Sphingosine 1-phosphate (S1P), a lysophospholipid, has gained relevance to multiple sclerosis through the discovery of FTY720 (fingolimod), recently approved as an oral treatment for relapsing forms of multiple sclerosis. Its mechanism of action is thought to be immunological through an active phosphorylated metabolite, FTY720-P, that resembles S1P and alters lymphocyte trafficking through receptor subtype S1P 1 . However, previously reported expression and in vitro studies of S1P receptors suggested that direct CNS effects of FTY720 might theoretically occur through receptor modulation on neurons and glia. To identify CNS cells functionally contributing to FTY720 activity, genetic approaches were combined with cellular and molecular analyses. These studies relied on the functional assessment, based on clinical score, of conditional null mouse mutants lacking S1P 1 in CNS cell lineages and challenged by experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. All conditional null mutants displayed WT lymphocyte trafficking that responded normally to FTY720. In marked contrast, EAE was attenuated and FTY720 efficacy was lost in CNS mutants lacking S1P 1 on GFAP-expressing astrocytes but not on neurons. In situ hybridization studies confirmed that astrocyte loss of S1P 1 was the key alteration in functionally affected mutants. Reductions in EAE clinical scores were paralleled by reductions in demyelination, axonal loss, and astrogliosis. Receptor rescue and pharmacological experiments supported the loss of S1P 1 on astrocytes through functional antagonism by FTY720-P as a primary FTY720 mechanism. These data identify nonimmunological CNS mechanisms of FTY720 efficacy and implicate S1P signaling pathways within the CNS as targets for multiple sclerosis therapies.ultiple sclerosis is an autoimmune disorder characterized by CNS demyelination, inflammation, and neurodegeneration (1). Current Food and Drug Administration (FDA)-approved therapies predominantly target immunological pathways through injectable agents. FTY720 (known clinically as fingolimod) is an oral drug that has shown efficacy in human multiple sclerosis clinical trials (2, 3), and in September 2010, received FDA approval as an oral therapy for relapsing forms of multiple sclerosis. The actions of FTY720 seem to involve at least two, paradoxically, opposite sphingosine 1-phosphate (S1P) receptor mechanisms operating in the immune system: agonism and functional antagonism. FTY720 is phosphorylated in vivo to produce the active metabolite FTY720-P, which is a nonselective S1P receptor agonist for four of the five known S1P receptors (4-6), and can function as an agonist in vivo (7). However, FTY720-P can also act as a functional antagonist, where bound S1P receptors are irreversibly internalized and degraded rather than recycled back to the cell surface as occurs with S1P ligands (8, 9); additionally, FTY720-P signaling may persist even after receptor internalization (10). The biological locus responsible for FTY720 efficac...

Every successful pregnancy requires proper embryo implantation. Low implantation rate is a major problem during infertility treatments using assisted reproductive technologies (ART) 1 . Here we report a new molecular influence on implantation through the lysophosphatidic acid (LPA) receptor LPA 3 2-4 . Targeted deletion of LPA 3 in mice resulted in significantly reduced litter size, which could be attributed to delayed implantation and altered embryo spacing. These two events led to delayed embryonic development, hypertrophic placentas shared by multiple embryos, and embryonic death. An enzyme demonstrated to influence implantation, cyclooxygenase-2 (COX-2) 5 , was downregulated in LPA 3 -deficient uteri during preimplantation. Down regulation of COX-2 led to reduced levels of prostaglandins that are critical for implantation 1 . Exogenous administration of the prostaglandins PGE 2 and cPGI into LPA 3 -deficient females rescued delayed implantation but did not rescue defects in embryo spacing. These data identify LPA 3 receptor-mediated signalling as a new influence on implantation and further indicate linkage between LPA signalling and prostaglandin biosynthesis.Multiple factors can adversely affect successful pregnancy. Two of these factors are failed synchronization between embryonic and endometrial development during implantation and occurrence of multiple gestations (especially monochorionic gestation), which can result in fetal demise 1,6-9 . These factors are particularly important for the clinical success and efficacy of ART. One molecular factor that has been previously implicated in female reproduction is the small, bioactive phospholipid LPA 10 . LPA has a range of influences that are mediated by at least four G protein-coupled receptors, LPA 1-4 2 . Deletion of LPA 1 and LPA 2 in mice revealed roles for these receptors in neural development, craniofacial formation, neuropathic pain, and altered cellular signalling, but without obvious effects on female reproduction 11-Correspondence and requests for materials should be addressed to J. Chun (e-mail:jchun@scripps.edu).. 6 These authors contributed equally to the work. Functional deletion of LPA 3 was achieved by replacing a fragment covering the untranslated region and the start codon in exon 2 with a neomycin-resistance gene in reverse orientation in R1 embryonic stem cells (supplementary Fig. S1). The LPA 3 -deficient mice were born with normal Mendelian frequency without sexual bias (supplementary Table S1), and appeared grossly normal (data not shown). However, LPA 3 -deficient females produced litter sizes of less than 50% compared to that from wild-type (WT) and LPA 3 heterozygote (Het) controls (supplementary Table S2), and showed a statistically significant prolongation of pregnancy (20.9±0.5 days, vs. 19.4±0.7 days in WT/Het controls, P<0.05). These phenotypes were independent of stud genotype, indicating defects in female reproduction. Supplementary InformationTowards determining whether LPA 3 deletion might directly affect the female...

Somatic gene recombination of amyloid precursor protein (APP) in human neurons has been identified, encompassing thousands of genomic variants occurring mosaically in normal and sporadic Alzheimer’s disease (AD) brains. Multiple sequencing strategies and junction-specific genomic in situ hybridization revealed APP recombination, lacking introns and having precise exonic junctions, termed genomic cDNAs (gencDNAs), often with multiple recombined junctions contained within a single nucleus. Most variants showed structural changes, particularly deletion of central exons with partial exons fused together, forming intra-exonic junctions, containing single nucleotide variations. APP is a causal gene mutated in forms of AD, and our studies identified variants enriched in sporadic AD neurons, including 10 mutations identical to those in published familial AD, yet arising somatically. Additional studies linked APP neuronal RNA transcription to the appearance of gencDNAs that could be preferentially transcribed to generate myriad gene variants contributing to diversity and function in the normal and diseased brain.

Schwann cell (SC) migration is an important step preceding myelination and remyelination in the peripheral nervous system, and can be promoted by peptide factors like neuregulins. Here we present evidence that a lipid factor, lysophosphatidic acid (LPA), influences both SC migration and peripheral myelination through its cognate G protein-coupled receptor (GPCR) known as LPA1. Ultrastructural analyses of peripheral nerves in mouse null-mutants for LPA1 showed delayed SC-to-axon segregation, polyaxonal myelination by single SCs, and thinner myelin sheaths. In primary cultures, LPA promoted SC migration through LPA1, while analysis of conditioned media from purified dorsal root ganglia neurons using HPLC/MS supported the production of LPA by these neurons. The heterotrimeric G-alpha protein, Gαi, and the small GTPase, Rac1, were identified as important downstream signaling components of LPA1. These results identify receptor mediated LPA signaling between neurons and SCs that promote SC migration and contribute to the normal development of peripheral nerves through effects on SC-axon segregation and myelination.