Neuroligin-3 interaction with CSPG4 regulates normal and malignant glial precursors through PIEZO1

Abstract

Glioma pathophysiology is robustly regulated by interactions with neurons. Key to these interactions is the role of neuroligin-3 (NLGN3), a synaptic adhesion molecule shed in response to neuronal activity^1-5 that functions as a paracrine factor crucial for glioma growth. Here, we elucidate the mechanistic pathway whereby shed NLGN3 interacts with glioma and their normal glial counterpart. NLGN3 interacts with Chondroitin Sulfate Proteoglycan 4 (CSPG4) on both glioma and healthy oligodendrocyte precursor cells (OPCs)^6-9, facilitating CSPG4 shedding by ADAM10. NLGN3-CSPG4 interactions and consequent shedding alter membrane tension, thereby activating PIEZO1 mechanosensitive channels and causing membrane depolarization. The NLGN3-CSPG4-PIEZO1 axis maintains OPCs in an undifferentiated, stem-like state and promotes glioma proliferation, underscoring important functional roles for the NLGN3-CSPG4-PIEZO1 axis in both healthy and malignant glial precursors.

Extended Data Fig. 1|. Biochemical analysis of NLGN3 interaction with Chondroitin Sulfate

a. Schematics of Size-Exclusion Chromatography (SEC) based coupling assay. NLGN3 and its substrates are mixed, run through the column, and the left-shift of the peak will be assessed. b. Binding assay of the NLGN3 with Chondroitin Sulfate (CS) and Heparin. Both Heparin and CS are binding to NLGN3, causing the peak to shift to the left. c. Dose-dependent interaction of NLGN3 and CS. d. Quantification of NLGN3-CS interaction shown in c.

Extended Data Fig. 2|. sNLGN3 induces ADAM10-dependent CSPG4 cleavage via activation of PIEZO1 channels

a. Scatterplots depicting tumor architecture of scRNA-seq produced from molecularly distinct patient glioma samples. From left to right are Histone H3K27M mutated diffuse midline glioma (DMG) and IDH1 wild-type glioblastoma (GBM). CSPG4 expression displayed in red across two classes of glioma. PIEZO1 and CSPG4 are both detected within the OPC-like fraction of these distinct glioma types. b. Violin plots of scaled gene expression level of mechanosensitive ion channels PIEZO1, PIEZO2, TRPC1 and TRPC6 in multiple patient biopsies of H3K27M mutant diffuse midline gliomas profiled by smart-seq2. All six biopsies are from Filbin et al. 2018. Counts were downloaded from GEO (GSE102130) and plotted following log normalization and scaling functions in Seurat (https://satijalab.org/seurat/). c. Western blot of PIEZO1 protein expression from lysates of SU-DIPG6 PIEZO1 WT and KO cells. d. Workflow depicting treatment of pediatric cortical GBM cells with recombinant NLGN3 ectodomains in the presence or absence of inhibitors of mechanosensitive ion channels (MSC) followed by culture medium concentration and western blot to detect shedding of CSPG4. e. Western blot of shed CSPG4 ectodomains from pediatric cortical GBM experiments depicted in d. f. Quantification of western blots (n (independent biological replicates of western blot) = 3, mean ± s.e.m., ordinary one-way ANOVA, ** P < 0.01, **** P < 0.0001).

Extended Data Fig. 3|. Whole-cell patch clamp characterization of PIEZO1 currents in glioma cells.

a. Representative traces of mechanosensitive currents recorded with cell-attached recording from WT pcGBM2 glioma cells vs CSPG4 KO cells, with and without NLGN3 treatment. b. Summary of cell-attached experiment performed in a (n (number of independent cells recorded) = 5, mean ± s.e.m.,). c. Schematics of whole-cell patch clamp recording of mechanosensitive currents from glioma cells. d. Representative traces of mechanosensitive currents measured from WT and PIEZO1 KO glioma cells. e. Summary of the current amplitude data represented in b. (n (number of independent cells recorded) = 5, mean ± s.e.m.,) f. (Top) Representative currents from glioma cells with the vehicle and NLGN3 treated conditions. (Bottom) Summary of the off kinetics of the cells as shown at the top. (n (number of independent cells recorded) = 3 for the vehicle and 4 for NLGN3, mean ± s.e.m., two-tailed students t-test, ** P < 0.01)

