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Bisphenol A (BPA) is a ubiquitously distributed endocrine disrupting chemical (EDC). BPA exposure in humans has been a matter of concern due to its increased application in the products of day to day use. BPA has been reported to cause toxicity in almost all the vital organ systems even at a very low dose levels. It crosses the blood brain barrier and causes neurotoxicity. We studied the effect of BPA on the cerebral cortex of C57BL/6J mice and examined whether BPA exposure alters the expression of axonal and myelin structural proteins. Male mice were dosed orally to 40 μg and 400 μg BPA/kg body weight for 60 days. BPA exposure resulted in memory loss, muscle coordination deficits and allodynia. BPA exposure also caused degeneration of immature and mature oligodendrocytes as evaluated by decreased mRNA levels of 2',3'-cyclic nucleotide 3' phosphodiesterase (CNPase), nestin, myelin basic protein (MBP) and myelin-associated glycoprotein-1 (MAG-1) genes revealing myelin related pathology. It was observed that subchronic BPA exposure caused neuroinflammation through deregulation of inflammatory cytokines mRNA and protein expression which further resulted into neurotoxicity through axonal as well as myelin degeneration in the brain. BPA also caused increased oxidative stress in the brain. Our study indicates long-term subchronic low dose exposure to BPA has the potential to cause axonal degeneration and demyelination in the oligodendrocytes and neurons which may have implications in neurological and neuropsychological disorders including multiple sclerosis (MS), neuromyelitis optica and others., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The recent report of 2',3'-cAMP isolated from rat kidney is the first proof of its biological existence, which revived interest in this mysterious molecule. 2',3'-cAMP serves as an extracellular adenosine source, but how it is degraded remains unclear. Here, we report that 2',3'-cAMP can be hydrolyzed by six phosphodiesterases containing three different families of hydrolytic domains, generating invariably 3'-AMP but not 2'-AMP. The catalytic efficiency (k(cat)/K(m)) of each enzyme against 2',3'-cAMP correlates with that against the widely used non-specific substrate bis(p-nitrophenyl)phosphate (bis-pNPP), indicating that 2',3'-cAMP is a previously unknown non-specific substrate for PDEs. Furthermore, we show that the exclusive formation of 3'-AMP is due to the P-O2' bond having lower activation energy and is not the result of steric exclusion at enzyme active site. Our analysis provides mechanistic basis to dissect protein function when 2',3'-cAMP hydrolysis is observed., (Copyright 2010 Elsevier Inc. All rights reserved.)

Angiostrongylus cantonensis causes severe central nervous system (CNS) infection leading to chronic demyelinating disease. The biological role of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in myelin-related disease requires investigation. CNP expression was investigated in the brain of mice at six time points 5-25 days post-inoculation with A. cantonensis. CNP was measured in brain tissue by quantitative reverse transcription polymerase chain reaction and by western blotting in the cerebrospinal fluid (CSF). The concentration of CNP in the CSF was significantly up-regulated during the severe phase of meningoencephalitis or meningitis as compared with controls. Immunohistochemical labelling localized CNP expression to the cytoplasm of oligodendrocytes. These results suggest that CNP in the CSF from mice with angiostrongylosis is an indicator of myelin destruction. Therefore, measurement of CNP in the CSF of patients with eosinophilic meningitis may be useful in clinical management.

