Microarray analysis of gene expression in proliferating Schwann cells: synergistic response of a specific subset of genes to the mitogenic action of heregulin plus forskolin

Cultured Schwann cells treated with heregulin growth factor require costimulation with a cyclic adenosine monophosphate-elevating agent to produce maximal cell proliferation. Gene chip expression analysis was used to identify genes that are induced or repressed when Schwann cells are treated with heregulin and/or forskolin. By utilizing arrays that contained 8799 probes, the expression of over 1000 genes was found to be significantly changed after 30 hr of treatment with heregulin, forskolin, or heregulin plus forskolin. Hierarchical clustering revealed groups of genes with distinct expression patterns. Of particular interest was a cluster of 140 genes that were up-regulated by heregulin plus forskolin but not by heregulin or forskolin alone. Many of the genes in this group have roles in cell division, such as cyclin B, cyclin D3, E2F-5, cdc 25B, polo-like kinase, and protein kinase C type III. These findings identify a profile of gene expression for Schwann cell proliferation.     

Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks

Genetic redundancy is a problem in gene targeting studies because functionally relevant sister proteins can compensate for the lack of protein product of a targeted gene. A molecular system is chosen in which it is hoped to demonstrate both the lack and presence of compensation after disruption of particular single genes. Mammals may not be able to compensate for the lack of heregulin, a single ligand for multiple ErbB receptors, however, compensation is expected when a single ErbB receptor is knocked out. To investigate this the heregulin-1, ErbB2, or ErbB3 locus was disrupted in a targeted manner and mice heterozygous for the mutation were analyzed. Heregulin and its receptors were shown to be involved in embryonic brain development and, more recently, in plastic changes associated with adult brain function in rodents. Although they have never been shown to play roles in mammalian behavior, it was decided to characterize the mice behaviorally using a battery of simple tests. Heregulin mutant mice exhibited elevated activity levels in the open field, showed improved rotorod performance, and finished T-maze spontaneous alternation task faster compared to control wild type littermates, findings that suggest a consistent hyperactivity across tests. ErbB2 and ErbB3 mutant mice, whose strain origin was identical to that of heregulin mutants, showed no sign of the behavioral alterations. It is suggested that the abnormalities seen in heregulin mutant mice are due to mutation at that locus and the lack of alterations seen in ErbB2 and ErbB3 mutant mice is the result of compensation by unaltered sister receptors.     

Proliferation of Schwann cells and regulation of cyclin D1 expression in an animal model of Charcot-Marie-Tooth disease type 1A

Overexpression of PMP22 is responsible for the most common form of inherited neuropathy, Charcot-Marie-Tooth disease (CMT) type 1A. The PMP22-transgenic rat (CMT rat) is an animal model of CMT1A, and its peripheral nerves show the characteristic features of ongoing demyelination and remyelination that is also seen in CMT1A patients. Since Schwann cell proliferation is a prominent feature of peripheral nerves in inherited peripheral neuropathies, we examined proliferation and the expression of cyclin D1 in CMT rats. D-type cyclins are required for the initial steps in cell division and nuclear import is crucial for the function of cyclin D1 in promoting cell proliferation. Like normal myelinating Schwann cells in wild-type rats, remyelinating Schwann cells in CMT rats show perinuclear cyclin D1 expression. Schwann cells with nuclear cyclin D1 expression, as well as proliferating Schwann cells, were both associated with demyelinated axonal segments. Supernumerary onion bulb Schwann cells, however, do not express cyclin D1 and were not proliferating. Thus, cyclin D1 expression and its subcellular localization correlate directly with distinct physiological states of Schwann cells in this animal model of CMT1A.     

Serum and forskolin cooperate to promote G1 progression in Schwann cells by differentially regulating cyclin D1, cyclin E1, and p27Kip expression

Proliferation of Schwann cells in vitro, unlike most mammalian cells, is not induced by serum alone but additionally requires cAMP elevation and mitogenic stimulation. How these agents cooperate to promote progression through the G1 phase of the cell cycle is unclear. We studied the integrative effects of these compounds on receptor-mediated signaling pathways and regulators of G1 progression. We show that serum alone induces strong cyclical expression of cyclin D1 and E1, 6 and 12 h after addition, respectively. Serum also promotes strong but transient erbB2, ERK, and Akt phosphorylation, but Schwann cells remain arrested in G1 due to high levels of the inhibitor, p27(Kip). Forskolin with serum promotes G1 progression in 22% of Schwann cells between 18 and 24 h by inducing a steady decline in p27(Kip) levels that reaches a nadir at 12 h coinciding with peak cyclin E1 expression. Forskolin also delays neuregulin-induced loss of erbB2 receptors allowing strong acute activation of PI3K, sustained erbB2 phosphorylation and G1 progression in 31% of Schwann cells. We find that the ability of forskolin to decrease p27(Kip) is associated with its ability to decrease Krox-20 expression that is induced by serum and further increased by neuregulin. Our results explain why serum is required but insufficient to stimulate proliferation and identify two routes by which forskolin promotes proliferation in the presence of serum and neuregulin. These findings provide insights into how G1 progression and, cell cycle arrest leading to myelination are regulated in Schwann cells.     

Differential Cyclin D1 Requirements of Proliferating Schwann Cells during Development and after Injury

Neurons regulate Schwann cell proliferation, but little is known about the molecular basis of this interaction. We have examined the possibility that cyclin D1 is a key regulator of the cell cycle in Schwann cells. Myelinating Schwann cells express cyclin D1 in the perinuclear region, but after axons are severed, cyclin D1 is strongly upregulated in parallel with Schwann cell proliferation and translocates into Schwann cell nuclei. During development, cyclin D1 expression is confined to the perinuclear region of proliferating Schwann cells and the analysis of cyclin D1-null mice indicates that cyclin D1 is not required for this type of Schwann cell proliferation. As in the adult, injury to immature peripheral nerves leads to translocation of cyclin D1 to Schwann cell nuclei and injury-induced proliferation is impaired in both immature and mature cyclin D1-deficient Schwann cells. Thus, our data indicate that the molecular mechanisms regulating proliferation of Schwann cells during development or activated by axonal damage are fundamentally different.     

