Cholinergic Neuronal Activity Promotes Diffuse Midline Glioma Growth through Muscarinic Signaling

Abstract

Neuronal activity promotes the proliferation of healthy oligodendrocyte precursor cells (OPC) and their malignant counterparts, gliomas. Many gliomas arise from and closely resemble oligodendroglial lineage precursors, including diffuse midline glioma (DMG), a cancer affecting midline structures such as the thalamus, brainstem and spinal cord. In DMG, glutamatergic and GABAergic neuronal activity promotes progression through both paracrine signaling and through bona-fide neuron-to-glioma synapses. However, the putative roles of other neuronal subpopulations - especially neuromodulatory neurons located in the brainstem that project to long-range target sites in midline anatomical locations where DMGs arise - remain largely unexplored. Here, we demonstrate that the activity of cholinergic midbrain neurons modulates both healthy OPC and malignant DMG proliferation in a circuit-specific manner at sites of long-range cholinergic projections. Optogenetic stimulation of the cholinergic pedunculopontine nucleus (PPN) promotes glioma growth in pons, while stimulation of the laterodorsal tegmentum nucleus (LDT) facilitates proliferation in thalamus, consistent with the predominant projection patterns of each cholinergic midbrain nucleus. Reciprocal signaling was evident, as increased activity of cholinergic neurons in the PPN and LDT was observed in pontine DMG-bearing mice. In co-culture, hiPSC-derived cholinergic neurons form neuron-to-glioma networks with DMG cells and robustly promote proliferation. Single-cell RNA sequencing analyses revealed prominent expression of the muscarinic receptor genes CHRM1 and CHRM3 in primary patient DMG samples, particularly enriched in the OPC-like tumor subpopulation. Acetylcholine, the neurotransmitter cholinergic neurons release, exerts a direct effect on DMG tumor cells, promoting increased proliferation and invasion through muscarinic receptors. Pharmacological blockade of M1 and M3 acetylcholine receptors abolished the activity-regulated increase in DMG proliferation in cholinergic neuron-glioma co-culture and in vivo. Taken together, these findings demonstrate that midbrain cholinergic neuron long-range projections to midline structures promote activity-dependent DMG growth through M1 and M3 cholinergic receptors, mirroring a parallel proliferative effect on healthy OPCs.

Figure 1.. Cholinergic neuronal activity-regulated modulation of oligodendrocyte precursor cells.

(A) Schematic of experimental paradigm for Optogenetic stimulation of cholinergic neurons in either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN). 5-week-old ChAT-IRES-Cre^+/wt × Ai230^flx/wt mice (P35–38) were stimulated for 30 minutes followed by perfusion after 3 hours. (B) Optogenetic stimulation of cholinergic neurons in LDT increases OPC proliferation (EdU⁺/Pdgfra⁺) in thalamus which is not seen after PPN stimulation (CTL (not stimulated), PPN stimulated, and LDT stimulated, n=5 mice/group). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (C) Optogenetic stimulation of cholinergic neurons in PPN increases OPC proliferation (EdU⁺/Pdgfra⁺) in pons (CTL, PPN, and LDT, n=5 mice). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (D) Confocal micrographs show thalamic OPC response after optogenetic stimulation of LDT (top image) and in pons after optogenetic stimulation of PPN (bottom image). ChRmine-oScarlet: green; Pdgfra: white; EdU: red, scale bars = 100um. (E) – (H) OPC response in (E) prefrontal cortex, (F) ventral tegmental area, (G) nucleus accumbens, and (H) hippocampus after optogenetic stimulation of cholinergic neurons (CTL, PPN, and LDT, n=5 mice/group). Unpaired two-tailed t-test; **p < 0.01, ns: non-significant. Data shown as mean, error bars indicate range.

Figure 2.. Interactions between cholinergic midbrain neurons and diffuse midline glioma.

(A) Schematic of experimental paradigm for optogenetic stimulation of cholinergic neurons in either laterodorsal tegmentum nucleus (LDT) or pedunculopontine nucleus (PPN) in mice bearing H3K27M DMG. Four-week-old ChAT-IRES-Cre^+/wt × Ai230^flx/wt mice (P28–30) were allografted with a H3K27M MADR tumour model into either the pons or thalamus, with optic ferrule placement into the LDT or PPN three weeks after allografting. Optogenetic stimulation for 30 minutes of the LDT or PPN was performed four weeks after allografting, followed by perfusion 24 hours after stimulation. (B) Proliferation index (EdU⁺/GFP⁺) of thalamus allografts in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=5 mice/group). Unpaired two-tailed t-test; ****p < 0.0001, ns: non-significant. Data shown as mean, error bars indicate range. (C) Proliferation index (EdU⁺/GFP⁺) of pons allografts in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=5 mice/group). Unpaired two-tailed t-test; ****p < 0.0001, ns: non-significant. Data shown as mean, error bars indicate range. (D) Confocal micrographs showing proliferating GFP⁺ tumour cells in the right thalamus in non-stimulated (“CTL”) (upper images) and LDT-stimulated (“LDT”) (bottom images) mice. GFP: green, EdU: red, scale bars = 100μm. (E) Proliferation index (EdU⁺/HNA⁺) of patient-derived pontine xenografts (SU-DIPG 17) in mice either stimulated in PPN or LDT or non-stimulated (“CTL”) (CTL, PPN, and LDT, n=4 mice/group). Unpaired two-tailed t-test; ***p < 0.001, ns: non-significant. Data shown as mean, error bars indicate range. (F) Confocal micrographs illustrate proliferating HNA⁺ glioma cells in non-stimulated (“CTL”) (upper images) and PPN-stimulated (“PPN”) (bottom images) mice after xenografting patient-derived diffuse midline glioma line (SU-DIPG17) into pons. HNA: green, EdU: red, scale bars = 100μm. (G) Schematic of experimental paradigm for investigating reciprocal signalling effects of tumour cells to cholinergic midbrain neurons. Four-week-old ChAT-IRES-Cre^+/wt × Ai230^flx/wt mice (P28–30) were either allografted with a H3K27 DMG or injected with buffered saline (HBSS) into pons and were perfused four weeks after surgery. Cholinergic neuronal activity of LDT and PPN were measured by cFos staining and normalized to four-week-old healthy ChAT-IRES-Cre^+/wt × Ai230^flx/wt mice. (H) An increased neuronal activity of cholinergic neurons (cFos⁺ in ChAT⁺ neurons) in LDT and PPN was observed in tumour-bearing mice when compared to healthy and saline-injected mice (healthy, n=5 mice; saline, PPN, and LDT, n=3 mice). Unpaired two-tailed t-test; *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Figure 3.. Direct effects of acetylcholine on diffuse midline glioma.

