Increased Ca2+ signaling in NRXN1α+/- neurons derived from ASD induced pluripotent stem cells.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a high co-morbidity of epilepsy and associated with hundreds of rare risk factors. NRXN1 deletion is among the commonest rare genetic factors shared by ASD, schizophrenia, intellectual disability, epilepsy, and developmental delay. However, how NRXN1 deletions lead to different clinical symptoms is unknown. Patient-derived cells are essential to investigate the functional consequences of NRXN1 lesions to human neurons in different diseases. Methods: Skin biopsies were donated by five healthy donors and three ASD patients carrying NRXN1α+/- deletions. Seven control and six NRXN1α+/- iPSC lines were derived and differentiated into day 100 cortical excitatory neurons using dual SMAD inhibition. Calcium (Ca2+) imaging was performed using Fluo4-AM, and the properties of Ca2+ transients were compared between two groups of neurons. Transcriptome analysis was carried out to undercover molecular pathways associated with NRXN1α+/- neurons. Results: NRXN1α+/- neurons were found to display altered calcium dynamics, with significantly increased frequency, duration, and amplitude of Ca2+ transients. Whole genome RNA sequencing also revealed altered ion transport and transporter activity, with upregulated voltage-gated calcium channels as one of the most significant pathways in NRXN1α+/- neurons identified by STRING and GSEA analyses. Conclusions: This is the first report to show that human NRXN1α+/- neurons derived from ASD patients' iPSCs present novel phenotypes of upregulated VGCCs and increased Ca2+ transients, which may facilitate the development of drug screening assays for the treatment of ASD.

Neurofilament light as an immune target for pathogenic antibodies.

Antibodies to neuronal antigens are associated with many neurological diseases including paraneoplastic neurological disorders, epilepsy, amyotrophic lateral sclerosis and multiple sclerosis. Immunization with neuronal antigens such as neurofilament light (NF-L), a neuronal intermediate filament in axons, has been shown to induce neurological disease and spasticity in mice. Also, although antibodies to NF-L are widely used as surrogate biomarkers of axonal injury in amyotrophic lateral sclerosis and multiple sclerosis, it remains to be elucidated if antibodies to NF-L contribute to neurodegeneration and neurological disease. To address this, we examined the pathogenic role of antibodies directed to NF-L in vitro using spinal cord co-cultures and in vivo in experimental autoimmune encephalomyelitis (EAE) and optic neuritis animal models of multiple sclerosis. Here we show that peripheral injections of antibodies to NF-L augmented clinical signs of neurological disease in acute EAE, increased retinal ganglion cell loss in experimental optic neuritis and induced neurological signs following intracerebral injection into control mice. The pathogenicity of antibodies to NF-L was also observed in spinal cord co-cultures where axonal loss was induced. Taken together, our results reveal that as well as acting as reliable biomarkers of neuronal damage, antibodies to NF-L exacerbate neurological disease, suggesting that antibodies to NF-L generated during disease may also be pathogenic and play a role in the progression of neurodegeneration.

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 co-expressed with SoxB2, and its downregulation phenocopied SoxB2 knockdown. We also show that SoxB2 and Hdac2 promote each other's 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.

Effect of sphingosine kinase modulators on interleukin-1ß release, sphingosine 1-phosphate receptor 1 expression and experimental autoimmune encephalomyelitis.

BACKGROUND AND PURPOSE: The sphingosine analogue, FTY720 (GilenyaR ), 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-1ß formation, sphingosine 1-phosphate levels and expression of S1P1 receptors. We also assessed the effect of compound 5 and ROMe in an experimental autoimmune encephalomyelitis (EAE) model in mice. EXPERIMENTAL APPROACH: We measured IL-1ß formation by macrophages, sphingosine 1-phosphate levels and expression levels of S1P1 receptors in vitro and clinical score in mice with EAE and the extent of 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-1ß release. These data suggest that 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 expression of cell surface S1P1 receptors in vitro. CONCLUSION AND IMPLICATIONS: This is the first demonstration that an activator of sphingosine kinase 1 (compound 5) and an inhibitor of sphingosine kinase 2 (ROMe, which also reduces cell surface S1P1 receptor expression) have an anti-inflammatory action in EAE.

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-1ß. 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.

Effect of ether glycerol lipids on interleukin-1ß 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-1ß release; either alone or in the presence of LPS. The stimulatory effect of sphingosine or 77-6 on LPS-stimulated IL-1ß 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-1ß 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.

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

The conclusion of a series of recent reports highlights the significant neurotoxic effects that rare earth elements, such as lanthanum (La), can have during neurodevelopment. These findings are, herein, combined and presented using a simplified schematic overview that provides a putative solution to the riddle of La-induced cognitive impairment. Our commentary also highlights potential targets for further investigations into the mechanisms which underpin La-induced neurotoxicity, with a focus on the crucial role of the hippocampus. Within this context, a central role for the cyclic-adenosine monophosphate signalling pathway is proposed.

The development of a ε-polycaprolactone scaffold for central nervous system repair.

Potential treatment strategies for the repair of spinal cord injury (SCI) currently favor 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 the central nervous system neural cell interaction and survival on such scaffolds before implantation. Previously, we demonstrated that aligned microgrooves 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 three-dimensional 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.

The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination.

It is currently thought that treatment for spinal cord injury (SCI) will involve a combined pharmacological and biological approach; however, testing their efficacy in animal models of SCI is time-consuming and requires large animal cohorts. For this reason we have modified our myelinating cultures as an in vitro model of SCI and studied its potential as a prescreen for combined therapeutics. This culture comprises dissociated rat embryonic spinal cord cells plated onto a monolayer of astrocytes, which form myelinated axons interspaced with nodes of Ranvier. After cutting the culture, an initial cell-free area appears persistently devoid of neurites, accompanied over time by many features of SCI, including demyelination and reduced neurite density adjacent to the lesion, and infiltration of microglia and reactive astrocytes into the lesioned area. We tested a range of concentrations of the Rho inhibitor C3 transferase (C3) and ROCK inhibitor Y27632 that have been shown to promote SCI repair in vivo. C3 promoted neurite extension into the lesion and enhanced neurite density in surrounding areas but failed to induce remyelination. In contrast, while Y27632 did not induce significant neurite outgrowth, myelination adjacent to the lesion was dramatically enhanced. The effects of the inhibitors were concentration-dependent. Combined treatment with C3 and Y27632 had additive affects with an enhancement of neurite outgrowth and increased myelination adjacent to the lesion, demonstrating neither conflicting nor synergistic effects when coadministered. Overall, these results demonstrate that this culture serves as a useful tool to study combined strategies that promote CNS repair.

Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells.

Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated.

Design, synthesis, and biological evaluation of enantiomeric beta-N-acetylhexosaminidase inhibitors LABNAc and DABNAc as potential agents against Tay-Sachs and Sandhoff disease.

N-Acetylhexosaminidases are of considerable importance in mammals and are involved in various significant biological processes. In humans, deficiencies of these enzymes in the lysosome, resulting from inherited genetic defects, cause the glycolipid storage disorders Tay-Sachs and Sandhoff diseases. One promising therapy for these diseases involves the use of beta-N-acetylhexosaminidase inhibitors as chemical chaperones to enhance the enzyme activity above sub-critical levels. Herein we describe the synthesis and biological evaluation of a potent inhibitor, 2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol (LABNAc), in a high-yielding 11-step procedure from D-lyxonolactone. The N-benzyl and N-butyl analogues were also prepared and found to be potent inhibitors. The enantiomers DABNAc and NBn-DABNAc were synthesised from L-lyxonolactone, and were also evaluated. The L-iminosugar LABNAc and its derivatives were found to be potent noncompetitive inhibitors of some beta-N-acetylhexosaminidases, while the D-iminosugar DABNAc and its derivatives were found to be weaker competitive inhibitors. These results support previous work postulating that D-iminosugar mimics inhibit D-glycohydrolases competitively, and that their corresponding L-enantiomers show noncompetitive inhibition of these enzymes. Molecular modelling studies confirm that the spatial organisation in enantiomeric inhibitors leads to a different overlay with the monosaccharide substrate. Initial cell-based studies suggest that NBn-LABNAc can act as a chemical chaperone to enhance the deficient enzyme's activity to levels that may cause a positive pharmacological effect. LABNAc, NBn-LABNAc, and NBu-LABNAc are potent and selective inhibitors of beta-N-acetylhexosaminidase and may be useful as therapeutic agents for treating adult Tay-Sachs and Sandhoff diseases.

Glycosphingolipid disorders of the brain.

Glycosphingolipids, comprising a ceramide lipid backbone linked to one/more saccharides, are particularly abundant on the outer leaflet of the eukaryotic plasma membrane and play a role in a wide variety of essential cellular processes. Biosynthesis and subsequently degradation of these lipids is tightly regulated via the involvement of numerous enzymes, and failure of an enzyme to participate in the metabolism results in storage of the enzyme's substrate, giving rise to a lysosomal storage disease. The characteristics, severity and onset of the disease are dependent on the enzyme deficient and the residual activity. Most lysosomal storage disorders found thus far are caused by a defect in the catabolic activity of a hydrolase, causing progressive accumulation of its substrate, predominantly in the lysosome. Storage of gangliosides, sialic acid containing glycosphingolipids, mostly found in the central nervous system, is a hallmark of neuronopathic forms of the disease, that include GM1 and GM2 gangliosidoses, Gaucher type II and III and Niemann-Pick C. Models for these diseases have provided valuable insight into the disease pathology and potential treatment methods.Treatment of these rare but severe disorders proves challenging due to restricted access of therapeutics through the blood-brain barrier. However, recent advances in enzyme replacement, bone marrow transplantation, gene transfer, substrate reduction and chaperon-mediated therapy provide great potential in treating these devastating disorders.

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 microM and 0.5 microM, 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.