Direct conversion of adult human skin fibroblasts into functional Schwann cells that achieve robust recovery of the severed peripheral nerve in rats

Direct conversion is considered a promising approach to obtain tissue-specific cells for cell therapies; however, this strategy depends on exogenous gene expression that may cause undesired adverse effects such as tumorigenesis. By optimizing the Schwann cell induction system, which was originally developed for trans-differentiation of bone marrow mesenchymal stem cells into Schwann cells, we established a system to directly convert adult human skin fibroblasts into cells comparable to authentic human Schwann cells without gene introduction. Serial treatments with beta-mercaptoethanol, retinoic acid, and finally a cocktail of basic fibroblast growth factor, forskolin, platelet-derived growth factor-AA, and heregulin-β1 (EGF domain) converted fibroblasts into cells expressing authentic Schwann cell markers at an efficiency of approximately 75%. Genome-wide gene expression analysis suggested the conversion of fibroblasts into the Schwann cell-lineage. Transplantation of induced Schwann cells into severed peripheral nerve of rats facilitated axonal regeneration and robust functional recovery in sciatic function index comparable to those of authentic human Schwann cells. The contributions of induced Schwann cells to myelination of regenerated axons and re-formation of neuromuscular junctions were also demonstrated. Our data clearly demonstrated that cells comparable to functional Schwann cells feasible for the treatment of neural disease can be induced from adult human skin fibroblasts without gene introduction. This direct conversion system will be beneficial for clinical applications to peripheral and central nervous system injuries and demyelinating diseases.     

Responses to cyclic AMP is impaired in the twitcher Schwann cells in vitro

Twitcher (twi/twi) is a murine model of genetic demyelinating disease globoid cell leukodystrophy (GLD). Available data suggest that demyelination in GLD is caused by degeneration or dysfunction of myelin-forming cells resulting from an accumulation of psychosine, a toxic substrate of galactosylceramidase and a potent inhibitor of protein kinase C (PKC). We investigated proliferation and differentiation of twi/twi Schwann cells in response to forskolin, an adenylate cyclase activator. In twi/twi Schwann cells isolated at the postnatal day (P) 10 prior to the onset of demyelination, proliferation and an expression of the surface galactocerebroside (galC) in response to forskolin were similar to those of +/+ mice. However, in twi/twi Schwann cells isolated from demyelinating sciatic nerves at P20 or P30, fewer numbers of cells expressed surface galC compared to age matched control (+/+) Schwann cells. In all Schwann cells, surface galC expression was lost after 3 days in vitro (DIV). However, with an administration of 50 μM forskolin in the media containing 1% fetal bovine serum (FBS) on the 4 DIV, surface galC could be reexpressed in all +/+ and P10 twi/twi Schwann cells but not in P20 or P30 twi/twi cells. In the media containing 10% FBS, forskolin also stimulated proliferation of Schwann cells from P10 twi/twi, and P10 and P30+/+ mice but not those from P30 twi/twi mice. These results are consistent with a metabolic perturbation of twi/twi Schwann cells that may be reflecting cellular dysfunctions by inhibition of the PKC.     

Differentiation of rat adipose tissue-derived stem cells into Schwann-like cells in vitro

In this study, we explored the competence of adipose-derived stem cells to differentiate into Schwann cells in vitro. Rat adipose-derived stem cells were sequentially treated with various factors beta-mercaptoethanol, all-trans-retinoic acid, followed by a mixture of forskolin, basic fibroblast growth factor, platelet-derived growth factor and heregulin. We found that differentiated adipose-derived stem cells displayed the morphology of Schwann cells. Western blotting and dual immunofluorescence staining confirmed that they produced proteins characteristic for Schwann cells, including S100 and glial fibrillary acidic protein. Furthermore, differentiated adipose-derived stem cells could enhance neurite outgrowth in coculture with sensory neurons. These results demonstrate that adipose-derived stem cells can differentiate into Schwann-like cells with morphological, phenotypic, and functional characteristics of Schwann cells.     

Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro

The use of cellular prostheses containing large populations of Schwann cells (SC) has been proposed as a future therapeutic approach in the repair of neural tissue. We have sought to define an efficient protocol for the harvest and expansion of human SC from mature human peripheral nerve. We evaluated SC proliferation occurring within fresh explants and studied the relationship between certain parameters (cell yield, purity, and rate of SC proliferation) and the conditions of maintenance of nerve explants prior to dissociation. In addition, we studied SC proliferation after dissociation in a variety of conditions. We observed that SC within explants divide at a low rate during the first 3 weeks following explantation; this proliferation falls to near zero during the fourth week. The cell yield, SC purity, and proliferation rate following dissociation were all increased when nerve explants were exposed to heregulin/forskolin for 2 weeks prior to dissociation. Electron microscopic analysis showed that heregulin/forskolin exerted trophic effects on SC within explants. Following dissociation, SC growth in heregulin/forskolin-containing medium was more rapid on laminin or collagen than on poly-L-lysine. These results provide new insights into human SC biology and suggest several procedural improvements for harvesting and expanding these cells. The new method we describe shortens our previous procedure by 4–6 weeks and provides a 30–50-fold increase in the number of SC obtained relative to the earlier procedure. © 1996 Wiley-Liss, Inc.