(A) Confocal micrographs showing cholinergic neurons generated from human induced pluripotent stem cells (hiPSCs) of a healthy 12-year-old male. MAP2: turquoise, ChAT: red, merged: white, scale bars = 100μm. (B) Representative confocal micrographs demonstrating dense neuron-to-glioma networks after co-culturing hiPSC-derived cholinergic neurons and DMG cells. MAP2: turquoise, Nestin: white, EdU: red, scale bars = 100μm. (C) Quantification of glioma cell proliferation (EdU⁺/Nestin⁺/MAP2⁻) when co-cultured with hiPSC-derived cholinergic neurons. Unpaired two-tailed t-test; ***p < 0.001. Data shown as mean, error bars indicate range. (D) Confocal images of cholinergic neuron–glioma co-culture with PSD95–RFP-expressing DMG cells (SU-DIPG13FL-PSD95). Yellow boxes indicate colocalizations of synapsin (presynaptic cholinergic neuron) and PSD95 (postsynaptic glioma cell expressing PSD95-RFP). Nestin: white, MAP2: turquoise, PSD95-RFP: red, synapsin: green, scale bars = 20μm (left) and 4μm (right). (E) 3-dimensional rendering of the image in (D), illustrating presynaptic cholinergic neuron (MAP2, turquoise, asterisk) with presynaptic puncta (green, synapsin) co-localizing with postsynaptic puncta (PSD95-RFP, red) expressed by postsynaptic glioma cell (nestin, white, arrow). (F) Proliferation index (EdU⁺/DAPI⁺) of a patient-derived DMG cell line (SU-DIPG17) after exposure to different concentrations of acetylcholine. (G) Representative confocal micrographs showing the proliferation of a patient-derived cell line without acetylcholine (upper images) and after exposure to 5μM acetylcholine (bottom images). DAPI: blue, EdU: red, scale bars = 100μm. (H) Proliferation index (EdU⁺/DAPI⁺) of a patient-derived cell line (SU-DIPG17) after exposure to various muscarinic and nicotinic receptor agonists (nicotine and muscarine) and antagonists (mecamylamine and scopolamine). One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; **p < 0.01, ns: non-significant. Data shown as mean, error bars indicate range. (I) 3D migration assay analysis comparing distance of glioma cell spread 72 h after seeding after exposure to acetylcholine. One-way analysis of variance (ANOVA) with Tukey’s post hoc analysis; *p < 0.05, **p < 0.01. Data shown as mean, error bars indicate range. (J) Representative images showing the glioma cell migration at timepoint zero and after 72h in control- and acetylcholine-treated wells. Scale bars = 1000μm.

Figure 4.. Cholinergic receptor gene expression and receptor mechanisms in gliomas.

(A) Heatmap of pseudo-bulk analysis of cholinergic receptors gene expression across central nervous system tumours from various studies with single-cell or single-nucleus RNA sequencing data. The red-marked boxes indicate studies with DMG samples. (B) Scatter plot correlating cholinergic receptor gene expression values with an OPC-like score in DMG samples. (C) Two-dimensional representation of the association between CHRM1 expression (red dots: centered value > 1) and the OPC-like (y axis) as well as OC-like and AC-like (x axis) scores for H3K27M DMGs. (D) Two-dimensional representation of the association between CHRM3 expression (red dots: centered value > 1) and the OPC-like (y axis) as well as OC-like and AC-like (x axis) scores for H3K27M DMGs. (E) Proliferation index (EdU⁺/Nestin⁺) of a patient-derived DMG cell line (SU-DIPG17) when co-cultured with hiPSC-derived cholinergic neurons and treated with M1 (VU0255035) and M3 (4-DAMP) receptor antagonists. Unpaired two-tailed t-test; **p < 0.01, ***p < 0.001. Data shown as mean, error bars indicate range. (F) Experimental paradigm (left) and proliferation index (EdU⁺/GFP⁺) of pontine allografts in mice optogenetically stimulated in PPN or non-stimulated controls (“CTL”), with or without administration of M1 (VU0255035) or M3 receptors (4-DAMP) pharmacological inhibitors prior to optogenetic stimulation of cholinergic neurons in mice bearing H3K27M DMG (MADR model) in the pons. The mice who did not receive M1 or M3 blockers were administered vehicle control. (n=4 mice/group in control and PPN-stimulated groups; n=3 mice/group in PPN-stimulated mice treated with M1-antagonist or M3-antagonist). The control group received vehicle control. Unpaired two-tailed t-test; ***p < 0.001, ****p < 0.0001. Data shown as mean, error bars indicate range.