Extended Data Fig. 4|. Treatment with Adam10 inhibitor decreases tumor burden in xenografted mouse model.

a. Paradigm for in vivo treatment with Adam10 inhibitor aderbasib in immunodeficient mice xenografted with a patient-derived glioblastoma cell line (“SF-232-HFC”) and the counting model for tumor proliferation and burden. b. Proliferation index in vehicle- and aderbasib-treated mice (n = 5 per group) in the xenografted area of the secondary motor cortex (“M2”), quantified by confocal microscopy of Ki67⁺/HNA⁺ cells. Data are presented as mean ± range. Two-tailed student’s t-test, ***P < 0.001 c. Proliferation index in vehicle- and aderbasib-treated mice (n = 5 per group) of migrated glioblastoma cells into the ipsilateral corpus callosum (“CC”), quantified by confocal microscopy of Ki67⁺/HNA⁺ cells. Data are presented as mean ± range. Two-tailed student’s t-test, ns = not significant d. Tumor burden in vehicle- and aderbasib-treated mice (n = 5 per group) in the xenografted area of the secondary motor cortex (“M2”), quantified by confocal microscopy as the number of HNA⁺ cells. Data are presented as mean ± range. Two-tailed student’s t-test, **P < 0.01 e. Tumor burden in vehicle- and aderbasib-treated mice (n = 5 per group) in the xenografted area of the ipsilateral corpus callosum (“CC”), quantified by confocal microscopy as the number of HNA⁺ cells. Data are presented as mean ± range. Two-tailed student’s t-test, ***P < 0.001 f. Representative images of xenografted glioblastoma cells in the secondary motor cortex (“M2”) in vehicle- and aderbasib-treated mice. Gray denotes myelin sheaths labeled by MBP; green denotes HNA⁺ glioma cells; red denotes EdU. Scale bar = 100 μm. g. Representative images of migrated glioblastoma cells into the ipsilateral corpus callosum (“CC”) in vehicle- and aderbasib-treated mice. Gray denotes myelin sheaths labeled by MBP; green denotes HNA⁺ glioma cells; red denotes EdU. Scale bar = 100 μm.

Extended Data Fig. 5|. Genetic depletion of CSPG4 disrupts NLGN3-induced growth of glioma cells in vitro

a. Workflow depicting NLGN3-induced EdU incorporation assay in the presence or absence of CSPG4 shedding blocker, ADAM10i b. Quantification of NLGN3-induced EdU incorporation from mouse NF1-associated optic glioma cells. NLGN3 treatment increases EdU incorporation relative to vehicle controls ((n (number of independent cells) = 5–11, mean ± s.e.m., p = 0.03, one-way ANOVA with Brown-Forsythe and Welch’s test). c. Schematics of cell proliferation assay using CellTiterGlo assay. d. Quantification of CellTiterGlo proliferation assay of WT and CSPG4 KO SU-DIPGXIII-Pons cells (n (number of independent biological trials) = 3, mean ± s.e.m.,, two-tailed student’s t-test, * P < 0.05, *** P < 0.001, **** P < 0.0001). Note that from day 3 that the proliferation rate (assessed by the raw luminescence) is significantly lower in the CSPG4 KO group.

Extended Data Fig. 6|. In vivo characterization of OPC proliferation state in NLGN3 KO mice.

a. Schematics for in vitro OPC electrophysiology. b. Representative traces of mechanosensitive currents measured from OPCs. c. Summary of mechanosensitive currents shown in b. (n (number of OPCs per group) = 5 for vehicle and 6 for NLGN3, mean ± s.e.m.) d. qPCR results for the rat OPCs post 48 hours after PDGFα withdrawal (n (number of independent trials) = 2) with and without NLGN3 treatment. e. Schematics of NLGN3 deletion from OPCs in vivo. Mice expressing cell type-specific (PDGFα), inducible Cre drivers were bred to Nlgn3^fl/fl mice. Tamoxifen was administered for 5 days beginning at P21. f. Representative confocal images of Pdgfra positive (white) and Olig2 positive cells in WT and NLGN3 KO animals. scale bar = 50 μm g. Quantification of total OPC counts in WT and NLGN3 groups, as shown in e. (n (number of independent biological replicate of animals) = 4, mean ± s.e.m., two-tailed student’s t-test, * P < 0.05).