Background: As human umbilical cord blood (UCB) is known to be a rich source of progenitor cells, the prospect of isolating a subset of these cells that could differentiate into cells of non-hematopoietic lineages suggests a therapeutic use for patients with inherited lysosomal and peroxisomal storage diseases currently treated with UCB transplantation., Methods: Oligodendrocyte-like cells were isolated from UCB by density-gradient centrifugation and expanded using selective media. We then characterized this population of cells using standard immunohistochemical staining methods for neural cell proteins and polymerase chain reaction (PCR) to detect RNA sequences for myelin basic protein (MBP). We also developed a functional assay demonstrating myelination of neurons in vitro., Results: Cells with oligodendrocyte-like morphology were reproducibly cultured ex vivo from fresh human UCB. Cells stained positively for multiple oligodendria cell markers (O1, MBP and CNPase) via immunohistochemical staining and flow cytometry. PCR confirmed the presence of MBP and CNPase mRNA. A further in vitro functional assay demonstrated the myelination of mature neuronal cells from the brain of a myelin-deficient murine model co-cultured with the oligodendrocyte-like cells., Discussion: After human UCB transplant, donor-derived cells have been noted to migrate to the brain over time. Although is not known whether these cells solely deliver enzyme replacement or a subset engrafts and differentiates into mature neural cells, the clinical improvements noted in these patients suggest a potential role for targeted cellular therapy. Oligodendrocyte-like cells isolated ex vivo and expanded from human UCB could provide a potential cellular therapy for patients with demyelinating or dismyelinating diseases.

Mechanisms of lesion repair in multiple sclerosis are incompletely understood. To some degree, remyelination can occur, associated with an increase of proliferating oligodendroglial cells. Recently, the expression of potassium channels has been implicated in the control of oligodendrocyte precursor cell proliferation in vitro. We investigated the expression of Kv1.4 potassium channels in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, a model of multiple sclerosis. Confocal microscopy revealed expression of Kv1.4 in AN2-positive oligodendrocyte precursor cells and premyelinating oligodendrocytes in vitro but neither in mature oligodendrocytes nor in the spinal cords of healthy adult mice. After induction of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, Kv1.4 immunoreactivity was detected in or around lesions already during disease onset with a peak early and a subsequent decrease in the late phase of the disease. Kv1.4 expression was confined to 2',3'-cyclic nucleotide 3'-phosphodiesterase-positive oligodendroglial cells, which were actively proliferating and ensheathed naked axons. After a demyelinating episode, the number of Kv1.4 and 2',3'-cyclic nucleotide 3'-phosphodiesterase double-positive cells was greatly reduced in ciliary neurotrophic factor knockout mice, a model with impaired lesion repair. In summary, the re-expression of an oligodendroglial potassium channel may have a functional implication on oligodendroglial cell cycle progression, thus influencing tissue repair in experimental autoimmune encephalomyelitis and multiple sclerosis.

Background: Previous adult stem cells studies have provided evidence that BM mesenchymal stem cells (MSC) exhibit multilineage differentiation capacity. These properties of MSC prompted us to explore the neural potential of MSC with a view to their use for the treatment of demyelinating disorders, such as multiple sclerosis. Indeed, issues such as the identification of a subset of stem cells that is neurally fated, methods of expansion and optimal stage of differentiation for transplantation remain poorly understood., Methods: In order to isolate mouse (m) MSC from BM, we used and compared the classic plastic-adhesion method and one depleting technique, the magnetic-activated cell sorting technique., Results: We established and optimized culture conditions so that mMSC could be expanded for more than 360 days and 50 passages. We also demonstrated that undifferentiated mMSC express the neural markers nestin, MAP2, A2B5, GFAP, MBP, CNPase, GalC, O1 under standard culture conditions before transplantation. The pluripotent stem cell marker Oct-4 and the embryonic stem cell marker Rex-1 are spontaneously expressed by untreated mMSC. The lineage-negative mMSC (CD5- CD11b- Ly-6G- Ter119- CD45R- c-kit/CD117-) overexpressed Oct-4, O1 and A2B5 in the first days of culture compared with the non-sorted MSC. Finally, we identified a distinct subpopulation of mMSC that is primed towards a neural fate, namely Sca-1+/nestin+ mMSC., Discussion: These results should facilitate the optimal timing of harvesting a neurally fated subpopulation of mMSC for transplantation into animal models of human brain diseases.

