Regulation der Kalzium Homöostase in Immunzellen und in Alzheimer-Modell Zelllinien

Ziel dieser Arbeit war es die Regulation von Kalziumsignalen genauer zu untersuchen und neue Gene zu identifizieren und charakterisieren die hierauf Einfluss haben. Ca2+ Signale regulieren genau kontrolliert verschiedene zelluläre Prozesse, wie u.a. Proliferation, Effektorfunktionen, Neurotransmitterabgabe oder Muskelkontraktionen. Dabei kann Ca2+ u.a. durch verschiedene Liganden-, speicher- oder spannungsgesteuerte Kanäle in die Zelle gelangen. In dieser Arbeit wurde ein neuer Regulator des speichergesteuerten Kalziumeinstroms (SOCE) identifiziert und charakterisiert. Bei STIM2.1, einer Spleißvariante des Kanal- Aktivators STIM2.2 (vorher STIM2), handelt es sich um eine um acht Aminosäuren längere Variante, die durch Retention und Translation eines zusätzlichen Exons entsteht. Bei dem hierdurch veränderten Bereich handelt es sich um die Kanal-aktivierende Domäne, der Interaktionsdomäne zwischen dem Aktivatorprotein und der Kanaluntereinheit Orai. Diagnostische PCRs und quantitative RT-PCRs zeigten, dass es sich bei STIM2.1 um eine ubiquitär exprimierte Variante handelt, wobei die höchste Expression im Vergleich zur konventionellen Variante STIM2.2 in unstimulierten T Zellen nachgewiesen wurde. Eine spleißspezifische siRNA Herunterregulation in CD4+ T Zellen enthüllte in Ca2+- Imaging Experimenten den inhibitorischen Effekt der neuen Spleißvariante auf SOCE. Eine heterologe Überexpression der Spleißvarianten zeigte, dass STIM2.1 im Gegensatz zu STIM2.2 nicht in der Lage ist die Kanalkomponenten Orai1 oder Orai2 zu aktivieren. Mit Hilfe von heterologen Koüberexpressionen in HEK-Orai1 stabilen Zelllinien wurde der dominant-negative Effekt von STIM2.1 sowohl auf STIM2.2 als auch auf STIM1 mediierten SOCE, sowie auf den endogenen SOCE in Jurkat T Zellen bestätigt. Anhand von FRET Analysen gelang es die fehlende Aktivierungsfähigkeit von STIM2.1 auf eine stark reduzierte Interaktion zwischen STIM2.1 und Orai1 zurückzuführen. Des Weiteren wurde mit beiden Spleißvarianten eine SILAC basierte Proteomanalyse zur Identifikation neuer STIM2 Interaktionspartner durchgeführt, um Regulatoren der STIM2 Funktion oder des speichergesteuerten Kalziumeinstroms zu finden. Dafür wurden Fusionskonstrukte aus STIM2.1 bzw. STIM2.2 mit APEX2 kloniert. Bei APEX2 handelt es sich um ein Enzym, das in Anwesenheit von H2O2 die Umwandlung von Biotin-Phenol in sein Radikal katalysiert, welches mit sich in der unmittelbaren Nähe befindenden Proteinen reagiert und so zur Biotinylierung dieser Proteine führt. Mittels Ca2+-Imaging, Westernblot Analyse und Immunzytochemie wurde sowohl die Funktionalität der STIM2-Konstrukte, als auch die lokal begrenzte Funktionalität von APEX2 bestätigt. Eine massenspektrometrische Analyse der biotinylierten Proteine ergab eine Reihe möglicher Hits, darunter je einen spleißspezifischen Hit. Die erfolgreich durchgeführte Proteomanalyse stellt einen guten Ausgangspunkt für eine zukünftige Validierung einiger der gefundenen Proteine als STIM2 Interaktionspartner dar. Der dritte Teil der Arbeit befasst sich mit neuronalem SOCE und dem spannunggesteuerten Kalziumeinstrom (VOCE) und dem Einfluss von APP und Präsenilin 1 auf diese beiden. Bei APP und Präsenilin 1 handelt es sich um Proteine, die vor allem im Bezug auf ihre Rolle in der Alzheimer Krankheit untersucht sind. Hier konnte gezeigt werden, dass eine Überexpression und ein Ausschalten von APP, ebenso wie ein Ausschalten von PS1 zu einem verringerten SOCE führt. Zudem erhöht der knock out von APP den spannungsgesteuerten Kalziumeinstrom, während die Überexpression von APP695 swedish diesen erniedrigte. qRT-PCR Daten sprechen dafür, dass Cav2.2 der hierfür verantwortliche Kanal ist. In PS1 knock out Zellen, die auch einen drastisch vergrößerten depolarisationsinduzierten Ca2+-Einstrom aufwiesen, konnten erhöhte relative mRNA Mengen von Cav2.2 und Cav3.1 gezeigt