Shizuka Takaku

High glucose-induced activation of the polyol pathway and changes of gene expression profiles in immortalized adult mouse Schwann cells IMS32

We investigated the polyol pathway activity and the gene expression profiles in immortalized adult mouse Schwann cells (IMS32) under normal (5.6 mm) and high (30 and 56 mm) glucose conditions for 7–14 days in culture. Messenger RNA and the protein expression of aldose reductase (AR) and the intracellular sorbitol and fructose contents were up‐regulated in IMS32 under high glucose conditions compared with normal glucose conditions. By employing DNA microarray and subsequent RT–PCR/northern blot analyses, we observed significant up‐regulation of the mRNA expressions for serum amyloid A3 (SAA3), angiopoietin‐like 4 (ANGPTL4) and ecotropic viral integration site 3 (Evi3), and the down‐regulation of aldehyde reductase (AKR1A4) mRNA expression in the cells under high glucose (30 mm) conditions. The application of an AR inhibitor, SNK‐860, to the high glucose medium ameliorated the increased sorbitol and fructose contents and the reduced AKR1A4 mRNA expression, while it had no effect on mRNA expressions for SAA3, ANGPTL4 or Evi3. Considering that the exposure to the high glucose (≥ 30 mm) conditions mimicking hyperglycaemia in vivo accelerated the polyol pathway in IMS32, but not in other previously reported Schwann cells, the culture system of IMS32 under those conditions may provide novel findings about the polyol pathway‐related abnormalities in diabetic neuropathy.

Spontaneously immortalized Schwann cells from adult Fischer rat as a valuable tool for exploring neuron–Schwann cell interactions

We established spontaneously immortalized Schwann cell lines from long‐term cultures of adult Fischer 344 rat dorsal root ganglia (DRG) and peripheral nerves. One of these cell lines, designated immortalized Fischer rat Schwann cells 1 (IFRS1), showed spindle‐shaped morphology; immunoreactivity for S100, p75 neurotrophin receptor (p75NTR), glial fibrillary acidic protein (GFAP), laminin, and vimentin; and mRNA expression of neurotrophic factors (NGF, GDNF, and CNTF), neurotrophin receptors (p75NTR, truncated TrkB, and TrkC), cell adhesion molecules (L1, NCAM, and N‐cadherin), myelin proteins [P0, PMP22, and myelin‐associated glycoprotein (MAG)], transcription factors (Krox20, Sox10, and Oct6), neuregulin‐1 receptors (ErbB2 and ErbB3), and an orphan G protein‐coupled receptor (Gpr126). Conditioned medium (CM) obtained from IFRS1 cells exhibited potent biological activity for the promotion of neuronal survival and neurite outgrowth of cultured adult rat DRG neurons. Furthermore, light and electron microscopic analyses revealed that IFRS1 cells were capable of myelinating neurites while in coculture with adult rat DRG neurons. These findings indicate that IFRS1 cells possess some biological properties of mature Schwann cells and that the coculture system with adult DRG neurons and IFRS1 cells can be a useful tool for the study of peripheral nerve degeneration and regeneration.

Ganglioside GD1a negatively regulates matrix metalloproteinase-9 expression in mouse FBJ cell lines at the transcriptional level

Mouse FBJ virus-induced osteosarcoma FBJ-S1 cells rich in GD1a are not readily metastatic, whereas FBJ-LL cells with low levels of GD1a are highly metastatic. GD1a was previously shown to suppress metastasis of mouse FBJ cells and to upregulate caveolin-1 and stromal interaction molecule 1 expression. The present study demonstrates that matrix metalloproteinase-9 (MMP-9) expression renders FBJ-LL cells invasive. MMP-9 is inversely regulated by GD1a, based upon four observations: MMP-9 mRNA content was 5 times higher in FBJ-LL cells than FBJ-S1 cells; a GD1a-re-expressing FBJ-LL cell variant produced through β1,4GalNAcT-1 cDNA transfection expressed lower levels of MMP-9; exogenous addition of GD1a to FBJ-LL cells decreased MMP-9 production in a dose- and time-dependent manner; and treatment of GD1a-rich cells with D-PDMP or siRNA targeting St3gal2 decreased GD1a expression, but augmented MMP-9 expression. This is the first report demonstrating that GD1a negatively regulates expression of MMP-9 at the transcriptional level.

Immortalized Adult Rodent Schwann Cells as In Vitro Models to Study Diabetic Neuropathy

We have established spontaneously immortalized Schwann cell lines from normal adult mice and rats and murine disease models. One of the normal mouse cell lines, IMS32, possesses some biological properties of mature Schwann cells and high proliferative activities. The IMS32 cells under hyperglycemic and/or hyperlipidemic conditions have been utilized to investigate the pathogenesis of diabetic neuropathy, especially the polyol pathway hyperactivity, glycation, increased oxidative stress, and reduced synthesis of neurotrophic factors. In addition to the mouse cell lines, our current study focuses on the characterization of a normal rat cell line, IFRS1, under normal and high glucose conditions. These Schwann cell lines can be valuable tools for exploring the detailed mechanisms leading to diabetic neuropathy and novel therapeutic approaches against that condition.

