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 Alzheimer's 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

Generation and diagnostic valorisation of monoclonal antibodies for human protein tau

The improved accessibility and quality of health care has resulted in a global ageing of the population. Given the fact that neurodegenerative disorders, of which Alzheimer s disease (AD) is the most prevalent, mainly affect the elderly, this represents a major socio-economic burden in need of attention. Although the etiology of many neurodegenerative diseases is not yet known, the aberrant aggregation of specific proteins in the brain is a common pathological hallmark found in many of these disorders. The main proteinaceous aggregates found in AD consist of extracellular deposits of amyloid-ß peptides (Aß), known as amyloid plaques, and intracellular aggregates of hyperphosphorylated protein tau in the form of paired helical filaments (PHF) and neurofibrillary tangles (NFT). These proteins have for several decades been the subject of scientific research focussed on unravelling the molecular mechanisms behind this devastating disease. The aggregation of protein tau into PHF and NFT occurs just prior to symptom onset and it reaches maximal levels at the late stage of AD, correlating with the severity of cognitive dysfunction. In contrast, Aß plaque pathology only shows a limited correlation to the clinical progression of AD and recent studies demonstrated that clearance of the Aß deposits in patients does not provide significant cognitive improvement. Consistently, protein tau is known to provoke neurodegeneration in the absence of amyloid pathology in a group of disorders known as tauopathies. It should be noted that the familial form of AD is associated with gene mutations leading to increased Aß42 production and aggregation as opposed to the sporadic form of AD where no gene mutations have thus far been discovered. Nevertheless, both Aß and tau are now widely used biomarkers in diagnostic tests for AD.Despite the advancements scientists have made, many questions, contradictions and hurdles still remain. These include the need for an accurate early diagnosis well before the appearance of clinical symptoms as well as the identification of the true toxic protein species involved in neurodegeneration. Both Aß and tau diagnostic tests are therefore in need of improvement, as is the general understanding of the molecular principles behind protein aggregation and toxicity. The latter subject has been intensively researched with the aid of different model systems, resulting in the description of many post translational modifications (PTMs) such as phosphorylation, truncation, oxidation, acetylation, glycosylation and conformational change as well as in vitro aggregation compounds. However, the relative contribution and spatio-temporal context of these PTMs in different neurological disorders is yet unknown, as is the in vivo relevance of the in vitro aggregation compounds. Moreover, recent advancements have shifted the focussing point from the insoluble protein aggregates to their soluble oligomeric counterparts. In the presentwork, generation of new monoclonal antibodies (mAbs) and insights based on the well documented humanized Saccharomyces cerevisiae model system is presented in order to investigate the molecular principles behind tau oligomerization as well as improve the currently available diagnostic tests.First, tau-expressing yeast strains were used to provide for the need of antigen (i.e. hyperphosphorylated, oligomeric tau) in antibody production and serve as a model for initial antibody characterization. The novel mAbs, called the ADx series, were shown to have distinct reactivities to monomeric and oligomeric tau. Additional validation of these mAbs on the THY-Tau22 mouse model and on AD patient brain extracts and slices pointed towards the existence of two distinct oligomeric tau species. Oligomeric species recognized by the high affinity ADx215 mAb, which was epitope mapped via peptide scanning and nuclear magnetic resonance (NMR) to the extreme N-terminus of tau including a non-phosphorylated Tyr18, did not significantly vary between different yeast strains, between 3- or 12-month old THY-Tau22 mice nor between AD patients of Braak stage 5 and 6, unlike the monomeric phosphorylation status of tau in these models (see Fig. 3.1 and Fig. 3.4). These data were supported by the apparent resistance of the ADx215 reactive tau oligomers to in vitro tau dephosphorylation suggesting that the formation of the ADx215 reactive tau oligomer was formed independently from appearance of certain AD-associated phospho-epitopes. In contrast, the new ADx210 mAb preferentially recognized tau monomers and oligomers when expressed in the pho85&#916; deletion strain, which is characterized by the presence of hyperphosphorylated tau and increased tau aggregation. The oligomeric reactivity diminished significantly upon tau dephosphorylation, indicating the hyperphosphorylated nature of these oligomers. Moreover, in AD patient brain extracts, the oligomeric reactivity closely followed monomeric tau hyperphosphorylation (see Fig. 3.1 and Fig. 3.4).The newly generated mAbs also showed a robust differentiation between cerebrospinal fluid (CSF) tau levels of control subjects and AD patients when tested in a Luminex xMAPTM based multiparametric test (p < 0.05), indicating their potential use as diagnostic tools. Based on these data, the applicability of the new mAbs in an enzyme linked immunosorbant assay (ELISA) was explored, resulting in the development of a new diagnostic total Tau ELISA test which was capable of accurately differentiating control subjects from AD patients based on CSF tau levels (p < 0.001) and has now been commercialized (see Fig. 3.8). In a second part, the purification of recombinantly expressed tau from the humanized yeast system was optimized. In parallel, the applicability of novel mAbs on multiple diagnostic platforms as well as the selection of an appropriate test calibrator was assessed. The quality of the tau antigen proved problematic at the antibody selection stage during thefirst immunization, as was the time needed to collect sufficient amounts of tau for antibody screening. Therefore, we provide evidence for optimization of tau purification from the humanized yeast model in terms of yield, purity and throughput. This goal was achieved by introduction of an affinity-tag-based purification step. While the previous ion exchange chromatography (IEC)-based purification method took almost 4 days to complete, was restricted to 2 liter yeast cultures and yielded approximately 10 - 15 µg of purified tau per liter of yeast culture, the novel nickel immobilized metal affinity capture chromatography (Ni-IMAC) method reduced the sample collection time to 2 days while yeast culture volumes could be upscaled to 10 liters with an approximate yield of 40 50 µg of purified tau per liter of yeast culture, opening the doors for industrial scale tau purification from the humanized yeast model (see Fig 4.5). Interestingly, assessment of the physiology of recombinantly expressed N- and C-terminally tagged tau (i.e. phosphorylation status, paperclip formation, oligomerization and aggregation as measured by sarkosyl insolubility) in yeast revealed that modification of the C-terminus of tau coincided with marked alterations in these characteristics, rendering it unsuited for tau purification but highlighting the importance of the C-terminus for these events (see Fig. 4.2 and Fig 4.3). Despite the improved purity of the sample, an immunogenic contaminant remained present in the purified tau samples. The identification and subsequent genetic deletion of ADH1 as the main contaminant in yeast based protein purification resulted in further process optimization.Additionally, the new purification method proved successful in providing sufficient amounts of antigen for antibody generation. One on the new mAbs generated using Ni-IMAC purified tau (ADx202) showed a similar epitope to a previously generated mAb (ADx201). Direct comparison of both antibodies in the new ELISA based total Tau test as well as on a multiparametric system using different calibrators was performed, showing a marked difference in their performance within the two tested platforms. While ADx201 was found to be most suited for an ELISA based test, ADx202 seemed to perform better in a multiparametric setup, regardless of the calibrator. This result highlights the importance of antibody validation on multiple platforms when developing a diagnostic test, in order to select the most optimal conditions (i.e. good concentration range of the measured marker, high affinity and specificity) (see Fig 4.12). Finally, the identification of distinct tau oligomeric species merited further investigation of molecular principles involved in the formation of these species. Different tau mutants were evaluated for their influence on the oligomerization potential and toxicity compared to native tau when expressed in the humanized yeast model. In addition, an in vitro compound study resulted in the identification of a new tau oligomerization compound.As expected, tau oligomerization was increased when native tau was expressed in the pho85&#916; strain as compared to the wild type BY4741, underlining the importance of tau hyperphosphorylation in the process of oligomer formation. Interestingly, the K280&#916; tau mutant, which is characterized by an increase of ß-sheet formation in the microtubule binding region (MTBR), showed a marked increase of ADx215 reactive tau oligomer formation, suggesting that this oligomeric species is in part stabilized by ß-sheets in the MTBR. A reduction of tau oligomerization was observed when the C-terminus was truncated at Asp421, a known caspase cleavage site. This data, together with the previously observed influence of the C-terminal modification of tau indicated the importance of an intact, unmodified C-terminus in tau paperclip formation and oligomerization (see Fig. 5.1). Additionally, assessment of a tau with a phospho-mimic Tyr18 mutation (Tyr18Asp) not only showed significant cytotoxicity during different growth phases of yeast, but also the formation of cytosolic tau inclusions in the late stationary phase of growth . Moreover, ADx210 reactivity was found to be upregulated in the pho85&#916; strain for the Tyr18 tau mutant, indicative of increased hyperphosphorylation dependent tau oligomer formation. This effect was solely due to the phospho-mimicking of Tyr18 as no significant alterations of other tau phospho-epitopes was detected (see Fig 5.2). Together, these observations point towards the formation of toxic tau oligomers carrying a phosphorylated Tyr18 residue during the earlier growth phases of yeast.In order to further optimize tau oligomerization (in light of subsequent immunizations), an in vitro compound study was performed to identify the most potent inducer of tau oligomerization. One compound, hexafluoro-isopropanol (HFIP), was selected and further optimized with regards to optimal concentration (5%), incubation time (24 hours) and temperature (25&#176;C) for in vitro tau oligomerization, resulting in up to 30% more oligomer formation after treatment (see Fig. 5.3 and Fig. 5.4). Interestingly, this compound was described to promote protein protein interactions, illustrating the principle of a critical concentration needed for tau oligomerization. These findings, together with the tau mutants described above, led to the development of a final tau purification and oligomerization protocol used to generate new high affinity oligomer specific mAbs.nrpages: 190status: publishe

