Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Biochemistry. Title from title-page of electronic thesis. Bibliography: leaves 141 - 176.

No / Nerve growth factor (NGF) has long been recognized as an important mast cell (MC) growth factor. To explore whether other neurotrophins (NTs) of the NGF family, which are widely expressed in mouse skin, affect the numbers and/or functions of MCs we examined the effects of NT-3 on neonatal skin MCs. We demonstrate that TrkC, the high affinity NT-3 receptor, is expressed by virtually all neonatal skin MCs in C57BL/6 mice, which indicates that MCs can respond to NT-3. Skin of neonatal and early postnatal NT-3-overexpressing mice (promoter: K14) displayed significantly and up to twofold increased numbers of MCs during the first 20 days after birth, as compared to wild-type mice. To check whether this increase in MC numbers in NT-3 transgenic mice reflects a higher rate of proliferation, we performed immunohistochemistry, which revealed that only 1-2% of all skin MCs both in NT-3-overexpressing and in wild-type controls showed Ki-67-positive nuclei, suggesting that the observed differences in the number of MCs do not reflect a higher rate of MC proliferation. Additionally, we show that the effect of NT-3 on the number of MCs is most likely to be stem cell factor (SCF)-independent, because NT-3 significantly downregulates secretion of SCF-protein in cultured dermal fibroblasts, as assessed by enzyme-linked immunosorbent assay. Numbers of skin MCs in neonatal TrkC-deficient mice were found to be modestly reduced, as compared to wild-type mice, indicating that NT-3 can modulate the number of MCs directly via TrkC, although TrkC does not seem to be essential for the number of basal MCs. To further analyze the effects of NT-3 on MCs, we stimulated skin organ culture of early postnatal C57BL/6 mouse skin with 5-50 ng/ml NT-3, which induced a significant increase in MC degranulation, as visualized by Giemsa staining. However, stimulation of isolated neonatal dermal skin MCs with NT-3 in vitro failed to result in MC activation, as measured by serotonin release. Our data suggest a role for NT-3 in the maturation of MCs, such as a TrkC-mediated stimulation of the differentiation of pre-existing, less mature MCs and/or by enhancing the migration of circulating MC precursors into the skin.

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by demyelination and loss of oligodendrocytes (OLGs), the CNS myelin-producing cells. Thus, understanding the mechanisms that control OLG development can provide valuable knowledge regarding remyelination therapies for MS. This disease is thought to result from an autoimmune attack towards myelin. FTY720, an immunomodulator under evaluation for MS treatment is a sphingosine-1-phosphate (S1P) analog. We found before that S1P plays a crucial role in the NT-3-mediated survival of OLGs, an observation that led us to investigate whether FTY720 could have any effect on these cells. Our studies demonstrate that FTY720 indeed has a direct effect on OLG progenitors, protecting them from apoptotic death through a mechanism involving ERK1/2 and Akt activation. However, another key finding of our study was that this drug arrested OLG differentiation, an effect counteracted by NT-3 which not only enhanced the survival of OLG progenitors but also stimulated their maturation. Furthermore, NT-3 induced an increase in myelin basic protein (MBP) levels in the absence of effects on MBP gene promoter activation or mRNA expression. These observations suggested that NT-3 up-regulated MBP levels by a posttranscriptional mechanism raising the question of whether this neurotrophin could have a more general positive effect on the expression of other OLG proteins. In agreement with this idea, we found that NT-3 also induced the expression of the myelin proteins MAG and MOG. Additionally, [35S]-Methionine labeling indicated a 50% increase in de novo protein synthesis following only a 15 min exposure to NT-3. Such a rapid increase in protein synthesis reinforced the idea that NT-3 plays a crucial role regulating protein expression by posttranscriptional mechanisms. In support of this possibility, we found that NT-3 stimulated the phosphorylation of the initiation factor eIF4E and its inhibitory partner 4EBP1, both essential players in mediating cap-dependent protein synthesis. This stimulation involved the activation of ERK1/2 and PI3K/mTOR mediated signaling pathways. To our knowledge, this is the first study on the regulation of translation initiation in OLGs and the first report describing the potential role of NT-3 as an activator of initiation.

