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Purpose This trial was conducted to compare effects of continuing versus withholding single-pill combination tablets consisting of angiotensin receptor blockers (ARBs) and calcium channel blockers (CCBs) on perioperative hemodynamics and clinical outcomes. Methods Patients undergoing minor abdominal or urological surgery (n = 106) were randomly assigned to Group C, in which ARB/CCB combination tablets were continued until surgery, or Group W, in which they were withheld within 24 h of surgery. Perioperative hemodynamics and clinical outcomes were compared between the Groups. Results The incidence of hypotension during anesthesia requiring repeated treatment with vasoconstrictors was higher in Group C than Group W (p = 0.0052). Blood pressure during anesthesia was generally lower in Group C than Group W (p p = 0.0246 and p = 0.0327, respectively). The incidence of postoperative hypertension did not differ between Groups (p = 0.3793). Estimated glomerular filtration rate (eGFR) on the preoperative day did not differ between Groups (p = 0.7045), while eGFR was slightly lower in Group C than Group W on the first and third postoperative days (p = 0.0400 and p = 0.0088, respectively), although clinically relevant acute kidney injury did not develop. Conclusions Continuing ARB/CCB combination tablets preoperatively in patients undergoing minor surgery increased the incidence of hypotension during anesthesia, increased requirements of vasoconstrictors to treat hypotension, and might deteriorate postoperative renal function, albeit slightly. These results suggest that withholding ARB/CCB tablets preoperatively is preferable to continuing them. Clinical trial registration This trial is registered with the Japan Registry of Clinical Trials (jRCT) at Japanese Ministry of Health, Labour, and Welfare (Trial ID: jRCT1031190027).

Perioperative Torsade de Pointes (TdP) is a potentially fatal arrhythmia. The association between the prolongation of corrected QT (QTc) interval and TdP has been well described, but prolonged QTc interval is likely not to be a sole determinant. The transmural dispersion of repolarization (TDR) represents regional heterogeneities in repolarization. Prolonged TDR is also considered responsible for TdP. Here we reviewed the current knowledge of perioperative TdP and their known mechanism along with risk factors including drugs. A number of perioperative drugs were reviewed for their effect on QT prolongation and TDR.

Pyruvate functions as a key molecule in energy production and as an antioxidant. The efficacy of pyruvate supplementation in diabetic retinopathy and nephropathy has been shown in animal models; however, its significance in the functional maintenance of neurons and Schwann cells under diabetic conditions remains unknown. We observed rapid and extensive cell death under high-glucose (> 10 mM) and pyruvate-starved conditions. Exposure of Schwann cells to these conditions led to a significant decrease in glycolytic flux, mitochondrial respiration and ATP production, accompanied by enhanced collateral glycolysis pathways (e.g., polyol pathway). Cell death could be prevented by supplementation with 2-oxoglutarate (a TCA cycle intermediate), benfotiamine (the vitamin B1 derivative that suppresses the collateral pathways), or the poly (ADP-ribose) polymerase (PARP) inhibitor, rucaparib. Our findings suggest that exogenous pyruvate plays a pivotal role in maintaining glycolysis–TCA cycle flux and ATP production under high-glucose conditions by suppressing PARP activity.

SUMMARYDiabetic peripheral neuropathy (DPN) is the most common chronic, progressive complication of diabetes mellitus. The main symptom is sensory loss; the molecular mechanisms are not fully understood. We found that Drosophila fed a high-sugar diet, which induces diabetes-like phenotypes, exhibit impairment of noxious heat avoidance. The impairment of heat avoidance was associated with shrinkage of the leg neurons expressing the Drosophila transient receptor potential channel Painless. Using a candidate genetic screening approach, we identified proteasome modulator 9 as one of the modulators of impairment of heat avoidance. We further showed that proteasome inhibition in the glia reversed the impairment of noxious heat avoidance, and heat-shock proteins and exosome secretion in the glia mediated the effect of proteasome inhibition. Our results establish Drosophila as a useful system for exploring molecular mechanisms of diet-induced peripheral neuropathy and propose that the glial proteasome is one of the candidate therapeutic targets for DPN.

Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies.

Autophagy is a catabolic process that removes damaged intracellular organelles components by lysosomal degradation, but it is also activated by oxidative stress. We previously reported that the docosahexaenoic acid (DHA) suppressed oxidative stress-induced autophagy and cell death in immortalized adult rat Schwann (IFRS1) cells. However, the detailed mechanism has not been clarified yet. Therefore, we aimed to elucidate the intracellular signal transduction pathway by which DHA suppresses oxidative stress-induced autophagy in IFRS1 cells. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in IFRS1 cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. The signal transduction of autophagy was assessed by immunoblotting of LC3, p62, AMP-activated protein kinase (AMPK) phosphorylation and UNC51-like kinase (ULK1) phosphorylation in IFRS1 cells. 50 μM tBHP increased the LC3-II/LC3-I ratio, and AMPK phosphorylation, whereas tBHP decreased the p62 protein expression, indicating that autophagy was induced by tBHP. Pretreatment with 10 μM DHA for 12 hours significantly decreased the LC3-II/LC3-I ratio and AMPK phosphorylation that were increased by tBHP, whereas DHA increased the p62 protein expression that was decreased by tBHP. p70S6Kinase and 4E-BP phosphorylation was not changed either by tBHP or by DHA. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome, but suppression of autolysosome was partial. These results suggest that DHA may prevent against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death through the AMPK-dependent signaling pathway in Schwann cells. Disclosure Y. Tatsumi: Research Support; Self; Daiichi Sankyo, Johnson & Johnson, Kyowa Kirin Co., Ltd., Lilly Diabetes, MSD K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Japan Tobacco Inc., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Co., Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly Japan K. K., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K. K., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. K. Sango: None. K. Kato: None. A. Kato: None. N. Niimi: None. H. Yako: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Eli Lilly Japan K. K., MDS Co., Ltd., Novartis Pharma K. K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K. K., Sumitomo Dainippon Pharma Co., Ltd. Funding Grant-in-Aid for Scientific Research (18K06763)

Aldose reductase (AR) is a member of the reduced nicotinamide adenosine dinucleotide phosphate (NADPH)-dependent aldo-keto reductase superfamily. It is also the rate-limiting enzyme of the polyol pathway, catalyzing the conversion of glucose to sorbitol, which is subsequently converted to fructose by sorbitol dehydrogenase. AR is highly expressed by Schwann cells in the peripheral nervous system (PNS). The excess glucose flux through AR of the polyol pathway under hyperglycemic conditions has been suggested to play a critical role in the development and progression of diabetic peripheral neuropathy (DPN). Despite the intensive basic and clinical studies over the past four decades, the significance of AR over-activation as the pathogenic mechanism of DPN remains to be elucidated. Moreover, the expected efficacy of some AR inhibitors in patients with DPN has been unsatisfactory, which prompted us to further investigate and review the understanding of the physiological and pathological roles of AR in the PNS. Particularly, the investigation of AR and the polyol pathway using immortalized Schwann cells established from normal and AR-deficient mice could shed light on the causal relationship between the metabolic abnormalities of Schwann cells and discordance of axon-Schwann cell interplay in DPN, and led to the development of better therapeutic strategies against DPN.

