SEMA4D upregulation signals neuronal stress and triggers reactive transformation of astrocytes

The close interaction and interdependence of glial cells and neurons allows for the possibility that glial 2 dysfunction contributes to and amplifies neurodegenerative pathology. Drivers of glial cell activation 3 may represent important targets to preserve normal homeostatic maintenance and modify disease 4 progression. We report here that semaphorin 4D (SEMA4D) is a trigger of astrocyte activation and is 5 upregulated in neurons under stress or damage, as characterized in brains from mouse models as well as 6 patients with Huntington’s and Alzheimer’s disease. These SEMA4D+ neurons are in close proximity to 7 reactive astrocytes that evidence distinctive morphologic changes and downregulation of glutamine 8 synthetase associated with reactive astrogliosis along with loss of some key normal astrocyte functions, 9 including glucose transport and glutamate recycling. It is further demonstrated that astrocytes express 10 cognate plexin receptors for SEMA4D and that binding to the ligand results in collapse of the actin 11 cytoskeleton and down regulation of glutamine synthetase, and glucose and glutamate transport. These 12 effects are prevented and reversed in cultures of iPSC-derived human astrocytes by antibody blockade of 13 SEMA4D. In vivo antibody neutralization of SEMA4D reduced reactive astrocyte phenotype and 14 prevented characteristic loss of GABAergic synapses in brains of CVN mice. These results suggest that 15 SEMA4D represents a novel driver of astrocyte transformation that is upregulated early during disease 16 progression and that antibody blockade of SEMA4D can preserve normal astrocyte and neuronal 17 function. 18 Mouse brain Whole mouse brains from heterozygote (KI) (CHDI-81003003) Huntington’s mice and wild-type (WT) littermate controls; collected at ~ 3, 6, 9 months of age were obtained from CRL Discovery Services. Three consecutive whole mouse brain coronal sections (5µm) from 3 subjects per age group (cases/controls) were used for Immunofluorescence staining of tissue sections were performed for mouse Invitrogen), NeuN Abcam), GFAP Abcam), Neuropeptide-Y (NPY; NBP-19808, Novus), sypatophysin (101004, SySy), VGLUT-1 (TA317309, Orogene), in accordance to manufacturer recommended concentrations combining host dependent secondary alexa fluor antibodies. Human brain sections: Human HD brain specimens were obtained from National Institute of Health, Neurobiobank (NBB) Brain and Tissue Repositories (protocol number -1105-HDPilotSep2018) through participating brain banks of The Mount Samples of human frontal cortex regions were obtained from inferior frontal gyrus including Brodmann area -BA 44-45 and parietal lobe regions from cortex, including Brodmann area- BA 1,2,3 and part of frontal cortex BA4. (F=female, M=male) study


