Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development

THE EXPRESSION PATTERN OF DEATH ASSOCIATED PROTEIN KINASE1 IN NORMAL DORSAL ROOT GANGLION NEURONS AND FOLLOWING PERIPHERAL NERVE INJURY

Death-associated protein kinase1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM) regulated serine/threonine kinase. An increasing body of evidence supports the significance of DAPK1 protein in cancer and CNS diseases. The role of DAPK in peripheral nerve regeneration and neuropathic pain remains completely unexplored. The aim of this study is to investigate the expression pattern of DAPK1 along with key proand anti-apoptotic cell signaling molecules (p53, Bax, Akt, ERK5, p38) and to verify the possibilities of DAPK1-NMDA NR2B relationship in dorsal root ganglion neurons (DRG) after 2 hours, 7 days and 14 days following a sciatic nerve injury. Gene expression analysis and immunohistochemistry were used to assess the effects of nerve injury. ATF3 was used as a neuronal injury marker. The results showed that DAPK1 mRNA was expressed and translated to functional protein in normal DRG neurons. Soon after a sciatic nerve injury (2 hours), DAPK1 was significantly up-regulated (p\u3c0.05, 2.2 fold) in the injured L4 and L5 DRG compared with the contralateral uninjured side. However, 7 days after axotomy a profound decrease was observed in the DAPK1 level, with a further reduction that reached its minimum level at 14 days postoperatively. In addition, 7 days after injury, most of the DAPK1 positive injured neurons (76.11%) were ATF3 positive, while after 14 days DAPK1 immunoreactivity significantly decreased (53.89%) in injured ATF3 positive neurons. Interestingly, DAPK1, p53 and Bax exhibited a similar expression pattern in axotomized lumbar DRG. The results also revealed that a sciatic nerve injury had no effects on the gene expression of ERK5, p38 and Akt at every time point. Moreover, NMDA NR2B mRNA expression increased after 7 days and continued to up-regulate significantly until 14 days postoperatively (p\u3c0.05, 3.6 fold). In contrast, our immunofluorescence results showed a decrease in the protein level in DRG neurons during this time period; but a strong positive NMDA NR2B immunoreactivety appeared in the satellite cells that surround the injured large-sized neurons in L4 and L5 DRG neurons. In addition, immunofluorescence double labelling revealed that DAPK1 and NMDA NR2B are co-localized in normal and injured DRG neurons. In conclusion, the down-regulations of DAPK1 following sciatic nerve injury, along with other vital pro-apoptotic players promoting neuronal survival might shed light on the mechanisms of peripheral nerve regeneration. The results also suggest that NMDA NR2B might modulate neuropathic pain through satellite cells, and not through neurons, 7 and 14 days after peripheral nerve injury

