Betty Soliven

FTY720 modulates human oligodendrocyte progenitor process extension and survival

FTY720, a sphingosine‐1‐phosphate (S1P) receptor agonist that crosses the blood–brain barrier, is a potential immuno‐therapy for multiple sclerosis. Our objective was to assess the effect of FTY720 on process extension, differentiation, and survival of human oligodendrocyte progenitor cells (OPCs), and link the functional effects with S1P receptor expression and signaling.

Fingolimod (FTY720) Enhances Remyelination Following Demyelination of Organotypic Cerebellar Slices

Remyelination, which occurs subsequent to demyelination, contributes to functional recovery and is mediated by oligodendrocyte progenitor cells (OPCs) that have differentiated into myelinating cells. Therapeutics that impact remyelination in the CNS could be critical determinants of long-term functional outcome in multiple sclerosis (MS). Fingolimod is a S1P receptor modulator in MS clinical trials due to systemic anti-inflammatory properties, yet may impact cells within the CNS by crossing the blood-brain barrier. Previous studies using isolated dissociated cultures indicate that neural cells express S1P receptors and respond to receptor engagement. Our objective was to assess the effects of fingolimod on myelin-related processes within a multicellular environment that maintains physiological cell-cell interactions, using organotypic cerebellar slice cultures. Fingolimod treatment had no impact on myelin under basal conditions. Fingolimod treatment subsequent to lysolecithin-induced demyelination enhanced remyelination and process extension by OPCs and mature oligodendrocytes, while increasing microglia numbers and immunoreactivity for the astrocytic marker glial fibrillary acidic protein. The number of phagocytosing microglia was not increased by fingolimod. Using S1P receptor specific agonists and antagonists, we determined that fingolimod-induced effects on remyelination and astrogliosis were mediated primarily through S1P3 and S1P5, whereas enhanced microgliosis was mediated through S1P1 and S1P5. Taken together, these data demonstrate that fingolimod modulates multiple neuroglial cell responses, resulting in enhanced remyelination in organotypic slice cultures that maintain the complex cellular interactions of the mammalian brain.

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers.

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol–anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo–glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo–glial interactions.

Functional consequences of S1P receptor modulation in rat oligodendroglial lineage cells

Fingolimod (FTY720) and its phosphorylated form FTY720P are modulators of sphingosine‐1‐phosphate (S1P) receptors, which are G‐protein coupled receptors linked to cell migration and vascular maturation. The efficacy of FTY720 in autoimmune diseases such as multiple sclerosis and its animal models has been attributed to its inhibition of lymphocyte trafficking to target organs. In this study, we examined the role of S1P receptors in cultured rat oligodendrocytes (OLGs) and OLG progenitor cells (OPCs) using the active phosphorylated form of FTY720. We found that (1) FTY720P improves the survival of neonatal rat OLGs during serum withdrawal, which is associated with the phosphorylation of extracellular signal regulated kinases (ERK1/2) and Akt; (2) FTY720P regulates OPC differentiation into OLGs in a concentration‐dependent manner; and (3) S1P receptors are differentially modulated by platelet‐derived growth factor (PDGF) resulting in downregulation of S1P5 and upregulation of S1P1 in OPCs. In addition, siRNA studies revealed that S1P1 participates in PDGF‐induced OPC mitogenesis. We conclude that S1P1 and S1P5 serve different functions during oligodendroglial development, and possibly during remyelination.

Proprioceptive Sensory Neuropathy in Mice with a Mutation in the Cytoplasmic Dynein Heavy Chain 1 Gene

Mice heterozygous for the radiation-induced Sprawling (Swl) mutation display an early-onset sensory neuropathy with muscle spindle deficiency. The lack of an H reflex despite normal motor nerve function in the hindlimbs of these mutants strongly suggests defective proprioception. Immunohistochemical analyses reveal that proprioceptive sensory neurons are severely compromised in the lumbar dorsal root ganglia of newborn Swl/+ mice, whereas motor neuron numbers remain unaltered even in aged animals. We have used positional cloning to identify a nine base-pair deletion in the cytoplasmic dynein heavy chain 1 gene (Dync1h1) in this mutant. Furthermore, we demonstrate that Loa/+ mice, which have previously been shown to carry a missense point mutation in Dync1h1 that results in late-onset motor neuron loss, also present with a severe, early-onset proprioceptive sensory neuropathy. Interestingly, in contrast to the Loa mutation, the Swl mutation does not delay disease progression in a motor neuron disease mouse model overexpressing a human mutant superoxide dismutase (SOD1G93A) transgene. Together, we provide in vivo evidence that distinct mutations in cytoplasmic dynein can either result in a pure sensory neuropathy or in a sensory neuropathy with motor neuron involvement.

Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells.

Glial cell line‐derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol‐linked receptor, GDNF family receptor (GFR) α1. In Ret‐negative cells, the alternative transmembrane coreceptor is the 140‐kDa isoform of neural cell adhesion molecule (NCAM) associated with a non‐receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRα1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF‐signaling in SCs. In addition to activation of extracellular signal‐regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP‐dependent protein kinase A and protein kinase C are involved in GDNF‐mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron–SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.