werden. Zusammengefasst gelang es in dieser Arbeit neue Regulationsmechanismen der Ca2+- Homöostase und hieran beteiligte Proteine zu identifizieren und charakterisieren. Zusätzlich wurde ein guter Ausgangspunkt für die Identifikation neuer STIM2 Interaktionspartner und somit möglichen weiteren Regulatoren der Ca2+-Homöostase geschaffen.Changes in intracellular calcium play a essential role in many signaling pathways. As a second messenger it controls proliferation, differentiation, muscle contraction, neurotransmitter release but also cell death. The spatio-temporal pattern of these signals, restricted and defined in amplitude, kinetics and frequency is crucial to control the outcome on a cellular level. In this work new mechanisms to fine tune calcium signals via different calcium entry mechanisms were identified. The main calcium entry in non-excitable cells is store operated calcium entry (SOCE). We found that in addition to the known molecular players, Orai1, Orai2 and Orai3 as the pore forming channel subunits and STIM1 and STIM2 (now STIM2.2) as the channel activator, a STIM2 splice variant, STIM2.1, exists. The new variant is slightly longer (8 aa) due to the retention of an additional exon within the interaction domain of STIM and Orai. The ubiquitous expressed variant is highly conserved within mammals and comparatively equal expression levels in comparison to STIM2.2 were found in naïve CD4+ T cells. Ca2+ imaging experiments in these cells revealed that STIM2.1 influences calcium entry via Orai in a substantial and unexpected way. Splice specific siRNA knock down experiments prooved that, instead of being an activator as its homologues, STIM2.1 influences SOCE in a dominant negative way. Heterologous overexpression of the new splice variant showed that STIM2.1 by itself is not able to active Orai1 and that co-overexpression with its homologues revealed its inhibitory effect on STIM2.2- and STIM1-mediated SOCE. The decreased ability to interact with the channel component Orai1 was shown by FRET experiments. To further identify regulators of calcium signals, and SOCE more specifically, both STIM2 splice variants were used to perform a SILAC based proteomic screen using mass spectrometry to determine new interaction partners. Thereby APEX2, an engineered ascorbate peroxidase with the ability to initiate biotinylation of nearby proteins in the presence of biotin-phenol and H2O2, was cloned into the C-terminal variable region of STIM2.1 and STIM2.2. Western blot analysis and immunocytochemistry were used to confirm the local action of APEX2 and functionality of the assay. Mass spectrometry analysis revealed many potential interaction partners, amongst others one specific prediction for each splice variant. First steps to try to validate two candidates were taken but have to be followed up. Neuronal SOCE seems to be principally mediated by STIM2 that had been shown to be downregulated in Alzheimer’s disease (AD) and thereby aggravating the disease. Stable AD model cell lines with knock outs of the familiar AD associated genes APP or Presenilin 1 or overexpressing APP were used to identify the influence of these proteins on store operated and voltage gated calcium channels. Whereas effects on SOCE were relatively small striking differences were identified analyzing Cav channels. Knockout of APP and PS1 increases voltage operated calcium entry (VOCE) most likely through Cav2.2 and Cav2.2 and Cav3.1 respectively whereas the overexpression of APP695 swedish decreases VOCE in line with a decrease in relative mRNA level of Cav2.2. Altogether this works identified and characterized a new modulator of SOCE and shows that APP and PS1, genes related with familiar AD, influence calcium signals by modifying SOCE and VOCE. Furthermore a successful screen to identify new STIM2 interaction partners was performed and sets the foundation to investigate their impact in STIM2 function and calcium signaling

