Kir6.2-deficient mice develop somatosensory dysfunction and axonal loss in the peripheral nerves.

Glucose-responsive ATP-sensitive potassium channels (KATP) are expressed in a variety of tissues including nervous systems. The depolarization of the membrane potential induced by glucose may lead to hyperexcitability of neurons and induce excitotoxicity. However, the roles of KATP in the peripheral nervous system (PNS) are poorly understood. Here, we determine the roles of KATP in the PNS using KATP-deficient (Kir6.2-deficient) mice. We demonstrate that neurite outgrowth of dorsal root ganglion (DRG) neurons was reduced by channel closers sulfonylureas. However, a channel opener diazoxide elongated the neurite. KATP subunits were expressed in mouse DRG, and expression of certain subunits including Kir6.2 was increased in diabetic mice. In Kir6.2-deficient mice, the current perception threshold, thermal perception threshold, and sensory nerve conduction velocity were impaired. Electron microscopy revealed a reduction of unmyelinated and small myelinated fibers in the sural nerves. In conclusion, KATP may contribute to the development of peripheral neuropathy.

A novel loss-of-function mutation of GATA3 (p.R299Q) in a Japanese family with Hypoparathyroidism, Deafness, and Renal Dysplasia (HDR) syndrome.

BACKGROUND: Hypoparathyroidism, deafness, and renal dysplasia (HDR) syndrome is a rare autosomal dominant disorder caused by mutations in the zinc finger transcription factor gene, GATA3. GATA3 has 2 zinc finger domains, which play an important role in the increase in target gene transcription activity. CASE PRESENTATION: A 50-year-old woman and her 27-year-old daughter were followed up because of hypoparathyroidism. They had bilateral sensorineural deafness. Abdominal computed tomography scanning revealed renal dysplasia in the mother, but no renal anomaly in the daughter. Direct sequencing of GATA3 gene revealed a novel heterozygous missense mutation at codon 299 (p.R299Q) in exon 4. This mutation is located at the junction between the 2 zinc fingers. The structure prediction showed that it caused a conformation change in this junction area, affecting the spatial position of the zinc fingers. Additionally, a more marked conformation change was observed in the N-terminal zinc finger region compared to that in the C-terminal region. Functional analysis of this mutant protein using an in vitro luciferase reporter assay system confirmed that the mutation abolished the enhancing effects of wild-type GATA3 on the promoter activity of the consensus GATA responsive element and that of human PTH gene. CONCLUSION: We identified a novel R299Q mutation in GATA3 in a Japanese family with HDR syndrome. We confirmed that R299Q is a loss-of-function mutation, due to the extensive conformational change in the zinc fingers of GATA3.

Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice.

BACKGROUND: Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted, a universally effective treatment of diabetic polyneuropathy has not yet been established. Recently, regenerative medicine studies in diabetic polyneuropathy using somatic stem/progenitor cell have been reported. However, the effectiveness of these cell transplantations was restricted because of their functional and numerical impairment in diabetic objects. Here, we investigated the efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived from mouse embryonic stem cells. METHODS AND RESULTS: Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improved several physiological impairments in diabetic polyneuropathy: hypoalgesia, delayed nerve conduction velocities, and reduced blood flow in sciatic nerve and plantar skin. Furthermore, pathologically, the capillary number to muscle fiber ratios were increased in skeletal muscles of transplanted hindlimbs, and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skin. Transplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injection sites. Moreover, several transplanted cells constructed chimeric blood vessels with recipient cells. CONCLUSIONS: These results suggest that transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabetic polyneuropathy.

Therapeutic efficacy of bone marrow-derived mononuclear cells in diabetic polyneuropathy is impaired with aging or diabetes.

