Maureen L. Condic

Ligand-induced changes in integrin expression regulate neuronal adhesion and neurite outgrowth

Receptors of the integrin family are expressed by every cell type and are the primary means by which cells interact with the extracellular matrix. The control of integrin expression affects a wide range of developmental and cellular processes, including the regulation of gene expression, cell adhesion, cell morphogenesis and cell migration. Here we show that the concentration of substratum-bound ligand (laminin) post-translationally regulates the amount of receptor (α6β1 integrin) expressed on the surface of sensory neurons. When ligand availability is low, surface amounts of receptor increase, whereas integrin RNA and total integrin protein decrease. Ligand concentration determines surface levels of integrin by altering the rate at which receptor is removed from the cell surface. Furthermore, increased expression of integrin at the cell surface is associated with increased neuronal cell adhesion and neurite outgrowth. These results indicate that integrin regulation maintains neuronal growth-cone motility over a broad range of ligand concentrations, allowing axons to invade different tissues during development and regeneration.

Alternative sources of pluripotent stem cells: scientific solutions to an ethical dilemma.

Stem cell researchers in the United States have faced a quagmire of uncertainty due to multiple factors: the ethical divide over the use of embryos for research, the lack of clarity in federal guidelines governing this research, the restrictive patent situation surrounding the generation of new human embryonic stem (HES) cell lines; and the limits on types of research eligible for federal funding. In this commentary, we describe how recent advances in derivation of hES cell-like lines may allow at least some of these uncertainties to be resolved. More importantly, we suggest that the derivation of hES cell-like lines by morally acceptable methods would not only avoid the corrosive effects of a protracted ethical debate over stem cell research, but would also allow U.S. researchers to access federal funds and compete on a more level international playing field.

Regulatory issues for personalized pluripotent cells

The development of personalized pluripotent stem cells for research and for possible therapies holds out great hope for patients. However, such cells will face significant technical and regulatory challenges before they can be used as therapeutic reagents. Here we consider two possible sources of personalized pluripotent stem cells: embryonic stem cells derived from nuclear transfer (NT‐ESCs) and induced pluripotent stem cells (iPSCs) derived from direct reprogramming of adult somatic cells. Both sources of personalized pluripotent stem cells face unique regulatory hurdles that are in some ways significantly higher than those facing stem cells derived from embryos produced by fertilization (ESCs). However, the outstanding long‐term potential of iPSCs and their relative freedom from the ethical concerns raised by both ESCs and NT‐ESCs makes direct reprogramming an exceptionally promising approach to advancing research and providing therapies in the field of regenerative medicine.

Integrin signaling is integral to regeneration.

The inability of the adult injured mammalian spinal cord to successfully regenerate is not well understood. Studies suggest that both extrinsic and intrinsic factors contribute to regeneration failure. In this review, we focus on intrinsic factors that impact regeneration, in particular integrin receptors and their downstream signaling pathways. We discuss studies that address the impact of integrins and integrin signaling pathways on growth cone guidance and motility and how lessons learned from these studies apply to spinal cord regeneration in vivo.

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin α subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Extracellular matrix in spinal cord regeneration: getting beyond attraction and inhibition.

