. <i>cerevisiae</i>.

<p>(<b>A</b>) Empty Vector, Wild-type (WT) 1AT and Z-1AT expressing cells were grown in inducible media and spotted onto nitrocellulose. After 48 hr of growth at 30°C, the cells were rinsed from the nitrocellulose with distilled water and then immunoblotted for 1AT. (<b>B</b>) Yeast screen methodology summary. The expression of Z-1AT is not toxic in yeast. As a consequence we proposed to identify genes whose presence is necessary for this absence of toxicity, the latter being revealed upon target gene deletion. The Euroscarf library of individual gene deletion mutants is transformed by expression plasmids for either Z-1AT, WT-1AT or the empty vector. Individual colonies are picked and plated in 96-well plates. The growth test is then initiated by inducing the expression of the recombinant 1AT proteins on specific media. Clones with defective growth are selected and the DNA barcode sequence for gene identification purposes. In brief, through this screen, we identified 171 KO strains in which Z-1AT toxicity was slightly changed compared to the empty vector. Then, we have tested these 171 KO strains for the toxicity induced by the WT-1AT expression and found 31 KO mutants in whom Z-1AT was more toxic than the WT-1AT. Finally, only 5 genes displayed a clear human ortholog. (<b>C</b>) Yeast cells transformed with empty vector, WT-1AT or Z-1AT were grown in liquid synthetic complete medium to log phase. Cells were spotted on plates containing SD medium (2% dextrose) to repress 1AT expression or SG medium (2% galactose) to induce 1AT expression, and were incubated at 30°C for 3 days. Shown is fourfold serial dilution starting with equal numbers of cells. Spotting assays for <i>HRD1</i> deletion strain (right panels) and the parental control strain (BY4247 –left panels) are shown.</p

Combined effect of HFE-H63D variant and ERAD impairment in WT and Z-1AT expressing cells.

<p><b>(A)</b> WT and Z-IB3 cells were treated with siRNA-mediated silencing of HERP or HRD1 for 72h and transfected with plasmid encoding HFE-H63D variant or pcDNA. At 48 h after transfection percentage of live cells for each conditions was determined. Data shown denote the -fold change relative to WT-IB3 cells (mean ± S.D., n = 3 independent experiments). In all panels asterisks indicate p < 0.05 as determined by two-tailed t test using WT-IB3 cells or siHERP Z-IB3 cells or siHRD1 Z-IB3 cells as reference. (<b>B)</b> Model for ERAD impairment and HFE-H63D mutation mediated liver toxicity associated with Z-1ATD. The model is adapted from Ordonez <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179369#pone.0179369.ref042" target="_blank">42</a>]. An insult resulting from the ERAD default increased the misfolded client proteins into the ER and this insult is effectively buffered by chaperone diffusion in WT-1AT cells. This prevents an UPR “hyperactivation” and the death of cells (‘‘ER stress resolved”). In contrast, in cells that accumulate Z-aggregates, the global ER environment is affected, which impairs chaperon access to misfolded proteins, thereby increasing the cell’s propensity to activate ER stress. An insult resulting from the ERAD default increased the misfolded client proteins, enhanced by the HFE-H63D mutation into the ER, and this insult is not effectively buffered by chaperone in Z-1AT cells. The cells are not anymore able to resolve the ER homeostasis (‘‘unresolved”) and therefore “hyperactivate” the UPR-mediated cell death.</p

Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.

<p>Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.</p

HERP-R50H stabilizes Herp endogenous and increases cell toxicity.