AIMS/INTRODUCTION: Recent studies have shown that cell transplantation therapies, such as endothelial precursor cells, bone marrow-derived mononuclear cells (BM-MNCs) and mesenchymal stem cells, are effective on diabetic polyneuropathy through ameliorating impaired nerve blood flow in diabetic rats. Here, we investigated the effects of BM-MNCs transplantation in diabetic polyneuropathy using BM-MNCs derived from adult (16-week-old) diabetic (AD), adult non-diabetic (AN) or young (8-week-old) non-diabetic (YN) rats. MATERIALS AND METHODS: BM-MNCs of AD and AN were isolated after an 8-week diabetes duration. The BM-MNCs were characterized using flow cytometry analysis of cell surface markers and reverse transcription polymerase chain reaction of several cytokines. BM-MNCs or saline were injected into hind limb muscles. Four weeks later, the thermal plantar test, nerve conduction velocity, blood flow of the sciatic nerve and capillary-to-muscle fiber ratio were evaluated. RESULTS: The number of CD29(+)/CD90(+) cells that host mesenchymal stem cells in BM-MNCs decreased in AD compared with AN or YN, and transcript expressions of basic fibroblast growth factor and nerve growth factor in BM-MNCs decreased in AD compared with AN or YN. Impaired thermal sensation, decreased blood flow of the sciatic nerve and delayed nerve conduction velocity in 8-week-diabetic rats were significantly ameliorated by BM-MNCs derived from YN, whereas BM-MNCs from AD or AN rats did not show any beneficial effect in these functional tests. CONCLUSIONS: These results show that cytokine production abilities and the mesenchymal stem cell population of BM-MNCs would be modified by aging and metabolic changes in diabetes, and that these differences could explain the disparity of the therapeutic efficacy of BM-MNCs between young and adult or diabetic and non-diabetic patients in diabetic polyneuropathy.

Mesenchymal stem cells ameliorate impaired wound healing through enhancing keratinocyte functions in diabetic foot ulcerations on the plantar skin of rats.

AIMS/HYPOTHESIS: Although the initial healing stage involves a re-epithelialization in humans, diabetic foot ulceration (DFU) has been investigated using rodent models with wounds on the thigh skin, in which a wound contraction is initiated. In this study, we established a rodent model of DFU on the plantar skin and evaluated the therapeutic efficacy of bone-marrow-derived mesenchymal stem cells (BM-MSCs) in this model. METHODS: The wounds made on the hind paws or thighs of streptozotocin induced diabetic or control rats were treated with BM-MSCs. Expression levels of phosphorylated focal adhesion kinase (pFAK), matrix metaroprotease (MMP)-2, EGF, and IGF-1, were evaluated in human keratinocytes, which were cultured in conditioned media of BM-MSCs (MSC-CM) with high glucose levels. RESULTS: Re-epithelialization initiated the healing process on the plantar, but not on the thigh, skin. The therapy utilizing BM-MSCs ameliorated the delayed healing in diabetic rats. In the keratinocytes cultured with MSC-CM, the decreased pFAK levels in the high glucose condition were restored, and the MMP2, EGF, and IGF-1 levels increased. CONCLUSIONS/INTERPRETATION: Our study established a novel rat DFU model. The impaired healing process in diabetic rats was ameliorated by transplantation of BM-MSCs. This amelioration might be accounted for by the modification of keratinocyte functions.

Sensory and motor physiological functions are impaired in gastric inhibitory polypeptide receptor-deficient mice.

AIMS/INTRODUCTION: Gastric inhibitory polypeptide (GIP) is an incretin secreted from the gastrointestinal tract after an ingestion of nutrients, and stimulates an insulin secretion from the pancreatic islets. Additionally, GIP has important roles in extrapancreatic tissues: fat accumulation in adipose tissue, neuroprotective effects in the central nervous system and an inhibition of bone resorption. In the current study, we investigated the effects of GIP signaling on the peripheral nervous system (PNS). MATERIALS AND METHODS: First, the presence of the GIP receptor (GIPR) in mouse dorsal root ganglion (DRG) was evaluated utilizing immunohistochemical analysis, western blotting and reverse transcription polymerase chain reaction. DRG neurons of male wild-type mice (WT) were cultured with or without GIP, and their neurite lengths were quantified. Functions of the PNS were evaluated in GIPR-deficient mice (gipr-/-) and WT by using current perception thresholds (CPTs), Thermal Plantar Test (TPT), and motor (MNCV) and sensory nerve conduction velocity (SNCV, respectively). Sciatic nerve blood flow (SNBF) and plantar skin blood flow (PSBF) were also evaluated. RESULTS: We confirmed the expression of GIPR in DRG neurons. The neurite outgrowths of DRG neurons were promoted by the GIP administrations. The gipr-/- showed impaired perception functions in the examination of CPTs and TPT. Both MNCV and SNCV were delayed in gipr-/- compared with these in WT. There was no difference in SNBF and PSBF between WT and gipr-/-. CONCLUSIONS: Our findings show that the GIP signal could exert direct physiological roles in the PNS, which might be directly exerted on the PNS.

The role of S100B in the interaction between adipocytes and macrophages.