Fig. 1 |. Unbiased screening identifies CSPG4 as a putative interacting partner of shed NLGN3

a. Schematic of glioma membrane isolation, solubilization, and affinity capture by immobilized NLGN3 ectodomains. NLGN3 affinity column is prepared by immobilizing biotinylated (c-terminal AviTag) NLGN3 ectodomains within a streptavidin resin b. Selected top hits from the NLGN3 affinity chromatography assay. Purified membrane proteins captured through NLGN3 affinity chromatography were analyzed by mass spectrometry. Note that both pediatric (DIPGXIII) and adult (MGG8) brain cancers show CSPG4 as a top hit. c. Cartoon representation of CSPG4 functional domains. The laminin-G (LNS) domains of CSPG4 are analogous to those of Neurexins (NRXNs), the canonical binding partners to NLGN3, followed by the signature chondroitin sugar-binding site in the middle of core repeat modules. d. Western blot of human CSF with anti-CSPG4 N-term and anti-NLGN3 antibodies. Control patient CSFs are all from patients with acute lymphocytic leukemia (ALL). Only CSF samples from diffuse midline glioma-bearing patients show both CSPG4 and NLGN3. e. Quantification of bands in d (n = the number of patients = 4/group, mean ± s.e.m).

Fig. 2 |. Shed NLGN3 treatment augments ADAM10-dependent CSPG4 cleavage.

a. Workflow depicting treatment of glioma cells or OPCs for 1 hour with recombinant NLGN3 ectodomains (to mimic naturally shed NLGN3) followed by conditioned medium (CM) concentration and western blot to detect shedding of CSPG4. b. Left: CSPG4 Western blot from diffuse intrinsic pontine glioma model SU-DIPG13 conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. CSPG4 exists as a ‘part-time’ proteoglycan and is detected as either a core protein (discrete band at ~268 kDa) or as a chondroitin sulfate proteoglycan (higher molecular weight smear). Treatment of conditioned medium with chondroitinase ABC removes chondroitin sulfate chains and collapses diffuse smears into discrete bands. A c-terminal specific CSPG4 antibody only detects protein in the cell lysate control lane, confirming that conditioned medium bands are shed ectodomains. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.003). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right: Quantification of CSPG4 shedding as fold change in protein band intensities in the left (top N-terminal blot) relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. c. Left: Western blot of CSPG4 shedding from IDH1 WT glioblastoma (GBM) model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0013). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = <0.0001). Right: Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the legend and written on the figures. d. Left: Western blot of CSPG4 shedding from NF1-mutant optic pathway glioma model conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0066). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0004). Right: Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the text and the figures. e. Left: Western blot of CSPG4 shedding from primary mouse oligodendrocyte precursor cell (OPC) conditioned medium after exposure to 100 nM sNLGN3 for 1-hour in the presence or absence of 5 uM ADAM10 inhibitor. NLGN3 treatment increases shedding relative to vehicle controls (p = 0.0011). NLGN3-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p = 0.0003) Right: Quantification of protein band intensities relative to vehicle-treated controls. n (independent biological replicates of western blot) = 3, mean ± s.e.m., ordinary one-way ANOVA, p-values are mentioned in the figures. f. Model depicting serial events occurring during NLGN3-induced CSPG4 ectodomain shedding by ADAM10 from glioma cells. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain.

Fig. 3 |. Membrane depolarization and actin depolymerization regulate ADAM10-dependent CSPG4 shedding

