The phosphoproteome during neuromuscular synapse formation

Die neuromuskuläre Synapse (NMS) überträgt Signale vom Nerv zur Skelettmuskulatur und reguliert damit die Bewegung dieser. Bildung und Erhaltung der NMS stehen in bedeutendem Zusammenhang mit der Signalübertragung, welche durch die Rezeptor-Tyrosinkinase MuSK induziert wird. MuSK wird autophosphoryliert und initiiert ihre Kinaseaktivität als Reaktion auf Agrin, welches vom Motoneuron sezerniert wird. Aktiviertes MuSK phosphoryliert nachgeschaltete Ziele, um eine Signalkaskade auszulösen. Diese Signalkaskade führt zu präsynaptischer Differenzierung, sowie zu postsynaptischer Spezialisierung, welche durch das Anreicherung der Acetylcholinrezeptoren (AChRs) charakterisiert ist. Beeinträchtigungen der MuSK Funktionalität führt zu einer akuten neuromuskulären Defizienz. Dies kann zum Beispiel bei Myasthenia gravis beobachtet werden oder führt zu perinatalem Tod bei MuSK defizienten Mäusen aufgrund von Atemstillstand. Um die phosphoproteomischen Zusammenhänge von MuSK Signalen zu untersuchen und identifizieren, wurde ein globaler und quantitativer Ansatz mittels Massenspektrometrie gewählt. Hierfür wurde ein Muskel-Zellkultursystem gewählt, um die postsynaptische Differenzierung nachzustellen. Insgesamt wurden 10183 Phosphopeptide identifiziert, von denen zumindest 203 2-fach hoch- oder runterreguliert waren. Regulierte Phosphopeptide wurden deren zeitlichem Profil entsprechend in vier unterschiedliche Cluster unterteilt. Innerhalb dieser Cluster wurde eine Überrepräsentation spezifischer Proteinklassen festgestellt, welche im Zusammenhang mit unterschiedlichen zellulären Funktionen stehen. Insbesondere wurde eine Anreicherung regulierter Phosphoproteinen festgestellt, welche eine Rolle bei posttranskriptionalen Mechanismen und zytoskelettaler Organisation spielen. Aufgrund der unverzichtbaren Rolle des Zytoskeletts bei dem AChR Clustering, wurde das zytoskelettale Regulationsprotein Palladin für weitere Studien identifiziert. Palladin existiert in mehreren Isoformen, welche während der Muskeldifferenzierung spezifisch hoch- oder runterreguliert werden. Um die Rolle von Palladin in Zusammenhang mit der MuSK Signalkaskade und dem AChR Clustering festzustellen, wurden Tet-ON Muskelzelllinien entwickelt. Diese unterdrückten Palladin in differenzierten Myotuben durch Doxycyclin-induzierte miRNA Expression. Ergebnisse zeigten, dass Palladin als Modulator postsynaptischer Differenzierung fungiert. Außerdem wurde durch eine MS Analyse eine neue Phosphoserin-Stelle (S751) entdeckt, welche während der „späten Agrin Stimulation“ hochreguliert ist. S751 ist Teil der Aktivierungsschleife der MuSK Kinasedomäne und. Phosphorylierung von S751 könnte direkten Einfluss auf die Kinaseaktivität haben. Die Phosphorylierung von S751 in Muskelzellen und Muskelgewebe wurde durch einen Antikörper gegen pS751 bestätigt. Um die funktionale Rolle von S751 zu untersuchen, wurde diese Stelle mutiert um Phosphorylierung entweder zu imitieren oder zu inaktivieren. Diese MuSK Mutanten wurden in Muskelzellen exprimiert. Folglich wurden MuSK Phosphorylierung, sowie AChR xiv Phosphorylierung und Clustering untersucht. Es konnte gezeigt werden, dass phosphoimitierende Mutanten von S751 die grundlegende MuSK Kinaseaktivität, AChR Phosphorylierung und AChR Clustergrößen steigern. Es wird angenommen, dass durch die Phosphorylierung von S751 eine Autoinhibition der MuSK Aktivierungsschleife freigesetzt wird. Dies fördert oder stabilisiert die MuSK Kinaseaktivität. Daher könnte die Phosphorylierung von S751 einen neuartigen Mechanismus zur Modulation der MuSK Kinaseaktivität während der Strukturbildung oder Erhaltung der NMS darstellen.The neuromuscular synapse (NMS) transfers signals from the nerve to the skeletal muscle, thereby regulating skeletal muscle movement. The formation and maintenance of the NMS are crucially linked to signal transduction events induced by the receptor tyrosine kinase MuSK. MuSK becomes autophosphorylated and initiates its kinase activity in response to the motor neuron-derived proteoglycan agrin. Activated MuSK phosphorylates downstream targets to induce a signaling cascade driving presynaptic differentiation as well as postsynaptic specialization characterized by the clustering of acetylcholine receptors (AChRs). Impaired MuSK function results in acute neuromuscular deficiencies as shown during myasthenia gravis or more severely in perinatal death in MuSK deficient mice due to respiratory failure. To identify and investigate the phosphoproteomic map of MuSK signaling, we performed a global and quantitative mass spectrometry approach using a muscle cell culture system modeling postsynaptic differentiation. We identified a total of 10183 phosphopeptides of which 203 were at least 2-fold up/down regulated. Regulated phosphopeptides were classified into four different clusters according to their temporal profiles. Within these clusters we found an overrepresentation of specific protein classes associated with different cellular functions. Particularly, we found an enrichment of regulated phosphoproteins involved in post-transcriptional mechanisms and cytoskeletal organization. Due to the indispensable role of the cytoskeleton in AChR clustering, I aimed to study the identified cytoskeletal regulator protein palladin. Palladin exists in multiple isoforms that are specifically up- or downregulated during muscle differentiation. In order to determine the role of palladin during MuSK signaling and AChR clustering, I generated Tet-ON muscle cell lines for subsequent doxycycline-inducible miRNA expression to silence palladin in differentiated myotubes. Results implicate palladin as a modulator of postsynaptic differentiation since AChR clustering is affected. Additionally, analysis of MS revealed a novel phosphoserine site, S751, which is upregulated during late agrin stimulation. S751 lies in the activation loop of the MuSK kinase domain and phosphorylation of S751 might directly influence kinase activity. An antibody against pS751 was generated and the phosphorylation of S751 in muscle cells and muscle tissue was reconfirmed with this antibody. To investigate the functional role of S751, it was mutated to either mimic or to block phosphorylation. MuSK mutants were expressed in muscle cells and subsequently, MuSK phosphorylation as well as downstream events, like AChR phosphorylation and clustering were assayed. As a result, I found that the phosphomimetic mutant of S751 increased basal MuSK kinase activity, AChR phosphorylation and AChR cluster size. I propose that the phosphorylation of S751 provides a mechanism to relief the autoinhibition of the MuSK activation loop, which could foster or stabilize MuSK kinase activity. Therefore, phosphorylation of S751 might represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMS maintenance.submitted by Bahar Z. CamurdanogluZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)464367