<p>(<b>A</b>) Immunoblot of 1AT, HRD1, BiP and Hsp90 protein expression in cell lysates following siRNA-mediated silencing of HERP and HRD1 in Z-IB3 cells. Tunicamycin (Tun) treatment at 2 μg/ml for 24 hours is used as positive control. Quantitative analyses of the immature (black bar graph) and mature (white bar graph) forms of Z-1AT after HERP and HRD1 silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicate <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference. (<b>B</b>) Effect of HRD1 and HERP silencing on steady state levels of Z-1AT in Z-IB3 cells. Cell homogenates were separated into insoluble (left) and soluble (right) fractions and these fractions were then subjected to immunoblot analysis for 1AT (top) and GAPDH (bottom). (<b>C</b>) Percentage of live cells was estimated as described in Materials and methods upon pcDNA, HERP-WT or HERP-R50H overexpression at 48 h after transfection. Data are presented as a ratio relative to control pcDNA empty vector (mean ± SD, n = 3 independent experiments). The asterisk indicates p < 0.05 as determined by two-tailed t-test using pcDNA empty vector as the reference. (<b>D</b>) Immunoblot of Flag tag (upper) and HERPUD1 (Herp) protein expression (lower) in cell lysates following cycloheximide (CHX) treatment. Black arrowhead indicates Flag-Herp expression bands and grey arrowhead indicates endogenous (Endo)-Herp expression bands. Z-IB3 cells were transfected with plasmids encoding the empty vector (pcDNA), HERP-WT, HERP-R50H and HERP-K61R mutation. At 48 h after transfection, 50 μg/ml cycloheximide (CHX) were added and the chase was performed in the absence or presence of CHX for the indicated times. Quantitative analysis of the cycloheximide-chase experiments for the Flag tag (left) and Herp endogenous (right) upon pcDNA, HERP-WT, HERP-R50H or HERP-K61R overexpression is shown. Data shown denote the -fold change relative to <i>t</i> = 0 (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels asterisks indicate <i>p</i> < 0.05 as determined by two-way ANOVA followed by Bonferroni post-test.</p

HRD1 silencing affects the viability, the traffic and secretion of human cells lines expressing the Z variant.

<p>(<b>A</b>) Viability is measured using GF-AFC Substrate. This compound can enter live cells where it is cleaved by the live-cell protease to release AFC. WT and Z-IB3 cells were treated as described in Material and methods. Quantitative analysis of candidate genes silencing on viability in WT or Z-IB3 cells (mean ± S.D., n = 3 independent experiments). The * indicates p < 0.05 as determined by two-tailed t-test using WT-IB3 cells as reference. (<b>B</b>) Immunoblot analysis of 1AT and Hsp90 protein expression in cell lysates and culture media following siRNA-mediated silencing of the candidate genes HDAC2 and HRD1 in WT and Z-IB3 cells. Traffic of the 1AT glycoprotein through the secretory pathway can be monitored by a change in its migration on SDS-PAGE in response to the processing of ER-acquired N-linked oligosaccharides (the immature form: I) during trafficking through the Golgi to generate the slower migrating, mature glycoform (Mat). The latter is secreted in the serum (secreted form: S) by the cell. Quantitative analyses of the immature, mature, and secreted forms of WT-1AT (black bar graph) and Z-1AT (white bar graph) after candidate genes silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated WT or Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicates <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference.</p

Performance of the Cepheid Methicillin-Resistant Staphylococcus aureus/S. aureus Skin and Soft Tissue Infection PCR Assay on Respiratory Samples from Mechanically Ventilated Patients for S. aureus Screening during the Phase 2 Double-Blind SAATELLITE Study.

We investigated the performance of the Xpert methicillin-resistant Staphylococcus aureus (MRSA)/S. aureus skin and soft tissue (SSTI) quantitative PCR (qPCR) assay in SAATELLITE, a multicenter, double-blind, phase 2 study of suvratoxumab, a monoclonal antibody (MAb) targeting S. aureus alpha-toxin, for reducing the incidence of S. aureus pneumonia. The assay was used to detect methicillin-susceptible S. aureus (MSSA) and MRSA in lower respiratory tract (LRT) samples from mechanically ventilated patients. LRT culture results were compared with S. aureus protein A () gene cycle threshold () values. Receiver operating characteristic (ROC) and Youden index were used to determine the cutoff for best separation of culture-S. aureus-negative and S. aureus-positive patients. Of 720 screened subjects, 299 (41.5%) were S. aureus positive by qPCR, of whom 209 had culture data: 162 (77.5%) were S. aureus positive and 47 (22.5%) were S. aureus negative. Culture results were negatively affected by antibiotic use and cross-laboratory variability. An inverse linear correlation was observed between values and quantitative S. aureus culture results. A value of 29 (≈2 × 10 CFU/mL) served as the best cutoff for separation between culture-negative and culture-positive samples. The associated area under the ROC curve was 83.8% (95% confidence interval [CI], 78 to 90%). Suvratoxumab provided greater reduction in S. aureus pneumonia or death than placebo in subjects with low S. aureus load ( ≥ 29; relative risk reduction [RRR], 50.0%; 90% CI, 2.7 to 74.4%) versus the total study population (RRR, 25.2%; 90% CI, -4.3 to 46.4%). The qPCR assay was easy to perform, sensitive, and standardized and provided better sensitivity than conventional culture for S. aureus detection. Quantitative PCR output correlated with suvratoxumab efficacy in reducing S. aureus pneumonia incidence or death in S. aureus-colonized, mechanically ventilated patients