Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3?. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3? in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3? to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting

Ultra-performance liquid chromatography-mass spectrometry for precise fatty acid profiling of oilseed crops

Oilseed crops are one of the most important sources of vegetable oils for food and industry. Nutritional and technical properties of vegetable oil are primarily determined by its fatty acid (FA) composition. The content and composition of FAs in plants are commonly determined using gas chromatography-mass spectrometry (GS-MS) or gas chromatography-flame ionization detection (GC-FID) techniques. In the present work, we applied ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) technique to FA profiling of sunflower and rapeseed seeds and compared this method with the GC-FID technique. GC-FID detected 11 FAs in sunflower and 13 FAs in rapeseed, while UPLC-MS appeared to be more sensitive, detecting about 2.5 times higher numbers of FAs in both plants. In addition to even-chain FAs, UPLC-MS was able to detect odd-chain FAs. The longest FA detected using GC-FID was an FA with 24 carbon atoms, whereas UPLC-MS could reveal the presence of longer FAs with the tails of up to 28 carbon atoms. Based on our results, we may conclude that UPLC-MS has great potential to be used for the assessment of FA profiles of oil crops

Quantitative Studien des Tau-Proteins und von Tauopathien

Tauopathies, such as Alzheimer’s Disease (AD), are a heterogeneous class of neurodegenerative disorders that exhibit a common pathological hallmark: the abnormal aggregation of aberrantly phosphorylated, misfolded tau protein inside neuronal and/or glial cells in the brain. Tau aggregation has been associated with tau gene missense mutations, changes in tau spliceform homeostasis, and many different post-translational modifications (PTMs). In particular, the pathological state of tau is often referred to as “hyperphosphorylated”, as phosphorylation is increased and present on several non-physiological sites in pathological conditions. Deciphering the role of tau phosphorylation and other PTMs has been a major goal in the field of neurodegeneration, as it may help in defining targets for prognostic and diagnostic approaches as well as clinically effective therapeutic strategies. Although current technologies, in particular mass spectrometry (MS)-based proteomics, have allowed for detailed mapping of tau phosphorylation and other PTMs, the disease-relevant sites remain elusive, as these qualitative studies yield limited information about functionality. Besides, only easily detectable modifications can be observed. In order to identify functionally relevant sites, the acquisition of comprehensive, quantitative data is indispensable. The aim of this thesis was the development of an analytical method to quantify tau and its PTMs in a precise manner. The developed assay, termed FLEXITau, is a sensitive MS-based targeted assay that measures unmodified tau peptide species relative to peptides from a heavy isotope-labeled standard, thus providing indirect but precise measure of the amount of modification present. In combination with traditional PTM mapping, site occupancy of individual sites can be calculated. The power of this assay was demonstrated by measuring the state of hyperphosphorylation of tau expressed in a cellular model for AD pathology, the Sf9 insect cells, mapping and calculating site occupancies for over 20 phosphorylations. In addition, FLEXITau was employed to define the tau PTM landscape present in AD post-mortem brain. The data confirmed that regions containing epitopes recognized by AD-specific antibodies are similarly modified in AD-tau and Sf9-tau. However other regions show large discrepancies in their PTM pattern, revealing limitations of Sf9-tau as a model system for tau pathology. Diagnosis of tauopathies in vivo as well as post-mortem is hampered by the clinicopathological heterogeneity of the diseases, the overlap of many pathological features, and the lack of accurate diagnostic tools. Thus, in the second part of this thesis, FLEXITau was applied to identify differences and similarities in the modification landscape of tau in human tissue from multiple tauopathies, namely AD, progressive supranuclear palsy (PSP), Pick’s disease (PiD), corticobasal degeneration (CBD), as well as non-demented individuals as a control group. The FLEXITau data showed that each condition presents with a unique molecular composition, i.e. a signature, determined by the tau PTM state and isoform distribution. Supervised machine learning approaches were then used to extract distinct peptide features from the FLEXITau data and train a computational classifier to distinguish each disease category. The diagnostic potential of the developed tool was corroborated by validation with an independent sample test