Glucagon-Like Peptide-1 Receptor Agonists as Potential Myelination-Inducible and Anti-Demyelinating Remedies.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) were developed as insulinotropic and anti-hyperglycemic agents for the treatment of type 2 diabetes, but their neurotrophic and neuroprotective activities have been receiving increasing attention. Myelin plays a key role in the functional maintenance of the central and peripheral nervous systems, and recent in vivo and in vitro studies have shed light on the beneficial effects of GLP-1RAs on the formation and protection of myelin. In this article, we describe the potential efficacy of GLP-1RAs for the induction of axonal regeneration and remyelination following nerve lesions and the prevention and alleviation of demyelinating disorders, particularly multiple sclerosis.

Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro.

Besides its insulinotropic actions on pancreatic ß 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.

Transplantation of dental pulp stem cells improves long-term diabetic polyneuropathy together with improvement of nerve morphometrical evaluation.

BACKGROUND: Although previous reports have revealed the therapeutic potential of stem cell transplantation in diabetic polyneuropathy, the effects of cell transplantation on long-term diabetic polyneuropathy have not been investigated. In this study, we investigated whether the transplantation of dental pulp stem cells (DPSCs) ameliorated long-term diabetic polyneuropathy in streptozotocin (STZ)-induced diabetic rats. METHODS: Forty-eight weeks after STZ injection, we transplanted DPSCs into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation (i.e., 52 weeks after STZ injection) the effects of DPSC transplantation on diabetic polyneuropathy were assessed. RESULTS: STZ-induced diabetic rats showed significant reductions in the sciatic motor/sensory nerve conduction velocity, increases in the current perception threshold, and decreases in capillary density in skeletal muscles and intra-epidermal nerve fiber density compared with normal rats, all of which were ameliorated by DPSC transplantation. Furthermore, sural nerve morphometrical analysis revealed that the transplantation of DPSCs significantly increased the myelin thickness and area. DPSC-conditioned media promoted the neurite outgrowth of dorsal root ganglion neurons and increased the viability and myelin-related protein expression of Schwann cells. CONCLUSIONS: These results indicated that the transplantation of DPSCs contributed to the neurophysiological and neuropathological recovery from a long duration of diabetic polyneuropathy.

Diagnostic accuracy of the anti-glutamic acid decarboxylase antibody in type 1 diabetes mellitus: Comparison between radioimmunoassay and enzyme-linked immunosorbent assay.

AIMS/INTRODUCTION: The distributer of the anti-glutamic acid decarboxylase antibody assay kit using radioimmunoassay (RIA) recently announced its discontinuation, and proposed an alternative kit using enzyme-linked immunosorbent assay (ELISA). The aim of the present study was to investigate the diagnostic values of the anti-glutamic acid decarboxylase antibody by RIA and ELISA among type 1 diabetes mellitus patients and control participants. MATERIALS AND METHODS: A total of 79 type 1 diabetes mellitus patients and 79 age-matched controls were enrolled and assessed using RIA and ELISA. Sensitivity, specificity, positive predictive values and negative predictive values were calculated for cut-off values (RIA = 1.5 U/mL and ELISA = 5.0 U/mL, respectively). Kappa coefficients were used to test for agreements between the RIA and ELISA methods regarding the diagnosis of type 1 diabetes mellitus. RESULTS: The sensitivity, specificity, positive predictive values, and negative predictive values for diagnosing type 1 diabetes mellitus were 57.0, 97.5, 95.7, and 69.4% by RIA, and 60.8, 100.0, 100.0 and 71.8% by ELISA, respectively. The diagnosis of type 1 diabetes mellitus using the RIA and ELISA methods showed substantial agreement with the kappa values of 0.74 for all participants, and of 0.64 for the acute type; however, there was moderate agreement with the kappa value of 0.56 for the slowly progressive type. CONCLUSIONS: The present study suggests that both anti-glutamic acid decarboxylase antibody by RIA and ELISA was useful for diagnosing type 1 diabetes mellitus. However, in the slowly progressive type, the degree of agreement of these two kits was poorer compared with those in all participants or in the acute type.

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.

Neurotrophic and neuroprotective properties of exendin-4 in adult rat dorsal root ganglion neurons: involvement of insulin and RhoA.

Glucagon-like peptide-1 (GLP-1) is thought to preserve neurons and glia following axonal injury and neurodegenerative disorders. We investigated the neurotrophic and neuroprotective properties of exendin (Ex)-4, a synthetic GLP-1 receptor (GLP-1R) agonist, on adult rat dorsal root ganglion (DRG) neurons and PC12 cells. GLP-1R was predominantly localized on large and small peptidergic neurons in vivo and in vitro, suggesting the involvement of GLP-1 in both the large and small sensory fiber functions. Ex-4 dose-dependently (1 ≤ 10 ≤ 100 nM) promoted neurite outgrowth and neuronal survival at 2 and 7 days in culture, respectively. Treatment with 100 nM Ex-4 restored the reduced neurite outgrowth and viability of DRG neurons caused by the insulin removal from the medium and suppressed the activity of RhoA, an inhibitory regulator for peripheral nerve regeneration, in PC12 cells. Furthermore, these effects were attenuated by co-treatment with phosphatidylinositol-3'-phosphate kinase (PI3K) inhibitor, LY294002. These findings imply that Ex-4 enhances neurite outgrowth and neuronal survival through the activation of PI3K signaling pathway, which negatively regulates RhoA activity. Ex-4 and other GLP-1R agonists may compensate for the reduced insulin effects on neurons, thereby being beneficial for the treatment of diabetic neuropathy.