INTRODUCTION The expression of matrix molecules in the CNS [1,2] and the role of matrix in development [3–5] and regeneration [6,7] have recently been the subject of excellent reviews. It is evident that matrix proteins, particularly proteoglycans, make major contributions to regenerative failure in the adult CNS. Although a number of growth inhibiting molecules are expressed in CNS myelin and on glia, recent data suggests that these molecules are not sufficient to prevent the regeneration of adult neurons. Adult neurons transplanted into intact [8] or degenerating [9] adult white matter tracts are able to robustly regenerate. Regeneration of adult neurons invariably fails when extending axons encounter the glial scar associated with the injury site, suggesting that molecules expressed in the scar play a critical role in adult regeneration failure. Following injury, a large number of extracellular matrix (ECM) proteins that are known to influence the regeneration of neurons in culture are up-regulated or altered in regions of scarring (Table 1). Many of these same molecules are up-regulated following injury in peripheral nerve, where regeneration occurs [10]. Historically, the thinking in the field of regeneration research has been dominated by the relatively simple view that specific matrix molecules either promote or inhibit the growth of neurons. Therapeutic approaches have been directed towards increasing the expression or the efficacy of the former and minimizing the negative impact of the latter. There are reasons to believe, however, that this view does not fully reflect the complexity of the problem. The relationship between growth cone motility and matrix adhesion is not simply a matter of more being better (see Fig. 1). In addition, the response of neurons to specific molecules no longer appears to be a fixed property of the molecule itself, but rather a complex interaction between the molecule, the precise molecular composition of the environment, the particular type of neuron interacting with that environment and the recent history of the neuron. Here we will briefly discuss the role of cell adhesion in growth-cone migration. We will then consider molecules typically thought to be either ‘growth-promoting’ or ‘growthinhibiting’, and review the evidence that suggests this molecular dualism is an inadequate description of how neurons interact with matrix molecules in development and regeneration. MOTILITYANDMATRIXADHESION Most matrix proteins that are considered to be growthpromoting also promote cell adhesion. Indeed, cell adhesion is thought to be a required first step for migration. The ability of adhesive matrix proteins to both promote and prevent the migration of non-neuronal cells has been well studied (Fig. 1). Theoretical calculations and empirical data indicate that non-neuronal cells will only migrate over a narrow range of matrix concentrations where cells are adhered strongly enough to generate traction, without being so strongly adhered that they are unable to change position [11,12]. Surprisingly, this well-established relationship between motility and adhesion has not been consistently applied to the study of growth-cone migration. In some situations, embryonic neurons appear to be a rare exception to the rule that migration will only occur within a narrow window of matrix concentrations. Growth cones of embryonic neurons efficiently migrate on concentrations of the ECM molecule laminin that vary over several orders of magnitude [13–15]. The ability of neurons to migrate over a wide range of laminin concentrations is due to a very unusual regulation of neuronal receptors for laminin [15]. Laminin receptors are down-regulated in response to high laminin concentrations, thereby reducing neuronal adhesion and allowing an intermediate level of attachment to be maintained over a wide range of absolute ligand concentrations. Laminin-receptor levels in embryonic neurons are modulated by desensitization, i.e., receptor is removed from the surface in response to high ligand levels by endocytosis. Thus, embryonic neurons, like all cells, migrate only at intermediate levels of attachment, but (at least on laminin substrata) embryonic neurons are able to dynamically adjust their levels of attachment by adjusting expression of receptors. While embryonic neurons are able to compensate for a wide range of laminin concentrations, the response of growth cones to other matrix molecules that utilize different receptor systems is unknown. There is evidence that the receptor molecule L1 (also known as Ng-CAM) participates in endocytotic recycling in growth cones [16–18], but whether the absolute levels of L1 compensate for availability of ligand to maintain a constant level of L1-mediated attachment is unknown. Nothing is known about the regulation of other receptors that promote neuronal adhesion to components of scar matrix. Moreover, very little is

Combined integrin activation and intracellular cAMP cause Rho GTPase dependent growth cone collapse on laminin-1.

Cyclic nucleotides regulate the response of both developing and regenerating growth cones to a wide range of guidance molecules through poorly understood mechanisms. It is not clear how cAMP levels are regulated or how they translate into altered growth cone behavior. Here, we show that intracellular cAMP levels are influenced by substrata and integrin receptors. We also show that growth cones require a substratum-specific balance between cAMP levels, integrin function and Rho GTPases to maintain motility and prevent collapse. Embryonic chick dorsal root ganglion neurons plated on different concentrations of laminin extend growth cones at similar speeds, yet have distinct levels of integrin expression, integrin activation and intracellular cAMP levels. Either increasing cAMP signaling or activating integrins enhances the rate of growth cone motility, but only on substrata where these two factors are endogenously low (i.e. low concentrations of laminin). Surprisingly, combining these two positive manipulations induces growth cone collapse and retraction on laminin but not on fibronectin. Collapse and retraction on laminin are Rho and Rac1 GTPase dependent and are associated with internalization of integrins, the primary receptors responsible for adhesion. These observations define a novel pathway through which cAMP influences growth cone motility and establish a link between integrins, cAMP and Rho GTPases in growth cones.

