Directional coupling of oligodendrocyte connexin-47 and astrocyte connexin-43 gap junctions.

Intercellular communication via gap junction channels between oligodendrocytes and between astrocytes as well as between these cell types is essential to maintain the integrity of myelin in the central nervous system. Oligodendrocyte gap junction connexin-47 (Cx47) is a key element in this crosstalk and indeed, mutations in human Cx47 cause severe myelin disorders. However, the permeation properties of channels of Cx47 alone and in heterotypic combination with astrocyte Cx43 remain unclear. We show here that Cx47 contains three extra residues at 5' amino-terminus that play a critical role in the channel pore structure and account for relative low ionic conductivity, cationic permselectivity and voltage-gating properties of oligodendrocyte-oligodendrocyte Cx47 channels. Regarding oligodendrocyte-astrocyte coupling, heterotypic channels formed by Cx47 with Cx43 exhibit ionic and chemical rectification, which creates a directional diffusion barrier for the movement of ions and larger negatively charged molecules from cells expressing Cx47 to those with Cx43. The restrictive permeability of Cx47 channels and the diffusion barrier of Cx47-Cx43 channels was abolished by a mutation associated with leukodystrophy, the Cx47P90S, suggesting a novel pathogenic mechanism underlying myelin disorders that involves alterations in the panglial permeation.

Ellagic acid protects from myelin-associated sphingolipid loss in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), the most common model for multiple sclerosis, is characterized by inflammatory cell infiltration into the central nervous system and demyelination. Previous studies have demonstrated that administration of some polyphenols may reduce the neurological alterations of EAE. In this work, we show that ellagic acid, a polyphenolic compound, is beneficial in EAE, most likely through stimulation of ceramide biosynthesis within the brain. EAE was induced in Lewis rats by injection of guinea-pig spinal cord tissue along with Freund's complete adjuvant containing Mycobacterium tuberculosis. Clinical signs first appeared at day 8 post-immunization and reached a peak within 3 days, coincident with reduction of myelin basic protein (MBP) in the cortex. Sphingolipids, the other major components of myelin, also decreased at the acute phase of EAE, both in the cerebral cortex and in the spinal cord. In rats receiving ellagic acid in the drinking water from 2 days before immunization, the onset of the disease was delayed and clinical signs were reduced. This amelioration of clinical signs was accompanied by sustained levels of both MBP and sphingolipid in the cortex, without apparent changes in infiltration of inflammatory CD3+ T-cells, microglial activation, or weight loss, which together suggest a neuroprotective effect of ellagic acid. Finally, in glioma and oligodendroglioma cells we demonstrate that urolithins, the ellagic acid metabolites that circulate in plasma, stimulate the synthesis of ceramide. Together these data suggest that ellagic acid consumption protects against demyelination in rats with induced EAE, likely by a mechanism involving sphingolipid synthesis.

Ultrasmall, water dispersible, TWEEN80 modified Yb:Er:NaGd(WO4)2 nanoparticles with record upconversion ratiometric thermal sensitivity and their internalization by mesenchymal stem cells.

This work presents the synthesis by coprecipitation of diamond shaped Yb:Er:NaGd(WO4)2 crystalline nanoparticles (NPs) with diagonal dimensions in the 5-7 nm × 10-12 nm range which have been modified with TWEEN80 for their dispersion in water, and their interaction with mesenchymal stem cells (MSCs) proposed as cellular NP vehicles. These NPs belong to a large family of tetragonal Yb:Er:NaT(XO4)2 (T = Y, La, Gd, Lu; X = Mo, W) compounds with green (2H11/2 + 4S3/2 â†’ 4I15/2) Er-related upconversion (UC) efficiency comparable to that of Yb:Er:ß-NaYF4 reference compound, but with a ratiometric thermal sensitivity (S) 2.5-3.5 times larger than that of the fluoride. At the temperature range of interest for biomedical applications (∼293-317 K/20-44 °C) S = 108-118 × 10-4 K-1 for 20 at%Yb:5 at%Er:NaGd(WO4)2 NPs, being the largest values so far reported using the 2H11/2/4S3/2 Er intensity ratiometric method. Cultured MSCs, incubated with these water NP emulsions, internalize and accumulate the NPs enclosed in endosomes/lysosomes. Incubations with up to 10 µg of NPs per ml of culture medium maintain cellular metabolism at 72 h. A thermal assisted excitation path is discussed as responsible for the UC behavior of Yb:Er:NaT(XO4)2 compounds.

Antiadipogenic effects of subthermal electric stimulation at 448 kHz on differentiating human mesenchymal stem cells.

