Dimitra Thomaidou

The neuron–glia signal β-neuregulin promotes Schwann cell motility via the MAPK pathway

Neuregulins constitute a family of related growth factors that play important roles in Schwann cell development and maturation. We investigated the involvement of β‐neuregulin in Schwann cell migration, using a simple in vitro bioassay. Pure Schwann cells were prepared from the sciatic nerves of 5‐day‐old rats and were grown in defined medium, with or without serum, until a monolayer of confluent cells was formed. A cell‐free area was then generated by inflicting a scratch resulting in a 1‐mm‐wide gap. Schwann cell migration within the gap was monitored microscopically at given time intervals and was quantified using an image analysis system. The extent of cell proliferation was estimated by BrdU incorporation, and cell migration was quantified both in the absence and presence of cytosine arabinoside. We found that, in the absence of serum, β‐neuregulin at a dose submaximal for proliferation increased the rate of Schwann cell migration by 84%. A more moderate effect was observed when β‐neuregulin was applied in the presence of serum which, however, is by itself responsible for increased Schwann cell motility. To assess the signal transduction pathways involved in this procedure we used one inhibitor of MAPK, PD098059, two inhibitors of PI‐3‐kinase, wortmannin, and LY0294002, and three different PKC inhibitors. Of these PD098059 inhibited the neuregulin‐induced enhancement in Schwann cell migration by 40%, the two PI‐3‐kinase inhibitors yielded an approximately 20% inhibition while the PKC inhibitors were ineffective. Our data indicate that the action of β‐neuregulin on Schwann cell motility is primarily mediated via the MAPK pathway. GLIA 34:39–51, 2001.

BM88 is a dual function molecule inducing cell cycle exit and neuronal differentiation of neuroblastoma cells via cyclin D1 down-regulation and retinoblastoma protein hypophosphorylation.

Control of cell cycle progression/exit and differentiation of neuronal precursors is of paramount importance during brain development. BM88 is a neuronal protein associated with terminal neuron-generating divisions in vivo and is implicated in mechanisms underlying neuronal differentiation. Here we have used mouse neuroblastoma Neuro 2a cells as an in vitro model of neuronal differentiation to dissect the functional properties of BM88 by implementing gain- and loss-of-function approaches. We demonstrate that stably transfected cells overexpressing BM88 acquire a neuronal phenotype in the absence of external stimuli, as judged by enhanced expression of neuronal markers and neurite outgrowth-inducing signaling molecules. In addition, cell cycle measurements involving cell growth assays, BrdUrd incorporation, and fluorescence-activated cell sorting analysis revealed that the BM88-transfected cells have a prolonged G1 phase, most probably corresponding to cell cycle exit at the G0 restriction point, as compared with controls. BM88 overexpression also results in increased levels of the cell cycle regulatory protein p53, and accumulation of the hypophosphorylated form of the retinoblastoma protein leading to cell cycle arrest, with concomitant decreased levels and, in many cells, cytoplasmic localization of cyclin D1. Conversely, BM88 gene silencing using RNA interference experiments resulted in acceleration of cell proliferation accompanied by impairment of retinoic acid-induced neuronal differentiation of Neuro 2a cells. Taken together, our results suggest that BM88 plays an essential role in regulating cell cycle exit and differentiation of Neuro 2a cells toward a neuronal phenotype and further support its involvement in the proliferation/differentiation transition of neural stem/progenitor cells during embryonic development.

Schwann cells engineered to express the cell adhesion molecule L1 accelerate myelination and motor recovery after spinal cord injury

Functional recovery after spinal cord lesion remains an important goal. A combination of inhibitory molecules and lack of appropriate permissive factors in the lesioned spinal cord results in failure of fiber tract reconnection and function. Experimental transplantation in rodent and primate models of CNS injuries has led to the idea that Schwann cells (SCs) are promising candidates for autologous transplantation to assist myelination of lesions and to deliver therapeutic agents in the CNS. In this study, we used retroviral transduction to genetically modify SCs from transgenic GFP-mice in order to overexpress the cell adhesion molecule L1, a protein promoting neurite outgrowth and implicated in myelination. SCs transduced to express L1 or its chimeric secreted form L1-Fc were mixed and grafted rostrally to the lesion site of adult mice immediately after spinal cord compression injury. Our results indicate that 3 weeks postoperatively, but not thereafter, mice transplanted with L1/L1-Fc-expressing SCs exhibited faster locomotor recovery as compared to animals which received SCs transduced with a control vector or no cells at all. Morphological analysis indicated that the accelerated functional recovery correlated with earlier and enhanced myelination by both grafted and host SCs. Moreover, increased sprouting of serotonergic fibers into and across the lesion site was observed in the L1/L1-Fc group as compared with controls. Our results suggest that transplantation of L1-overexpressing SCs enhances early events in spinal cord repair after injury and may be considered in combinatorial strategies together with other regeneration-promoting molecules.

