The development of a ε-polycaprolactone (PCL) scaffold for CNS repair

Potential treatment strategies for the repair of spinal cord injury (SCI) currently favour a combinatorial approach incorporating several factors, including exogenous cell transplantation and biocompatible scaffolds. The use of scaffolds for bridging the gap at the injury site is very appealing although there has been little investigation into CNS neural cell interaction and survival on such scaffolds before implantation. Previously we demonstrated that aligned micro-grooves 12.5-25 µm wide on ε-polycaprolactone (PCL) promoted aligned neurite orientation and supported myelination. In this study we identify the appropriate substrate and its topographical features required for the design of a 3D scaffold intended for transplantation in SCI. Using an established myelinating culture system of dissociated spinal cord cells, recapitulating many of the features of the intact spinal cord, we demonstrate that astrocytes plated on the topography secrete soluble factors(s) that delay oligodendrocyte differentiation but do not prevent myelination. However, as myelination does occur after a further 10-12 days in culture this does not prevent the use of PCL as a scaffold material as part of a combined strategy for the repair of SCI

Effect of ether glycerol lipids on interleukin-1β release and experimental autoimmune encephalomyelitis

We have assessed the effect of two ether glycerol lipids, 77-6 ((2S, 3R)-4-(Tetradecyloxy)-2-amino-1,3-butanediol) and 56-5 ((S)-2-Amino-3-O-hexadecyl-1-propanol), which are substrates for sphingosine kinases, on inflammatory responses. Treatment of differentiated U937 macrophage-like cells with 77-6 but not 56-5 enhanced IL-1β release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1β release was reduced by pretreatment of cells with the caspase-1 inhibitor, Ac-YVAD-CHO, thereby indicating a role for the inflammasome. The enhancement of LPS-stimulated IL-1β release in response to sphingosine, but not 77-6, was reduced by pretreatment of cells with the cathepsin B inhibitor, CA074Me, indicating a role for lysosomal destabilization in the effect of sphingosine. Administration of 56-5 to mice increased disease progression in an experimental autoimmune encephalomyelitis model and this was associated with a considerable increase in the infiltration of CD4+ T-cells, CD11b+ monocytes and F4/80+ macrophages in the spinal cord. 56-5 and 77-6 were without effect on the degradation of myc-tagged sphingosine 1-phosphate 1 receptor in CCL39 cells. Therefore, the effect of 56-5 on EAE disease progression is likely to be independent of the inflammasome or the sphingosine 1-phosphate 1 receptor. However, 56-5 is chemically similar to platelet activating factor and the exacerbation of EAE disease progression might be linked to platelet activating factor receptor signaling

Role of sphingosine 1-phosphate receptors, sphingosine kinases and sphingosine in cancer and inflammation

Sphingosine kinase (there are two isoforms, SK1 and SK2) catalyses the formation of sphingosine 1-phosphate (S1P), a bioactive lipid that can be released from cells to activate a family of G protein-coupled receptors, termed S1P1-5. In addition, S1P can bind to intracellular target proteins, such as HDAC1/2, to induce cell responses. There is increasing evidence of a role for S1P receptors (e.g. S1P4) and SK1 in cancer, where high expression of these proteins in ER negative breast cancer patient tumours is linked with poor prognosis. Indeed, evidence will be presented here to demonstrate that S1P4 is functionally linked with SK1 and the oncogene HER2 (ErbB2) to regulate mitogen-activated protein kinase pathways and growth of breast cancer cells. Although much emphasis is placed on SK1 in terms of involvement in oncogenesis, evidence will also be presented for a role of SK2 in both T-cell and B-cell acute lymphoblastic leukemia. In patient T-ALL lymphoblasts and T-ALL cell lines, we have demonstrated that SK2 inhibitors promote T-ALL cell death via autophagy and induce suppression of c-myc and PI3K/AKT pathways. We will also present evidence demonstrating that certain SK inhibitors promote oxidative stress and protein turnover via proteasomal degradative pathways linked with induction of p53-and p21-induced growth arrest. In addition, the SK1 inhibitor, PF-543 exacerbates disease progression in an experimental autoimmune encephalomyelitis mouse model indicating that SK1 functions in an anti-inflammatory manner. Indeed, sphingosine, which accumulates upon inhibition of SK1 activity, and sphingosine-like compounds promote activation of the inflammasome, which is linked with multiple sclerosis, to stimulate formation of the pro-inflammatory mediator, IL-1β. Such compounds could be exploited to produce antagonists that diminish exaggerated inflammation in disease. The therapeutic potential of modifying the SK-S1P receptor pathway in cancer and inflammation will therefore, be reviewed

