We established spontaneously immortalized Schwann cell lines from long-term cultures of adult mouse dorsal root ganglia and peripheral nerves. One of the cell lines, designated IMS32, responded to mitogenic stimuli by platelet-derived growth factor (PDGF)-BB, acidic and basic fibroblast growth factors (aFGF, bFGF), and transforming growth factors (TGF)-beta 1 and -beta 2, as determined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. Furthermore, conditioned media (CM) obtained from IMS32 cells showed mitogenic activity for both IMS32 cells and long-term cultured Schwann cells. Western blot analysis revealed TGF-beta-like molecule in the CM, and the activity was absorbed with anti-TGF-beta neutralizing antibody. Reverse transcription followed by polymerase chain reaction (RT-PCR) of IMS32 RNA revealed that these cells expressed TGF-beta 1, -beta 2, and -beta 3 transcripts. When rat pheochromocytoma PC12 cells were incubated with the CM, they developed neurite growth. Coculture of PC12 and IMS32 cells also showed neurite growth of PC12 cells. RNA transcripts of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), ciliary neurotrophic factor (CNTF), and glial cell line-derived neurotrophic factor (GDNF) were detected from IMS32 cells by RT-PCR. In these, we sequenced the mouse GDNF cDNA coding region and observed 97% and 90% homologies to corresponding rat and human cDNA sequences, respectively. These results indicate that the immortalized Schwann cell line mitotically responds to various growth factors and secretes autocrine and paracrine growth-promoting activities in vitro.

The physiological roles of sonic hedgehog (Shh) have been intensively characterized in development of various organs. However, their functions in adult tissues have not been fully elucidated. We investigated the expression and the potential function of Shh in crush‐injured adult rat sciatic nerves. The Shh expression was up‐regulated in Schwann cells adjacent to the injured site. The time‐course analyses of various neurotrophic factors revealed the up‐regulation of Shh mRNA followed by that of brain‐derived neurotrophic factor (BDNF) mRNA. The continuous administration of cyclopamine, a hedgehog signal inhibitor, to the injured site suppressed the increase of BDNF expression and deteriorated the survival of motor neurons in lumbar spinal cord. Treatment of exogenous Shh in cultured Schwann cells enhanced the BDNF expression. The BDNF promoter activity (exon I and II) was increased in IMS32 cells co‐transfected with Shh and its receptor Smoothened. These findings imply that the up‐regulated expression of Shh in Schwann cells may play an important role in injured motor neurons through the induction of BDNF.

We have constructed a chitosan nonwoven nanofiber mesh tube consisting of oriented fibers by the electrospinning method. The efficacy of oriented nanofibers on Schwann cell alignment and positive effect of this tube on peripheral nerve regeneration were confirmed. The physical properties of the chitosan nanofiber mesh sheets prepared by electrospinning with or without fiber orientation were characterized. Then, immortalized Schwann cells were cultured on these sheets. Furthermore, the chitosan nanofiber mesh tubes with or without orientation, and bilayered chitosan mesh tube with an inner layer of oriented nanofibers and an outer layer of randomized nanofibers were bridgegrafted into rat sciatic nerve defect. As a result of fiber orientation, the tensile strength along the axis of the sheet increased. Because Schwann cells aligned along the nanofibers, oriented fibrous sheets could exhibit a Schwann cell column. Functional recovery and electrophysiological recovery occurred in time in the oriented group as well as in the bilayered group, and approximately matched those in the isograft. Furthermore, histological analysis revealed that the sprouting of myelinated axons occurred vigorously followed by axonal maturation in the isograft, oriented, and bilayered group in the order. The oriented chitosan nanofiber mesh tube may be a promising substitute for autogenous nerve graft.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat spinal motoneurons. The seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed, and AxCAhGDNF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or PBS was inoculated in C7 vertebral foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of beta-galactosidase activity in lesioned spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned spinal motoneurons and expression of virus-induced human GDNF mRNA transcripts in the lesioned spinal cord tissue. Nissl-stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned ventral horn motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with spinal cord injury and motor neuron diseases.

Two missense mutations (P123H and V70M) of ␤-synuclein (␤-syn), the homologue of ␣-syn, have been recently identified in dementia with Lewy bodies. However, the mechanism through which these mutations influence the pathogenesis of dementia with Lewy bodies is unclear. To investigate the role of the ␤-syn mutations in neurodegeneration, each mutant was stably transfected into B103 neuroblastoma cells. Cells overexpressing mutated ␤-syn had eosinophilic cytoplasmic inclusion bodies immunopositive for mutant ␤-syn, and electron microscopy revealed that these cells were abundant in various cytoplasmic membranous inclusions resembling the histopathology of lysosomal storage disease. Consistent with these findings, the inclusion bodies were immunopositive for lysosomal markers, including cathepsin B, LAMP-2, GM2 ganglioside, and ATP13A2, which has recently been linked to PARK9. Notably, formation of these lysosomal inclusions was greatly stimulated by co-expression of ␣-syn, was dependent on the phosphorylation of ␣-syn at Ser-129, and was more efficient with the A53T familial mutant of ␣-syn compared with wild type. Furthermore, the inclusion formation in cells overexpressing mutant ␤-syn and transfected with ␣-syn was significantly suppressed by treatment with autophagy-lysosomal inhibitors, which were associated with impaired clearance of syn proteins and enhanced apoptosis, indicating that formation of lysosomal inclusions might be protective. Collectively, the results demonstrated unambiguously that overexpression of ␤-syn mutants (P123H and V70M) in neuroblastoma cells results in an enhanced lysosomal pathology. We suggest that these missense mutations of ␤-syn might play a causative role in stimulating neurodegeneration.