set, where it achieved accuracies of 80% for PSP, 92% for PiD, 94% for CBD and controls, and 96% for AD. Altogether these findings show that FLEXITau is a versatile assay to analyze tau PTMs with unprecedented precision, with application to a wide range of biological and clinical questions. Thus in the future it may have a significant impact on the characterization, diagnosis and prognosis of tauopathies and could lay the ground for novel disease-modifying and preventative therapeutics.Tauopathien, wie z.B. die Alzheimer-Krankheit (AK), sind eine heterogene Klasse von neurodegenerativen Erkrankungen, die ein gemeinsames pathologisches Merkmal aufweisen: die Ablagerung von abnormal phosphoryliertem Tau-Protein in Neuronen oder Gliazellen im Gehirn. Die Aggregation von Tau ist mit Punktmutationen des Tau-Gens, Veränderungen in der Tau-Isoform-Homöostase und vielen verschiedenen post-translationalen Modifikationen (PTMs) assoziiert. Insbesondere wird der pathologische Zustand vom Tau-Protein oft als "hyperphosphoryliert" bezeichnet, da Phosphorylierung im pathologischen Zustand im erhöhten Maße und an unphysiologischen Stellen auftritt. Die Entschlüsselung der Rolle von Tau-Phosphorylierung und anderen PTMs ist ein wichtiges Ziel im Forschungsbereich der Neurodegeneration, da diese zur Bestimmung von molekularen Targets für prognostische und diagnostische Ansätze sowie für therapeutische Strategien benutzt werden könnte. Obwohl derzeitige Technologien, insbesondere die Massenspektrometrie (MS)-basierte Proteomik, eine detaillierte Kartierung von Phosphorylierungsstellen und anderen Tau-PTMs ermöglicht haben, ist es derzeit nicht eindeutig, welche PTMs krankheitsrelevant sind, da diese qualitativen Studien nur begrenzt Informationen über die Funktionalität der einzelnen Modifikationen liefern. Zudem können nur leicht nachweisbare Veränderungen mit diesen Techniken beobachtet werden. Um funktionsrelevante PTMs zu identifizieren, ist es unentbehrlich, umfassende und quantitative Daten zu gewinnen. Das Ziel dieser Arbeit war die Entwicklung eines Analyseverfahrens, das Tau und seine PTMs in einer präzisen Art und Weise quantifizieren kann. Der entwickelte Assay, FLEXITau genannt, ist ein empfindlicher MS-basierter Test, der unveränderte Peptide von Tau (also ohne PTMs) im quantitativen Vergleich zu schweren Tau-Peptiden misst. Diese stammen von einem Protein-Standard, der mit schweren Isotopen markiert ist. Hierdurch kann indirekt, aber präzise, der Bruchteil der modifizierten Peptide abgeleitet werden. In Kombination mit traditionellen PTM-Analysen kann die Stelle der Modifikation teilweise mit Aminosäure-genauer Auflösung bestimmt werden. Um die Verwendbarkeit des Assays zu testen, wurde hyperphosphoryliertes Tau in Sf-9-Insektenzellen, einem Zellmodell für AK, exprimiert und dessen Tau-PTM Muster analysiert. Hierbei wurden über 20 Sf-9-Tau Phosphorylierungsstellen lokalisiert und deren Besetzung berechnet. Zudem wurde FLEXITau dazu benutzt, das Tau-PTM Muster vergleichsweise in AK-post-mortem Gehirnproben zu bestimmen. Die Ergebnise bestätigten, dass Regionen, die Erkennungssequenzen für AK-spezifische Antikörper enthalten, im Sf-9-Tau tatsächlich ähnlich stark modifiziert sind wie im AK-Tau. Allerdings zeigten andere Regionen große Unterschiede in ihrem PTM-Muster, was offenbart, dass das Sf-9-Tau nur beschränkt als Modellsystem für AK-Tau geeignet ist. Die differentielle Diagnose von Tauopathien, sowohl klinisch als auch post-mortem, wird erschwert durch Uneindeutigkeit der Symptome, durch Überlappung vieler pathologischer Merkmale, aber auch durch mangelnde Zuverlässigkeit der aktuellen Diagnose-Methoden. Daher wurde im zweiten Teil dieser Arbeit FLEXITau angewandt, um Unterschiede und Ähnlichkeiten im Modifikationsmuster von Tau in menschlichem post-mortem-Gehirngewebe aus mehreren Tauopathien zu identifizieren, und zwar von Patienten mit AK, progressiver supranukleärer Blickparese (PSP), Pick-Krankheit (PiK), cortikobasaler Degeneration (CBD) sowie von Personen ohne Demenzerkrankung (Kontrolle). Die FLEXITau Daten zeigten, dass jede Krankheit eine eindeutige molekulare Zusammensetzung von Tau aufweist, eine Art Signatur, bestimmt durch die Verteilung von Tau-PTMs und Isoformen. Durch „überwachtes maschinelles Lernen“ (engl. supervised machine learning) wurden bestimmte Peptidmerkmale aus den FLEXITau-Daten herausgefiltert und dazu verwendet, einen Klassifizierungs-Algorithmus zu erstellen, der in der Lage ist, jede Krankheitskategorie von den anderen zu unterscheiden. Die Leistungsfähigkeit der entwickelten Methode zur Diagnose von Tauopathien wurde mit einem unabhängigen post-mortem-Datensatz validiert, und erreichte Genauigkeiten von 80% für PSP, 92% für PiK, 94% für CBD und Kontrollen, sowie 96% für AK. Insgesamt zeigen diese Ergebnisse, dass FLEXITau ein vielseitiger Test ist, der Tau-PTMs mit neuartiger Präzision misst, und so auf eine Vielzahl biologischer und klinischer Fragen angewendet werden kann

