Ann De Vos

Protein folding diseases and neurodegeneration: Lessons learned from yeast

Budding yeast Saccharomyces cerevisiae has proven to be a valuable model organism for studying fundamental cellular processes across the eukaryotic kingdom including man. In this respect, complementation assays, in which the yeast protein is replaced by a homologous protein from another organism, have been very instructive. A newer trend is to use the yeast cell factory as a toolbox to understand cellular processes controlled by proteins for which the yeast lacks functional counterparts. An increasing number of studies have indicated that S. cerevisiae is a suitable model system to decipher molecular mechanisms involved in a variety of neurodegenerative disorders caused by aberrant protein folding. Here we review the current knowledge gained by the use of so-called humanized yeasts in the field of Huntingtons, Parkinsons and Alzheimers diseases.

THE DECOMPOSITION OF COPPER OXALATE TO METALLIC COPPER IS WELL SUITED FOR CHECKING THE INERT WORKING-CONDITIONS OF THERMAL-ANALYSIS EQUIPMENT

Thermal analysis (TA) techniques require careful control of some experimental parameters. One of these parameters is the gaseous atmosphere in which the experiment is carried out. Particularly at high temperatures, even very small amounts of oxygen can drastically influence the experiment so that totally different results are obtained compared with those in a completely inert atmosphere. The conduction of inert gases such as argon or (below SOO’C) nitrogen over an oxygen absorbent before entering the TA equipment is a necessary but not sufficient condition for carrying out the experiment in a totally oxygen-free atmosphere. One has to take care that the TA equipment is completely free of the oxygen that is inevitably inside the equipment after loading. Even flushing with the inert gas for 30 min at a flow of 50 ml min-’ before starting the experiment might be insufficient to remove all the residual air if the gas inlet and gas outlet are not placed in the right positions. This is shown in the following experiment. Figure 1 shows the decomposition of 30.543 mg copper oxalate; the equipment was flushed with argon for 30 min at a rate of 50 ml mine1 before starting the experiment and the flushing was continued during the experiment at the same flow-rate. In an inert atmosphere the decomposition goes to metallic copper (theoretical remaining weight, 41.9%). As shown by the weight increase between 300 and 6OO”C, there is an oxidation of copper (theoretical remaining weight is 52.5% if 100% CuO)! The

Serine‐409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast

Unraveling the biochemical and genetic alterations that control the aggregation of protein tau is crucial to understand the etiology of tau-related neurodegenerative disorders. We expressed wild type and six clinical frontotemporal dementia with parkinsonism (FTDP) mutants of human protein tau in wild-type yeast cells and cells lacking Mds1 or Pho85, the respective orthologues of the tau kinases GSK3β and cdk5. We compared tau phosphorylation with the levels of sarkosyl-insoluble tau (SinT), as a measure for tau aggregation. The deficiency of Pho85 enhanced significantly the phosphorylation of serine-409 (S409) in all tau mutants, which coincided with marked increases in SinT levels. FTDP mutants tau-P301L and tau-R406W were least phosphorylated at S409 and produced the lowest levels of SinT, indicating that S409 phosphorylation is a direct determinant for tau aggregation. This finding was substantiated by the synthetic tau-S409A mutant that failed to produce significant amounts of SinT, while its pseudophosphorylated counterpart tau-S409E yielded SinT levels higher than or comparable to wild-type tau. Furthermore, S409 phosphorylation reduced the binding of protein tau to preformed microtubules. The highest SinT levels were found in yeast cells subjected to oxidative stress and with mitochondrial dysfunction. Under these conditions, the aggregation of tau was enhanced although the protein is less phosphorylated, suggesting that additional mechanisms are involved. Our results validate yeast as a prime model to identify the genetic and biochemical factors that contribute to the pathophysiology of human tau.

C-terminal neurogranin is increased in cerebrospinal fluid but unchanged in plasma in Alzheimer's disease

Biomarkers monitoring synaptic degeneration/loss would be valuable for Alzheimers disease (AD) diagnosis. Postsynaptic protein neurogranin may be a promising cerebrospinal fluid (CSF) biomarker but has not yet been evaluated as a plasma biomarker.

The decomposition of the oxalate precursor and the stability and reduction of the YBa2Cu4O8 superconductor studied by TG coupled with FTIR and by XRD

The 124 superconductor YBa2Cu4O8 was prepared from the oxalate precursor Y2(C2O4)3. ·4BaC2O4·8CuC2O4·xH2O at one atmosphere oxygen pressure. In O2 the precursor decomposes in one step at 300°C and more gradually (300°–600°C) in Ar. The stability of the superconductor is strongly dependent on the gas atmosphere: in O2 and in air there is no significant weight change as long as the temperature does not exceed 800°C, whereas in a 1% O2-99%N2 mixture decomposition starts at about 670°C with the formation of CuO and YBa2Cu3Ox withx<7. The reduction of YBa2Cu4O8 in a 5% H2-95% Ar mixture takes place in at least four major steps with formation of products such as Y2O3, BaO, Cu2O, Cu, BaY2O4 and Ba4Y2O7.ZusammenfassungAus dem Oxalatpräkursor Y2(C2O4)34BaC2O48CuC2O4·xH2O wurde der 124 Supraleiter YBa2Cu4O8 hergestellt. In Sauerstoff zersetzt sich der Präkursor bei 300°C in einem Schritt, in Argon nach und nach (300°–600°C). Die Stabilität des Supraleiters hängt stark von der Atmosphärenzusammensetzung ab: in Sauerstoff und in Luft gibt es bis 800C keinen bemerkenswerten Gewichtsverdust, während in einem Gemisch aus 1% O2 und 99% N2 die Zersetzung bei etwa 670°C beignnt, wobei CuO und YBa2Cu3Ox mitx gebildet wird. Die Reduktion von YBa2Cu4O8 in einem 5%H2-95%Ar-Gemisch erfolgt in mindestens vier Hauptschritten, wobei Produkte wie Y2O3, BaO, Cu2O, Cu, BaY2O4 und Ba4Y2O7 gebildet werden.