Der Einfluss von Tyrosin-Phosphorylierung auf den Dscam1-Signalweg

The Drosophila Dscam1 gene can be spliced into thousands of different isoforms, providing the basis for a cell surface code: Each cell expresses a distinct subset of 10-50 isoforms, rendering its surface uniquely recognizable. The importance of Dscam1 for axonal and dendritic patterning has been demonstrated in numerous in vivo assays. However, surprisingly little is known regarding the signaling pathway of the Dscam1 receptor. This dissertation describes my efforts to understand the molecular mechanisms of neuronal self-recognition. My dissertation is divided into three chapters: The first two chapters consist of two published papers to which I have contributed during my time in the neuronal wiring laboratory. They demonstrate that the Dscam1 receptor is indispensable for the axonal patterning of mechanosensory neurons in the ventral nerve cord of the fly. In contrast to its role in uniform dendritic patterning, it is critical to regulate Dscam1 signaling in some sub-compartments of the outgrowing axons. Such spatial regulation of Dscam1 signaling by the novel ligand Slit and tyrosine-phosphorylation allows the formation of complicated neurite patterns. Dscam1 tyrosine phosphorylation is negatively modulated by the receptor tyrosine phosphatase RPTP69D. In chapter 3, I summarize the results of a combination of proteomic screens. They were aimed at unraveling the Dscam1 signaling complex and at identifying tyrosine phosphorylated proteins that are regulated by Dscam1 signaling. These results link the Dscam1 receptor directly to the actin and tubulin cytoskeleton and suggest that the receptor is capable of physically recruiting components of the translational machinery to the membrane. Furthermore, I found the cytoplasmic domain to be associated with components of the cellular endomembrane system, suggesting that receptor internalization might be an important regulatory mode, fine-tuning the signaling response.Das Drosophila Dscam1-Gen kann in Tausende Isoformen translatiert werden. Diese stellen die Grundlage für einen Zelloberflächencode dar: Jede Zelle exprimiert eine individuelle Kombination von 10-50 Isoformen. Dies verleiht der Zelloberfläche eine einzigartige Identität. Der Belang von Dscam1 für die Bildung von Neuriten-Verzweigungen ist in vivo überzeugend demonstriert worden. Allerdings ist wenig über den Dscam1-Signalweg bekannt. Diese Dissertation dokumentiert meine Bemühungen, die molekularen Mechanismen der neuronalen Selbsterkennung zu verstehen. Meine Dissertation gliedert sich in drei Teile: Die ersten beiden Teile umfassen zwei bereits publizierte Studien, zu denen ich als Autorin beigetragen habe. Diese zeigen, dass Dscam1 unentbehrlich für die Bildung axonaler Verzweigungen von mechanosensorischen Neuronen in der ventralen Nervenschnur der Fliege ist. Im Gegensatz zur Bildung gleichmäßiger dendritischer Muster ist es wichtig, das Dscam1-Signal in Unterregionen des auswachsendenden Axons zu regulieren. Nur wenn solch eine lokale Kontrolle des Dscam1 Signalweges durch den neu identifizierten Liganden Slit und Tyrosin-Phosphorylierung gewährleistet ist, können sich komplexe axonale Verzweigungen bilden. Die Phosphorylierung des Dscam1-Rezeptors wird durch die Rezeptor-Tyrosin-Phosphatase RPTP69D negativ reguliert. Der dritte Teil meiner Dissertation befasst sich mich mit proteomischen Experimenten. Sie waren darauf ausgerichtet Proteine zu identifizieren, die auf ein Dscam1-Signal mit der Veränderung ihres Phosphorylierungs-Status reagieren. Aus den Ergebnissen lässt sich eine direkte Verbindung zwischen Dscam1 und dem Zytoskelett ableiten. Sie legen des weiteren nahe, dass der Rezeptor auch Komponenten der Translationsmaschinerie an die Membran rekrutiert. Die Assoziation von Dscam1 mit Vesikelkomponenten legt auch nahe, dass Endozytose ein wichtiger Modus der feinabgestimmten Signalregulierung sein könnte

Neurotrophic factor regulation of gene expression in primary sensory neurons of the mouse

In this study, the expression of several sensory neuron specific/predominant genes, and the effects of neurotrophic factors upon them were studied in embryonic, postnatal and adult mouse sensory neurons. In the embryonic mouse, NGF/TrkA signalling was shown to be essential for the expression of mRNAs encoding substance P and the sodium channels Navl.8 and Navl.9 in DRG and trigeminal ganglia. Differential regulation of the two isoforms of calcitonin gene related peptide (CGRP) mRNA was apparent in the DRG with a requirement of NGF/TrkA signalling for expression of a, but not p CGRP. This was not reflected in the trigeminal ganglia. Postnatally, experiments revealed that NGF/TrkA signalling within the DRG and trigeminal ganglia is 1) essential for expression of SP, ocCGRP, pCGRP, Navl.8, Navl.9 mRNAs, 2) possibly required for expression of the neuropeptide galanin and the capsaicin receptor vanilloid receptor 1 (VR1) mRNAs, 3) not required for pituitary adenylate cyclase-activating peptide (PACAP) mRNA. Conversely, within the nodose ganglia, expression of Navl .8 and Navl .9 mRNAs did not require NGF/TrkA signalling. No regulation of all aforementioned genes by neurotrophin-3 (NT-3) was observed in trigeminal, nodose or dorsal root ganglia. In the adult mouse, DRG cultures were utilised to study gene regulation by the neurotrophic factors NGF, artemin and macrophage stimulating protein (MSP). Expression of SP, aCGRP, pCGRP, Navl.8, Navl.9 and VR1 mRNAs all showed a decrease following 96 hours in culture that was inhibited by presence of MSP (50ng/ml), NGF (lOng/ml) or artemin (lOng/ml). PACAP, galanin, damage induced neuronal endopeptidase (DINE) and activating transcription factor 3 (ATF3) mRNAs increased over time, but neurotrophic factors could impede such increases. No axotomy or neurotrophic factor-induced effects were observed for P2X3, Navl.6 or Navl.7 mRNAs. Interestingly the additional presence of leukaemia inhibitory factor (LIF) opposed NGF, MSP and artemin-induced effects on PCGRP, SP, VR1 and galanin mRNAs, whilst enhancing effects on PACAP and DINE transcripts

