Untersuchungen zur Funktion der Knorpelproteine Ucma und Matrilin-1 im Zebrafisch

UCMA (im Zebrafisch Ucma) und Matrilin-1 sind zwei „nicht kollagene“ Proteine der Extrazellulärmatrix des Knorpels, deren Funktion bisher nur unzureichend aufgeklärt wurde. In der vorliegenden Arbeit wurde der Zebrafisch als Modellorganismus herangezogen um Expression und Funktion beider Proteine während der Zebrafisch Embryonalentwicklung zu untersuchen. UCMA, auch GRP genannt, ist ein vor kurzem entdecktes Mitglied der Familie der Glutamat γ-carboxylierten Proteine und wird hauptsächlich im Knorpel exprimiert. Im Zebrafisch sind aufgrund einer zusätzlichen Genomduplikation zwei Kopien des UCMA Gens vorhanden, ucmaa und ucmab, die sich jeweils auf den Chromosomen 25 und 4 befinden. Genstruktur, alternatives Spleißen und Proteinsequenz von UCMA sind zwischen Säugetieren und Zebrafisch stark konserviert und beide Formen von Zebrafisch Ucma sind in skelettalen Geweben exprimiert. Ucmaa wurde ab 18 hpf im sich entwickelnden Notochord detektiert. Außerdem wurde es in den sich entwickelnden Kraniofazialknorpeln gefunden. Ucmab Expression hingegen konnte nur schwach in spezifischen Knorpeln des Kraniofazialskeletts, jedoch nicht im Notochord und erst ab 96 hpf detektiert werden. Der „knockdown“ von Ucmaa führt zu schweren Wachstumsdefekten und Störungen der Skelettbildung. Im Knorpel von Morphanten findet sich weniger Aggrecan und Kollagen II. Vergleichbare Defekte konnten nach Unterdrückung der Glutamat γ-Carboxylierung durch Warfarin beobachtet werden, ein Hinweis darauf, dass die posttranslationale Modifikation essentiell für die Funktion von Ucmaa ist und die fehlende Glutamat γ-Carboxylierung an der Entstehung der sogenannten „Warfarin Embryopathien“ und ähnlichen skelettalen Krankheiten des Menschen beteiligt sein kann. Matrilin-1 ist das prototypische Mitglied der Familie der Matriline und in Mensch und Maus hauptsächlich in Knorpel exprimiert. Matn-1 weist während der Entwicklung des Zebrafisches eine mehrphasige Expression auf. Während der frühen Expression, die bei ca. 15 hpf ihren Höhepunkt erreicht, ist Matn-1 im ganzen Zebrafischembryo mit Ausnahme des Notochords exprimiert. Während der späten Expression, die ab etwa 64 hpf beginnt, kann Matn-1 hauptsächlich im Knorpel detektiert werden. Der „knockdown“ von Matn-1 führt, ähnlich wie jener von Ucmaa, sowohl zu Wachstumsdefekten und Störungen bei der Bildung der Kraniofazialknorpel, als auch zum Verlust von Aggrecan und Kollagen II. Der Verlust von Matn-1 hat während der frühen und der späten Expression unterschiedliche Auswirkungen. In der frühen Expressionsphase ändert sich die Zellmorphologie nicht, jedoch können ER-Stress und Apoptose nachgewiesen werden. Während der zweiten Expressionsphase führt der Verlust von Matn-1 zu ausgeprägten morphologischen Veränderungen des endoplasmatischen Retikulums von Chondrozyten. Anzeichen für ER-Stress gibt es nicht, dagegen kann Autophagie nachgewiesen werden. Ein weiterer Hinweis auf eine Störung von Synthese und Sekretion durch den „knockdown“ von Matn-1 ergibt sich aus der Behandlung von Matn-1 Morphanten mit dem Proteasominhibitor Bortezomib. Diese Behandlung erhöhte die Phänotypenrate signifikant, so dass anzunehmen ist, dass die betroffenen Zellen in begrenztem Umfang in der Lage sind den Matn-1 „knockdown“ mit einem erhöhten Proteinabbau zu kompensieren

Factor associated with neutral sphingomyelinase activity mediates navigational capacity of leukocytes responding to wounds and infection:live imaging studies in zebrafish larvae

Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (kACT) after Ca2+ activation and of force decay (τREL−1) during relaxation upon Ca2+ removal, isometric force at maximal (Fmax) or partial Ca2+ activations, and force response to an external stretch applied to the relaxed myofibril (Fpass). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that kACT, Fmax, Ca2+ sensitivity of the force, and Fpass were comparable and τREL−1 was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease–related mutations in sarcomeric proteins in the zebrafish model

Matrilin-1 Is Essential for Zebrafish Development by Facilitating Collagen II Secretion

Background: Matrilin-1 is an abundant cartilage extracellular matrix protein. Results: Morpholino knockdown of matrilin-1 in zebrafish results in growth defects, disturbed craniofacial cartilage formation, and decreased collagen II deposition. Conclusion: Matrilin-1 is indispensible for zebrafish development, presumably by facilitating secretion of collagen II. Significance: These results challenge the concept that matrilins only function as extracellular adaptor proteins. Matrilin-1 is the prototypical member of the matrilin protein family and is highly expressed in cartilage. However, gene targeting of matrilin-1 in mouse did not lead to pronounced phenotypes. Here we used the zebrafish as an alternative model to study matrilin function in vivo. Matrilin-1 displays a multiphasic expression during zebrafish development. In an early phase, with peak expression at about 15 h post-fertilization, matrilin-1 is present throughout the zebrafish embryo with exception of the notochord. Later, when the skeleton develops, matrilin-1 is expressed mainly in cartilage. Morpholino knockdown of matrilin-1 results both in overall growth defects and in disturbances in the formation of the craniofacial cartilage, most prominently loss of collagen II deposition. In fish with mild phenotypes, certain cartilage extracellular matrix components were present, but the tissue did not show features characteristic for cartilage. The cells showed endoplasmic reticulum aberrations but no activation of XBP-1, a marker for endoplasmic reticulum stress. In severe phenotypes nearly all chondrocytes died. During the early expression phase the matrilin-1 knockdown had no effects on cell morphology, but increased cell death was observed. In addition, the broad deposition of collagen II was largely abolished. Interestingly, the early phenotype could be rescued by the co-injection of mRNA coding for the von Willebrand factor C domain of collagen II1a, indicating that the functional loss of this domain occurs as a consequence of matrilin-1 deficiency. The results show that matrilin-1 is indispensible for zebrafish cartilage formation and plays a role in the early collagen II-dependent developmental events

Erratum : Methylglyoxal modification of Na v 1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy (Nature Medicine (2012) 18 (926-933))

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Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Nav1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Nav1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Nav1.8 knockout (Scn10−/−) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy