Köln, University, Diss., 2003.

Οι πρωτεογλυκάνες, μόρια της εξωκυττάριας ουσίας, αλληλεπιδρούν μεταξύ τους και με άλλα μορφορυθμιστικά μόρια (γλυκοπρωτεΐνες, ιντεγκρίνες) καθώς επίσης και με αυξητικούς παράγοντες (FGF, TGF-β, Wnt, hedgehog). Οι πρωτεογλυκάνες ενεργοποιούν ποικίλα μονοπάτια μεταγωγής σήματος και επάγουν πλειοτροπικές αποκρίσεις από τα κύτταρα καθώς συμμετέχουν στον έλεγχο της κυτταρικής προσκόλλησης, μετανάστευσης και πολλαπλασιασμού, διαφοροποίησης και επιβίωσης κυττάρων και της μορφογένεσης ιστών και οργάνων. Η agrin, πρωτεογλυκάνη θειικής ηπαράνης, έχει ανιχνευτεί στις βασικές μεμβράνες των εγκεφάλου, νευρομυϊκής σύναψης, πνεύμονα και νεφρού. Έκφραση της agrin δεν έχει μελετηθεί στο πρώιμο έμβρυο. Στην παρούσα εργασία μας, με τη μέθοδο της RT-PCR και του ανοσοφθορισμού, μελετήσαμε πότε αρχίζει να εκφράζεται και πού εκφράζεται η agrin κατά τη διάρκεια της ανάπτυξης του πρώιμου εμβρύου όρνιθας από το στάδιο Χ (μορίδιο) μέχρι το ΗΗ17 (29 σωμίτες/οργανογένεση). Το mRNA της agrin πρωτοανιχνεύτηκε, σε χαμηλά επίπεδα, στο στάδιο ΗΗ1-ΗΗ2 (προχωρημένο βλαστίδιο) και η έκφρασή του ρυθμίζεται αναπτυξιακά. Η έκφραση του mRNA της agrin ήταν υψηλή στο στάδιο ΗΗ3 (μέσα σταδίου γαστριδίου), χαμηλότερη στο στάδιο ΗΗ4-5 (πρώιμο νευρίδιο), υψηλή στο στάδιο ΗΗ8-9 (5-6 σωμίτες) και χαμηλότερη στο στάδιο ΗΗ10 (10 σωμίτες). Ένταση φθορισμού της πρωτεΐνης agrin ανιχνεύτηκε και στις τρεις βλαστικές στιβάδες στο στάδιο ΗΗ4 (γαστρίδιο), καθώς και στο νευροεπιθήλιο στο στάδιο ΗΗ8 (4 σωμίτες). Στο στάδιο ΗΗ11 (13 σωμίτες) ο φθορισμός της agrin ήταν ισχυρός στο νευρικό σωλήνα και στους σωμίτες, ενώ ανιχνεύτηκε αξιοσημείωτη ένταση στο άνω τοίχωμα του εντέρου και στα προκαρδιακά κύτταρα. Στο στάδιο ΗΗ17, ο φθορισμός της agrin ήταν έντονος στο νευροεπιθήλιο του νευρικού σωλήνα, ενώ ανιχνεύτηκε και στις βασικές μεμβράνες του διεγκεφάλου και μυελεγκεφάλου. Ένταση έκφρασης της agrin εντοπίστηκε στο φακό και στον αμφιβληστροειδή του οφθαλμού και στο μυοτόμο, αλλά όχι στο δερμοτόμο και σκληροτόμο των σωμιτών. Έντονη ήταν η έκφραση της agrin στο ενδοκάρδιο και στο μυοκάρδιο στην καρδία και στα τοιχώματα του εντέρου. Επίσης, μελετήσαμε το ρόλο της agrin, μέσω αναστολής της λειτουργίας της, με τη χρήση μονοκλωνικών αντισωμάτων έναντι αυτής. Τα αποτελέσματά μας έδειξαν ότι η απουσία της πρωτεΐνης agrin προκαλεί ανωμαλίες στην επιθηλιοποίηση κυρίως των σωμιτών, του καρδιακού σωλήνα και του εντέρου. Επίσης, τα αποτελέσματά μας έδειξαν ότι η agrin φαίνεται να συμμετέχει στα σηματοδοτικά μονοπάτια που καθοδηγούν τα κύτταρα των νευρικών κρηπίδων κατά τη μετανάστευσή τους. Η agrin αρχίζει να εκφράζεται με την έναρξη των μορφογενετικών κινήσεων της γαστριδίωσης, ρυθμίζεται αναπτυξιακά και είναι σημαντικός ο ρόλος της στη διαφοροποίηση κυττάρων και στη μορφογένεση ιστών και οργάνων στο πρώιμο έμβρυο. / Agrin is a heparan sulfate proteoglycan and has been studied extensively in the late embryonic and postnatal nervous system and muscle. Agrin seems to play a critical role in the mediation of axonal growth and path finding. We studied the expression of agrin by RT-PCR and immunofluorescence in the chick embryo from stages X (morula) to HH17 (29 somites). Agrin mRNA was first detectable at low levels at stage HH1-HH2 (late blastula) and its expression was developmentally regulated. Expression of agrin mRNA was high at stage HH3 (intermediate streak), lower at stage HH4-5 (head process), high at stage HH8-9 (5-6 somites) and lower at stage HH10 (10 somites). Agrin protein fluorescence was strong in the three germ layers at stage HH4 (definitive streak) and was strong in the neuroepithelium at stage HH8 (4 somites). At stage HH11 (13 somites), agrin fluorescence was strong in the neural tube and somites and was intense in the gut roof plate and in the heart primordia. By stage HH17, agrin fluorescence was strong in the neuroepithelium of neural tube and was detected in the laminal surface of the diencephalon and myelencephalon. Agrin expression was strong in the lens and retina in the eye, was strong in the myotome but was not expressed in the dermotome and sclerotome in the somites, was strong in myocardium and endocardium in the heart and in the gut walls. Immunodetection of agrin was intense in neuroepithelium and mesenchymal tissues as they epithelialize. Inhibition of function of agrin by blocking antibodies showed that agrin is crucial for maintaining basement membrane integrity which mediates the epithelialization especially in somites, heart tube and gut. It is also possible that agrin participates in the signaling pathways that guide neural crest cell migration.