Skin Tissue Models

Skin Tissue Models provides a translational link for biomedical researchers on the interdisciplinary approaches to skin regeneration. As the skin is the largest organ in the body, engineered substitutes have critical medical application to patients with disease and injury - from burn wounds and surgical scars, to vitiligo, psoriasis and even plastic surgery. This volume offers readers preliminary description of the normal structure and function of mammalian skin, exposure to clinical problems and disease, coverage of potential therapeutic molecules and testing, skin substitutes, models as study platforms of skin biology and emerging technologies. The editors have created a table of contents which frames the relevance of skin tissue models for researchers as platforms to study skin biology and therapeutic approaches for different skin diseases, for clinicians as tissue substitutes, and for cosmetic and pharmaceutical industries as alternative test substrates that can replace animal models. Offers descriptions of the normal structure/function of mammalian skin, exposure to clinical problems, and more Presents coverage of skin diseases (cancer, genodermatoses, vitiligo and psoriasis) that extends to clinical requirements and skin diseases in vitro models Addresses legal requirements and ethical concerns in drugs and cosmetics in vitro testing Edited and authored by internationally renowned group of researchers, presenting the broadest coverage possible. © 2018 Elsevier Inc. All rights reserved.(undefined)info:eu-repo/semantics/publishedVersio

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin

Computational Interrogation of Transcriptional and Post-Transcriptional Mechanisms Regulating Dendritic Development

The specification and modulation of cell-type specific dendritic morphologies plays a pivotal role in nervous system development, connectivity, structural plasticity, and function. Regulation of gene expression is controlled by a wide variety of cellular and molecular mechanisms, of which two major types are transcription factors (TFs) and microRNAs (miRNAs). In Drosophila, dendritic complexity of dendritic arborization (da) sensory neurons of the peripheral nervous system are known to be regulated by two transcription factors Cut and Knot, although much remains unknown about the molecular mechanisms and regulatory networks via which they regulate the final arbor shape through spatio-temporal modulation of dendritic development and dynamics. Here we use bioinformatics analysis of transcriptomic data to identify putative genomic targets of these TFs with a particular emphasis on those that effect neuronal cytoskeletal architecture. We use transcriptomic, as well as data from various genomic and protein interaction databases, to build a weighted functional gene regulatory network for Knot, to identify the biological pathways and downstream genes that this TF regulates. To corroborate bioinformatics network predictions, knot putative targets, which classify into neuronal and cytoskeletal functional groups, have been experimentally validated by in vivo genetic perturbations to elucidate their role in Knot-mediated Class IV (CIV) dendritogenesis. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression, however identification of biologically-relevant target genes for this epigenetic regulatory mechanism remains a significant challenge. To address this knowledge gap, we have developed a novel R based tool, IntramiR-ExploreR, that facilitates integrated discovery of miRNA targets by incorporating target databases and novel target prediction algorithm to arrive at high confidence intragenic miRNA target predictions. We have explored the efficacy of this tool using D.melanogaster as a model organism for bioinformatics analyses and functional validation, and identified targets for 83 intragenic miRNAs. Predicted targets were validated, using in vivo genetic perturbation. Moreover, we are constructing interaction maps of intragenic miRNAs focusing on neural tissues to uncover regulatory codes via which these molecules regulate gene expression to direct cellular development

Progenitor cells in auricular cartilage demonstrate promising cartilage regenerative potential in 3D hydrogel culture