Upregulation of galectin-3 in immortalized Schwann cells IFRS1 under diabetic conditions.

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.

Rho-kinase regulation of TNF-α-induced nuclear translocation of NF-κB RelA/p65 and M-CSF expression via p38 MAPK in mesangial cells.

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rho-kinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-α promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rho-kinase-dependent manner. Consistent with this observation, TNF-α-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNA-facilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-κB RelA/p65 and subsequent DNA binding activity, with no significant effects on IκBα degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.

Adenoviral expression of TDP-43 and FUS genes and shRNAs for protein degradation pathways in rodent motoneurons in vitro and in vivo.

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding transactivation response (TAR) DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. In the present study, we produced recombinant adenovirus vectors encoding wild type and mutant TDP-43 and FUS, and those encoding short hairpin RNAs (shRNAs) for proteasome (PSMC1), autophagy (ATG5), and endosome (VPS24) systems to investigate whether the coupled gene transductions in motoneurons by these adenoviruses elicit ALS pathology. Cultured neurons, astrocytes and oligodendrocytes differentiated from adult rat neural stem cells and motoneurons derived from mouse embryonic stem cells were successfully infected with these adenoviruses showing cytoplasmic aggregate formation. When these adenoviruses were injected into the facial nerves of adult rats, exogenous TDP-43 and FUS proteins were strongly expressed in facial motoneurons by a retrograde axonal transport of the adenoviruses. Co-infections of adenovirus encoding shRNA for PSMC1, ATG5 or VPS24 with TDP-43 or FUS adenovirus enhanced cytoplasmic aggregate formation in facial motoneurons, suggesting that impairment of protein degradation pathways accelerates formation of TDP-43 and FUS-positive aggregates in ALS.

Fasudil inhibits ER stress-induced VCAM-1 expression by modulating unfolded protein response in endothelial cells.

The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress.

Rho-kinase inhibition prevents the progression of diabetic nephropathy by downregulating hypoxia-inducible factor 1α.

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

Myelination in coculture of established neuronal and Schwann cell lines.

Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 µg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 µg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

Clinical results for tocilizumab over one year in the clinical setting as assessed by CDAI (clinical disease activity index): CRP at week 12 and MMP-3 at week 24 are predictive factors for CDAI.

To investigate the clinical results of 1 year tocilizumab (TCZ) treatment of rheumatoid arthritis patients in clinical practice by using the clinical disease activity index (CDAI). Thirty-one patients with inadequate response to DMARDs, including methotrexate (MTX), or TNF inhibitors received TCZ (8 mg every 4 weeks). The clinical responses were measured using the 28-joint disease activity score (DAS28-ESR) and CDAI. Matrix metalloproteinase-3 (MMP-3) was assessed as a serological biomarker. Mean baseline DAS28-ESR was 5.96, decreasing to 2.89 at week 52 with a remission rate (DAS28-ESR < 2.6) of 35.5%. Mean baseline CDAI was 28.4, decreasing to 10.2 at week 52 with a remission rate (CDAI ≤ 2.8) of 22.6%. Of patients whose CRP levels had fallen to below the limit of detection by week 12, 65.2% achieved remission or low disease activity as assessed by CDAI at week 52. Median baseline MMP-3 level was 165.7 ng/mL, decreasing to 79.5 ng/mL at week 52. A positive correlation was seen between CDAI at week 52 and MMP-3 level from week 12 onward. About 50% of the patients treated with TCZ in clinical practice achieved a low disease activity level at week 52 as assessed by CDAI, which does not include acute-phase proteins. Our results suggested that CRP levels falling to below the limit of detection by week 12 and MMP-3 ≤ 80.6 ng/mL at week 24 could predict low disease activity or remission at week 52 as assessed by CDAI.

Survival after total joint arthroplasty in patients with rheumatoid arthritis. Comparison of the postoperative life expectancies and survival by initial operative years: 1970s and 1980s group versus 1990s group.

Abstract A total of 867 patients with rheumatoid arthritis (RA) underwent 1764 total joint arthroplasties (total hip arthroplasty and/or total knee arthroplasty) over three decades from 1970 to 1999. A survey on their postoperative survival was conducted in December 2000 in which these patients were divided into two groups. The patients who had their initial arthroplasty in the period 1970 to 1989 were classified as the "70s and 80s group," and those who had their initial arthroplasty from 1990 to 1999 were classified as the "90s group." Using the Kaplan-Meier method, their cumulative survival rates were compared. The survival rates of the 70s and 80s group, consisting of 433 patients, were 84.8% at the 5th postoperative year, 60.1% at the 10th year, and 45.3% at the 15th year. Although the survey period of the 90s group is shorter than that of the 70s and 80s group, their survival rates were 90.2% at the 5th year and 84.3% at the 10th year. At present, the life expectancies of the 90s group are good, and their cumulative survival rate is significantly greater than that of the 70s and 80s group (P < 0.01). It seems that this improvement has been contributed to by the recent increase in the number of RA patients with good prognoses and the well-timed application of arthroplasty, which decreases the number of patients with poor risk.

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro.

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.