Sox1 Maintains the Undifferentiated State of Cortical Neural Progenitor Cells via the Suppression of Prox1-Mediated Cell Cycle Exit and Neurogenesis†‡§

Neural stem/progenitor cells maintain their identity via continuous self‐renewal and suppression of differentiation. Gain‐of‐function experiments in the chick revealed an involvement for Sox1‐3 transcription factors in the maintenance of the undifferentiated neural progenitor (NP) identity. However, the mechanism(s) employed by each factor has not been resolved. Here, we derived cortical neural/stem progenitor cells from wild‐type and Sox1‐null mouse embryos and found that Sox1 plays a key role in the suppression of neurogenic cell divisions. Loss of Sox1 leads to progressive depletion of self‐renewing cells, elongation of the cell cycle of proliferating cells, and significant increase in the number of cells exiting the cell cycle. In proliferating NP cells, Sox1 acts via a prospero‐related homeobox 1 (Prox1)‐mediated pathway to block cell cycle exit that leads to neuronal differentiation in vivo and in vitro. Thus, our results demonstrate that Sox1 regulates the size of the cortical NP pool via suppression of Prox1‐mediated neurogenic cell divisions. STEM CELLS 2011;29:89–98

Soluble forms of NCAM and F3 neuronal cell adhesion molecules promote Schwann cell migration: identification of protein tyrosine phosphatases ζ/β as the putative F3 receptors on Schwann cells

Neural cell adhesion molecule (NCAM) and F3 are both axonal adhesion molecules which display homophilic (NCAM) or heterophilic (NCAM, F3) binding activities and participate in bidirectional exchange of information between neurones and glial cells. Engineered Fc chimeric molecules are fusion proteins that contain the extracellular part of NCAM or F3 and the Fc region of human IgG1. Here, we investigated the effect of NCAM‐Fc and F3‐Fc chimeras on Schwann cell (SC) migration. Binding sites were identified at the surface of cultured SCs by chimera coated fluorospheres. The functional effect of NCAM‐Fc and F3‐Fc binding was studied in two different SC migration models. In the first, migration is monitored at specific time intervals inside a 1‐mm gap produced in a monolayer culture of SCs. In the second, SCs from a dorsal root ganglion explant migrate on a sciatic nerve cryosection. In both systems addition of the chimeras significantly increased the extent of SC migration and this effect could be prevented by the corresponding anti‐NCAM or anti‐F3 blocking antibodies. Furthermore, antiproteoglycan‐type protein tyrosine phosphatase ζ/β (RPTPζ/β) antibodies identified the presence of RPTPζ/β on SCs and prevented the enhancing effect of soluble F3 on SC motility by 95%. The F3‐Fc coated Sepharose beads precipitated RPTPζ/β from SC lysates. Altogether these data point to RPTPζ/β is the putative F3 receptor on SCs. These results identify F3 and NCAM receptors on SC as potential mediators of signalling occurring between axons and glial cells during peripheral nerve development and regeneration.

BM88/CEND1 coordinates cell cycle exit and differentiation of neuronal precursors

During development, coordinate regulation of cell cycle exit and differentiation of neuronal precursors is essential for generation of appropriate number of neurons and proper wiring of neuronal circuits. BM88 is a neuronal protein associated in vivo with terminal neuron-generating divisions, marking the exit of proliferative cells from the cell cycle. Here, we provide functional evidence that BM88 is sufficient to initiate the differentiation of spinal cord neural precursors toward acquisition of generic neuronal and subtype-specific traits. Gain-of-function approaches show that BM88 negatively regulates proliferation of neuronal precursors, driving them to prematurely exit the cell cycle, down-regulate Notch1, and commit to a neuronal differentiation pathway. The combined effect on proliferation and differentiation results in precocious induction of neurogenesis and generation of postmitotic neurons within the ventricular zone. The dual action of BM88 is not recapitulated by the cell cycle inhibitor p27Kip1, suggesting that cell cycle exit does not induce differentiation by default. Mechanistically, induction of endogenous BM88 by forced expression of the proneural gene Mash1 indicates that BM88 is part of the differentiation program activated by proneural genes. Furthermore, BM88 gene silencing conferred by small interfering RNA in spinal cord neural progenitor cells enhances cell cycle progression and impairs neuronal differentiation. Our results implicate BM88 in the synchronization of cell cycle exit and differentiation of neuronal precursors in the developing nervous system.