The sensitivity of murine spermiogenesis to miglustat is a quantitative trait: a pharmacogenetic study

BACKGROUND: A major event in the post-meiotic development of male germ cells is the formation of the acrosome. This process can be perturbed in C57BL/6 mice by administration of the small molecule miglustat (N-butyldeoxynojirimycin, NB-DNJ). The miglustat-treated mice produce morphologically abnormal spermatozoa that lack acrosomes and are poorly motile. In C57BL/6 mice, miglustat can be used to maintain long-term reversible infertility. In contrast, when miglustat was evaluated in normal men, it did not affect spermatogenesis. To gain more insight into this species difference we have now evaluated the reproductive effects of miglustat in rabbits, in multiple mouse strains and in interstrain hybrid mice. METHODS: Male mice of 18 inbred strains were administered miglustat orally or via miniosmotic pumps. Rabbits were given the compound in their food. Fourth-generation interstrain hybrid mice, bred from C57BL/6 and FVB/N mice (which differ in their response to miglustat), also received the drug. Data on fertility (natural mating), sperm motility and morphology, acrosome status, and serum drug levels were collected. RESULTS: In rabbits the drug did not induce aberrations of sperm shape or motility, although the serum level of miglustat in rabbits far exceeded the level in C57BL/6 mice (8.4 μM and 0.5 μM, respectively). In some strains of the Swiss and Castle lineages of inbred mice miglustat did not cause infertility, severe morphological sperm aberrations or reduced sperm motility. In these strains miglustat only had milder effects. However, miglustat strongly disturbed acrosome and sperm nucleus development in AKR/J and BALB/c mice and in a number of C57BL/6-related strains. The consequences of drug administration in the interstrain hybrid mice were highly variable. Judging by the number of grossly abnormal spermatozoa, these genetically heterogeneous mice displayed a continuous range of intermediate responses, distinct from either of their parental strains. CONCLUSION: The effects of miglustat on spermatogenesis in mice are strain-dependent, while in rabbits the drug is ineffective. Evaluation of interstrain hybrid mice indicated that the sensitivity of spermatogenesis to miglustat is a quantitative trait. These studies pave the way for identifying the genetic factors underlying the strain/species differences in the effect of miglustat

Effect of sphingosine kinase modulators on interleukin-1β release, sphingosine 1-phosphate receptor 1 expression and experimental autoimmune encephalomyelitis : FTY720 analogues and inflammation

Background and Purpose: The sphingosine analogue, FTY720 (Gilenya®) alleviates clinical disease progression in multiple sclerosis. Here we variously assessed the effects of an azide analogue of (S)-FTY720 vinylphosphonate (compound 5; a sphingosine kinase 1 activator), (R)-FTY720 methyl ether (ROMe, a sphingosine kinase 2 inhibitor) and RB-020 (a sphingosine kinase 1 inhibitor and sphingosine kinase 2 substrate) on IL-1beta formation, sphingosine 1-phosphate levels and S1P1 expression. We also assessed the effect of compound 5 and ROMe in an experimental autoimmune encephalomyelitis (EAE) model. Experimental Approach: We measured IL-1beta formation by macrophages, sphingosine 1-phosphate levels and S1P1 expression levels in vitro and clinical score and inflammatory cell infiltration into the spinal cord in vivo. Key Results: Treatment of differentiated U937 macrophages with compound 5, RB-020 or sphingosine (but not ROMe) enhanced IL-1beta release. This data suggests these compounds might be pro-inflammatory in vitro. However, compound 5 or ROMe reduced disease progression and infiltration of inflammatory cells into the spinal cord in EAE and ROMe induced a reduction in CD4+ and CD8+ T-cell levels in the blood (lymphopenia). Indeed, ROMe induced a marked decrease in cell surface S1P1 expression in vitro. Conclusion and Implications: This is the first demonstration that an activator of SK1 (compound 5) and an inhibitor of SK2 (ROMe, which also reduces cell surface S1P1 expression) have an anti-inflammatory action in EAE

Lysosomal storage and pathologenesis in a novel in vitro cellular model of Sandhoff disease