Yeast as a Model for Alzheimer's Disease: Latest Studies and Advanced Strategies

The yeast Saccharomyces cerevisiae, a unicellular eukaryotic model, has enabled major breakthroughs in our understanding of a plethora of cellular and molecular processes. Today, a 're-invention' of its use in fundamental and applied research is paving the way for a better understanding of the mechanisms causing neurodegeneration. The increasing emergence of neurodegenerative disorders is becoming more and more problematic in our ageing society. Most prevalent is Alzheimer's disease (AD), affecting more than 35 million people worldwide (Abbott, Nature 475, S2-S4, 2011) and causing an enormous burden on a personal and communal level. The disease is characterized by two major pathological hallmarks: extracellular amyloid plaques consisting mainly of deposits of amyloid β (Aβ) peptides, and intracellular neurofibrillary tangles (NFTs), consisting mainly of aggregates of hyperphosphorylated tau protein. Despite the huge importance of thoroughly understanding the underlying molecular mechanisms of neurodegeneration, progress has been slow. However, multiple complementary research methods are proving their value, particularly with the work done with S. cerevisiae, which combines well-established, fast genetic and molecular techniques with the ability to faithfully capture key molecular aspects of neurodegeneration. In this review chapter, we focus on the considerable progress made using S. cerevisiae as a model system for Alzheimer's disease.status: publishe

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models: IMPACT ON TAU OLIGOMERIZATION AND DIAGNOSTICS

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