Introduction
Glial cells are increasingly recognized for their important contribution to onset and progression of 21 neurodegenerative diseases 1 . Studies from multiomic, epidemiologic, clinical and animal models have 22 advanced the understanding of interactions between neurons and glial cells and the role of 23 neuroinflammatory mechanisms. Astrocytes in particular have been increasingly recognized as key 24 contributors to pathology and progression of AD, HD, PD, ALS and other slowly progressive 25 neurodegenerative diseases. Astrocytes play a critical role in the integration of neural synaptic networks 26 and are well-positioned to couple energy metabolism with synaptic activity. Fine cytoplasmic processes 27 of astrocytes cradle synapses and express glutamate receptors while astrocyte endfeet express glucose 28 transporter and completely cover brain capillaries. Astrocytes are extremely responsive to disruptions in 29 their microenvironment, as occurs in injury and neurodegenerative disorders, with morphological and 30 functional changes referred to as reactive astrogliosis and astrocytopathy 2 . Such astrocytic 31 transformation may be a key pathological driver of disease, as reactive astrogliosis has been shown to 32 precede neuronal loss and to be sufficient to trigger disease phenotype in animal models 3 . Astrogliosis 33 is characterized by gain of inflammatory processes 4 , as well as loss of normal homeostatic functions. 34 Dysregulation of normal functions include impairment in glucose uptake and neurotransmitter recycling 35 due to downregulation of key receptors and enzymes in the respective pathways, including Glut1, 36 MCT4, and glycolytic enzymes that regulate metabolic activity 5,6 and EAAT2 and glutamine 37 synthetase (GS) required for recycling glutamate and GABA transmitters 7 . Cytoskeletal rearrangements 38 are another hallmark of astrogliosis characterized by upregulation of glial fibrillary acidic protein 39 (GFAP) and hypertrophic cell bodies with retracted short processes and loss of fine processes 8 . These 40 morphologic changes may influence astroglial pathogenesis via disruption of normal functions that 41 depend on cytoplasmic projections, including neuro-glial and glial-endothelial networks. 42 43 Semaphorin 4D (SEMA4D) regulates the actin cytoskeleton through small membrane Rho GTPases [9][10][11] . 44 The actin cytoskeleton controls cell morphology and the ability to extend projections required for cell 45 migration and for direct interactions with other cells. In the CNS, SEMA4D signals through its high 46 affinity plexin receptors (PLXNB1, PLXNB2) (i) to activate glial cells 12,13 , (ii) to inhibit migration and 47 differentiation of glial progenitor cells that can replace damaged oligodendrocytes and replenish 48 astrocytes 9,10,14,15 , and (iii) to disrupt endothelial tight junctions that are required for the integrity of the 49 blood-brain barrier (BBB) 12 . 50 We report here that neurons upregulate SEMA4D during underlying Huntington's and Alzheimer's 51 disease progression and that this is associated with inflammatory transformation of astrocytes as 52 reflected in altered morphology and downregulation of GS expression. Glutamine synthetase is 53 predominantly expressed by normal astrocytes in the brain and is a key enzyme in recycling glutamate 54 and GABA neurotransmitters. Upregulation of SEMA4D in neurons was detected in brain sections of an 55 HD transgenic model (Q175) as well as in staged HD patient brain autopsy samples. Similarly, 56 upregulated SEMA4D was detected in AD brain autopsy sections and in CVN-AD mice. Astrocytes are 57 shown to express the high affinity plexin-B1 receptor for SEMA4D and interaction with the ligand 58 triggers reversible depolymerization of F-actin and collapse of the cytoskeleton. A number of 59 observations of our own and others further suggest that SEMA4D/plexin-B1 signaling affects the ability 60 of mature astrocytes to extend cytoplasmic projections required for their normal functions including 61 glucose transport through direct interaction with brain capillaries [16][17][18][19] and glutamate recycling through 62 projections that cradle synapses. Therefore, blockade of SEMA4D signaling may serve to normalize 63 astrocytic and neuronal functions and slow neurodegenerative disease progression. We previously 64 reported that an anti-SEMA4D antibody ameliorated neurodegenerative processes in YAC128 HD 65 transgenic mice 20 .

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Here, we further elucidate mechanism of action and demonstrate that antibody blockade prevented or 67 reversed SEMA4D-induced changes to astrocyte morphology and function in vitro and in vivo. In the 68 CVN mouse model of AD, treatment with anti-SEMA4D antibody restored normal astrocyte 69 morphology and reversed neuronal dysfunction, as evidenced by preventing characteristic loss of 70 GABAergic synapses. We will report elsewhere results of a phase 2 study that indicates clinical benefit 71 of SEMA4D blockade in Huntington's disease. Upregulation of SEMA4D in neurons during Huntington's and Alzheimer's disease progression 75 As in the case of other slowly progressive neuroinflammatory/neurodegenerative diseases, chronic 76 activation of inflammatory glial cells is believed to play a key role in the underlying progression of 77 Huntington's disease 3,[21][22][23][24] . We have previously reported that SEMA4D triggers depolymerization of the 78 actin cytoskeleton in glial progenitor cells and impedes their migration 12 . Similar effects on the 79 cytoskeleton of mature astrocytes are characteristic of astrogliosis and could profoundly impact their 80 normal function. Additionally, we previously reported that treatment with anti-SEMA4D antibody 81 ameliorates neurodegenerative processes in YAC128 HD transgenic mice 20 . We, therefore, investigated 82 expression of SEMA4D in neural tissue during HD progression and potential expression of cognate 83 receptors on astrocytes. SEMA4D is here shown to be upregulated in neurons of HD transgenic Q175 84 mice ( Fig. 1a, b), increasing with age as disease progresses. Upregulation of SEMA4D can be detected 85 even at 3 months of age (Fig. 1a, b), prior to disease symptom onset, which typically occurs ~ 5 months 86 of age in this mouse model. A significant reduction in the number of NeuN+ neurons at 9 months of age 87 provides evidence of neuronal loss with increasing SEMA4D expression and disease progression.