Yeast as a Model System to Study Tau Biology

Hyperphosphorylated and aggregated human protein tau constitutes a hallmark of a multitude of neurodegenerative diseases called tauopathies, exemplified by Alzheimers disease. In spite of an enormous amount of research performed on tau biology, several crucial questions concerning the mechanisms of tau toxicity remain unanswered. In this paper we will highlight some of the processes involved in tau biology and pathology, focusing on tau phosphorylation and the interplay with oxidative stress. In addition, we will introduce the development of a human tau-expressing yeast model, and discuss some crucial results obtained in this model, highlighting its potential in the elucidation of cellular processes leading to tau toxicity.

Automated iodimetric determination of Cu+, Cu2+ and Cu3+ in the superconductor YBCO using a modified Gran plot technique

Abstract An automated potentiometric determination of the oxygen content of the superconductor YBCO is described. The end- point of the titration is found using a linear Gran plot technique. Suitable equations are derived by which the number of titration points can be considerably reduced and the titration can be stopped even before the equivalence point is reached. Automation of the iodimetric titration can be performed in an easy manner. During the titration a good estimate of the equivalence volume can be calculated using only two titration points.

The Cerebrospinal Fluid Neurogranin/BACE1 Ratio is a Potential Correlate of Cognitive Decline in Alzheimer's Disease.

Background: In diagnosing Alzheimer’s disease (AD), ratios of cerebrospinal fluid (CSF) biomarkers, such as CSF Aβ1-42/tau, have an improved diagnostic performance compared to the single analytes, yet, still a limited value to predict cognitive decline. Since synaptic dysfunction/loss is closely linked to cognitive impairment, synaptic proteins are investigated as candidate CSF AD progression markers. Objective: We studied CSF levels of the postsynaptic protein neurogranin and protein BACE1, predominantly localized presynaptically, and their relation to CSF total-tau, Aβ1-42, Aβ1-40, and Aβ1-38. All six analytes were considered as single parameters as well as ratios. Methods: Every ELISA involved was based on monoclonal antibodies, including the BACE1 and neurogranin immunoassay. The latter specifically targets neurogranin C-terminally truncated at P75, a more abundant species of the protein in CSF. We studied patients with MCI due to AD (n = 38) and 50 dementia due to AD patients, as well as age-matched cognitively healthy elderly (n = 20). A significant subset of the patients was followed up by clinical and neuropsychologically (MMSE) examinations for at least one year. Results: The single analytes showed statistically significant differences between the clinical groups, but the ratios of analytes indeed had a higher diagnostic performance. Furthermore, only the ratio of CSF neurogranin trunc P75/BACE1 was significantly correlated with the yearly decline in MMSE scores in patients with MCI and dementia due to AD, pointing toward the prognostic value of the ratio. Conclusion: This is the first study demonstrating that the CSF neurogranin trunc P75/BACE1 ratio, reflecting postsynaptic/presynaptic integrity, is related to cognitive decline.

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.

Detection and quantification of novel tau/phospho-tau epitopes in CSF using a multiplex assay approach

internationally accepted criteria, and AD cases were further classified for the presence of cerebrovascular disease (CBVD). Controls with no history of stroke and cognitive impairment were selected from the Singapore Epidemiology of Eye Disease program and matched by race, gender and 5year age groups. Retinal vascular parameters (retinal vascular caliber, fractal dimension and tortuosity) were assessed using a semi-automated computer-based program. Due to its skewed distribution, retinal vessel tortuosity was log-transformed. Logistic regression models were constructed adjusting for gender, age, hypertension, diabetes and hypercholesterolemia status. Results: A total of 156 AD cases (98 without CBVD and 58with CBVD), 27 vascular dementia cases, and 493 controls were included in this preliminary analysis. Narrower arteriolar caliber was associated with VaD (multivariable-adjusted odds ratio (OR) per standard deviation (SD) decrease: 2.41; 95%CI: 1.13-5.14) and AD with CBVD (OR per SD decrease: 1.84; 95%CI: 1.05-3.23). Narrower venular caliber (OR per SD decrease: 1.72; 95%CI: 1.11-2.68), decreased arteriolar fractal dimension (OR per SD decrease: 1.29; 95%CI: 1.03-1.61) and venular fractal dimension (OR per SD increase: 1.36; 95%CI: 1.08-1.72) were associated with only AD without CBVD. However, increased arteriolar and venular tortuosity was associated with all three subtypes of dementia. Conclusions: A sparser retinal microvascular network was associated with AD, whereas narrower arterioles were associated with dementia linked to CBVD, and tortuous retinal vessels were associated with all three subtypes of dementia. This suggests that retinal vascular parameters may potentially useful in assessing the contribution of microvascular pathology to the different subtpyes of dementia.