Cyclical and Dose-Dependent Responses of Adult Human Mature Oligodendrocytes to Fingolimod

Fingolimod is a sphingosine-1-phosphate (S1P) analogue that has been used in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis. Fingolimod readily accesses the central nervous system, raising the issue of its direct effects on neural cells. We assessed the effects of active fingolimod on dissociated cultures of mature, myelin-producing oligodendrocytes (OLGs) derived from adult human brain. Human OLGs express S1P receptor transcripts in relative abundance of S1P5>S1P3>S1P1, with undetectable levels of S1P4. Low doses of fingolimod (100 pmol/L to 1 nmol/L) induced initial membrane elaboration (2 days), subsequent retraction (4 days), and recurrence of extension with prolonged treatment (8 days). Higher doses (10 nmol/L to 1 mumol/L) caused the opposite modulation of membrane dynamics. Retraction was rescued by co-treatment with the S1P3/S1P5 pathway antagonist, suramin, and was associated with RhoA-mediated cytoskeletal signaling. Membrane elaboration was mimicked using the S1P1 agonist SEW2871. Fingolimod rescued human OLGs from serum and glucose deprivation-induced apoptosis, which was reversed with suramin co-treatment and mimicked using an S1P5 agonist. High doses of fingolimod induced an initial down-regulation of S1P5 mRNA levels relative to control (4 hours), subsequent up-regulation (2 days), and recurrent down-regulation (8 days). S1P1 mRNA levels were inversely regulated compared with S1P5. These results indicate that fingolimod modulates maturity- and species-specific OLG membrane dynamics and survival responses that are directly relevant for myelin integrity.

Mutations in MUSK causing congenital myasthenic syndrome impair MuSK–Dok-7 interaction

We describe a severe congenital myasthenic syndrome (CMS) caused by two missense mutations in the gene encoding the muscle specific receptor tyrosine kinase (MUSK). The identified MUSK mutations M605I and A727V are both located in the kinase domain of MuSK. Intracellular microelectrode recordings and microscopy studies of the neuromuscular junction conducted in an anconeus muscle biopsy revealed decreased miniature endplate potential amplitudes, reduced endplate size and simplification of secondary synaptic folds, which were consistent with postsynaptic deficit. The study also showed a striking reduction of the endplate potential quantal content, consistent with additional presynaptic failure. Expression studies in MuSK deficient myotubes revealed that A727V, which is located within the catalytic loop of the enzyme, caused severe impairment of agrin-dependent MuSK phosphorylation, aggregation of acetylcholine receptors (AChRs) and interaction of MuSK with Dok-7, an essential intracellular binding protein of MuSK. In contrast, M605I, resulted in only moderate impairment of agrin-dependent MuSK phosphorylation, aggregation of AChRs and interaction of MuSK with Dok-7. There was no impairment of interaction of mutants with either the low-density lipoprotein receptor-related protein, Lrp4 (a co-receptor of agrin) or with the mammalian homolog of the Drosophila tumorous imaginal discs (Tid1). Our findings demonstrate that missense mutations in MUSK can result in a severe form of CMS and indicate that the inability of MuSK mutants to interact with Dok-7, but not with Lrp4 or Tid1, is a major determinant of the pathogenesis of the CMS caused by MUSK mutations.

A genetic mouse model of adult-onset, pervasive central nervous system demyelination with robust remyelination

Adult-onset demyelinating disorders of the central nervous system represent the most common neurological abnormalities in young adults. Nevertheless, our understanding of disease pathogenesis and recovery in demyelinating disorders remains incomplete. To facilitate investigation into these processes, we have developed a new mouse model system that allows for the induction of dipththeria toxin A subunit expression in adult oligodendrocytes, resulting in widespread oligodendrocyte loss and demyelination of the central nervous system. These mice develop severe ataxia and tremor that correlates with impaired axonal conduction in the spinal cord. Strikingly, these animals fully recover from their motor and physiological defects and display extensive oligodendrocyte replenishment and widespread remyelination. This model system demonstrates the robust reparative potential of myelin in the central nervous system and provides a promising model for the quantitative assessment of therapeutic interventions that promote remyelination.

Ceramide Inhibits Inwardly Rectifying K+ Currents via a Ras- and Raf-1-Dependent Pathway in Cultured Oligodendrocytes

Ceramide is a lipid mediator implicated in apoptosis induced by proinflammatory cytokines in many cell types, including oligodendrocytes (OLGs). To determine whether ceramide modulates transmembrane signaling events in OLGs, we studied its effect on intracellular Ca2+ (Cai), resting membrane potential and inwardly rectifying K+currents (IKir) in cultured neonatal rat OLGs. We report here that (1) exposure to C2-ceramide (cer) rarely increases OLG Cai, whereas sphingosine elicits sustained increase in Cai; (2) cer causes OLG depolarization, an effect mimicked by sphingosine-1-phosphate but not by sphingosine; and (3) cer, but not its inactive analog dihydroceramide, inhibits OLG IKir. The cer effect is attenuated by Ras antibody Y13-259, by protein kinase C inhibitory peptide (19–36), and by suppression of c-Raf-1 expression with antisense raf-1 oligonucleotides. We conclude that cer-induced OLG depolarization is mediated via inhibition ofIKir by a Ras- and raf-1-dependent pathway, which results in the phosphorylation of the inward rectifier K+ channel protein.