Neural crest motility and integrin regulation are distinct in cranial and trunk populations

The neural crest is a transient cell population that travels long distances through the embryo to form a wide range of derivatives. The extensive migration of the neural crest is highly unusual and incompletely understood. We examined the ability of neural crest cells (NCCs) to migrate under different conditions in vitro. Unlike most motile cell types, avian NCCs migrate efficiently on a wide range of fibronectin concentrations. Strikingly, the migration of NCCs on laminin depends on the axial level from which the crest is derived. On high concentrations of laminin, cranial NCCs migrate at approximately twice the rate of trunk NCCs and show greater persistence, a higher percentage of migratory cells, and a less organized cytoskeleton. The difference in migration between cranial and trunk neural crest is not due to transcriptional differences in integrin mRNA, but rather to differences in posttranslational regulation. Overexpression of a single integrin is sufficient to significantly slow the migration velocity of cranial neural crest cultured on high laminin densities. These results demonstrate that neural crest cells accommodate a wide range of ECM concentrations in vitro and suggest that differences in integrin regulation along the anterior-posterior axis may contribute to differences in neural crest migration and cell fate.

Alternative Sources of Pluripotent Stem Cells: Ethical and Scientific Issues Revisited

Stem cell researchers in the United States continue to face an uncertain future, because of the changing federal guidelines governing this research, the restrictive patent situation surrounding the generation of new human embryonic stem cell lines, and the ethical divide over the use of embryos for research. In this commentary, we describe how recent advances in the derivation of induced pluripotent stem cells and the isolation of germ-line-derived pluripotent stem cells resolve a number of these uncertainties. The availability of patient-matched, pluripotent stem cells that can be obtained by ethically acceptable means provides important advantages for stem cell researchers, by both avoiding protracted ethical debates and giving U.S. researchers full access to federal funding. Thus, ethically uncompromised stem cells, such as those derived by direct reprogramming or from germ-cell precursors, are likely to yield important advances in stem cell research and move the field rapidly toward clinical applications.

Adaptation of Sensory Neurons to Hyalectin and Decorin Proteoglycans

Proteoglycans are abundantly expressed in the pathways of developing and regenerating neurons, yet the responses of neurons to specific proteoglycans are not well characterized. We have shown previously that one chondroitin sulfate proteoglycan (CSPG), aggrecan, is potently inhibitory to sensory axon extension in short-term assays and that over time, embryonic neurons adapt to aggrecan-mediated inhibition through the transcriptional upregulation of integrin expression (Condic et al., 1999). Here, we have compared the response of embryonic sensory neurons to structurally distinct CSPGs that belong to either the hyalectin (or lectican) family of large, aggregating proteoglycans or the decorin (or small leucine-rich proteoglycan) family of smaller proteoglycans. Both of these structurally diverse proteoglycan families are expressed in developing embryos and inhibit outgrowth of embryonic sensory neurons in short-term cultures. These results document a previously uncharacterized inhibitory function for the decorin-family proteoglycan biglycan. Interestingly, embryonic neurons adapt to these diverse proteoglycans over time. Adaptation is associated with upregulation of select integrin α subunits in a proteoglycan-specific manner. Overexpression of specific integrin α subunits improves neuronal regeneration on some but not all decorin-family CSPGs, suggesting that neurons adapt to inhibition mediated by closely related proteoglycans using distinct mechanisms. Our findings indicate that CSPGs with diverse core proteins and distinct numbers of chondroitin sulfate substitution sites mediate a similar response in sensory neurons, suggesting that increased integrin expression may be an effective means of promoting axonal regeneration in the presence of diverse inhibitory proteoglycan species in vivo.