We previously reported that the docosahexaenoic acid (DHA) induced the antioxidant enzyme, heme oxygenase-1 (HO-1) through the nuclear factor erythroid 2-related factor 2 (Nrf2) and protected immortalized adult mouse Schwann (IMS32) cells from oxidative stress. Whereas autophagy is the process by which intracellular components are degraded by the lysosomes, but it also involved in stabilization of Nrf2 and transcriptional activation of Nrf2 target genes. Therefore, autophagy is thought to be closely related to oxidative stress, which is one major cause of diabetic neuropathy. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) for 3 hours decreased cell viability measured by MTT assay in immortalized adult rat Schwann (IFRS1) cells, while pretreatment with 10 μM DHA for 12 hours significantly protected tBHP-induced cytotoxicity. Autophagy was assessed by immunoblotting of LC3B and p62. 50 μM tBHP increased the LC3B-II/LC3B-I ratio and decreased the p62 expression in IFRS1 cells, indicating that autophagy was induced by tBHP. DHA pretreatment alleviated tBHP-induced autophagy. Autophagosome and autolysosome were induced by 50 μM tBHP in IFRS1 cells, as demonstrated by DAPRed and DALGreen staining. DHA pretreatment significantly reduced the production of tBHP-induced autophagosome and autolysosome. These results suggest that oxidative stress induces autophagy and promotes cell death in Schwann cells and that treatment with DHA may protect against diabetic neuropathy by attenuating oxidative stress-induced autophagy and cell death in Schwann cells. Disclosure Y. Tatsumi: None. A. Kato: None. K. Sango: None. T. Himeno: None. M. Kondo: None. S. Tsunekawa: None. Y. Kato: None. H. Kamiya: Speaker’s Bureau; Self; Astellas Pharma Inc., AstraZeneca K.K., Boehringer Ingelheim K.K., Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. J. Nakamura: Research Support; Self; Astellas Pharma Inc., Boehlinger Ingelheim Japan Co., Ltd., Daiichi Sankyo, Eli Lilly Japan K.K., Japan Tobacco Inc., Kaken Pharmaceutical Co., Ltd., Kowa Company, Ltd., Kyowa Hakko Kirin Co., Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Novartis Pharma K.K., Novo Nordisk Pharma Ltd., Ono Pharmaceutical Co., Ltd., Otsuka Pharmaceutical Co., Ltd., Pfizer Japan Inc., Sanofi K.K., Sanwa Kagaku Kenkyusho, Shionogi & Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker’s Bureau; Self; Abbott Japan Co., Ltd., ARKRAY, Astellas Pharma Inc., AstraZeneca K.K., Boehlinger Ingelheim Japan Co., Ltd.,, Daiichi Sankyo, Eli Lilly Japan K.K., Fukuda Denshi, Kissei Pharmaceutical Co., Ltd., Kowa Company, Ltd., Mitsubishi Tanabe Pharma Corporation, MSD K.K., Mylan, Novartis Pharma K.K., Novo Nordisk Pharma Ltd, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho, Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited, Terumo Medical Corporation. K. Kato: None. Funding Japan Society for the Promotion of Science (18K06763)

May-Hegglin anomaly (MHA) is an inherited autosomal dominant disorder characterized by giant platelets and inclusion bodies in granulocytes, and thrombocytopenia. There is no consensus on the perioperative management of this disorder. We report a case involving a patient with MHA who was perioperatively managed without platelet transfusion for cervical laminectomy and laminoplasty. In our case, the platelet count was measured to be 0.6 × 104/µL using an automatic blood cell counter. Peripheral blood smear and genetic test analyses were performed, leading to a definitive diagnosis of MHA. However, clot retraction, serotonin release, and platelet aggregation were normal. Total intravenous anesthesia with propofol and remifentanil, in combination with intermittent injection of fentanyl, was administered. The total blood loss volume was 300 mL, and perioperative course was uneventful. Visual platelet count and platelet function were preserved in this case, although platelet or red blood cell transfusion was not performed. No bleeding tendency was observed during perioperative management.

Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5–7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10 ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12–16 h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50 µg/mL), BDNF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the βIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot–Marie–Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.

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.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

A large variety of drugs have been reported to cause peripheral neuropathies as dose-limiting adverse effects; however, most of them primarily affect axons and/or neuronal cell bodies rather than Schwann cells and/or myelin sheaths. In this chapter, we focus on the drugs that seem to elicit the neuropathies with schwannopathy and/or myelinopathy-predominant phenotypes, such as amiodarone, dichloroacetate, and tumor necrosis factor-α antagonists. Although the pathogenesis of demyelination induced by these drugs remain largely obscure, the recent in vivo and in vitro studies have implicated the involvement of metabolic abnormalities and impaired autophagy in Schwann cells and immune system disorders in the disruption of neuron-Schwann cell contact and interactions.

Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported, however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

There is growing evidence that galectin-1 (GAL-1) and galectin-3 (GAL-3) are involved in the process of axonal regeneration after injury and the pathogenesis of peripheral neuropathies, but the precise roles of these galectins in the peripheral nervous system (PNS) remain unclear. In this paper, we summarized the distribution and possible functions of GAL-1 and GAL-3 in the PNS at mature stages under normal and pathological conditions. Predominant expression of GAL-1 and GAL-3 in isolectin B4 (IB4)-binding small non-peptidergic sensory neurons in dorsal root ganglia (DRG) accords with the involvement of these molecules in nociceptive and thermoceptive functions and GDNF-induced neurite outgrowth. Up-regulation of GAL-1 mRNA/protein in the neurons and Schwann cells upon peripheral nerve injury suggests its active participation in axonal regeneration; both GAL-1 in the reduced and oxidized forms appear to be key molecules for normal onset of Wallerian degeneration and subsequent neurite outgrowth, but the latter lacks lectin activity and may promote the process by stimulating macrophages rather than directly acting on neurons. GAL-3 deficient mice exhibited the acceleration in the process of Wallerian degeneration, but there is room for argument on its pathological roles in axonal regeneration. Up-regulation of GAL-3 protein in immortalized Schwann cells IFRS1 under diabetic conditions and its cytoprotective functions on the cells suggest that it may act as a factor against the progression of diabetic neuropathy, but further evidence is needed to support this idea. All these findings have shed light on both physiological and pathological roles of GAL-1 and GAL-3 in the PNS, and further evidence will be helpful to develop novel strategies for functional restoration after peripheral nerve injury and therapeutic efficacy against diabetic and other neuropathies.

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 large variety of drugs have been reported to cause peripheral neuropathies as dose-limiting adverse effects; however, most of them primarily affect axons and/or neuronal cell bodies rather than Schwann cells and/or myelin sheaths. In this chapter, we focus on the drugs that seem to elicit the neuropathies with schwannopathy and/or myelinopathy-predominant phenotypes, such as amiodarone, dichloroacetate, and tumor necrosis factor-α antagonists. Although the pathogenesis of demyelination induced by these drugs remain largely obscure, the recent in vivo and in vitro studies have implicated the involvement of metabolic abnormalities and impaired autophagy in Schwann cells and immune system disorders in the disruption of neuron-Schwann cell contact and interactions.

A 57-year-old Japanese man was admitted to the hospital with back pain and fever, multiple lung nodules, and abdominal aortic aneurysm (AAA). Laboratory tests performed at admission showed an increased proteinase 3 anti-neutrophil cytoplasmic antibody (PR3-ANCA) level. Video-associated thoracoscopic lung biopsy was performed; pathologic examination showed granulation tissue with necrosis and multinucleated giant cells. The diagnosis of granulomatosis with polyangiitis (GPA) was confirmed on the basis of the clinical presentation, laboratory findings, and lung biopsy. All symptoms were ameliorated, and the serum level of PR3-ANCA declined following treatment with prednisolone and cyclophosphamide. Although the association of GPA with AAA is rare, GPA may be included among the large vessel vasculitides that can give rise to aortic aneurysm.

DNA polymerase κ (Pol κ) is a specialized DNA polymerase involved in translesion DNA synthesis. Although its bypass activities across lesions are well characterized in biochemistry, its cellular protective roles against genotoxic insults are still elusive. To better understand the in vivo protective roles, we have established a human cell line deficient in the expression of Pol κ (KO) and another expressing catalytically dead Pol κ (CD), to examine the cytotoxic sensitivity to 11 genotoxins including ultraviolet C light (UV). These cell lines were established in a genetic background of Nalm-6-MSH+, a human lymphoblastic cell line that has high efficiency for gene targeting, and functional p53 and mismatch repair activities. We classified the genotoxins into four groups. Group 1 includes benzo[a]pyrene diolepoxide, mitomycin C, and bleomycin, where the sensitivity was equally higher in KO and CD than in the cell line expressing wild-type Pol κ (WT). Group 2 includes hydrogen peroxide and menadione, where hypersensitivity was observed only in KO. Group 3 includes methyl methanesulfonate and ethyl methanesulfonate, where hypersensitivity was observed only in CD. Group 4 includes UV and three chemicals, where the chemicals exhibited similar cytotoxicity to all three cell lines. The results suggest that Pol κ not only protects cells from genotoxic DNA lesions via DNA polymerase activities, but also contributes to genome integrity by acting as a non-catalytic protein against oxidative damage caused by hydrogen peroxide and menadione. The non-catalytic roles of Pol κ in protection against oxidative damage by hydrogen peroxide are discussed.

