Establishment of a spontaneously immortalized Schwann cell line IKARS1 from aldose reductase-deficient mice

Abstract

Aldose reductase (AR), the first enzyme of the polyol pathway, converts glucose to sorbitol, and galactose to galactitol. Sorbitol is further converted to fructose by sorbitol dehydrogenase (SDH). The increased glucose flux into the polyol pathway via AR in Schwann cells and subsequent accumulation of sorbitol and fructose is recognized as a major contributing factor for the pathogenesis of diabetic neuropathy. The accumulation of galactitol as a consequence of galactose feeding in animals can be a cause of 'galactosemic' neuropathy. In contrast to these pathogenic roles, AR contributes to osmoregulation and aldehyde detoxification under normoglycemic conditions; however, little is known about its physiological roles in the peripheral nervous system (PNS). We established spontaneously immortalized Schwann cell lines from long-term cultures of aldose reductase (AR)-deficient and normal C57BL/6 mouse dorsal root ganglia (DRG) and peripheral nerves. These cell lines, designated as immortalized knocked out AR Schwann cells 1 (IKARS1) and 1970C3, showed distinct Schwann cell phenotypes, such as spindle-shaped morphology and immunoreactivity for S100 and p75 low-affinity neurotrophin receptor. Conditioned medium obtained from these cells exhibited potent biological activity for the promotion of survival and neurite outgrowth of cultured adult mouse DRG neurons. Exposure to 25 mM of galactose for 24 h resulted in a drastic increase in intracellular galactitol contents in 1970C3, but not in IKARS1. However, we failed to detect significant up-regulation of sorbitol and fructose contents by exposure to high glucose (30 mM) for 24 h in the both cell lines . By using DNA microarray and subsequent real time RT-PCR analyses, we observed significant down-regulation of mRNA expression for the polyol pathway-related enzymes, such as SDH and ketohexokinase, in IKARS1 as compared with 1970C3. In contrast, significant up-regulation of mRNA expression for aldo-keto reductases Akr1b7 and Akr1b8 in IKARS1 as compared with 1970C3 was detected. Furthermore, exposure to the reactive aldehydes, such as 3-deoxyglucosone (0.5 mM), methylglyoxal (0.25 mM) and 4-hydroxynonenal (2.5 μM), significantly (>2.5 fold) up-regulated the mRNA expression for Akr1b7 in IKARS1, but not in 1970C3. Because we observed no significant differences between the two cell lines in the viability after exposure to these aldehydes, the detoxification function might be taken over by Akr1b7 and other aldo-keto reductases in the absence of AR. These Schwann cell lines can be useful tools for the study of polyol metabolism and functional roles of AR in the PNS.