Muscle spindle fibers are specialized stretch receptors that allow the perception and coordination of limb movement. Differentiation of muscle spindles is initiated by signals derived from the in growing Ia sensory neurons during development. The sensory neuron secretes neuregulin which binds and signals through the ErbB receptors to initiate a signaling cascade. This cascade results in the expression of a specific repertoire of genes, one of which is the transcription factor Egr3, which is necessary in the development of muscle spindles. Signaling occurs efficiently when the postsynaptic receptors are clustered into large aggregates in apposition to an innervating nerve. Using what is known about acetylcholine receptor clustering at neuromuscular junctions as a model, this study shows the importance of the basal lamina proteins agrin and laminin and their shared receptor ??-dystroglycan in aggregating ErbB receptors at sensory synapses. The study also shows that signaling through these receptors subsequently results in increased expression of Egr3, the transcription factor critical to muscle spindle fiber differentiation. Using an ??-dystroglycan silenced culture, it is shown that ??-dystroglycan is necessary to induce neuregulin, laminin and agrin induced Egr3. In these same myotube cultures there is also a reduced number of AChR-ErbB3 colocalized aggregates and this is not rescued with the addition of laminin. Taken together, these results suggest an essential role for basal lamina components and ??-dystroglycan, molecules that are crucial in acetylcholine receptor aggregation at neuromuscular junctions, in the induction of the transcription factor Egr3, a critical transcription factor involved in muscle spindle fiber differentiation.