Le récepteur à activité tyrosine kinase TrkC est un récepteur aux neurotrophines qui contrôle la mise en place des neurones proprioceptifs au cours du développement du système nerveux. En présence de son ligand, la neurotrophine‐3 (NT‐3), TrkC transmet un signal de survie, tandis qu’en absence des facteurs trophiques les neurones vont mourir via l’apoptose. Cependant, les mécanismes moléculaires de ce programme de mort ne sont pas connus. D'autre part, TrkC peut également accomplir un rôle proapoptotique dans le cancer. En effet, son expression est un facteur de bon pronostic pour le neuroblastome, la tumeur solide extra crânienne la plus courante chez l'enfant. Ce paradoxe apparent pourrait s’expliquer par la notion de récepteur à dépendance : ces récepteurs induisent la mort cellulaire en situation d’absence de ligand, alors que la présence de leur ligandinhibe leur activité proapoptotique. Nous avons fourni la preuve que parmi les récepteurs aux neurotrophines, seul TrkC est un récepteur à dépendance. Son activité nécessite un clivage par des caspases, générant un fragment proapoptotique. Par ailleurs, nous avons mis en évidence la surproduction autocrine de NT‐3 dans une fraction de neuroblastomes à haut risque, permettant aux cellules malignes de contourner le mécanisme de contrôle antitumoral de TrkC. De plus, nous avons démontré que la perturbation de l'interaction TrkC/NT‐3 induit la mort des cellules de neuroblastomein vitro et empêche la croissance tumorale et les métastases dans des modèles aviaires et murins. Ces travaux établissent les bases d’une potentielle stratégie thérapeutique s'appuyant sur la restauration d'une voie d’apoptose fonctionnelle induite par TrkC dépourvu du ligand / The tyrosine kinase receptor TrkC is a neurotrophin receptor that assures an adequate establishment of proprioceptive neurons during nervous system development. Upon neurotrophin‐3 (NT‐3) binding TrkC transduces a classic survival signal, while in absence of trophic support a program of apoptotic cell death will take place. However the molecular mechanisms leading to neuron cell death are not understood. On the other hand, although TrkC was first identified as an oncogene, it may also accomplish a proapoptotic role in cancer. Indeed its expression has been correlated with a good prognosis of Neuroblastoma, the most common extracranial solid tumor of early childhood. Thisapparent paradox could be explained by the dependence receptor notion: these receptors induce apoptotic cell death in settings of absence of ligand, whereas the presence of their ligand inhibits this proapoptotic activity. We provide evidence here that among neurotrophin receptors, only TrkC is a dependence receptor and its activity relies on the caspase‐mediated cleavage of its intracellular domain, which allows the release of a proapoptotic fragment. Moreover, we show that an autocrine production of NT‐3 concerns a large fraction of aggressive neuroblastoma and provides a selective advantage by allowing malignant cells to overcome with TrkC antitumoral control. We demonstrate that the disruption of the TrkC/NT‐3 interaction triggers neuroblastoma cell death in vitro andprevents tumor growth and metastasis in avian and murine models. This work set the basis for analternative anticancer therapeutic strategy relying on the reengagement of a cell death program mediated by unbound TrkC

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

No description available.