The 448 kHz capacitive­resistive electric transfer (CRET) is an electrothermal therapy currently applied in anticellulite and antiobesity treatments. The aim of the present study was to determine whether exposure to the CRET electric signal at subthermal doses affected early adipogenic processes in adipose­derived stem cells (ADSC) from human donors. ADSC were incubated for 2 or 9 days in the presence of adipogenic medium, and exposed or sham­exposed to 5 min pulses of 448 kHz electric signal at 50 µA/mm2 during the last 48 h of the incubation. Colorimetric, immunofluorescence, western blotting and reverse transcription­quantitative polymerase chain reaction assays were performed to assess adipogenic differentiation of the ADSC. Electric stimulation significantly decreased cytoplasmic lipid content, after both 2 and 9 days of differentiation. The antiadipogenic response in the 9 day samples was accompanied by activation of mitogen­activated protein kinase kinase 1/2, decreased expression and partial inactivation of peroxisome proliferator­activated receptor (PPAR) Î³, which was translocated from the nucleus to the cytoplasm, together with a significant decrease in the expression levels of the PPARG1 gene, perilipin, angiopoietin­like protein 4 and fatty acid synthase. These results demonstrated that subthermal stimulation with CRET interferes with the early adipogenic differentiation in ADSC, indicating that the electric stimulus itself can modulate processes controlling the synthesis and mobilization of fat, even in the absence of the concomitant thermal and mechanical components of the thermoelectric therapy CRET.

Power-Frequency Magnetic Field Inhibits Adipogenic Differentiation in Human ADSC.

BACKGROUND/AIMS: Semicircular lipoatrophy (SL) is an idiopathic condition characterized by atrophy of subcutaneous fatty tissue. Although several studies have suggested a possible association between SL and occupational exposure to power frequency magnetic fields (MF), no mechanism has been proposed so far that explains an influence of these fields on adipogenesis. METHODS: The study investigates the effects of a 50 Hz, 100 µT MF on the adipogenesis of stem cells isolated from human adipose tissue (ADSC). Cells were plated in Petri dishes and either exposed intermittently to the field for 42 hours or sham-exposed. RESULTS: Field exposure significantly reduced lipid accumulation within the cells, revealed in Oil Red O stained samples by spectrophotometry and colorimetry. Early cell passages were particularly sensitive to the effect: 30.40 ± 5.77% and 47.96 ± 12.47% below controls in the spectrophotometric and colorimetric assays, respectively. Such antiadipogenic effect was accompanied by significant changes in the expression of key effectors/regulators of early adipogenesis: PPARx03B3;, p-ERK1/2 and Sox9, indicating that at least the ERK/PPARx03B3; signaling pathway could be involved in the effect. CONCLUSIONS: These results constitute an experimental support to the hypothesis that power frequency MF can be one of the factors involved in the etiology of SL.

Regulation of connexin hemichannel activity by membrane potential and the extracellular calcium in health and disease.

Connexins are thought to solely mediate cell-to-cell communication by forming gap junction channels composed of two membrane-spanning hemichannels positioned end-to-end. However, many if not all connexin isoforms also form functional hemichannels (i.e., the precursors of complete channels) that mediate the rapid exchange of ions, second messengers and metabolites between the cell interior and the interstitial space. Electrical and molecular signaling via connexin hemichannels is now widely recognized to be important in many physiological scenarios and pathological conditions. Indeed, mutations in connexins that alter hemichannel function have been implicated in several diseases. Here, we present a comprehensive overview of how hemichannel activity is tightly regulated by membrane potential and the external calcium concentration. In addition, we discuss the genetic mutations known to alter hemichannel function and their deleterious effects, of which a better understanding is necessary to develop novel therapeutic approaches for diseases caused by hemichannel dysfunction. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

Levodopa analgesia in experimental neuropathic pain.

Levodopa has been shown to produce analgesia in various clinical and experimental settings, but its use for chronic pain treatment has not been established. We have undertaken a study of the antiallodynic actions of levodopa in a rat model of painful mononeuropathy. When administered systemically, levodopa produced a decrease in tactile and cold allodynia lasting at least 3h. Direct intrathecal (i.t.) levodopa injection at lumbar levels produced a similar, though shorter, antiallodynic effect. This effect was blocked by the D2-type receptor antagonist sulpiride, which supports the involvement of the spinal dopaminergic system in the analgesic action of levodopa on neuropathic pain. These results provide experimental support on the antiallodynic effect of levodopa in neuropathic pain and suggest that at least part of the analgesic action takes place in the spinal cord and involves dopaminergic D2-type receptors.

Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells.

