Etablierung konditionaler Knock-out Mäuse für ERAB (endoplasmic reticulum-associated amyloid ß-peptide binding protein) : funktionelle Analysen zur Bedeutung für die T-Zellentwicklung und den Stoffwechsel

Toleranz gegenüber körpereigenem Gewebe wird durch Deletion/Inaktivierung selbstreaktiver T-Zellen erreicht. Sind Mechanismen der Selbst-Toleranz unwirksam, können T-Zellreaktionen gegen körpereigene Antigene zu Autoimmunerkrankungen führen. Dabei können T-Effektorzellen lokale Entzündungsreaktionen hervorrufen und Gewebezellen schädigen. Eine Klärung der molekularen Mechanismen, die zur Brechung von Toleranz führen, ist für die Entwicklung neuer Therapiekonzepte bei Autoimmunerkrankungen und zur Eliminierung von Tumoren von großem Interesse. Erste Studien unseres Kooperationspartners D. Stern zur Funktion des Xchromosomalen ERAB („endoplasmic reticulum-associated amyloid β-peptide binding protein“) deuteten darauf hin, dass dieses Protein in Entzündungsreaktionen und in der T-Zell-Apoptose involviert sein könnte. In der vorliegenden Arbeit wurden daher konditionale Knock-out Mäuse für ERAB hergestellt. Der komplette Knock-out in den Mäusen zeigte einen letalen Phänotyp. Eine gestörte Expression des ERAB Gens durch das Neomyzin-Resistenz-Gen führte zu Bewegungsstörungen und Krämpfen der Tiere, zu einer erhöhten Apoptoserate in den Organen Milz, Thymus, Darm, Gehirn, Hoden, Leber und Niere und schließlich zum Tod der Tiere im Alter von ca. 5 Monaten. Männchen dieser ERAB/neo Linie zeigten einen Phänotyp, der vergleichbar mit den Auswirkungen einer Isoleucinstoffwechselkrankheit des Menschen ist, die durch Punktmutationen im entsprechenden menschlichen Gen hervorgerufen wird. Eine ERAB-Defizienz ausschließlich in Endothelzellen und hämatopoetischen Stammzellen führte zu ähnlichen Symptomen wie bei ERAB/neo Männchen beobachtet wurden. Jedoch starben die Tiere erst im Alter von 6-7 Monaten. Die beiden erhaltenen Knock-out Weibchen dieser Linie hingegen sind lebensfähig. Der Grund für den Tod der männlichen Tiere konnte noch nicht gefunden werden. In ersten Experimenten mit T-zellspezifischen Knock-out Tieren für ERAB konnte eine normale T-Zellentwicklung und eine verzögerte Thymusrückbildung nachgewiesen werden. Glucocorticoidinduzierte, aber nicht CD95-vermittelte Apoptose führte in ERAB-defizienten Thymozyten zu einer erhöhten Apoptoserate im Vergleich zu Wildtyp-Thymozyten. Da ERAB bei Alzheimer- und Parkinson-Erkrankungen eine wichtige Rolle spielt, sind die hier generierten Knock-out Mäuse für ERAB auch für entsprechende neurologische Untersuchungen sehr interessant

A non-enzymatic function of 17 beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required

Endothelial Microparticles From Acute Coronary Syndrome Patients Induce Premature Coronary Artery Endothelial Cell Aging and Thrombogenicity: Role of the Ang II/AT1 Receptor/NADPH Oxidase-Mediated Activation of MAPKs and PI3-Kinase Pathways

International audienceBackground: Microparticles (MPs) have emerged as a surrogate marker of endothelial dysfunction and cardiovascular risk. This study examined the potential of MPs from senescent endothelial cells (ECs) or from patients with acute coronary syndrome (ACS) to promote premature EC aging and thrombogenicity. Methods: Primary porcine coronary ECs were isolated from the left circumflex coronary artery. MPs were prepared from ECs and venous blood from patients with ACS (n=30) and from healthy volunteers (n=4) by sequential centrifugation. The level of endothelial senescence was assessed as senescence-associated β-galactosidase activity using flow cytometry, oxidative stress using the redox-sensitive probe dihydroethidium, tissue factor activity using an enzymatic Tenase assay, the level of target protein expression by Western blot analysis, platelet aggregation using an aggregometer, and shear stress using a cone-and-plate viscometer. Results: Senescence, as assessed by senescence-associated β-galactosidase activity, was induced by the passaging of porcine coronary artery ECs from passage P1 to P4, and was associated with a progressive shedding of procoagulant MPs. Exposure of P1 ECs to MPs shed from senescent P3 cells or circulating MPs from ACS patients induced increased senescence-associated β-galactosidase activity, oxidative stress, early phosphorylation of mitogen-activated protein kinases and Akt, and upregulation of p53, p21, and p16. Ex vivo, the prosenescent effect of circulating MPs from ACS patients was evidenced only under conditions of low shear stress. Depletion of endothelial-derived MPs from ACS patients reduced the induction of senescence. Prosenescent MPs promoted EC thrombogenicity through tissue factor upregulation, shedding of procoagulant MPs, endothelial nitric oxide synthase downregulation, and reduced nitric oxide–mediated inhibition of platelet aggregation. These MPs exhibited angiotensin-converting enzyme activity and upregulated AT1 receptors and angiotensin-converting enzyme in P1 ECs. Losartan, an AT1 receptor antagonist, and inhibitors of either mitogen-activated protein kinases or phosphoinositide 3-kinase prevented the MP-induced endothelial senescence. Conclusions: These findings indicate that endothelial-derived MPs from ACS patients induce premature endothelial senescence under atheroprone low shear stress and thrombogenicity through angiotensin II–induced redox-sensitive activation of mitogen-activated protein kinases and phosphoinositide 3-kinase/Akt. They further suggest that targeting endothelial-derived MP shedding and their bioactivity may be a promising therapeutic strategy to limit the development of an endothelial dysfunction post-ACS

A non-enzymatic function of 17beta-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival

Deficiency of the mitochondrial enzyme 2-methyl-3-hydroxybutyryl-CoA dehydrogenase involved in isoleucine metabolism causes an organic aciduria with atypical neurodegenerative course. The disease-causing gene is HSD17B10 and encodes 17beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer's disease. Here we show that clinical symptoms in patients are not correlated with residual enzymatic activity of mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10(-/-) mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. This finding indicates that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to defects in general mitochondrial function. Therefore alternative therapeutic approaches to an isoleucine-restricted diet are required