Stefaan Wera

Characterization of α-synuclein aggregation and synergistic toxicity with protein tau in yeast

A yeast model was generated to study the mechanisms and phenotypical repercussions of expression of α‐synuclein as well as the coexpression of protein tau. The data show that aggregation of α‐synuclein is a nucleation–elongation process initiated at the plasma membrane. Aggregation is consistently enhanced by dimethyl sulfoxide, which is known to increase the level of phospholipids and membranes in yeast cells. Aggregation of α‐synuclein was also triggered by treatment of the yeast cells with ferrous ions, which are known to increase oxidative stress. In addition, data are presented in support of the hypothesis that degradation of α‐synuclein occurs via autophagy and proteasomes and that aggregation of α‐synuclein disturbs endocytosis. Reminiscent of observations in double‐transgenic mice, coexpression of α‐synuclein and protein tau in yeast cells is synergistically toxic, as exemplified by inhibition of proliferation. Taken together, the data show that these yeast models recapitulate major aspects of α‐synuclein aggregation and cytotoxicity, and offer great potential for defining the underlying mechanisms of toxicity and synergistic actions of α‐synuclein and protein tau.

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.

Protein phosphatase 2A on track for nutrient-induced signalling in yeast

Early studies identified two bona fide protein phosphatase 2A (PP2A)‐encoding genes in Saccharo‐myces cerevisiae, designated PPH21 and PPH22. In addition, three PP2A‐related phosphatases, encoded by PPH3, SIT4 and PPG1, have been identified. All share as much as 86% sequence similarity at the amino acid level. This review will focus primarily on Pph21 and Pph22, but some aspects of Sit4 regulation will also be discussed. Whereas a role for PP2A in yeast morphology and cell cycle has been readily recognized, uncovering its function in yeast signal transduction is a more recent breakthrough. Via their interaction with phosphorylated Tap42, PP2A and Sit4 play a pivotal role in target of rapamycin (TOR) signalling. PPH22 overexpression mimics overactive cAMP–PKA (protein kinase A) signalling and PP2A and Sit4 might represent ceramide signalling targets. The methylation of its catalytic subunit stabilizes the heterotrimeric form of PP2A and might counteract TOR signalling. We will show how these new elements could lead us to understand the role and regulation of PP2A in nutrient‐induced signalling in baker’s yeast.

Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells.

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65α) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (ΔG = −55.9 kcal/mol) in the 5′-untranslated region (5′-UTR). Deletion of the 5′-UTR allows efficient translation of the PR65α message in vitro and overexpression in COS-1 cells. Insertion of the 5′-UTR into the β-galactosidase leader sequence dramatically inhibits translation of the β-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65α expression vector lacking the 5′-UTR, express high levels of PR65α, accumulating in both nucleus and cytoplasm. PR65α overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M.(1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.

Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast

Abstract The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP3) in yeast is rapidly transformed into IP4 and IP5 by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP5 into IP6. In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP3 accumulation upon glucose addition was completely absent in the plc1Δ strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP2 and generation of IP3, seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP3 signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

NIPP-1, a nuclear inhibitory subunit of protein phosphatase-1, has RNA-binding properties

NIPP-1 is a nuclear inhibitory subunit of protein phosphatase-1 with structural similarities to some proteins involved in RNA processing. We report here that baculovirus-expressed recombinant NIPP-1 displays RNA-binding properties, as revealed by North-Western analysis, by UV-mediated cross-linking, by RNA mobility-shift assays, and by chromatography on poly(U)-Sepharose. NIPP-1 preferentially bound to U-rich sequences, including RNA-destabilizing AUUUA motifs. NIPP-1 also associated with single-stranded DNA, but had no affinity for double-stranded DNA. The binding of NIPP-1 to RNA was blocked by antibodies directed against the COOH terminus of NIPP-1, but was not affected by prior phosphorylation of NIPP-1 with protein kinase A or casein kinase-2, which decreases the affinity of NIPP-1 for protein phosphatase-1. The catalytic subunit of protein phosphatase-1 did not bind to poly(U)-Sepharose, but it bound very tightly after complexation with NIPP-1. These data are in agreement with a function of NIPP-1 in targeting protein phosphatase-1 to RNA.

Analysis and modification of trehalose 6-phosphate levels in the yeast Saccharomyces cerevisiae with the use of Bacillus subtilis phosphotrehalase.