Glutamate is an excitatory amino acid that serves important functions in mammalian brain development through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/ kainate receptor stimulation. Neural stem cells with self-renewal and multilineage potential are a useful tool to study the signals involved in the regulation of brain development. We have investigated the role played by AMPA/kainate receptors during the differentiation of neural stem cells derived from fetal rat striatum. The application of 1 and 10 microM kainic acid increased significantly the phosphorylation of the cyclic AMP response element binding protein (CREB), raised bromodeoxyuridine incorporation in O4-positive oligodendrocyte precursors, and increased the number of O1-positive cells in the cultures. Increased CREB phosphorylation and proliferation were prevented by the AMPA receptor antagonist 4-4(4-aminophenyl)-1,2-dihydro-1-methyl-2-propylcarbamoyl-6,7-methylenedioxyphthalazine (SYM 2206) and by protein kinase A and protein kinase C inhibitors. Cultures treated with 100 microM kainic acid showed decreased proliferation, a lower proportion of O1-positive cells, and apoptosis of O4-positive cells. None of these effects were prevented by SYM 2206, suggesting that kainate receptors take part in these events. We conclude that AMPA receptor stimulation by kainic acid promotes the proliferation of oligodendrocyte precursors derived from neural stem cells through a mechanism that requires the activation of CREB by protein kinase A and C. In the neurons derived from these cells, either AMPA or kainate receptor stimulation produces neuritic growth and larger cell bodies.

Intrastriatal infusion of liver growth factor stimulates dopamine terminal sprouting and partially restores motor function in 6-hydroxydopamine-lesioned rats.

Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and may be useful for neuroregenerative therapies. The aim of this work was to investigate the effects of the intrastriatal (IS) infusion of LGF in the 6-hydroxydopamine rat model of Parkinson's disease. Tyrosine hydroxylase-positive innervation was significantly increased in the dopamine-denervated striatum of rats receiving intrastriatal LGF infusions (160 ng/day/rat x 15 days) as compared with a vehicle-infused group. There was no evidence of dopaminergic neurogenesis in the striatum or substantia nigra in any experimental group at the times studied. However, in those animals undergoing IS-LGF infusion for 48 hr, we found a significant increase in both microglial proliferation and in the number of microglial cells that acquired the ameboid morphology. This is characteristic of activated microglia/macrophages that has been reported to play an important role in dopamine terminal sprouting. In summary, our study shows that IS infusion of LGF stimulates the outgrowth of tyrosine hydroxylase-positive terminals in the striatum of 6-hydroxydopamine-treated rats. As apomorphine-induced rotational behavior was also reduced in these animals, we propose LGF as a novel factor that, when delivered to the striatum, may be useful in the treatment of Parkinson's disease.

Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C.

Neural stem cells (NSC) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nervous tissue. In vitro experimental conditions can differentiate these cells into specific neuronal phenotypes. In the present study, we describe the combined effect of basic fibroblast growth factor (bFGF) and dibutyryladenosine 3',5'-cyclic monophosphate (dbcAMP) on the differentiation of fetal rat striatal NSC into tyrosine hydroxylase-positive cells. Tyrosine hydroxylase induction was accompanied by the activation of ERK1/ERK2 mitogen-activated protein kinase and was inhibited by the ERK1/ERK2 pathway blocker PD98059, suggesting that ERK activation may be important for this process. In addition, protein kinase C (PKC) was shown to be required for tyrosine hydroxylase protein expression. The inhibition of PKC by staurosporin, as well as its downregulation, decreased the ability of bFGF+dbcAMP to generate tyrosine hydroxylase-positive cells. Moreover, the PKC activator phorbol 12-myristate 13-acetate (PMA) together with bFGF and dbcAMP led to a significant increase in phospho-ERK1/ERK2 levels, and the percentage of beta-tubulin III-positive cells that expressed tyrosine hydroxylase increased by 3.5-fold. PMA also promoted the phosphorylation of the cyclic AMP response element binding protein that might contribute to the increase in tyrosine hydroxylase-positive cells observed in bFGF+dbcAMP+PMA-treated cultures. From these results, we conclude that the manipulation in vitro of NSC from rat fetal striatum with bFGF, cyclic AMP analogs, and PKC activators promotes the generation of tyrosine hydroxylase-positive neurons.

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours.

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.

A procedure for direct lumbar puncture in rats.

We have set out to establish a fast, simple and innocuous method for repeatedly obtaining cerebrospinal fluid (CSF) samples from rats that are undergoing different experimental procedures or suffering pathological conditions. Here, we report a method that has been optimized to repeatedly collect 30-50 microl of CSF in rats by direct lumbar puncture using a procedure that generally takes 15-20 min to perform and presents very little hazard to the animal. The rats are anaesthetized with isofluorane and placed on a board in such a way that the spine is curved at the level of the L3-L5 vertebrae. After performing a small incision in the skin of the back, a neonatal lumbar puncture needle is introduced into the intrathecal space and CSF is passively collected in the needle cup (facilitating maneuvers are described herein). Moreover, we have further adapted this method to permit the intrathecal delivery of pharmacological agents and cell suspensions. In such experiments, behavioral tests can be conducted 10-15 min after the intrathecal injection and the activity of the implanted cells can be assessed by sampling lumbar CSF at later times.