Clinical and histological outcome of MOG_35–55 EAE in <i>ngr1^−/−.</i>

<p>Data represent mean ± SEM. *p&lt;0.05, **p&lt;0.01. Wild Type (WT) and Nogo Receptor 1 deficient (<i>ngr1^−/−)</i> values are shown for the peak (18 days post-immunization, dpi) and the chronic (45dpi) stage of MOG peptide (MOG_35–55 )-EAE.</p

Peripheral immune response of <i>ngr1-/-</i> mice to rMOG.

<p>(<b>A</b>) <i>In vitro</i> proliferative response of <i>ngr1-/-</i> and WT splenocytes stimulated with rMOG or anti-CD3/CD28 at 18 and 45 days post immunization (dpi). <i>ngr1-/-</i> splenocytes showed an equivalent proliferative response to that of WT splenocytes. (<b>B</b>) Quantification of pro-inflammatory (INF-γ, TNFα, Interleukin (IL)-2, IL-17A and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines in supernatants derived from rMOG and anti-CD3/CD28 stimulated splenocytes cultures. <i>ngr1</i> deficiency had no impact on splenocyte cytokine production at either time points analyzed. Data represent mean ± SEM (n = 3-5). (<b>C</b>) Serum rMOG-specific IgG, IgG1, IgG2b and IgM antibody response as determined by ELISA. Data represent mean ± SEM (n = 2-6).</p

Immune-phenotype and bone marrow (BM) progenitor status of naïve <i>ngr1-/-</i> and WT mice.

<p>(<b>A</b>) Comparative flow cytometric analysis of single cell suspension from spleen, BM, thymus, blood, lymph nodes and central nervous system (CNS) associated mononuclear cells. Proportion and total number of CD3⁺CD8⁺ and CD3⁺CD4⁺ T cells, B220⁺ B cells, Gr-1⁺ granulocytes and F4/80 (Gr-1^loF4/80⁺) monocyte/macrophage are shown. Data represent mean ± SEM (n = 8-11). *p&lt;0.05 Mann-Whitney test. (<b>B</b>) Quantification of BM-derived colony number in naïve <i>ngr1-/-</i> and WT mice<b>.</b> After 8 days on methylcellulose culture, BM-derived progenitors from <i>ngr1-/-</i> animals were capable of producing granulocytes (G); macrophages (M) and mixed (GM) colonies at comparable numbers to WT mice. Bars represent mean colony number/plate ± SEM (n = 3).</p

Immune-phenotype of <i>ngr1-/-</i> mice during EAE induced with rMOG.

<p><i>ngr1-/-</i> and WT animals were immunized with rMOG and the mononuclear cells isolated at 18 (peak) and 45 (chronic) days post-immunization (dpi) were analyzed by flow cytometry. Proportion and total number of lymphocytes, granulocytes and monocyte/macrophages of bone marrow (BM; <b>A</b>); spleen, (<b>B</b>); lymph nodes, (<b>C</b>); thymus (<b>D</b>) and central nervous system (CNS; <b>E</b>) are shown. No significant differences were found between <i>ngr1-/-</i> and WT for all organs and time points examined. Data represent mean ± SEM (n = 6-11). (<b>F</b>) Further analysis of microglia (CD45^loCD11b⁺) and macrophages (CD45^hiCD11b⁺) in the CNS of <i>ngr1-/-</i> and WT mice was performed. <i>ngr1-/-</i> animals presented an increased proportion of microglial cells at 18 dpi and a decreased number of macrophages at 45 dpi (n = 3-4; *p&lt; 0.05 Mann-Whitney test).</p

Clinical and histological outcome of rMOG EAE in <i>ngr1^−/−.</i>

<p>Data represent mean ± SEM. *p≤0.05. Wild Type (WT) and Nogo Receptor 1 deficient (<i>ngr1^−/−</i>) values are shown for the peak (18 days post-immunization, dpi) and the chronic (45dpi) stage of recombinant MOG-EAE.</p

Reduced severity of MOG₃₅_–55 peptide-EAE in <i>ngr1-/-</i> mice.

<p>(<b>A</b>) EAE was induced by immunization with MOG_35–55 peptide and animals were scored daily for disease clinical manifestations. <i>ngr1-/-</i> mice presented a less severe clinical disease than WT mice. Data were pooled from 2 independent experiments (n = 11-13; mean ± SEM). ***p&lt;0.05, two-way ANOVA. (<b>B</b>) Representative lumbar-thoracic spinal cord sections stained with hematoxylin-eosin for inflammation, luxol fast blue for demyelination and Bielschowsky silver impregnation for axonal damage. Histological examination was performed at 18 and 45 days post-immunization (dpi). Compared to WT controls, a trend towards reduced inflammation, demyelination and axonal damage could be observed in <i>ngr1-/-</i> spinal cords (magnification 20X, scale bar = 200 µm). (<b>C-D</b>) Flow cytometric analysis of spleen <b>(C)</b> and central nervous system (CNS) (<b>D</b>) mononuclear cells at 18 and 45 dpi. The proportion and number of immune cell populations analyzed did not differ significantly between <i>ngr-/-</i> and WT mice. Data represent mean ± SEM (n = 3-5).</p

α-MSH (100 nM) stimulated 2-Deoxy Glucose uptake and TBC1D1 S237, T596 and S700 phosphorylation +/- H89 in dissected soleus explants from WT mice.

<p>A: Soleus muscle was dissected, stimulated and 2-DG was measured as described (n indicated in the individual bars). B: Phosphorylation of TBC1D1 was measured in soleus muscle using WB as described. TBC1D1 S237, T596 and S700 phosphorylation is normalized to total TBC1D1. Findings are shown as a representative immunoblot and pooled data quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001). Data generated in the experiment are only obtained from experiment day 4.</p

α-MSH stimulated 2-Deoxy Glucose uptake in soleus and extensor digitorum longus (EDL) muscle explants.

<p>3.A: soleus explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). Data is presented as the mean ± SEM of pooled data from a series of experiments (see individual bars). 3.B: EDL explants from WT and AMPK KD mice were stimulated with α-MSH (100 nM). 3.C: Phosphorylation of Akt was measured in soleus explants after α-MSH-stimulation (100 nM). Data is presented as the mean ± SEM. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH (100 nM) stimulated TBC1D1 S237 and T596 phosphorylation in WT and AMPK KD mice.

<p>TBC1D1 S237 and T596 phosphorylation sites were measured in soleus muscle using WB as described (n indicated in the individual bars). Phosphorylation of TBC1D1 S237 and T596 is normalized to total TBC1D1. Findings are shown as representative immunoblots and pooled data is quantified in bar graphs as arbitrary units. 2-way RM ANOVA was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle). ^# indicates a significant effect of genotype (^#p < 0.05, ^##p < 0.01, ^###p < 0.001).</p

α-MSH induced phosphorylation of AMPK in differentiated L6 myotubes and in soleus explants dissected from lean and diet-induced obese (DIO) mice.

<p>A: Phosphorylation of AMPK in lean and DIO mice in basal conditions in soleus muscle explants (n=4). 2.B: α-MSH-phosphorylation of AMPK in lean and DIO soleus muscle explants (n=6). 2.C: Phosphorylation of AMPK after α-MSH-stimulation (100 nM) in differentiated L6 myotubes (n=4). AMPK phosphorylation is normalized to total AMPK. Unpaired one-tailed t-test was used to calculate statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle)</p