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. We established a spontaneously immortalized Schwann cell line derived from familial amyloidotic polyneuropathy transgenic mice. Conditioned medium from the cells contained variant transthyretin (TTR), and inhibited neurite outgrowth of neurons. TTR aggregates were observed in the Schwann cells and satellite cells of aged mice. Proteasome inhibition induced TTR aggregates as aggresomes in the cultured cells. These results support the hypothesis that Schwann cells contribute to neurodegeneration in familial amyloidotic polyneuropathy (FAP).

A spontaneously immortalized adult Fischer rat Schwann cell line IFRS1 retains the characteristic features of normal Schwann cells, and can be a useful tool for the study of diabetic neuropathy. In the present study, we examined the effects of high glucose and 3-deoxyglucosone (3-DG) on the viability and the protein expression of advanced glycation endproducts (AGE)-binding proteins, such as galectin-3 (GAL-3) and receptor for AGE (RAGE) in IFRS1 cells. Exposure to 30mM of glucose or 0.2mM of 3-DG for 7 days failed to impair the IFRS1 cell viability, but significantly upregulated the expression of GAL-3. The same exposure tended to increase the expression of RAGE, but the changes were not significant. The high glucose-induced upregulation of GAL-3 was attenuated by cotreatment with 0.2mM of an anti-glycated agent aminoguanidine or 20nM of an anti-oxidant trans-resveratrol. In addition, treatment of IFRS1 cells with 1μg/ml of recombinant GAL-3 for 48h resulted in the upregulation of B-cell lymphoma 2 (Bcl-2) and the downregulation of 4-hydroxynonenal (4HNE). These findings suggest the involvement of GAL-3 in the glycation and oxidative stress under diabetic conditions and its cytoprotective role in Schwann cells.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

Translesion DNA synthesis (TLS) is an important pathway that avoids genotoxicity induced by endogenous and exogenous agents. DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in TLS but its protective roles against DNA damage in vivo are still unclear. To better understand these roles, we have established knock-in mice that express catalytically-inactive Polk and crossbred them with gpt delta mice, which possess reporter genes for mutations. The resulting mice (inactivated Polk KI mice) were exposed to mitomycin C (MMC), and the frequency of point mutations, micronucleus formation in peripheral erythrocytes, and γH2AX induction in the bone marrow was determined. The inactivated Polk KI mice exhibited significantly higher frequency of mutations at CpG and GpG sites, micronucleated cells, and γH2AX foci-positive cells than did the Polk wild-type (Polk + ) mice. Recovery from MMC-induced DNA damage, which was evaluated by γH2AX induction, was retarded in embryonic fibroblasts from the knock-in mice when compared to those from the Polk + mice. These results suggest that Polk mediates TLS, which suppresses point mutations and DNA double-strand breaks caused by intra- and interstrand cross-links induced by MMC treatment. The established knock-in mice are extremely useful to elucidate the in vivo roles of the catalytic activity of Polk in suppressing DNA damage that was induced by a variety of genotoxic stresses.

