Fig. 5

SEMA4D triggers receptor-mediated astrocyte reactivity, including changes in astrocyte morphology, expression of key transporters for glutamate recycling and energy metabolism and impairs astrocyte function of glucose uptake. a Primary human astrocyte cultures were stained for PLXN receptors (blue), compared to isotype control antibodies (red). Antibody blocking effect was determined by incubation with rSEMA4D or control protein, in presence/absence of anti-SEMA4D antibody/VX15 (human IgG4) or isotype-matched control antibodies for 48 h. Cultures were stained for b EAAT-2, and c GLUT-1 and MCT-4 transporters. d In a separate experiment, blocking of receptors was assessed using anti-PLXNB1 (mouse IgG1) and/or anti-PLXNB2 (mouse IgG2a) or isotype-matched control antibodies and the same conditions as above. Quantification is shown as; mean + SEM of replicates for each treatment. e Glucose uptake was measured in human astrocyte cultures treated as above. rSEMA4D was added at time 0 and antibodies were added at t = 0 (solid lines and circles) for inhibition or t = 24 h (dotted lines and triangles) to evaluate reversal of activity. Quantification for each condition is shown as average + SEM from 3 wells/condition/timepoint. f Morphologic changes showing length and number of primary processes. For b to f, multivariate regression analysis was performed to determine the effect treatment conditions and dependent variables with treatment type. The significance levels are reported with Tukey post hoc tests for multivariate analysis