Schwann cells contribute to neurodegeneration in transthyretin amyloidosis.

Familial amyloidotic polyneuropathy (FAP) is one of the transthyretin (TTR) amyloidoses characterized by extracellular amyloid deposits and peripheral nerve involvement. Recently, we found significant expression of the TTR gene in Schwann cells of the peripheral nervous system. We hypothesized that local expression of variant TTR in Schwann cells may contribute to neurodegeneration in FAP. Schwann cells derived from the dorsal root ganglia (DRG) of transgenic mice expressing variant human TTR in a mouse null background were cultured long term to obtain spontaneously immortalized cell lines. We established an immortalized Schwann cell line, TgS1, derived from the transgenic mice. TgS1 cells synthesized variant TTR and secreted it into the medium. As sensory neuropathy usually arises early in FAP, we examined the effect of the conditioned medium derived from TgS1 cells on neurite outgrowth from DRG sensory neurons. Conditioned medium derived from TgS1 cells inhibited neurite outgrowth from the sensory neurons. TTR deposition in the DRG of aged transgenic mice was investigated by immunohistochemistry. TTR aggregates were observed in the cytoplasm of Schwann cells and satellite cells. Proteasome inhibition induced TTR aggregates as aggresomes in TgS1 cells. In conclusion, local variant TTR gene expression in Schwann cells might trigger neurodegeneration in FAP.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone‐induced schwannopathy

Amiodarone hydrochloride (AMD), an anti‐arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor‐primed PC12 cells. Treatment with AMD (1, 5, and 10 μm) induced time‐ and dose‐dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2‐related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3‐II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD‐induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time‐ and dose‐dependent manner. These findings suggest that AMD‐induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

A spontaneously immortalized Schwann cell line from aldose reductase-deficient mice as a useful tool for studying polyol pathway and aldehyde metabolism

The increased glucose flux into the polyol pathway via aldose reductase (AR) is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy, whereas little is known about the functional significance of AR in the peripheral nervous system. Spontaneously immortalized Schwann cell lines established from long‐term cultures of AR‐deficient and normal C57BL/6 mouse dorsal root ganglia and peripheral nerves can be useful tools for studying the physiological and pathological roles of AR. These cell lines, designated as immortalized knockout AR Schwann cells 1 (IKARS1) and 1970C3, respectively, demonstrated distinctive Schwann cell phenotypes, such as spindle‐shaped morphology and immunoreactivity to S100, p75 neurotrophin receptor, and vimentin, and extracellular release of neurotrophic factors. Conditioned media obtained from these cells promoted neuronal survival and neurite outgrowth of cultured adult mouse dorsal root ganglia neurons. Microarray and real‐time RT‐PCR analyses revealed significantly down‐regulated mRNA expression of polyol pathway‐related enzymes, sorbitol dehydrogenase and ketohexokinase, in IKARS1 cells compared with those in 1970C3 cells. In contrast, significantly up‐regulated mRNA expression of aldo‐keto reductases (AKR1B7 and AKR1B8) and aldehyde dehydrogenases (ALDH1L2, ALDH5A1, and ALDH7A1) was detected in IKARS1 cells compared with 1970C3 cells. Exposure to reactive aldehydes (3‐deoxyglucosone, methylglyoxal, and 4‐hydroxynonenal) significantly up‐regulated the mRNA expression of AKR1B7 and AKR1B8 in IKARS1 cells, but not in 1970C3 cells. Because no significant differences in viability between these two cell lines after exposure to these aldehydes were observed, it can be assumed that the aldehyde detoxification is taken over by AKR1B7 and AKR1B8 in the absence of AR.

Myelination in cocultures of established neuronal and Schwann cell lines

O2-H-1-2 Zic2 hypomorphic mutant mice as a schizophrenia model and ZIC2 mutations identified in schizophrenia patients Minoru Hatayama 1 , Akira Ishiguro 1, Yoshimi Iwayama 2, Noriko Takashima 1, Kazuto Sakoori 1, Tomoko Toyota 2, Yayoi Nozaki 1, Yuri S. Odaka 1, Kazuyuki Yamada 3, Takeo Yoshikawa 2, Jun Aruga 1 1 Lab. for Behavioral and Developmental Disorders, BSI, Riken, Japan 2 Laboratory for Molecular Psychiatry, BSI, RIKEN, Wako, Japan 3 Support Unit for Animal Experiments, BSI, RIKEN, Wako, Japan