Coordination of cell cycle exit and differentiation of neuronal progenitors

During development, co-ordinate regulation of cell cycle exit and differentiation of neuronal precursors is essential for generation of appropriate number of neurons and proper wiring of neuronal circuits. Recent studies have identified some of the molecules implicated in the regulation of these cellular events, but the complex machinery that orchestrates these processes into a coherent developmental program remains unclear. BM88/Cend1 is a neuronal protein associated in vivo with terminal neuron-generating divisions, marking the exit of proliferative cells from the cell cycle. Genetic studies in neural cell lines, neural stem/progenitor cells using the neurosphere system and in the developing chicken neural tube in vivo have shown that BM88/Cend1 is a dual function molecule co-ordinating cell cycle exit and differentiation of neuronal progenitors. These studies have thus shed light on a molecular determinant that participates, along with other known and possibly still unknown regulators, in the complex processes by which a progenitor cell becomes a mature neuron.

BM88/Cend1 Expression Levels Are Critical for Proliferation and Differentiation of Subventricular Zone-Derived Neural Precursor Cells

Neural stem cells remain in two areas of the adult mammalian brain, the subventricular zone (SVZ) and the dentate gyrus of the hippocampus. Ongoing neurogenesis via the SVZ‐rostral migratory stream pathway maintains neuronal replacement in the olfactory bulb (OB) throughout life. The mechanisms determining how neurogenesis is restricted to only a few regions in the adult, in contrast to its more widespread location during embryogenesis, largely depend on controlling the balance between precursor cell proliferation and differentiation. BM88/Cend1 is a neuronal lineage‐specific regulator implicated in cell cycle exit and differentiation of precursor cells in the embryonic neural tube. Here we investigated its role in postnatal neurogenesis. Study of in vivo BM88/Cend1 distribution revealed that it is expressed in low levels in neuronal precursors of the adult SVZ and in high levels in postmitotic OB interneurons. To assess the functional significance of BM88/Cend1 in neuronal lineage progression postnatally, we challenged its expression levels by gain‐ and loss‐of‐function approaches using lentiviral gene transfer in SVZ‐derived neurospheres. We found that BM88/Cend1 overexpression decreases proliferation and favors neuronal differentiation, whereas its downregulation using new‐generation RNA interference vectors yields an opposite phenotype. Our results demonstrate that BM88/Cend1 participates in cell cycle control and neuronal differentiation mechanisms during neonatal SVZ neurogenesis and becomes crucial for the transition from neuroblasts to mature neurons when reaching high levels.

Lentivirus-mediated expression of insulin-like growth factor-I promotes neural stem/precursor cell proliferation and enhances their potential to generate neurons

J. Neurochem. (2010) 115, 460–474.

Cell Adhesion Molecules in Gene and Cell Therapy Approaches for Nervous System Repair

The inability of the central nervous system (CNS) to efficiently repair damages results in severe functional impairment after trauma or neurodegenerative/demyelinating diseases. Regeneration failure is attributed to inhibitory molecules creating a nonpermissive environment for axonal regrowth, and dictates the necessity for the development of novel therapeutic strategies. An emerging approach for improving regeneration is the use of gene therapy to manipulate cell adhesion molecule expression in experimental animal models of degeneration. Alternatively, cell transplantation to replace lost neurons and the grafting of myelinating cells to repair demyelinating lesions are promising approaches for treating CNS injuries and demyelination. Schwann cells (SCs), oligodendrocyte progenitors, olfactory ensheathing cells and embryonic and neural stem cells have been shown to form myelin after transplantation into the demyelinated CNS. The repair capacity of the peripheral nervous system (PNS) is much higher, but there is still a limit to the amount of nerve loss that can be bridged after injury, and longer nerve gaps call for the use of conduits populated with living cells. In both cases, the interaction of grafted cells with the host environment is of paramount importance for the incorporation and functional integration of these cells and the manipulation of cell adhesion molecules is an attractive approach towards achieving this goal. In this review we summarize data from the recent literature regarding the manipulation of cell adhesion molecule expression towards CNS and PNS repair and discuss the prospects for future therapeutic applications.