In order to elucidate molecular mechanisms underlying brain dysfunction in offspring exposed to ethanol in utero, subclinical doses of ethanol that do not have apparent structural effect on the offspring were administered intraperitoneally to pregnant rats at various gestational stages. We measured the activity of membrane marker enzymes and the level of mRNA of myelin proteins of the offspring brain. The activity of a myelin specific enzyme, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) decreased in ethanol-exposed offspring. This effect was not related to the period of gestation or the dose of ethanol. Perikaryonal enzymes, acetylcholinesterase and Na+, K(+)-ATPase, were significantly affected in groups exposed to ethanol at early fetal stage and in high doses. Expression of mRNAs of CNP and myelin basic proteins decreased significantly in the ethanol-treated group, with abnormal developmental profile suggesting a relationship with delayed myelination in offspring exposed to ethanol in utero. The present findings suggest that in spite of the low doses of ethanol that do not cause clinical symptoms in the offspring, prenatal exposure to ethanol affects the level of mRNA of membrane enzyme proteins in the offspring brain, consequently causing a corresponding reduction in enzyme activity, that may lead to neuronal dysfunction. In a separate study, blood ethanol levels were found to reach a maximum level within 30 min after injection and be undetectable after 5 to 10 h. No accumulation effects due to daily injection were observed.

cAMP acts as a primary signal and is regulated by a secreted cyclic nucleotide phosphodiesterase (PDE) throughout development in Dictyostelium discoideum. Expression of the PDE gene (pde) is controlled by promoters specific to vegetative growth (prV), aggregation (prA), or late development (prL). Promoter-containing regions were individually fused to the pde coding sequence. After transformation multiple copies of each construct led to overexpression of PDE mRNA and enzyme activity with the temporal profile expected of each promoter. Overexpression of PDE from prV and prA altered the timing of aggregation compared to control transformants, but the final morphology was normal. Control transformants showed delayed aggregation compared to nontransformed cells. Cells that overexpressed PDE from prL aggregated like the control transformants, but no fruiting bodies were formed. Individual promoter regions were fused to the beta-galactosidase gene (lacZ). Cells that expressed prA-lacZ were dispersed throughout aggregation fields and mounds. Cells that expressed prL-lacZ were first seen distributed homogeneously throughout tight and tipped mounds. In slugs most of these cells are localized in the anterior region. During culmination, cells that expressed the prL-lacZ construct became incorporated into the stalk and were seen in the upper and lower cups surrounding the spore mass.

Recent work has greatly expanded our knowledge of the structure, regulation and diversity of enzymes involved in the synthesis and degradation of cyclic nucleotides. This review focuses on recent work that provides insight into the structure and function of the cyclases and phosphodiesterases that regulate cyclic nucleotide metabolism. Particular emphasis is given to the roles played by multiple isoforms of each enzyme system.

A second cDNA for mouse brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase (cDNAII) encoding 420 amino acids was isolated. The only difference from the cDNA obtained previously (cDNAI), which encodes 400 amino acids, was the 5'-end 53 bp. Comparison of the sequence of the mouse gene with the sequence of cDNAII indicates alternative splicing within exon 1. The 5'-end sequence of cDNAII was found in a region of the gene upstream of exon 1 and a second transcription initiation site was identified.

The mouse 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene was isolated from a mouse gene library. Restriction endonuclease mapping and DNA sequencing analysis revealed that this gene is about 6 kb long and is separated into three exons by two introns. The transcription initiation site was identified. The mouse cDNA of 2374 bp was obtained and used for the screening and analysis of the gene.

A cDNA of 1762 base pairs was obtained from a cDNA library of human brain by immunoscreening, and the nucleotide sequence of the cDNA was determined. The complete amino acid sequence of human 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced from the nucleotide sequence of the cDNA. Human enzyme was found to contain 401 amino acids including initiation methionine and have a molecular weight of 45,098. RNA blot hybridization revealed a single mRNA band at the position of about 3000 bases. DNA blot hybridization suggested that a single-copy 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene exists per haploid genome.