Humans possess multiple specialized DNA polymerases that continue DNA replication beyond a variety of DNA lesions. DNA polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N2-deoxyguanine (BPDE-N2-dG) DNA adducts in an almost error-free manner. In the previous work, we changed the amino acids close to the adducts in the active site and examined the bypass efficiency. The substitution of alanine for phenylalanine 171 (F171A) enhanced by 18-fold in vitro, the efficiencies of dCMP incorporation opposite (−)- and (+)-trans-anti-BPDE-N2-dG. In the present study, we established human cell lines that express wild-type Pol κ (POLK+/−), F171A (POLK F171A/−) or lack expression of Pol κ (POLK−/−) to examine the in vivo significance. These cell lines were generated with Nalm-6, a human pre-B acute lymphoblastic leukemia cell line, which has high efficiency for gene targeting. Mutations were analyzed with shuttle vectors having (−)- or (+)-trans-anti-BPDE-N2-dG in the supF gene. The frequencies of mutations were in the order of POLK−/− > POLK+/− > POLK F171A/− both in (−)- and (+)-trans-anti-BPDE-N2-dG. These results suggest that F171 may function as a molecular brake for bypass across BPDE-N2-dG by Pol κ and raise the possibility that the cognate substrates for Pol κ are not BP adducts in DNA but may be lesions in DNA induced by endogenous mutagens.

Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.

Matrix metalloproteinases (MMPs) that are secreted by activated T cells play a significant role in degradation of the extracellular matrix around the blood vessels and facilitate autoimmune neuroinflammation; however, it remains unclear how MMPs act in lesion formation and whether MMP-targeted therapies are effective in disease suppression. In the present study, we attempted to treat experimental autoimmune encephalomyelitis (EAE) by administration of small interfering RNAs (siRNAs) for MMP-2, MMP-9, and minocycline, all of which have MMP-inhibiting functions. Minocycline, but not siRNAs, significantly suppressed disease development. In situ zymography revealed that gelatinase activities were almost completely suppressed in the spinal cords of minocycline-treated animals, while significant gelatinase activities were measured in the EAE lesions of control animals. However, MMP-2 and MMP-9 mRNAs and proteins in the spinal cords of treated rats were unexpectedly upregulated. At the same time, mRNA for tissue inhibitors of MMPs (TIMP)-1 and -2 were also upregulated. The EnzChek Gelatinase/Collagenase assay using tissue containing native MMPs and TIMPs demonstrated that gelatinase activity levels in the spinal cords of treated rats were suppressed to the same level as those in normal spinal cord tissues. Finally, double immunofluorescent staining demonstrated that MMP-9 immunoreactivities of treated rats were almost the same as those of control rats and that MMP-9 and TIMP-1 immunoreactivities were colocalized in the spinal cord. These findings suggest that minocycline administration does not suppress MMPs at mRNA and protein levels but that it suppresses gelatinase activities by upregulating TIMPs. Thus, MMP-targeted therapies should be designed after the mechanisms of candidate drugs have been considered.

The objective of this study is to clarify developmental transformations in social perception through a developmental survey which focuses on social thinking abilities and judgment abilities in junior high school students. This survey and analysis, while assuming social thinking abilities and judgment abilities in the context of social sciences to be composed of the five elements of actual judgment abilities, inductive inference abilities, deductive reasoning abilities, social judgment abilities and critical thinking abilities, discussed the following two points. (1) Developmental tendencies in junior high school students’ social thinking abilities and judgment abilities; (2) the relationship of the several abilities which comprise social thinking abilities and judgment abilities. In the results of the analysis of data from junior high school students, it was observed that there was a tendency for deductive reasoning abilities, social judgment abilities and critical thinking abilities to rise in line with students’ progress through the school grades, with this extension being particularly marked between the second grade and third grade. The study found a sequential order in the degree of difficulty of the abilities, proceeding from inductive inference abilities and deductive reasoning abilities to social judgment and then from social judgment to critical thinking abilities, as well as mutual relationships between the different abilities.