The anatomy of the cranial peripheral nervous system has been studied for over a century, yet surprisingly little is known about how the nerves are guided to their targets. The study of the development of these nerves has important implications for our understanding of craniofacial anomalies and possible treatments for both injury and genetic disorders of nerve development such as Goldenhar-Gorlin syndrome. We have discovered that retinoic acid (RA) may play a role in the development of the trigeminal nerve. Inhibition of retinoic acid receptors (RAR) results in trigeminal nerves that become unbundled or defasciculated in the eye region. To further understand how RA is affecting trigeminal development we searched for genes downregulated in response to RAR inhibition by the inhibitor BMS-453 and have identified neurotrophin-3 (NT-3), activated leukocyte cell adhesion molecule (ALCAM) and Semaphorin 4B (Sema4B). We have analyzed the expression patterns of Sema4B and NT-3 by in situ hybridization and have found NT-3 expression in the eye and Sema4B in the embryonic target of the trigeminal nerve, lens of the eye and in the pharyngeal arches. ALCAM has been analyzed via qRT-PCR and its transcription is downregulated just prior to the observed defasciculation phenotype. The pattern of expression of these genes combined with known expression of NT-3 receptors allows us to suggest a model whereby RA signaling regulates Sema4B, ALCAM and NT-3, which support the survival, guidance and fasciculation of the trigeminal nerve. This work has the potential to better understanding of the complex nature of cranial nervous system development.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Emerging trends in cell-therapy based tissue repair have focused on the renewable source of adult stem cells including human bone marrow-derived mesenchymal stromal cells (hMSCs). Due to immunomodulatory properties as well as a potential to differentiate into cells characteristic of all three germ layers, hMSCs provide a source of immature cells for utilization in cell-therapy based treatments. Marrow isolated adult multilineage inducible (MIAMI) cells are a homogeneous sub-population of hMSCs which maintain self-renewal potential during ex vivo expansion, in addition to efficiently undergoing trans-differentiation into neuron-like cells in vitro. Even though hMSCs have the potential to be used for neural tissue repair, the molecular mechanisms by which they are stimulated to become neuron-like cells have not been fully characterized. Therefore the work described herein focuses on the molecular mechanisms by which MIAMI cells undergo NT-3 dependent neuronal commitment. MIAMI cells express both the full length (FL-) and tyrosine kinase deficient (TKd-) isoforms of the NTRK3 receptor, the primary NT-3 receptor, at the protein level. NT-3 stimulation of MIAMI cells during neuronal commitment induced the phosphorylation of FL-NTRK3, degradation of TKd-NTRK3, downstream activation of the Mek1/2-Erk1/2 signaling cascade, and subsequent up-regulation of a limited number of pro-neuronal genes. These findings were verified using chemical inhibitors to block NTRK autophosphorylation (K252a) and Erk1/2 activation (U0126). TKd-NTRK3 is hypothesized to activate Rac1 upon NT-3 stimulation. Rac1 was found to suppress NT-3 stimulated Erk1/2 phosphorylation, as well as downstream gene expression, as determined using a Rac1 chemical inhibitor. Further characterization confirmed that Rac1b is the predominant Rac1 isoform in MIAMI cells. Rac1b siRNA mediated knock-down resulted in increased expression of the pro-neuronal genes NGN2, MAP2, NFH and NFL during NT-3 stimulation via regulation of Mek1/2-Erk1/2. Rac1b is also involved in NT-3 stimulated cell proliferation, as well as repression of CCND1 and CCNB1 mRNA expression. In an attempt to enhance neuronal differentiation of MIAMI cells, EGF and bFGF were used to pretreat MIAMI cells prior to NT-3 stimulated neuronal commitment. EGF/bFGF pretreatment increased NTRK3 and NTRK1 protein levels along with NT-3 stimulated Erk1/2 phosphorylation. In addition, bFGF versus EGF/bFGF pretreatment restricted the expression of the pro-neuronal transcription factors Ngn2 and Prox1 versus the neural stem cells self-renewal transcription factor Musashi-1, respectively. The culmination of this work provides a model for the NT-3 induced neuronal commitment of MIAMI cells in vitro, as well as insight into the neurogenic potential of MSCs for future applications in cell-therapy based tissue repair.

Thesis (M.S.)--Miami University, Dept. of Zoology, 2004. / Title from first page of PDF document. Includes bibliographical references (p. 36-47).