Metabolic and amyloid PET network reorganization in Alzheimer's disease: differential patterns and partial volume effects

Alzheimer’s disease (AD) is a neurodegenerative disorder, considered a disconnection syndrome with regional molecular pattern abnormalities quantifiable by the aid of PET imaging. Solutions for accurate quantification of network dysfunction are scarce. We evaluate the extent to which PET molecular markers reflect quantifiable network metrics derived through the graph theory framework and how partial volume effects (PVE)-correction (PVEc) affects these PET-derived metrics 75 AD patients and 126 cognitively normal older subjects (CN). Therefore our goal is twofold: 1) to evaluate the differential patterns of [18F]FDG- and [18F]AV45-PET data to depict AD pathology; and ii) to analyse the effects of PVEc on global uptake measures of [18F]FDG- and [18F]AV45-PET data and their derived covariance network reconstructions for differentiating between patients and normal older subjects. Network organization patterns were assessed using graph theory in terms of “degree”, “modularity”, and “efficiency”. PVEc evidenced effects on global uptake measures that are specific to either [18F]FDG- or [18F]AV45-PET, leading to increased statistical differences between the groups. PVEc was further shown to influence the topological characterization of PET-derived covariance brain networks, leading to an optimised characterization of network efficiency and modularisation. Partial-volume effects correction improves the interpretability of PET data in AD and leads to optimised characterization of network properties for organisation or disconnection

Einfluss verschiedener Schwefeldünger und unterschiedlicher Düngungshöhen auf den Ertrag von Kleegras

Im ökologischen Pflanzenbau haben kleinkörnige Leguminosen einen hohen Schwefelbedarf. Da der atmosphärische Schwefeleintrag in den letzten Jahrzehnten deutlich verringert wurde, stellt sich die Frage, ob S im Kleegras ein limitierender Nährstoff ist. Hierzu wurden Feldversuche an insgesamt vier Standorten durchgeführt. Die Standzeit betrug zwei Hauptnutzungsjahre mit einer Ansaat im August/September. Die sieben verschiedenen Düngungsvarianten wurden zu jedem Hauptnutzungsjahr gedüngt. Im Einzelnen wurden Magnesiumsulfat (20 und 40 kg S/ha im zeitigen Frühjahr), Calciumsulfat (40 kg S/ha im zeitigen Frühjahr) und elementarer Schwefel (20 und 40 kg S/ha mit Herbstausbringung, 40 kg S/ha im zeitigen Frühjahr sowie 2 x 20 kg S/ha ab der Saat) neben einer Kontrolle ohne Düngung geprüft. Durch eine Schwefelgabe erzielte das Kleegras in beiden Hauptnutzungsjahren immer einen Mehrertrag. Im ersten Hauptnutzungsjahr fiel der Mehrertrag nach Sulfatdünger höher aus als nach den Varianten mit elementarem Schwefel. Im zweiten Hauptnutzungsjahr lag zwischen den Düngungsformen kein Unterschied mehr vor. In beiden Hauptnutzungsjahren wurde nach der Gabe von 40 kg S/ha ein höherer Ertrag als nach einer Applikation von 20 kg S/ha festgestellt

Targeted alpha therapy in vivo: direct evidence for single cancer cell kill using 149Tb-rituximab

This study demonstrates high-efficiency sterilisation of single cancer cells in a SCID mouse model of leukaemia using rituximab, a monoclonal antibody that targets CD20, labelled with terbium-149, an alpha-emitting radionuclide. Radio-immunotherapy with 5.5MBq labelled antibody conjugate (1.11GBq/mg) 2 days after an intravenous graft of 5·106 Daudi cells resulted in tumour-free survival for >120 days in 89% of treated animals. In contrast, all control mice (no treatment or treated with 5 or 300µg unlabelled rituximab) developed lymphoma disease. At the end of the study period, 28.4%±4% of the long-lived daughter activity remained in the body, of which 91.1% was located in bone tissue and 6.3% in the liver. A relatively high daughter radioactivity concentration was found in the spleen (12%±2%/g), suggesting that the killed cancer cells are mainly eliminated through the spleen. This promising preliminary in vivo study suggests that targeted alpha therapy with 149Tb is worthy of consideration as a new-generation radio-immunotherapeutic approac