We have established spontaneously immortalized Schwann cell lines from long-term cultures of adult Fischer rat peripheral nerves. One of them, termed as immortalized Fischer rat Schwann cells 1 (IFRS1), has been shown to retain distinct Schwann cell phenotypes, such as spindle-shaped morphology with expression of glial cell markers (S100, glial fibrillary acidic protein and p75 low affinity neurotrophin receptor) and transcription factors (Krox20, Oct6 and SOX10), synthesis and secretion of neurotrophic factors and cytokines, and fundamental ability to myelinate neurites in cocultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Consequently, IFRS1 cells and their cocultures with neuronal cells can be beneficial tools for exploring the precise mechanisms of myelination as a result of neuron-Schwann cell interactions and the pathogenesis of demyelinating neuropathies.Keywords¾immortalized Schwann cells; coculture; myelination; demyelination

Human cells possess multiple specialized DNA polymerases (Pols) that bypass a variety of DNA lesions which otherwise would block chromosome replication. Human polymerase kappa (Pol κ) bypasses benzo[a]pyrene diolepoxide-N(2)-deoxyguanine (BPDE-N(2)-dG) DNA adducts in an almost error-free manner. To better understand the relationship between the structural features in the active site and lesion bypass by Pol κ, we mutated codons corresponding to amino acids appearing close to the adducts in the active site, and compared bypass efficiencies. Remarkably, the substitution of alanine for phenylalanine 171 (F171), an amino acid conserved between Pol κ and its bacterial counterpart Escherichia coli DinB, enhanced the efficiencies of dCMP incorporation opposite (-)- and (+)-trans-anti-BPDE-N(2)-dG 18-fold. This substitution affected neither the fidelity of TLS nor the efficiency of dCMP incorporation opposite normal guanine. This amino acid change also enhanced the binding affinity of Pol κ to template/primer DNA containing (-)-trans-anti-BPDE-N(2)-dG. These results suggest that F171 functions as a molecular brake for TLS across BPDE-N(2)-dG by Pol κ and that the F171A derivative of Pol κ bypasses these DNA lesions more actively than does the wild-type enzyme.

Although intravenous immunoglobulin (IVIG) has been reported to improve the status of expanded disability status scale (EDSS) of multiple sclerosis (MS) patients and reduce the annual relapse rate, some studies did not find its beneficial effects. In the present study, using an animal model for MS, we found that prophylactic, but not therapeutic, treatment successfully suppressed the disease development. During the search for factors involved in the disease suppression by IVIG, we obtained evidence suggesting that IVIG exerts its function, at least in part, by suppressing activation of matrix metalloproteinases (MMP)-2 and -9. Gelatin zymography revealed that gelatinase activities were suppressed by IVIG treatment in the spinal cord, but not in plasma. This finding raises the possibility that IVIG blocks MMP activities at the interface between the blood stream and CNS. With in situ zymography, we also observed that gelatinase activities were expressed mainly in astrocytes in the inflamed spinal cord of control rats and that this expression was attenuated by the treatment. These findings provide useful information to set optimal conditions for IVIG treatment of MS and to obtain more beneficial effects.

Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human Poleta and Polkappa proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in Poleta was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type Poleta) to 7 : 1 (R61K). Similarly, Tyr112 in Polkappa was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type Polkappa) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in Poleta, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of Poleta and Polkappa might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA.

In mammalian cells, DNA polymerase beta (Polbeta) and poly(ADP-ribose) polymerase-1 (PARP-1) have been implicated in base excision repair (BER) and single-strand break repair. Polbeta knockout mice exhibit extensive neuronal apoptosis during neurogenesis and die immediately after birth, while PARP-1 knockout mice are viable and display hypersensitivity to genotoxic agents and genomic instability. Although accumulating biochemical data show functional interactions between Polbeta and PARP-1, such interactions in the whole animal have not yet been explored. To study this, we generate Polbeta(-/-)PARP-1(-/-) double mutant mice. Here, we show that the double mutant mice exhibit a profound developmental delay and embryonic lethality at mid-gestation. Importantly, the degree of the neuronal apoptosis was dramatically reduced in PARP-1 heterozygous mice in a Polbeta null background. The reduction was well correlated with decreased levels of p53 phosphorylation at serine-18, suggesting that the apoptosis depends on the p53-mediated apoptosis pathway that is positively regulated by PARP-1. These results indicate that functional interactions between Polbeta and PARP-1 play important roles in embryonic development and neurogenesis.