Sandhoff disease is a devastating autosomal recessive GM2 gangliosidosis in which a deficiency of β-hexosaminidase results in lysosomal storage of the enzyme's substrates, including GM2 and GA2 glycolipid as well as glycoprotein-derived oligosaccharides (OS). The effect of individual storage products on the induction of neurodegeneration in the gangliosidoses has not been well defined. Currently there is no valid in vitro cellular model for studying this disease so the aim of this work was to chemically induce the disease phenotype using a potent, reversible β- hexosaminidase inhibitor, N-benzyl-2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol (NBnLABNAc, SRI). Multiple cell lines, both human and mouse-derived were studied, and following SRI treatment, murine RAW264.7 macrophage-like cells exhibited the Sandhoff phenotype, in agreement with glycoconjugate storage found in mouse models, and were used for further study. The structures of the glycosphingolipids (GSLs) and OS, stored due to β-hexosaminidase inhibition, as well as the cellular localization were determined by normal phase HPLC and mass spectrometry, and subcellular fractionation and chemical extraction of the cytosol and lysosome, respectively. These stored lysosomal β-hexosaminidase substrates resulted in a tightly regulated inflammatory response where caspase-dependent apoptosis was prevented. It remained unclear which of the glycoconjugates were responsible for triggering this cytokine-mediated state, so substrate (glycolipid) reduction therapeutics Nbutyldeoxynojirimycin (NB-DNJ) and N-butyldeoxygalactonojirimycin (NB-DGJ) were administered to SRI-induced storage cells. Reduced GSL storage levels were observed but OS accumulation, although changed in structural composition, did not decrease, as predicted from the mechanism of action of each of the imino sugar inhibitors used. Both imino sugars restored GM2 levels whereas the GA2 glycolipid and OS levels remained elevated but the inflammatory response was normalized, implicating the sole contributor to the pathogenesis of Sandhoff disease is GM2 ganglioside. However, NBDNJ treatment resulted in an inflammatory response at high concentrations, presumably due to α-glucosidase-mediated inhibition of protein folding, demonstrating the greater potential of NB-DGJ in treating Sandhoff disease and possibly other related disorders. Besides substrate reduction therapy, chaperone-mediated therapy (CMT) has promise in treating lysosomal storage disorders such as Sandhoff disease. Following administration of an inhibitor of the deficient enzyme to act as a molecular chaperone that stabilises the conformation of the mutant enzyme, ER associated degradation is avoided, improving traffic to the lysosome. The partial increase in enzyme activity using CMT may be sufficient to adjust the critical threshold enzyme activity to levels where GSL storage in the lysosome is reduced to non-pathological concentrations. Cells derived from human Sandhoff and Tay-Sachs patients were assayed for β-hexosaminidase activity, GSL and OS storage levels. However, despite the lack of β-hexosaminidase activity, GM2 and GA2 glycolipid levels were negligible, possibly due to the cellular origin. Following treatment with SRI, elevated activity of β-hexosaminidase did not result in significant amelioration of the GSL storage levels. In terms of OS storage and enzyme activity, SRI induced differential effects depending on the residual β-hexosaminidase activity present, demonstrating the limitations of this compound in enhancing the total activity of β-hexosaminidase in these cells exhibiting GM2 gangliosidosis.</p

Methodological accuracy and firm interpretation of enzymatic analysis: the usefulness of Bisswanger's "Practical Enzymology"

No abstract available

The extent and the nature of the cholinergic contribution to the hepatic encephalopathy-induced cognitive impairment

No abstract available

Erratum to: Lanthanum-induced neurotoxicity: solving the riddle of its involvement in cognitive impairment?

Due to an unfortunate oversight, a mistake was made in the Fig. 1 legend (p. 2033). Readers should note that the elements in blue colour are reported to be up-regulated/activated/increased, while the elements highlighted in red colour are reported to be down-regulated/inhibited/decreased following in vivo exposure to La3+, not the other way round as falsely indicated

An evolutionarily conserved soxb-hdac2 crosstalk regulates neurogenesis in a cnidarian

SoxB transcription factors and histone deacetylases (HDACs) are each major players in the regulation of neurogenesis, but a functional link between them has not been previously demonstrated. Here, we show that SoxB2 and Hdac2 act together to regulate neurogenesis in the cnidarian Hydractinia echinata during tissue homeostasis and head regeneration. We find that misexpression of SoxB genes modifies the number of neural cells in all life stages and interferes with head regeneration. Hdac2 was coexpressed with SoxB2, and its downregulation phe-nocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other\u27s transcript levels, but Hdac2 counteracts this amplification cycle by deacetylating and destabilizing SoxB2 protein. Finally, we present evidence for conservation of these interactions in human neural progenitors. We hypothesize that crosstalk between SoxB transcription factors and Hdac2 is an ancient feature of metazoan neurogenesis and functions to stabilize the correct levels of these multifunctional proteins