OBJECTIVE: The S100 calcium binding protein B (S100B) implicated in brain inflammation acts via the receptor of advanced glycation end products (RAGE) and is also secreted from adipocytes. We investigated the role of S100B in the interaction between adipocytes and macrophages using a cell-culture model. DESIGN AND METHODS: RAW264.7 macrophages (RAW) were stimulated by recombinant S100B to observe alterations in TNF-α and M1 markers; 3T3-L1 adipocytes (L1) were stimulated by TNF-α to examine S100B secretion. RAW and L1 were then mutually stimulated with conditioned media of each other, or co-cultured. The effects of S100B silencing or a RAGE-neutralizing antibody were also investigated. RESULTS: S100B upregulated TNF-α and M1 markers in RAW, and TNF-α augmented S100B secretion from L1. L1 conditioned media stimulated TNF-α secretion from RAW, and RAW conditioned media increased S100B secretion from L1. The co-culture of RAW and L1 increased TNF-α, S100B, and the expression of M1 markers and the MCP-1 receptor CCR2. The silencing of S100B or RAGE neutralization significantly ameliorated TNF-α hypersecretion from RAW that were stimulated with L1 conditioned media. CONCLUSIONS: Thus, S100B as an adipokine may play a role in the interaction between adipocytes and macrophages to establish a vicious paracrine loop.

Mesenchymal stem cell-like cells derived from mouse induced pluripotent stem cells ameliorate diabetic polyneuropathy in mice.

BACKGROUND: Although pathological involvements of diabetic polyneuropathy (DPN) have been reported, no dependable treatment of DPN has been achieved. Recent studies have shown that mesenchymal stem cells (MSCs) ameliorate DPN. Here we demonstrate a differentiation of induced pluripotent stem cells (iPSCs) into MSC-like cells and investigate the therapeutic potential of the MSC-like cell transplantation on DPN. RESEARCH DESIGN AND METHODS: For induction into MSC-like cells, GFP-expressing iPSCs were cultured with retinoic acid, followed by adherent culture for 4 months. The MSC-like cells, characterized with flow cytometry and RT-PCR analyses, were transplanted into muscles of streptozotocin-diabetic mice. Three weeks after the transplantation, neurophysiological functions were evaluated. RESULTS: The MSC-like cells expressed MSC markers and angiogenic/neurotrophic factors. The transplanted cells resided in hindlimb muscles and peripheral nerves, and some transplanted cells expressed S100 ß in the nerves. Impairments of current perception thresholds, nerve conduction velocities, and plantar skin blood flow in the diabetic mice were ameliorated in limbs with the transplanted cells. The capillary number-to-muscle fiber ratios were increased in transplanted hindlimbs of diabetic mice. CONCLUSIONS: These results suggest that MSC-like cell transplantation might have therapeutic effects on DPN through secreting angiogenic/neurotrophic factors and differentiation to Schwann cell-like cells.

Transplantation of neural crest-like cells derived from induced pluripotent stem cells improves diabetic polyneuropathy in mice.

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of 16-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced growth factors: nerve growth factor, neurotrophin 3, vascular endothelial growth factor, and basic fibroblast growth factor. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of growth factors and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

Beneficial effects of exendin-4 on experimental polyneuropathy in diabetic mice.

OBJECTIVE: The therapeutic potential of exendin-4, an agonist of the glucagon-like peptide-1 receptor (GLP-1R), on diabetic polyneuropathy (DPN) in streptozotocin (STZ)-induced diabetic mice was investigated. RESEARCH DESIGN AND METHODS: The presence of the GLP-1R in lumbar dorsal root ganglion (DRG) was evaluated by immunohistochemical analyses. DRG neurons were dissected from C57BL6/J mice and cultured with or without Schwann cell-conditioned media in the presence or absence of GLP-1 (7-37) or exendin-4. Then neurite outgrowth was determined. In animal-model experiments, mice were made diabetic by STZ administration, and after 12 weeks of diabetes, exendin-4 (10 nmol/kg) was intraperitoneally administered once daily for 4 weeks. Peripheral nerve function was determined by the current perception threshold and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood flow (SNBF) and intraepidermal nerve fiber densities (IENFDs) also were evaluated. RESULTS: The expression of the GLP-1R in DRG neurons was confirmed. GLP-1 (7-37) and exendin-4 significantly promoted neurite outgrowth of DRG neurons. Both GLP-1R agonists accelerated the impaired neurite outgrowth of DRG neurons cultured with Schwann cell-conditioned media that mimicked the diabetic condition. At the doses used, exendin-4 had no effect on blood glucose or HbA(1c) levels. Hypoalgesia and delayed MNCV and SNCV in diabetic mice were improved by exendin-4 without affecting the reduced SNBF. The decreased IENFDs in sole skins of diabetic mice were ameliorated by exendin-4. CONCLUSIONS: Our findings indicate that exendin-4 ameliorates the severity of DPN, which may be achieved by its direct actions on DRG neurons and their axons.