In the yeast Saccharomyces cerevisiae, trehalose is synthesized by the trehalose synthase complex in two steps. The Tps1 subunit catalyses the formation of trehalose 6-phosphate (Tre6P), which is dephosphorylated by the Tps2 subunit. Tps1 also controls sugar influx into glycolysis; a tps1 deletion strain is therefore unable to grow on glucose. It is unclear whether this regulatory function of Tps1 is mediated solely by Tre6P or also involves the Tps1 protein. We have developed a novel sensitive and specific assay method for Tre6P. It is based on the conversion of Tre6P into glucose and glucose 6-phosphate with purified phosphotrehalase from Bacillus subtilis. The glucose formed is measured with the glucose-oxidase/peroxidase method. The Tre6P assay is linear in the physiological concentration range. The detection limit, including the entire extraction procedure, is 15 nmol, corresponding to an intracellular concentration of 100 microM. To modify Tre6P levels in vivo, we expressed B. subtilis phosphotrehalase in yeast. The enzyme is functional because it rescues the temperature-sensitive growth defect of a tps2Delta strain and drastically lowers Tre6P levels in this strain. However, phosphotrehalase expression remains without effect on Tre6P levels in wild-type strains, as opposed to overexpression of Tps2. Because Tps2 is part of the Tre6P synthase (TPS) complex and because this complex is destabilized in tps2 deletion strains, these results can be explained if Tre6P is sequestered within the TPS complex in wild-type cells. The very low levels of Tre6P in cells overexpressing Tps2 have a limited effect on sugar phosphate accumulation and do not prevent growth on glucose. Taken together, our results support a model in which the regulatory function of Tps1 on sugar influx is mediated both by the Tps1 protein and by Tre6P.

Virosome-based active immunization targets soluble amyloid species rather than plaques in a transgenic mouse model of Alzheimer’s disease

Active vaccination with amyloid peptides shows promise for the treatment and prevention of Alzheimer’s disease (AD). Several studies in transgenic mouse models of AD have revealed the potency of vaccination to prevent or even clear amyloid plaques from mouse brain. However, the idea that soluble oligomeric species of β-amyloid (Aβ), rather than plaques, trigger the disease has gained momentum, and current active vaccination strategies affect the levels of total or soluble brain Aβ little or not at all. We describe an active vaccination method based on Aβ1-16 presented on the surface of virosomes, which triggered a dramatic decrease in both soluble Aβ40 (75% reduction; p=0.01) and soluble Aβ42 (62% reduction; p=0.03) in a double transgenic mouse model of AD. Whereas Aβ40 and Aβ42 levels in the insoluble fraction tended to be reduced (by 30% and 27%, respectively), the number of thioflavine-S-positive amyloid plaques was not affected. The high specific antibody responses, obtained without eliciting T-cell reactivity, demonstrate that immunostimulating reconstituted influenza virosomes are a promising antigen carrier system against the neuropathology of AD.

Preparation of Cyclo-Phen-Type Ligands: Chelators of Metal Ions as Potential Therapeutic Agents in the Treatment of Neurodegenerative Diseases

Many recent studies have shown the major role played by metal ions (copper, zinc, iron, ...) in the modification of the folding and/or the aggregation of proteins that leads to serious pathologies. Several neurodegenerative diseases (Alzheimer’s disease, spongiform encephalopathies, Parkinson’s disease, Huntington’s disease, ...) involve similar disastrous interactions between metal ions and proteins. In the case of Alzheimer’s disease, the pathology is associated with the aggregation of b-amyloid peptides (Ab) in the brain, which leads to the formation of amyloid plaques. The accumulation of redox-active metal ions in these amyloid plaques is probably responsible for the oxidative stress which induces neuronal lesions in the brain that result in irreversible loss of intellectual faculties. The use of a metal ligand like Clioquinol led to improvements in patients suffering from Alzheimer’s disease and indicated that therapeutic approaches are possible with metal ion chelators in neurodegenerative diseases. Among Cu/Zn chelators capable of solubilizing Ab from post mortem brain tissue, interesting results were obtained with bathophenanthroline and bathocuproine, two ligands based on the 1,10phenanthroline structure. However, due to the presence of sulfate residues, these chelators are too hydrophilic to cross the blood brain barrier; consequently their use in vivo cannot be envisaged.

Laboratory validation of a lateral flow device for the detection of CyHV-3 antigens in gill swabs

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.