Comparison of the T-tubule system in adult rat ventricular and atrial myocytes, and its role in excitation-contraction coupling and inotropic stimulation

Narrow, tubular, inward projections of the sarcolemma ('T-tubules') are an established feature of adult mammalian ventricular myocytes that enables them to generate the whole-cell Ca2+ transients and produce coordinated contraction. Loss of T-tubules can occur during ageing and under pathological conditions, leading to altered cardiac excitation-contraction coupling. In contrast to adult ventricular cells, atrial myocytes do not generally express an extensive T-tubule system at any stage of development, and therefore rely on Ca2+ channels around their periphery for the induction of Ca2+ signalling and excitation-contraction coupling. Consequently, the characteristics of systolic Ca2+ signals in adult ventricular and atrial myocytes are temporally and spatially distinct. However, although atrial myocytes do not have the same regularly spaced convoluted T-tubule structures as adult ventricular cells, it has been suggested that a proportion of adult atrial cells have a more rudimentary tubule system. We examined the structure and function of these atrial tubules, and explored their impact on the initiation and recovery of Ca2+ signalling in electrically paced myocytes. The atrial responses were compared to those in adult ventricular cells that had intact T-tubules, or that had been chemically detubulated. We found that tubular structures were present in a significant minority of adult atrial myocytes, and were unlike the T-tubules in adult ventricular cells. In those cells where they were present, the atrial tubules significantly altered the on-set, amplitude, homogeneity and recovery of Ca2+ transients. The properties of adult atrial myocyte Ca2+ signals were different from those in adult ventricular cells, whether intact or detubulated. Excitation-contraction coupling in detubulated adult ventricular myocytes, therefore, does not approximate to atrial signalling, even though Ca2+ signals are initiated in the periphery of the cells in both of these situations. Furthermore, inotropic responses to endothelin-1 were entirely dependent on T-tubules in adult ventricular myocytes, but not in atrial cells. Our data reveal that that the T-tubules in atrial cells impart significant functional properties, but loss of these tubular membranes does not affect Ca2+ signalling as dramatically as detubulation in ventricular myocytes

FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease.

Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as "hyperphosphorylated". Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies

FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease

Tauopathies, including Alzheimer's disease (AD), are associated with the aggregation of modified microtubule associated protein tau. This pathological state of tau is often referred to as "hyperphosphorylated". Due to limitations in technology, an accurate quantitative description of this state is lacking. Here, a mass spectrometry-based assay, FLEXITau, is presented to measure phosphorylation stoichiometry and provide an unbiased quantitative view of the tau post-translational modification (PTM) landscape. The power of this assay is demonstrated by measuring the state of hyperphosphorylation from tau in a cellular model for AD pathology, mapping, and calculating site occupancies for over 20 phosphorylations. We further employ FLEXITau to define the tau PTM landscape present in AD post-mortem brain. As shown in this study, the application of this assay provides mechanistic understanding of tau pathology that could lead to novel therapeutics, and we envision its further use in prognostic and diagnostic approaches for tauopathies

Diaminothiazoles modify Tau phosphorylation and improve the tauopathy in mouse models.

Although Tau accumulation is a feature of several neurodegenerative conditions, treatment options for these conditions are nonexistent. Targeting Tau kinases represents a potential therapeutic approach. Small molecules in the diaminothiazole class are potent Tau kinase inhibitors that target CDK5 and GSK3β. Lead compounds from the series have IC50 values toward CDK5/p25 and GSK3β in the low nanomolar range and no observed toxicity in the therapeutic dose range. Neuronal protective effects and decreased PHF-1 immunoreactivity were observed in two animal models, 3×Tg-AD and CK-p25. Treatment nearly eliminated Sarkosyl-insoluble Tau with the most prominent effect on the phosphorylation at Ser-404. Treatment also induced the recovery of memory in a fear conditioning assay. Given the contribution of both CDK5/p25 and GSK3β to Tau phosphorylation, effective treatment of tauopathies may require dual kinase targeting

Cell adhesion-signaling components are required for SV40 infection. Integrins, in addition to GM1 lipids, are required for SV40 binding and infection.

<p>(A) A targeted siRNA screen reveals several structural and signaling components of cell adhesion to regulate the SV40 infectious route. A set of four siRNAs against 263 genes was applied in A431 human epithelial cells and virus infection was assessed by the presence of nuclear large T-antigen. Low-resolution imaging and image processing with the CellProfiler analysis software were subsequently performed. A Support Vector Machine (SVM)-based classification method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055799#pone.0055799-Rm1" target="_blank">[47]</a> was then used to determine percentage of infection upon siRNA treatment. The table shows the genes that reduced (red shades) or enhanced (green shades) SV40 infection with different strength when knocked down. The values in the boxes represent the number of different siRNAs that gave a similar phenotype. (B) Epistasis analysis between Cav1, GRAF1, and Ezrin. A431 cells were treated with siRNA against each one of these genes or combinations of two. Two or three siRNAs were used per gene. Cells were subsequently treated with SV40 and infection levels were assessed by the presence of nuclear T-antigen. The graph shows values pooled from the individual infection indices. p-values: 1.3×10⁻⁴ (Ezrin-siRNA, Cav1-siRNA), 0.39 (Ezrin-siRNA, GRAF1-siRNA). (C) Blocking integrin α2 function with an antibody reduces SV40 infection, similar to siRNA-mediated knock down. A431 cells were pre-incubated with 0.02 µg/µL of blocking antibody 20 min prior to infection (p-values 1×10^−4–7×10⁻⁴). (D) siRNA against integrins α2 and β1 reduces binding of SV40 at the surface of A431 cells. Binding was performed at cold for 2 h and binding capacity was determined by immunoblotting for the presence of the major capsid protein VP1 in cell extracts. Signal intensity was quantified by the ImageJ software and standard deviation corresponds to two independent experiments. (E) SV40 binds onto the surface of various cell lines with different intensity; GM1-deficient cells retain the ability to bind SV40. Quantification of signal intensity from two independent experiments was performed as in (D). (F) SV40-like particles (VLPs) can bind cells that lack its native receptor GM1 in a dose-dependent manner. (G) SV40 can bind cells that lack its native receptor GM1 via integrins. GM95 cells were treated with siRNA against integrin α2 and SV40 binding was determined by the abundance of VP1 protein, as described in (D). (H) Integrins can serve as binding sites for SV40. Integrin α2β1 was immunoprecipitated from A431 cells pretreated for 2 h with SV40 in the cold, and the VP1 protein was detected in the immunocomplex by immunoblotting (red box). Transferrin receptor was used as a negative control.</p