Neuromuscular synapse formation is driven by two nerve-derived molecules, agrin and neuregulin. Agrin is believed to signal through a Muscle Specific Kinase (MuSK)/Lrp4 receptor complex to cluster existing acetylcholine receptors (AChRs) in the postsynaptic membrane via a rapsyn-mediated mechanism, while neuregulin signals via ErbB kinase receptors to induce synaptic gene transcription. Recent studies suggest that neuregulin-ErbB signalling may also cross-signal to the agrin-MuSK pathway to modulate agrin’s ability to cluster AChRs. This thesis aimed to further elucidate this idea. Results of this thesis present two novel findings. First, there is a direct interaction between two tyrosine kinase signalling pathways at the neuromuscular synapse and second, neuregulin plays an important role in modulating, modifying and refining AChRs at developing synapses. Here I show that neuregulin can modulate two distinct processes. In the presence of agrin, neuregulin was able to potentiate both agrin-induced AChR clustering and agrin-induced AChR cluster dispersal, and this modulation by neuregulin occurred independently of any transcriptional mechanism. In vitro, I observed a marked effect by neuregulin on the number and size of AChR clusters that were induced by agrin. Treatment of myotubes for 4hrs with agrin and neuregulin led to a significant potentiation in agrin-induced AChR clustering compared to agrin treatment alone. Neuregulin on its own had no measurable effect on AChR clustering. When incubation times were much longer (12hrs), neuregulin promoted a further significant decrease in AChR cluster number compared to agrin treatment alone. Thus at 12hrs, rather than inhibit AChR clustering, as has been previously suggested, neuregulin promoted the dispersal of AChRs from pre-existing agrin-induced clusters. Follow-up in vivo studies into the potentiating ability of neuregulin in agrin-induced AChR clustering showed that the injection of exogenous neuregulin into developing mouse sternomastoid musculature led to an increase in the size of AChRs. Collectively, these data suggest interactions between the signalling pathways initiated by agrin and neuregulin. Subsequent investigation into the second messengers downstream of agrin-MuSK and neuregulin-ErbB signalling revealed that cyclin-dependent kinase 5 (Cdk5) and Shp2 played a role in neuregulin’s modulation of AChR cluster formation and dispersal. It appears that neuregulin enhances the phosphorylation status of MuSK by inhibiting the Shp2-dependent negative feedback loop on MuSK phosphorylation, thereby leading to an increase in AChR cluster numbers. By contrast, the way in which neuregulin disperses agrin-induced AChR clusters seems to occur partially, via a Cdk5 signalling-dependent mechanism. While it is accepted that neuregulin acts in a transcriptional manner during neuromuscular synapse formation, real-time PCR and immunoblot results suggest that transcriptional regulation was not involved in neuregulin’s modulation of agrin-induced AChR clustering.

Myasthenia gravis ist eine Autoimmunerkrankung, die durch Störung der Erregungsübertragung an der neuromuskulären Endplatte zu einer Schwäche der Muskulatur führt. In dieser Arbeit wird die Rolle von Cortactin und Agrin als potentielle neue Antigene von Autoantikörpern bei Myasthenia gravis untersucht. Die detektierten Antikörper werden charakterisiert und die klinischen Merkmale der Patient*innen ausgewertet. / Myasthenia gravis is an autoimmune disease that leads to muscle weakness through an impaired signal transmission at the neuromuscular junction. This publication examines the role of cortactin and agrin as new potential antigens of autoantibodies in myasthenia gravis. The detected antibodies are characterized and the clinical features of the patients are evaluated.

No description available.

I detta arbetet så analyserar jag soundet ifrån tre framgångsrika musikproducenter inom popmusiken samt återskapar deras sound på mina egna låtar. Detta görs genom en grundlig analys i tre steg. Resultatet blev tillfredställande då jag har fått ett par ”aha-upplevelser” som direkt höjt kvalitén på mina produktioner.

<p>Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.</p><p>Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length. </p><p>In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies. </p><p>In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.</p> / Dissertation