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To determine whether similar changes in expression of neuronal SEMA4D characterizes human disease 89 progression, we examined autopsy samples obtained from the NIH brain bank of 3 subjects each 90 representative of normal control, and HD pathological stage 0, stage 1, and stage 2. Staining for 91 SEMA4D and the neuronal marker HuC/HuD (Fig. 1c) shows statistically significant upregulation of 92 SEMA4D in neurons (Fig. 1d) and evidence of neuronal loss (Fig. 1e)  To determine if SEMA4D upregulation is a common underlying pathogenic mechanism in another 99 neurodegenerative disease, we evaluated autopsy sections from subjects with AD. Consistent with 100 observations in HD, we observed significant upregulation of SEMA4D and a reduction in the neuronal 101 density in numerous regions of interest in AD brains compared to normal controls. Significant changes 102 in the frontal cortex, temporal lobe and thalamus of AD affected subjects are shown in Figure 2.  (Fig 4). Furthermore, the close proximity of GS-positive endfeet of stained astrocytes (purple) to 113 HuC/HuD-positive neurons (green or yellow with SEMA4D co-stain) observed in normal healthy brain 114 tissue appears to be disrupted in the HD brain (Fig. 4a). Consistent with observations in HD subjects, 115 we observed significant reduction in GS expression and morphologic changes associated with reactive 116 astrogliosis in numerous regions of interest in AD brains as well (Fig 4c). Significant changes in the 117 frontal cortex, temporal lobe and thalamus of AD affected subjects compared to normal controls are 118 shown in Figure 4d. Collectively, these morphological changes and downregulation of GS are 119 consistent with a reactive astrocyte phenotype and coincident with disease progression and increasing 120 upregulation of SEMA4D in neurons.

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Astrocytes express plexin-B1 receptors for SEMA4D and ligand triggers morphological 122 transformation and reduced metabolic function. 123 To determine whether SEMA4D upregulation in neurons can trigger astrocyte transformation, we 124 investigated whether astrocytes express plexin-B1 receptors and whether binding of SEMA4D impacts 125 morphologic and functional changes associated with astrogliosis. As shown in Figure 5a, plexin-B1 126 receptors are highly expressed in purified astrocytes, and recombinant SEMA4D rapidly triggers 127 approximately 60% depolymerization of F-actin after only one hour of exposure in vitro. As would be 128 expected, addition of SEMA4D reduces the amount of astrocyte cytoplasmic projections that 129 spontaneously extend in culture which is, however, reversible and recovers following addition of 130 SEMA4D blocking antibody at 20 hrs (Fig. 5b). Similarly, SEMA4D induced morphologic changes in 131 primary human astrocyte cultures, as evidenced by shortened length and reduced number of primary 132 neurite branches (Fig. 5c). These effects were significantly inhibited by addition of blocking antibody to 133 SEMA4D. Extensive astrocytic branching facilitates interactions with neuronal junctions and brain 134 capillaries and is, therefore, critical to astrocyte function.

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Glutamate recycling and glucose transport are important normal functions of astrocytes and may afford 136 the opportunity to couple synaptic activity with brain energy metabolism. Both functions are 137 significantly disrupted upon reactive astrocyte transformation 6,31 . Excitatory amino acid transporter 138 (EAAT)-2 is one of the major astrocytic glutamate transporters, regulating glutamate uptake at the 139 synaptic cleft in order to maintain homeostasis and potentially prevent adventitious signaling and 140 excitotoxic activity. Binding of rSEMA4D to PLXNB1 receptor expressed on primary human astrocytes 141 significantly reduced expression of EAAT-2 glutamate transporter (Fig. 6a). As expected, anti-142 SEMA4D antibody (VX15) alone has no effect on EAAT-2 expression. In contrast, blocking antibody 143 (VX15) significantly inhibited effects of SEMA4D and restored EAAT-2 to near control levels. To 144 determine effects of SEMA4D on astrocyte energy metabolism, expression of glucose transporter 145 (GLUT-1), necessary for glucose uptake by astrocytes, and monocarboxylate transporter (MCT) 4, the 146 astrocytic transporter of lactate for diffusion to neurons, were evaluated. Significant reduction in 147 expression of GLUT-1 and MCT4 on astrocytes was observed following incubation with recombinant 148 SEMA4D (Fig. 6b). As expected, anti-SEMA4D antibody (VX15) alone has no effect on GLUT-1 and 149 MCT4 expression, while blocking antibody (VX15) significantly inhibited effects of SEMA4D binding 150 to PLXNB1 receptor on astrocytes.

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To further demonstrate effects of SEMA4D signaling on astrocytic functional activity, we assessed the 152 ability of astrocytes to transport glucose in presence of SEMA4D (Fig. 6c). SEMA4D reduced glucose 153 uptake in cultured astrocytes by 24 hours, while SEMA4D antibody (VX15) alone had no effect. 154 However, in the presence of rSEMA4D, VX15 blocked binding to inhibit the reduction of glucose 155 uptake by astrocytes. Addition of SEMA4D antibody 24 hours after rSEMA4D treatment reversed the 156 SEMA4D-induced conversion of astrocytes to hypometabolic state.

