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. 2022 Aug 6;19(1):200.
doi: 10.1186/s12974-022-02509-8.

Semaphorin 4D is upregulated in neurons of diseased brains and triggers astrocyte reactivity

Elizabeth E Evans  1 , Vikas Mishra  1 , Crystal Mallow  1 , Elaine M Gersz  1 , Leslie Balch  1 , Alan Howell  1 , Christine Reilly  1 , Ernest S Smith  1 , Terrence L Fisher  1 , Maurice Zauderer  2   3
Affiliations

Semaphorin 4D is upregulated in neurons of diseased brains and triggers astrocyte reactivity

Elizabeth E Evans et al. J Neuroinflammation. .

Abstract

Background: The close interaction and interdependence of astrocytes and neurons allows for the possibility that astrocyte dysfunction contributes to and amplifies neurodegenerative pathology. Molecular pathways that trigger reactive astrocytes may represent important targets to preserve normal homeostatic maintenance and modify disease progression.

Methods: Semaphorin 4D (SEMA4D) expression in the context of disease-associated neuropathology was assessed in postmortem brain sections of patients with Huntington's (HD) and Alzheimer's disease (AD), as well as in mouse models of HD (zQ175) and AD (CVN; APPSwDI/NOS2-/-) by immunohistochemistry. Effects of SEMA4D antibody blockade were assessed in purified astrocyte cultures and in the CVN mouse AD model. CVN mice were treated weekly from 26 to 38 weeks of age; thereafter mice underwent cognitive assessment and brains were collected for histopathology.

Results: We report here that SEMA4D is upregulated in neurons during progression of neurodegenerative diseases and is a trigger of reactive astrocytes. Evidence of reactive astrocytes in close proximity to neurons expressing SEMA4D is detected in brain sections of patients and mouse models of HD and AD. We further report that SEMA4D-blockade prevents characteristic loss of GABAergic synapses and restores spatial memory and learning in CVN mice, a disease model that appears to reproduce many features of AD-like pathology including neuroinflammation. In vitro mechanistic studies demonstrate that astrocytes express cognate receptors for SEMA4D and that ligand binding triggers morphological variations, and changes in expression of key membrane receptors and enzymes characteristic of reactive astrocytes. These changes include reductions in EAAT-2 glutamate transporter and glutamine synthetase, key enzymes in neurotransmitter recycling, as well as reduced GLUT-1 glucose and MCT-4 lactate transporters, that allow astrocytes to couple energy metabolism with synaptic activity. Antibody blockade of SEMA4D prevented these changes and reversed functional deficits in glucose uptake.

Conclusions: Collectively, these results suggest that SEMA4D blockade may ameliorate disease pathology by preserving normal astrocyte function and reducing the negative consequences of reactive astrogliosis.

Keywords: Alzheimer’s disease; Disease models; Huntington’s disease; Metabolic reprogramming; Neurodegeneration; Neurotransmitter recycling; Pathogenesis; Reactive astrocytes; Semaphorin.

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Conflict of interest statement

M. Zauderer, E.E. Evans, T.L. Fisher, V. Mishra, L. Balch, C. Mallow, A. Howell, E. Gersz, C. Reilly and E.S. Smith are employees of Vaccinex, Inc. and own stock and/or stock options in the company.

Figures

Fig. 1
Fig. 1
SEMA4D is progressively upregulated in HD neurons in parallel with evidence of astrocyte activation in Q175 KI HD mice. a Q175 knock-in mouse model of HD exhibits age-dependent upregulation and colocalization of SEMA4D in cortical neurons. b Quantification of SEMA4D expression and number of NeuN+ neurons in Q175 model with three mice/age group. Quantification analysis was performed on the entire coronal section of each mouse; mean + SEM of the sum of integrated fluorescence intensity/mouse/age group is shown. c Representative images of GFAP staining of caudoputamen at × 20 magnification. d Fractal dimension analysis of GFAP+ astrocytes in mouse frontal cortex were obtained and quantified from coronal sections (~ 5 µM); significant changes in brains of Q175 mice at 9.3 months of age observed, where data are presented as mean of all astrocytes + SEM. Group differences and statistical significance were determined using two-way ANOVA, where significance is reported at 95% confidence intervals and p-values are Bonferroni-adjusted and are indicated by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig. 2
Fig. 2
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. 3
Fig. 3
SEMA4D is upregulated in neurons of human AD; changes in astrocyte GS expression and morphology are indicative of astrogliosis. Human brain autopsy sections from non-diseased human and AD, with representations from thalamus, temporal lobe, and frontal cortex were processed for SEMA4D, Huc/HuD (neuronal cell body), GFAP (astrocytes), and GS (astrocyte cell body and processes). Representative images of human temporal lobe sections stained for a SEMA4D and Huc/HuD and b GS, and c GFAP staining; soma is circled in binary image conversion. Fractal dimension analysis of GFAP+ astrocytes demonstrate significant changes between non-diseased (“normal”) and AD individuals. (Note different scales shown in a–c) d Quantification of SEMA4D in HuC/HuD+ cells, HuC/HUD+ cell density, and GS expression 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
Fig. 4
SEMA4D antibody treatment inhibits reactive astrocytes and restores neuronal and cognitive deficits in CVN mouse model of AD. CVN and WT mice were treated in vivo with anti-SEMA4D “aS4D” or mouse IgG1 isotype control antibody “CTRL”. a CA1 hippocampal region of CVN and wild type mice were stained GFAP and b fractal dimension analysis and c GS expression in GFAP+ cells demonstrates significant changes in brains of CVN mice compared to wild type, which is restored following treatment with anti-SEMA4D antibody. d The hippocampal region was stained with anti-somatostatin antibody or anti-Neuropeptide-Y (NPY) to identify specific subsets of inhibitory neurons. No effects on excitatory synapses were observed in diseased mice (as determined by Synaptophysin and VGLUT-1 staining). Percentages were quantified for all animals (n = 9–13/group) and normalized to total area scanned; group mean + SEM are shown. For b–d, group differences and statistical significance were determined using Kruskal–Wallis H test and subsequent pairwise comparisons were performed using Dunn's procedure with a Bonferroni correction for multiple comparisons. e Latency was assessed in Radial Arm Water Maze at week 36. Group means of all animals + SEM for each trial block is shown. Statistical significance was determined by 2way mixed ANOVA with repeated measures
Fig. 5
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
Fig. 6
Fig. 6
Schematic model for effects of SEMA4D on astrogliosis. In keeping with published data of others regarding changes in reactive astrocyte function, it is suggested that SEMA4D is upregulated in stressed neurons and binds to PLXN-B1/B2 receptors to trigger reactive astrocytes, characterized by morphologic reorganization of the cytoskeleton and retraction of dendritic processes, downregulation of glucose and lactate transporters (GLT1 and MCT), downregulation of glutamate receptor (EAAT2), and reduction in glutamine synthetase (GS), key functional transporters and enzymes in astrocytes. SEMA4D-induced reactive changes indicated by red arrows. Dysfunctional transport and conversion of metabolic and neurotransmitter substrates may reduce astrocytic neuroprotection mechanisms and impair recycling glutamate to glutamine in astrocytes and glutamine to glutamate and GABA in neurons. Image created with BioRender.com; adapted from Bélanger, Allaman, Magistretti. Cell Metab. 2011
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