Agrin and neuregulin are neurotrophic molecules well known for their roles at synapses in the peripheral and central nervous systems. The expression of these two molecules is not restricted to these sites however, as they are broadly expressed across multiple organ systems. What roles do agrin and neuregulin play within these alternate systems; what is the function of these molecules outside the nervous system? Here I investigate potential roles for agrin and neuregulin in inflammation. Inflammation is an immediate response by innate immune cells, primarily macrophages, to infection and is characterized by the synthesis of pro-inflammatory mediators. The innate immune system possesses multiple redundant mechanisms to locally control inflammation. The neuro-immune axis is one means of control. Often called the cholinergic anti-inflammatory pathway, it acts to regulate local inflammation via nerve-secreted acetylcholine signaling through the homopentameric α7 nicotinic acetylcholine receptors (α7nAChR) present on macrophages. Both agrin and neuregulin have been independently described to share an intricate relationship with acetylcholine receptors (AChR) in the nervous system. Agrin is best known for its role in AChR aggregation at the neuromuscular junction while neuregulin has related roles in AChR transcription, cell survival, communication and differentiation. Based on the common characteristics of synapses in the nervous and immune systems we were curious to see if agrin and neuregulin played analogous roles on macrophages. Here we show that agrin and its receptor dystroglycan are expressed on RAW264.7 macrophages. In addition, agrin treated macrophages demonstrate increased endogenous agrin and α7nAChR expression. By blocking α-dystroglycan (α-DG), a receptor for agrin, with an anti-α-DG antibody we further saw a reduction in agrin expression. We also show that agrin is able to aggregate surface α7nAChRs and transmembrane agrin co-localizes with α7nAChRs therein. Agrin appears to induce approximately a 15-fold increase in anti-inflammatory cytokine IL-10 in macrophages but does not increase pro-inflammatory cytokine TNF-α or IL-6 synthesis. Agrin-treated macrophages challenged with LPS, a potent activator of inflammation, exhibit a 57% decrease in IL-6. Macrophages treated with agrin also exhibit a 4-fold increase in STAT3, a regulator of anti-inflammatory action. The potential anti-inflammatory effects of agrin in the periphery parallel previous work describing the effects of neuregulin in the brain. Previous work completed by our lab suggests a role for neuregulin in augmenting the expression of α7nAChRs on microglia, the macrophages of the brain, but not in peripheral macrophages. Here we show that treatment of LPS challenged microglia with neuregulin produces an 88% decrease in IL-6 and a 33% decrease in TNF-α. These results indicate both agrin and neuregulin are able to induce an increase in α7nAChRs and augment the synthesis of pro- and anti-inflammatory cytokines in their respective systems. These results also further the support the evidence of neuro-immune crosstalk in the immune system. Taken together these results present two novel players in inflammatory regulation by macrophages in the periphery and CNS.

The nervous system requires rapid, efficient, and accurate transmission between cells for proper functioning. Synapses are the predominant structures through which such vital communication occurs. How synapses are formed, maintained, and eliminated are questions of fundamental importance. At the nerve-muscle synapse, formation of the postsynaptic apparatus is directed by agrin. The hallmark activity of agrin is the aggregation of acetylcholine receptors (AChRs) into dense clusters opposite the presynaptic nerve terminal. Early events in the agrin signal transduction cascade include activation of the receptor tyrosine kinase MuSK and tyrosine phosphorylation of AChRs, but how these events lead to AChR cluster formation is unknown. Using the calcium buffer BAPTA, we demonstrate that intracellular calcium fluxes are necessary for agrin-induced formation of AChR clusters. However, clamping calcium fluxes before agrin stimulation does not alter agrin-induced phosphorylation of either MuSK or AChRs, indicating that this calcium-dependent step occurs downstream of both MuSK and AChR phosphorylation. These results identify a new step in the agrin signaling pathway required for the formation of AChR clusters. We show that intracellular calcium fluxes also play an important role in stabilizing AChR clusters. Clamping intracellular calcium fluxes results in rapid dispersal of AChR clusters and dephosphorylation of both MuSK and AChRs, even if agrin is continually present. Furthermore, the protein tyrosine phosphatase inhibitor pervanadate inhibits both the dispersal and dephosphorylation, indicating a role for a tyrosine phosphatase in AChR cluster dispersal. Our data indicate that AChR clusters are maintained by agrin/MuSK-induced intracellular calcium fluxes that tonically inhibit a tyrosine phosphatase localized to AChR clusters. Our findings also show that distinct molecular mechanisms mediate the formation and the dispersal of agrin-induced AChR clusters. The work presented here expands our understanding of synaptic differentiation in several ways. First, I characterized a new, calcium-dependent step required for the formation of agrin-induced AChR clusters. Next, I showed that postsynaptic specializations must be actively maintained, and describe a molecular mechanism that stabilizes AChR clusters. Finally, dispersal and formation of AChR clusters occurs by distinct pathways. Our understanding of the mechanisms regulating the formation and modulation of synapses will help us to better understand how the nervous system develops and responds to the world around us.