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Based on these results, we surmised that SEMA4D upregulation during disease progression could 159 activate astrocytes and degrade their ability to perform normal functions including glucose metabolism 160 and neurotransmitter recycling (Fig. 6d). To assess the therapeutic potential of blocking SEMA4D to 161 restore these functions, SEMA4D antibody treatment was evaluated in the CVN mouse model of AD, 162 which displays characteristics of AD progression, including reactive gliosis, amyloid deposition, 163 phosphorylated tau protein, spatial memory impairments, and significant neuronal death in hippocampal, 164 cortical, and thalamic regions [32][33][34] . Mice were treated weekly starting at 26 weeks of age, around the 165 time of symptom onset, and brains were collected at 41 weeks of age, when major pathologic changes 166 including glial activation and neuronal degeneration have occurred. SEMA4D upregulation (data not 167 shown) and astrocyte morphologic transformation was confirmed in the CVN mice (Fig 7). Treatment 168 with SEMA4D blocking antibody preserved normal morphology (Fig. 7 a, b) in hippocampal astrocytes 169 of CVN mice. Astrocytic glutamine synthetase expression was significantly reduced in the hippocampus 170 of CVN mice compared to wild type mice (p=0.0054), however no significant difference was observed 171 in CVN mice treated with SEMA4D blocking antibody compared to wild type controls (p=0.9276). It 172 has been reported that disruption of the GS-dependent conversion of glutamate to glutamine cycle in 173 reactive astrocytes has a differential effect on loss of inhibitory GABAergic relative to excitatory 174 glutamatergic signaling, It was suggested that this may be due to differences in transmitter pools at 175 inhibitory versus excitatory synapses or alternative sources of glutamine for glutamate synthesis 7 .

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Dysfunction of inhibitory synapses was observed in CVN mice, as evidenced by loss of somatostatin 177 and neuropeptide Y (NPY), markers of inhibitory synapses in the interneurons of the subiculum and 178 dentate gyrus. No effects on excitatory or total synapses were observed in diseased mice, as determined 179 by vesicular glutamate transporter 1 (VGLUT-1) and synaptophysin expression (Fig 7c). These results 180 demonstrate that SEMA4D blockade prevented the differential loss of inhibitory synapses seen in CVN 181 mice that could be an important factor in disrupting neural circuits.  What mechanism(s) give rise to disease associated pathogenic changes in astrocytes? We report here 204 that during underlying disease progression in both HD and AD transgenic mice and patients, SEMA4D 205 is upregulated in neurons (Fig. 1, 2). We further demonstrate that astrocytes express high affinity plexin-206 B1 receptors for SEMA4D (Fig. 5a). Binding of recombinant SEMA4D to purified GFAP+ astrocytes in 207 vitro triggers significant depolymerization of F-actin (Fig. 5a) and restricts the ability to extend 208 cytoplasmic projections which is, however, reversible and can be recovered following addition of 209 SEMA4D blocking antibody (Fig. 5b, 5c). These results suggest that in the presence of SEMA4D the 210 ability of astrocytes to perform normal functions that depend on cytoplasmic projections, for example, 211 interaction with brain capillaries to facilitate glucose transport and perisynaptic cytoplasmic projections 212 that express glutamate receptors responsible for recycling 80% of free glutamate, would be degraded. 213 Indeed, we observed rapid morphologic changes within one hour of in vitro treatment with SEMA4D, 214 while functional effects of SEMA4D on dendritic extension are observed at 10-24 hours. Concurrent 215 with these morphologic changes, we observed downregulation of astrocyte glucose transporter GLUT-1 216 and lactate transporter MCT4 at 24-48 hours. Importantly, these changes perturbed normal astrocytic 217 function, as evidenced by downregulation of glucose uptake, which was reversible with antibody 218 blockade of SEMA4D (Fig. 6). Glutamate transport was also regulated by SEMA4D, as evidenced by   Glutamine synthetase is a key enzyme in glutamate recycling and is predominantly expressed by 235 astrocytes in the brain. Downregulation of GS has been previously described as a marker of 236 inflammatory activation of astrocytes 7,28 . To determine whether reactive transformation of astrocytes is 237 a feature of disease progression, we examined glutamine synthetase (GS) expression in GFAP+ cells and 238 determined that there is striking reduction in GS expression with advancing disease (Fig. 4). We 239 confirmed that this was accompanied by morphological changes associated with reactive astrogliosis in 240 HD and AD (Fig. 3, 4). Inhibitory synapses are especially vulnerable to changes in glutamine levels 7

