S1P₃ and SphK1 are expressed by reactive astrocytes and macrophages in MS lesions.

<p>Immunofluorescence co-localization studies confirmed that S1P₃ is predominately expressed by reactive astrocytes (A). SphK1 is also expressed on reactive astrocytes (B), but the major cell type expressing SphK1 is macrophage in lesions and perivascular cuffs (C–D). Scale bars are 10 µm.</p

S1P₃ and SphK1 mRNAs and protein are upregulated in rat primary astrocytes by LPS stimulation.

<p>Primary astrocytes were incubated in culture medium for 5 or 24 h with LPS (100 ng/ml). The mRNA levels of S1P₁ (A), S1P₃ (B), SphK1 (C) and SphK2 (D) were assessed after 5 h and 24 h incubation. Quantitative PCR results are shown as the percentage expression of HKG (GAPDH) and represent mean ± SEM of three independent experiments. LPS mediated sustained upregulation of S1P₃ (E–F) and SphK1 (G–H) as shown by Western blots of plasma membrane fractions. Primary rat astrocytes were incubated with LPS (100 ng/ml) in serum-free medium containing 0.25% BSA for 12 and 48 h. Representative immunoblots are shown. Graphs represent the mean ± SEM of three independent experiments and are reported as protein expression normalized to actin, expressed as fold change over basal level. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01 <u>vs</u>. respective control.</p

S1P₃ contributes to the S1P-induced CXCL1 release by primary astrocytes.

<p>Astrocytes were pre-treated with or without LPS (100 ng/ml) for 12 h in serum-free medium and then stimulated or not with S1P (1 µM), a S1P₃ agonist (Compound 20, 10 µM) or a S1P₁ agonist (AUY954, 10 µM) for 5 h. The graph shows the effect of LPS pre-treatment on the release of CXCL1 by S1P, S1P₃ or S1P₁ agonists. Data are a pool of two independent experiments each performed in six replicates ± SEM. One-Way ANOVA followed by Bonferroni's multiple comparison test: **p<0.01, ***p<0.001.</p

SphK1 is activated in response to LPS.

<p>Serum-deprived astrocytes were incubated for 30 min with 100 ng/ml LPS in the presence or absence of SKI (10 µg/ml). 100 µg of cell extracts were used to determine SphK1 activity by thin layer chromatography. Data represents the mean ± SEM of three independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01.</p

Increased S1P₃-mediated ERK-1/2, but not Akt signaling by LPS.

<p>Astrocytes were serum-deprived during the 12 h treatment with LPS and were then stimulated for 20 min with 10 µM S1P₃ agonist (Compound 20) or 1 µM AUY954. The results show the relative ERK-1/2 phosphorylation (A, C ) and Akt phosphorylation (B, D) ± SEM normalized against actin for three (A, B) and two (C, D) independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001.</p

S1P₃ and SphK1 are upregulated in MS lesions.

<p>(A, B): Immunohistochemical peroxidise staining shows that S1P₃ receptor and SphK1 enzyme expressions are increased in a chronic-active MS lesion which was located in parietal subventricular white matter. S1P₃ expression is strong on reactive astrocytes in this lesion border (C), in the lesion (D) and on perivascular cells (G), but is weak in normal control brain white matter (H). SphK1 expression is increased in reactive astrocytes (E) and in macrophages in this MS lesion (F). A particularly high expression of SphK1 is seen in perivascular inflammatory cells (I), whereas its expression is very low in normal control brain white matter (J). This Figure shows representative stainings. Scale bars are 200 µm for A and B, and 20 µm for C–J. The sections were counterstained with haematoxylin.</p

LPS-induced astrocyte migration is SphK1-dependent, but proliferation-independent.

<p>Astrocyte were treated with increasing concentrations of S1P (10 nM, 100 nM, 1000 nM) (A) or LPS (1 ng/ml, 10 ng/ml, 100 ng/ml) (B), and cell proliferation was measured using a [³H]- thymidine uptake assay. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (A) *p<0.05 <u>vs</u>. control, ***p<0.001 <u>vs</u>. control. Astrocytes were pre-treated or not with SKI (10 µg/ml, 1 h) and then stimulated with LPS (100 ng/ml) (C) or stimulated with S1P (500 nM) and LPS (100 ng/ml), respectively, in the presence or absence of 10 µM antimitotic treatment (D). The graphs show the SKI-mediated inhibition of LPS-induced migration (C) and the influence of antimitiotic treatment on S1P- or LPS-induced migration after 48 h incubation (D). The surface of area covered by GFAP immunoreactivity is plotted. Each scratch was evaluated with an average of four photographs, and each treatment group represented five to six replicates. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (C) *p<0.05, **p<0.01; (D) *p<0.05 **p<0.01 <u>vs</u>. respective control.</p

Ten-point standardized rating scale for EAE clinical score monitoring.

<p>The final score for each animal is determined by the addition of all the above mentioned categories. Note: Score  =  15 for dead animal.</p

Disease course of mice undergoing EAE after immunization with rat MOG35-55.

<p>*Mice were immunized <i>s.c.</i> with rat MOG ₃₅₋₅₅. Pertussis toxin was administered <i>i.p.</i> at day 0 and day 2. 12 animals per group were included in this study. Neurological impairment was monitored using a ten-point standardized rating scale and expressed as Mean ± Standard Deviation. Spinal cord and cerebellum were taken at day14 (D14), day17 (D17) and day28 (D28) in MOG-induced (n = 4) and SHAM mice (n = 4). The body weight was monitored over the disease time course and the body weight loss ± SEM was reported. Disease onset occurred at day 10 and reached a maximum at day 22.</p

Identification of VHY potential substrates using phospho-peptides arrays.

<p>Each graphic correspond to the results expressed as optical density in arbitrary unites (a.u.) arising from two different arrays representing 720 different phospho-peptides. An arbitrary threshold allowed for the selection of the three phospho-peptides hits of each array namely (1) PDGFR-β (Platelet-derived Growth Factor Receptor beta); MK13 (MAPKinase 13/p38MAPKdelta); ATF2 (Activating Transcription Factor 2); SNX6 (Sorting Nexin 6); IF (Intrinsic factor); ErbB3 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 3). Arrays were ran at pH6 with an enzyme concentration of 2 ng/mL.</p