Labeling of DOTA-conjugated HPMA-based polymers with trivalent metallic radionuclides for molecular imaging

Background In this work, the in vitro and in vivo stabilities and the pharmacology of HPMA-made homopolymers were studied by means of radiometal-labeled derivatives. Aiming to identify the fewer amount and the optimal DOTA-linker structure that provides quantitative labeling yields, diverse DOTA-linker systems were conjugated in different amounts to HPMA homopolymers to coordinate trivalent radiometals Me(III)* = gallium-68, scandium-44, and lutetium-177. Results Short linkers and as low as 1.6% DOTA were enough to obtain labeling yields > 90%. Alkoxy linkers generally exhibited lower labeling yields than alkane analogues despite of similar chain length and DOTA incorporation rate. High stability of the radiolabel in all examined solutions was observed for all conjugates. Labeling with scandium-44 allowed for in vivo PET imaging and ex vivo measurements of organ distribution for up to 24 h. Conclusions This study confirms the principle applicability of DOTA-HPMA conjugates for labeling with different trivalent metallic radionuclides allowing for diagnosis and therapy

DNA double strand breaks as predictor of efficacy of the alpha-particle emitter Ac-225 and the electron emitter Lu-177 for somatostatin receptor targeted radiotherapy

Key biologic effects of the alpha-particle emitter Actinium-225 in comparison to beta-particle emitter Lutetium-177 labeled somatostatin analogue DOTATOC in vitro and in vivo were studied to evaluate the significance of H2AX-foci formation and its downstream effects. To determine relative biological effectiveness (RBE) between the two isotopes somatostatin expressing AR42J cells were incubated with Ac-225-DOTATOC and Lu-177-DOTATOC up to 48 h and viability was analyzed using the MTT assay. DNA double strand breaks were quantified after immunofluorescence staining of H2AX. Cell cycle was analyzed by flow cytometry. In vivo, uptake of both radiolabeled somatostatin-analogues into subcutaneous AR42J tumors and number of cells displaying H2AX-foci were measured. Therapeutic efficacy was assayed by monitoring tumor growth after treatment with activities translated from in vitro cytotoxicity. Ac-225-DOTATOC was synthesized with specific activities between 0.2-0.4 MBq/µg and radiochemical purity of > 90%. ED50 values were 30 kBq/ml after 24 h and 14 kBq/ml after 48 h. Lu-177-DOTATOC displayed radiochemical purity of >95% and ED50 values of 10 MBq/ml after 48 h. Number of DNA double strand breaks increased with increasing concentration of Ac 225 DOTATOC and Lu-177-DOTATOC similarly, if a factor of approximately 700 of Lu-177 activities over Ac-225 activities was applied. Already 24 h after incubation with 2.5, 5, and 10 kBq/ml Ac 225 DOTATOC cell cycle studies showed an increment of the percentage of tumor cells in G2/M phase up to 60%. After 72 h an apoptotic subG1 peak was also detectable. Tumor uptake for both radio peptides at 48 h was identical with 7.5 %ID/g, though overall number of cells with H2AX-foci was higher for tumors treated with 48 kBq Actinium-225-DOTATOC than tumors treated with 30 MBq Lutetium-177-DOTATOC (35% vs. 21%). Tumors with a mean volume of 0.34 ml reached exponential tumor growth after 25 days (44 kBq Ac-225-DOTATOC), after 21 days (34 MBq Lu-177-DOTATOC) and after 5 days (control). Thus H2AX-foci displayed the key parameter after irradiation with similar downstream effects for beta and alpha irradiation.JRC.E.5-Nuclear chemistr

Functional Imaging of Pheochromocytoma with 68Ga-DOTATOC and 68C-HED in a Genetically Defined Rat Model of Multiple Endocrine Neoplasia