The reconstruction of auricular deformities is a very challenging surgical procedure that could benefit from a tissue engineering approach. Nevertheless, a major obstacle is presented by the acquisition of sufficient amounts of autologous cells to create a cartilage construct the size of the human ear. Extensively expanded chondrocytes are unable to retain their phenotype, while bone marrow-derived mesenchymal stromal cells (MSC) show endochondral terminal differentiation by formation of a calcified matrix. The identification of tissue-specific progenitor cells in auricular cartilage, which can be expanded to high numbers without loss of cartilage phenotype, has great prospects for cartilage regeneration of larger constructs. This study investigates the largely unexplored potential of auricular progenitor cells for cartilage tissue engineering in 3D hydrogels

Protection à long terme du système nerveux : étude de facteurs extrinsèques chez C. elegans

Tout au long de la vie d’un organisme, l’architecture du système nerveux est mise à l’épreuve par des processus de maturation, de croissance, de stress mécaniques et de vieillissement. Bien que certaines molécules de maintenance de l’organisation des ganglions et fascicules neuronaux aient été identifiés chez le nématode C. elegans, les mécanismes assurant la protection à long terme de l’architecture du système nerveux restent mal compris. Chez les mutants de maintenance neuronale sax-7/L1CAM, certaines structures neuronales se développent initialement normalement, mais se désorganisent avec le temps. Un criblage génétique effectué au laboratoire a indiqué l’implication du gène mig-6/Papiline dans la maintenance neuronale: la perte de fonction de mig-6 supprime la désorganisation neuronale progressive des mutants sax-7. De plus, l’organisation neuronale des mutants mig-6 est mieux préservée dans un contexte de stress mécanique que chez le type sauvage. Un équilibre entre l'adhésion cellulaire et la flexibilité du milieu semble donc clé. Par ailleurs, les cellules gliales sont en relation étroite avec les neurones, mais leur implication dans la maintenance neuronale reste inexplorée. Ainsi, lors de ces travaux, la question principale est d’étudier la contribution de la matrice extracellulaire et de cellules gliales dans un contexte de maintenance de l’architecture du système nerveux chez C. elegans. Les résultats révèlent que MIG-6/Papiline régule l’état de la matrice extracellulaire en modifiant l’organisation du collagène IV, un composant abondant et conservé des membranes basales. Cette modification du collagène IV semble compenser les défauts d’adhésion cellulaire présents chez les mutants de maintenance sax-7/L1CAM et contrer un déplacement des ganglions neuronaux lors d’un stress mécanique accru. L’exploration de cellules gliales en contexte de maintenance neuronale a mis en évidence certains défauts des mutants de maintenance sax-7/L1CAM. Comprendre les principes généraux du maintien de l'architecture et de la connectivité neuronale pourrait aider à identifier des facteurs clés influençant l'apparition et la progression de neuropathologies.Throughout life, the architecture of the nervous system is challenged by processes of maturation, growth, mechanical stress and aging. Although neuronal maintenance mechanisms of ganglia and fascicles organization involving conserved factors have been identified in the nematode C. elegans, little is known about processes that aim for the long-term protection of the nervous system architecture. In sax-7/L1CAM neuronal maintenance mutants, some neuronal ganglia and fascicles initially develop normally, but become disorganized over time. A genetic screen performed in the laboratory indicated the involvement of mig-6/Papilin in neuronal maintenance: loss of mig-6 function suppresses progressive neuronal disorganization in sax-7 mutants. Moreover, the neuronal organization of mig-6 mutants is better preserved under mechanical stress than in the wild-type strain. A balance between the adhesion of neurons to their environment and the flexibility of the surrounding extracellular matrix thus seems of importance. Furthermore, glial cells are closely related to neurons, but their involvement in the maintenance of the organization of neuronal structures remains unexplored. The main question of this work is to study the contribution of the extracellular matrix and of two types of glial cells in the context of maintenance of the nervous system architecture in C. elegans. Our results reveal that MIG-6/Papilin regulates the state of the extracellular matrix by altering the organization of collagen IV, an abundant and conserved component of basement membranes, thus compensating for cell adhesion defects in sax-7/L1CAM maintenance mutants and counteracting a neural ganglia displacement upon increased mechanical stress. Our exploration of glial cells in the context of neuronal maintenance also revealed defects in sax-7/L1CAM maintenance mutants. Understanding the general principles of maintenance of neuronal architecture and connectivity could help identify key factors influencing the onset and progression of neuropathologies