Fig 2: Ionized calcium-binding adaptor protein-1 (Iba1)+ cells in brains of C57BL/6 (WT) and Mecp2T158A/y mice. (A) Representative images of immunohistochemistry Iba1 staining in the striatum of age-matched mice from the 8-week cohort, C57BL/6 (WT), and Mecp2T158A/y treated with isotype control (P) or anti-SEMA4D antibody (T). Lower panel represents high-magnification images of Iba1+ cells with various morphologies; arrowhead = ramified, white arrow = amoeboid, yellow arrow = amoeboid with stout. (B) Representative images of immunohistochemistry Iba1 staining in the outer cortex including pia mater. (C–H) Quantification of Iba1+ cell density in the striatum, outer cortex and pons. For immunohistochemistry, three consecutive mouse brain sagittal serial sections (10 µm), 25 µm apart, from each mouse, were evaluated to determine the number of Iba1+ cells. Three sections/mouse were averaged for each mouse, and group means are an average of 6 mice/group. Data are expressed as group mean ± standard error of the mean. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001.

Fig 3: SEMA4D is increasingly upregulated in neurons during human HD disease progression; changes in astrocyte GS expression and morphology are indicative of astrogliosis. Human brain autopsy sections from non-diseased “normal” human and HD pathological stage 0, 1, and 2 were processed for SEMA4D, Huc/HuD (neuronal cell body), GFAP (astrocytes), and GS (astrocyte cell body and processes). Brain regions included frontal cortex, parietal lobe, and striatum (caudate/putamen). a Representative images of frontal lobe illustrate upregulation of neuronally expressed SEMA4D (100-micron scale shown). Quantification of b SEMA4D expression in HucHUD+ cells and c HucHUD+ neuron density. d GFAP staining of frontal lobe are shown; soma is circled in binary image conversion. Fractal dimension analysis of GFAP+ astrocytes demonstrate significant changes between non-diseased and HD. Group differences and statistical significance was determined by an independent-samples t-test. e GS expression is reduced and retracted astrocytic endfeet are observed in HD, compared to normal. (20-micron scale shown). f Quantification of GS in GFAP+ cells. Each bar represents autopsy tissue from one individual; mean of the sum of integrated fluorescence intensity of 3 consecutive sections/individual + SEM is shown for each subject/condition. Group differences and statistical significance was determined using one-way ANOVA with Tukey post hoc analysis

Fig 4: Effects of anti-semaphorin 4D (SEMA4D) antibody on the cytoskeleton. (A) Immunofluorescence shows α-tubulin (red) and nuclei (blue) in Mecp2T158A/y mixed glial culture exposed to recombinant semaphorin 4D (rSEMA4D) or phosphate-buffered saline (PBS) and treated with anti-SEMA4D or isotype control (placebo) antibody for 1 h. (B) Mean intensity of α-tubulin staining in GFAP+ (not shown) astrocytes was quantified and expressed as mean ± standard error of the mean. *** p < 0.001.

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