a. Workflow depicting treatment of IDH1 WT GBM (MGG8) cells for 1 hour with the potassium channel blocker 4-Aminopyridine (4-AP) followed by culture medium concentration and western blot to detect shedding of CSPG4. b. Left: Representative western blot of shed CSPG4 ectodomains from experiment depicted in a. 4-AP treatment increases shedding relative to vehicle controls. Note that 4-AP-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p < 0.0001) Right: Quantification of western blots. (n (independent biological replicates of western blot) = 4, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. c. Workflow depicting blue light stimulation of Channelrhodopsin-2 (ChR2)-expressing rat OPCs followed by culture medium concentration and western blot to detect shedding of CSPG4. d. Left: Representative current of ChR2-expressing OPCs. (Light power density = 1.0 mW/mm², wavelength = 470 nm, duration = 1 second). Right: Representative western blot of shed CSPG4 ectodomains from experiment depicted in c (pulse width = 25 ms, frequency = 10 Hz). Membrane depolarization by ChR2 stimulation increases shedding relative to no blue light stimulation controls. e. Workflow depicting treatment of IDH1 WT GBM cells (MGG8) for 1-hour with the actin polymerization inhibitor cytochalasin D followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Left: Western blot of shed CSPG4 ectodomains from experiment depicted in e. Cytochalasin D treatment increases shedding relative to vehicle controls (p = 0.0175). Cytochalasin D-induced shedding of CSPG4 is blocked by pre-treating cells with 5 uM ADAM10 inhibitor (p < 0.0001) Right: Quantification of western blots (n (independent biological replicates of western blot) = 7, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Cartoon diagram summarizing the treatments of 4-AP, ChR2, and cytochalasin D, that are inducing membrane depolarization. h. Model depicting CSPG4 shedding through NLGN3 interaction that implicates an ion channel that is sensitive to membrane stress to depolarize the membrane and activate ADAM10.

Fig. 4|. sNLGN3 induces ADAM10-dependent CSPG4 cleavage via activation of PIEZO1 channels

a. Representative images of glioma cells colored based on the lifetime of fluorescent tension sensor FlippeR-TR. The fluorescence probe works by specifically targeting the plasma membrane, and the longer lifetime corresponds to increased membrane tension. b. Quantification of the lifetimes measured from the experiment shown in a. (n (number of independent biological samples used for imaging) = 6 – 10, mean ± s.e.m., two-tailed t-test, **** P < 0.0001, n.s. = not significant) c. Scatterplots depicting tumor architecture of scRNA-seq produced from molecularly distinct patient glioma samples. From left to right are Histone H3K27M mutated diffuse midline glioma (DMG) and IDH1 wild-type glioblastoma (GBM). PIEZO1 expression displayed in red across two classes of glioma. Note that PIEZO1 is detected within the OPC-like fraction of these distinct glioma types. d. left: Confocal image of immunostaining in SU-DIPGXIII-FL xenografted mouse brain slice. (PIEZO1 = red, GFP (marker for glioma cells) = green, scale bar = 10 μm) right: 3-dimensional reconstructions of glioma cell surface showing PIEZO1 localization. Patient-derived DIPG cells (SU-DIPGXIII-FL; nestin, blue) co-localize with PIEZO1 puncta (red). Scale bar, 3 μm. e. Workflow depicting treatment of glioma cells with recombinant NLGN3 ectodomains in the presence or absence of modulators of mechanosensitive ion channels (MSC) followed by culture medium concentration and western blot to detect shedding of CSPG4. f. Western blot of shed CSPG4 ectodomains from experiment depicted in c. Treatment with Yoda1 (PIEZO1 agonist), increases shedding relative to vehicle controls (p = 0.0464). In contrast, treating cells with MSC blockers gadolinium (Gd³⁺) or the spider toxin GsMTx4 prevent NLGN3-induced shedding of CSPG4 (p = 0.0008 and p < 0.0001, respectively) Right: Quantification of western blots (n (independent biological replicates of western blot) = 11, mean ± s.e.m., ordinary one-way ANOVA, p-values are written on the figures. g. Model depicting CSPG4 shedding via ADAM10 activation that is downstream of NLGN3 interaction and ensuing membrane depolarization through PIEZO1 channel activation. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Fig. 5 |. Blockade of CSPG4 shedding by PIEZO1 prevents NLGN3-induced changes to glioma cells.