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and are also known to be critically affected in AD. NPY and somatostatin, produced mainly by 242 GABAergic interneurons, are associated with cognitive and emotional processes and appear to play an 243 important role in learning and memory 43,44 . Hippocampal NPY+ neurons are strongly affected in early 244 stages of AD pathology and significantly reduced in brains of AD patients 45 and CVN mice 46 . In the 245 CVN model, treatment with SEMA4D blocking antibodies prevented transformation to the astrocytic 246 reactive phenotype, restored expression of GS responsible for recycling glutamine stores to the neurons, 247 and thereby inhibited the loss of inhibitory NPY+ and somatostatin+ neurons (Fig. 7).

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Here, we provide evidence of SEMA4D regulation of astrocytic function, but we also consider the  Strategies that target these pathogenic processes have the potential to be broadly applicable to multiple 285 devastating diseases.    shown. Mean phalloidin-positive area/cell in a field of ~ 300 cells was quantified using ImagePro software in each of five separate culture wells/condition. b. SEMA4D inhibits process extension and migratory function of astrocytes; pepinemab reverses effects. Cell-free area in Radius 24-well Cell Migration Assay (Cell Biolabs) was determined following culture of purified astrocytes for the indicated time in the presence or absence of recombinant SEMA4D (15 mcg/ml), added at time 0. SEMA4D antibody "VX15" or isotype control antibody "CTRL Ig" (50 ug/ml) was added at time = 20 hours to determine whether the effect is reversible. Results in replicate wells (n=6) at each time point are normalized to cell-free area at time 0. Statistical significance was determined using two-way ANOVA. c. iPSC-derived human astrocytes were cultured in presence of rSEMA4D or control protein rEGFR. Antibody blocking effect was determined by incubation with rSEMA4D or control protein rEGFR (5 ug/ml), in presence/absence of anti-SEMA4D antibody/VX15 or isotype control human IgG4 antibody (25 ug/ml) for 48 hours. Morphologic changes in primary branch length and number of branches were quantified. Statistical significance was determined using Dunnet's multiple comparisons test, compared to control at each time point. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. b c d Figure 6: Astrocytic functions of glutamate and glucose transport are mediated by SEMA4D signaling and are reversible with antibody blockade of SEMA4D. iPSC-derived human astrocytes cultures were treated as in Figure 5 and stained for a. glutamate transporter (EAAT-2), and b. glucose (GLUT-1) and lactate (MCT-4) transporters. Antibody blockade of SEMA4D restored transporter expression. c. Glucose uptake was measured in human astrocyte cultures following incubation with rSEMA4D or control protein rEGFR (25 ug/ml), in presence/absence of anti-SEMA4D antibody/VX15 or isotype control human IgG4 antibody (125 ug/ml) in triplicate wells for 48 hours (circles). To evaluate reversal of activity, rSEMA4D was added at time 0 and antibodies were added at t=24 hr (squares). Quantification for each condition is shown as average+SEM from 3 wells/condition/timepoint. Statistical significance was determined using Dunnet's multiple comparisons test, compared to control at each time point. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. d. Proposed SEMA4D effects on astrogliosis and neuronal support. SEMA4D is upregulated in stressed neurons and binds to PLXNB1 receptor to trigger reactive astrogliosis, 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 receptors and enzymes in astrocytes; activation induced changes indicated by red arrows. Dysfunctional transport and conversion of metabolic and neurotransmitter substrates reduces astrocytic neuroprotection mechanisms and impairs recycling glutamate to glutamine in astrocytes and glutamine to glutamate and GABA in neurons. Astrocytic regulation of neuronal loss and reduced synaptic function contribute to pathogenic mechanisms in neurodegenerative disease. Image created with BioRender.com. : SEMA4D antibody treatment reverses astrocyte activation and synaptic function in AD mice. a. CA1 hippocampal region of CVN and wild type mice were stained GFAP and b. fractal dimension analysis demonstrates significant changes in brains of CVN mice compared to wild type, which is restored following treatment with anti-SEMA4D antibody. c. The hippocampal region was stained with anti-somatostatin antibody or anti-Neuropeptide-Y (NPY) to identify specific subsets of inhibitory neurons that begin to degenerate during early AD pathogenesis. 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; values for each mouse, along with group mean and standard error bars are shown. *p<0.05 and ***p<0.005 by 1-way ANOVA with Bonferroni's Multiple Comparison Test.