Comparison of the planimetry and point-counting methods for estimating kidney volume using magnetic resonance imaging

Introduction: Kidney volume (KV) is an important parameter for clinical assessment of patients with diabetes or renal artery stenosis and for assessment of kidney transplant candidates. The purpose of this study was to compare KV estimations obtained by using the Cavalieri principle combined with point-counting and planimetry techniques. In addition, we evaluated the results to construct a confidence interval value for KV according to a new approach

Estimation of cerebral surface area using vertical sectioning and magnetic resonance imaging: A stereological study

Stereological techniques using isotropic uniform random and vertical uniform random sections have been used for surface area estimation. However, there are a few studies in which the surface area of the brain is estimated using the vertical section technique in a stereological approach. The objective of the current study was to apply the vertical section technique using cycloid test probes for estimation of cerebral surface area in magnetic resonance imaging (MRI). In this study, cerebral surface areas were estimated in a total of 13 young subjects (6 males, 7 females) who were free of any neurological symptoms and signs. The means (+/-S.D.) of the surface areas were 1619.92 +/- 140.97 cm(2), 1625.69 +/- 147.58 cm(2) and 1674.69 +/- 160.60 cm(2) for 36, 18 and 12 vertical sections, respectively. The mean coefficient of error obtained by applying cycloid test lines that use a 2.8-cm ratio of area associated with each cycloid was estimated at 0.05). in addition, the three models correlated well with each other. From these results, it is concluded that the vertical section technique is an unbiased, efficient and reliable method and is ideally suited to in vivo examination of MRI data for estimating the surface area of the brain. Hence, we suggest that estimation of surface area using MRI and stereology may be clinically relevant for assessing cortical atrophy as well as for investigating the structure and function of cerebral hemispheres. (C) 2009 Elsevier B.V. All rights reserved.Stereological techniques using isotropic uniform random and vertical uniform random sections have been used for surface area estimation. However, there are a few studies in which the surface area of the brain is estimated using the vertical section technique in a stereological approach. The objective of the current study was to apply the vertical section technique using cycloid test probes for estimation of cerebral surface area in magnetic resonance imaging (MRI). In this study, cerebral surface areas were estimated in a total of 13 young subjects (6 males, 7 females) who were free of any neurological symptoms and signs. The means (+/-S.D.) of the surface areas were 1619.92+/-140. 97 cm (2), 1625.69+/-147. 58 cm(2) and 1674.69+/-160. 60 cm(2) for 36, 18 and 12 vertical sections, respectively. The mean coefficient of error obtained by applying cycloid test lines that use a 2. 8-cm ratio of area associated with each cycloid was estimated at 0.05). In addition, the three models correlated well with each other. From these results, it is concluded that the vertical section technique is an unbiased, efficient and reliable method and is ideally suited to in vivo examination of MRI data for estimating the surface area of the brain. Hence, we suggest that estimation of surface area using MRI and stereology may be clinically relevant for assessing cortical atrophy as well as for investigating the structure and function of cerebral hemispheres

Scientific Reports / MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.(VLID)468870

EGFR Controls Hair Shaft Differentiation in a p53-Independent Manner

Biological Sciences; Cell Biology; Developmental Biology; Stem Cells ResearchSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction

Inherited deficiency in ether lipids, a subgroup of phospholipids whose biosynthesis needs peroxisomes, causes the fatal human disorder rhizomelic chondrodysplasia punctata. The exact roles of ether lipids in the mammalian organism and, therefore, the molecular mechanisms underlying the disease are still largely enigmatic. Here, we used glyceronephosphate O-acyltransferase knockout (Gnpat KO) mice to study the consequences of complete inactivation of ether lipid biosynthesis and documented substantial deficits in motor performance and muscle strength of these mice. We hypothesized that, probably in addition to previously described cerebellar abnormalities and myelination defects in the peripheral nervous system, an impairment of neuromuscular transmission contributes to the compromised motor abilities. Structurally, a morphologic examination of the neuromuscular junction (NMJ) in diaphragm muscle at different developmental stages revealed aberrant axonal branching and a strongly increased area of nerve innervation in Gnpat KO mice. Post-synaptically, acetylcholine receptor (AChR) clusters colocalized with nerve terminals within a widened endplate zone. In addition, we detected atypical AChR clustering, as indicated by decreased size and number of clusters following stimulation with agrin, in vitro. The turnover of AChRs was unaffected in ether lipid-deficient mice. Electrophysiological evaluation of the adult diaphragm indicated that although evoked potentials were unaltered in Gnpat KO mice, ether lipid deficiency leads to fewer spontaneous synaptic vesicle fusion events but, conversely, an increased post-synaptic response to spontaneous vesicle exocytosis. We conclude from our findings that ether lipids are essential for proper development and function of the NMJ and may, therefore, contribute to motor performance. Read the Editorial Highlight for this article on page 46