a. (Left) Schematics of cell-attached patch clamp recording of mechanosensitive currents from glioma cells. (Right) The representative waveforms of mechanosensitive currents measured with 10 mmHg and 30 mmHg pressure inputs. b. Representative traces of mechanosensitive currents (pressure input = 40 mmHg, 1 second) with vehicle (black), PIEZO1 agonist Yoda1 (cyan), NLGN3 (red) and PIEZO1 blocker GsMTx4 (brown). c. Summary of mechanosensitive currents shown in b. (n (number of independent biological samples) = 6 – 8, mean ± s.e.m., Kruskal-Wallis test, ** P < 0.01, *** P < 0.001, n.s. = not significant) d. Schematics of xenografting of SU-DIPGVI WT/PIEZO1 KO into immunocompromised mice followed by assessment of proliferation with Ki67+/HNA staining. e. Representative confocal images of WT and PIEZO1 KO brain slices explained in d. (white = Ki67, red = HNA, white arrows denote the overlapping cells with both Ki67 and HNA stained). f. Summary of tumor proliferation (g, %Ki67+) in WT and PIEZO1 KO samples. (n (number of independent biological samples) = 5, mean ± s.e.m., two-tailed t-test, * P < 0.05). g. Volcano plot of differentially expressed genes comparing WT and PIEZO1 KO MGG8 cells. Genes enriched in PIEZO1 KO relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in PIEZO1 KO cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p-value). h. Gene ontology analysis of genes that were significantly depleted in PIEZO1 KO cells relative to WT. Enriched programs all center around sterol biosynthesis and metabolism. Of note, FABP7 is amongst the most strongly down-regulated genes in PIEZO1 KO cells and is reported to function in the maintenance of both glioma stem cells and healthy OPCs. i. Cartoon model depicting mechanism of NLGN3-induced CSPG4 shedding and functional consequences. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.

Fig. 6 |. sNLGN3-induced CSPG4 shedding regulates OPC cell state

a. Workflow depicting EdU incorporation and detection in rat OPCs following treatment with NLGN3. b. Representative images of EdU incorporation in primary rat OPCs after 4-hour treatments. c. Quantification of cell proliferation depicted in a. (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. **** P < 0.0001) d. Schematic depicting differentiation and morphological changes of OPCs upon mitogen withdrawal in vitro. e. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3. f. Quantification of cells at each morphological stage following 48-hours of treatment. ( n (number of independent trials) = 4, two-way ANOVA with multiple comparisons and Bonferroni correction, not significant) Note that PDGFAA treated and NLGN3-treated cells are not significantly different regarding progression to stage 3 morphology as assessed by two-way ANOVA with multiple comparisons and Bonferroni correction. g. Representative images of OPCs following 48-hour mitogen withdrawal in the presence or absence of supplemental sNLGN3 and ADAM10 inhibition. h. Quantification of OPC (CSPG4 positive) to OL (MBP positive) ratio in cells depicted in g (n (independent biological replicates) = 3) mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparisons correction. p-values are written on the figures). i. PCA plot of bulk RNA-seq produced from rat OPCs that were cultured for 48 hours under PDGFAA withdrawal and supplementation with NLGN3 and/or inhibitors of CSPG4 shedding. Vehicle and NLGN3-treated cells are distinct in PC space from the inhibitor-treated samples. j. Volcano plot of differentially expressed genes comparing NLGN3-treated rat OPCs against PDGFAA withdrawal alone. Genes enriched in NLGN3-treated relative to WT cells are plotted with positive log2 fold change. Conversely, genes that are depleted in NLGN3-treated cells relative to WT are plotted as negative log2 fold change. Genes are colored according to log2 fold change and adjusted p-value thresholds (gray = non-significant by either metric, green = log2 fold change of 1 or greater, blue = adjusted p-value of 10e6 or greater, red = gene exceeds thresholds for fold change and p-value). k. Heatmap of NLGN3-induced genes from Venkatesh et al 2017. Primary rat OPCs were cultured for 48 hours without PDGFAA to initiate differentiation and NLGN3 was supplemented in half the cultures. Duplicate vehicle and NLGN3-treated samples were profiled by bulk RNA-seq. TPM values for each condition were averaged and then the log2 fold change was calculated between NLGN3-treated and vehicle control samples. These values were plotted such that zero appears as white on a symmetrical axis that ranges from −4 in blue and 4 in red. l. Model depicting CSPG4 shedding via PIEZO1 opening and ADAM10 activation. These events impede OPC differentiation in vitro upon growth factor withdrawal. Red circular sector = ADAM10; Green rectangle = shed NLGN3; blue = CSPG4 ectodomain; light blue = PIEZO1.