Rats affected by the MENX multitumor syndrome develop pheochromocytoma (100%). Pheochromocytomas are uncommon tumors and animal models are scarce, hence the interest in MENX rats to identify and preclinically evaluate novel targeted therapies. A prerequisite for such studies is a sensitive and noninvasive detection of MENXassociated pheochromocytoma. We performed positron emission tomography (PET) to determine whether rat pheochromocytomas are detected by tracers used in clinical practice, such as 68Ga-DOTATOC (somatostatin analogue) or 11C-Hydroxyephedrine (HED), a norepinephrine analogue. We analyzed four affected and three unaffected rats. The PET scan findings were correlated to histopathology and immunophenotype of the tumors, their proliferative index, and the expression of genes coding for somatostatin receptors or the norepinephrine transporter. We observed that mean 68Ga-DOTATOC standard uptake value (SUV) in adrenals of affected animals was 23.3 ± 3.9, significantly higher than in control rats (15.4 ± 7.9; P = .03). The increase in mean tumor-to-liver ratio of 11C-HED in the MENX-affected animals (1.6 ± 0.5) compared to controls (0.7 ± 0.1) was even more significant (P = .0016). In a unique animal model, functional imaging depicting two pathways important in pheochromocytoma biology discriminated affected animals from controls, thus providing the basis for future preclinical work with MENX rats

PSA and PSA Kinetics Thresholds for the Presence of 68Ga-PSMA-11 PET/CT-Detectable Lesions in Patients with Biochemical Recurrent Prostate Cancer

68Ga-PSMA-11 positron-emission tomography/computed tomography (PET/CT) is commonly used for restaging recurrent prostate cancer (PC) in European clinical practice. The goal of this study is to determine the optimum time for performing these PET/CT scans in a large cohort of patients by identifying the prostate-specific-antigen (PSA) and PSA kinetics thresholds for detecting and localizing recurrent PC. This retrospective analysis includes 581 patients with biochemical recurrence (BC) by definition. The performance of 68Ga-PSMA-11 PET/CT in relation to the PSA value at the scan time as well as PSA kinetics was assessed by the receiver-operating-characteristic-curve (ROC) generated by plotting sensitivity versus 1-specificity. Malignant prostatic lesions were identified in 77%. For patients that were treated with radical prostatectomy (RP) a PSA value of 1.24 ng/mL was found to be the optimal cutoff level for predicting positive and negative scans, while for patients previously treated with radiotherapy (RT) it was 5.75 ng/mL. In RP-patients with PSA value <1.24 ng/mL, 52% scans were positive, whereas patients with PSA ≥1.24 ng/mL had positive scan results in 87%. RT-patients with PSA <5.75 ng/mL had positive scans in 86% and for those with PSA ≥5.75 ng/mL 94% had positive scans. This study identifies the PSA and PSA kinetics threshold levels for the presence of 68Ga-PSMA-11 PET/CT-detectable PC-lesions in BC patients

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

BACKGROUND: Alpha-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for alpha-particle emitting isotopes facilitating selective tumor therapies. PRINCIPAL FINDINGS: A dimer of the vascular tumor homing peptide F3 was chemically coupled to the alpha-emitter (213)Bi ((213)Bi-DTPA-[F3](2)). We found (213)Bi-DTPA-[F3](2) to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of (213)Bi-DTPA-[F3](2) we treated mice bearing intraperitoneally growing xenograft tumors with (213)Bi-DTPA-[F3](2). In a tumor prevention study between the days 4-14 after inoculation of tumor cells 6x1.85 MBq (50 microCi) of (213)Bi-DTPA-[F3](2) were injected. In a tumor reduction study between the days 16-26 after inoculation of tumor cells 6x1.85 MBq of (213)Bi-DTPA-[F3](2) were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found (213)Bi-DTPA-[F3](2) to accumulate in tumors but only low activities were found in control organs except for the kidneys, where (213)Bi-DTPA-[F3](2) is found due to renal excretion. CONCLUSIONS/SIGNIFICANCE: In conclusion we report that (213)Bi-DTPA-[F3](2) is a novel tool for the targeted delivery of alpha-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology