Joelle Rosseels

The minimum domain of Pho81 is not sufficient to control the Pho85–Rim15 effector branch involved in phosphate starvation-induced stress responses

The phosphate regulatory mechanism in yeast, known as the PHO pathway, is regulated by inorganic phosphate to control the expression of genes involved in the acquisition of phosphate from the medium. This pathway is also reported to contribute to other nutritional responses and as such it affects several phenotypic characteristics known also to be regulated by protein kinase A, including the transcription of genes involved in the general stress response and trehalose metabolism. We now demonstrate that transcription of post-diauxic shift (PDS)-controlled stress-responsive genes is solely regulated by the Pho85–Pho80 complex, whereas regulation of trehalose metabolism apparently involves several Pho85 cyclins. Interestingly, both read-outs depend on Pho81 but, while the previously described minimum domain of Pho81 is sufficient to sustain phosphate-regulated transcription of PHO genes, full-length Pho81 is required to control trehalose metabolism and the PDS targets. Consistently, neither the expression control of stress-regulated genes nor the trehalose metabolism relies directly on Pho4. Finally, we present data supporting that the PHO pathway functions in parallel to the fermentable growth medium- or Sch9-controlled pathway and that both pathways may share the protein kinase Rim15, which was previously reported to play a central role in the integration of glucose, nitrogen and amino acid availability.

Tau monoclonal antibody generation based on humanized yeast models: impact on Tau oligomerization and diagnostics.

Background: Oligomers of protein Tau are associated with neurodegenerative diseases. Results: New antibodies were generated and validated that recognize different degrees of oligomerization of protein Tau. Conclusion: Low order and higher order oligomers differ in C-terminal Tau phosphorylation and reflect consecutive stages in disease progression. Significance: Antibodies recognizing Tau oligomers provide insight into disease etiology and are promising diagnostic tools. A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr18. For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

Digital ELISA for the quantification of attomolar concentrations of Alzheimer's disease biomarker protein Tau in biological samples

The close correlation between Tau pathology and Alzheimers disease (AD) progression makes this protein a suitable biomarker for diagnosis and monitoring of the disorder evolution. However, the use of Tau in diagnostics has been hampered, as it currently requires collection of cerebrospinal fluid (CSF), which is an invasive clinical procedure. Although measuring Tau-levels in blood plasma would be favorable, the concentrations are below the detection limit of a conventional ELISA. In this work, we developed a digital ELISA for the quantification of attomolar protein Tau concentrations in both buffer and biological samples. Individual Tau molecules were first captured on the surface of magnetic particles using in-house developed antibodies and subsequently isolated into the femtoliter-sized wells of a 2 × 2 mm2 microwell array. Combination of high-affinity antibodies, optimal assay conditions and a digital quantification approach resulted in a 24 ± 7 aM limit of detection (LOD) in buffer samples. Additionally, a dynamic range of 6 orders of magnitude was achieved by combining the digital readout with an analogue approach, allowing quantification from attomolar to picomolar levels of Tau using the same platform. This proves the compatibility of the presented assay with the wide range of Tau concentrations encountered in different biological samples. Next, the developed digital assay was applied to detect total Tau levels in spiked blood plasma. A similar LOD (55 ± 29 aM) was obtained compared to the buffer samples, which was 5000-fold more sensitive than commercially available ELISAs and even outperformed previously reported digital assays with 10-fold increase in sensitivity. Finally, the performance of the developed digital ELISA was assessed by quantifying protein Tau in three clinical CSF samples. Here, a high correlation (i.e. Pearson coefficient of 0.99) was found between the measured percentage of active particles and the reference protein Tau values. The presented digital ELISA technology has great capacity in unlocking the potential of Tau as biomarker for early AD diagnosis.