Abstract Neuronal activity robustly drives glioma progression, mediated via paracrine and synaptic mechanisms and offering novel therapeutic avenues for a disease with a devastating prognosis. While the activity-dependent regulation of diffuse midline glioma, H3K27-mutant (DMG-H3K27) and IDH-wildtype glioblastoma has been investigated thoroughly, the interaction of neurons with diffuse hemispheric glioma, H3G34-mutant (DHG-H3G34) remains to be elucidated. Recent transcriptomic analyses suggest a distinct cell-of-origin for DHG-H3G34, demonstrating these tumors primarily resemble early interneuron lineage cells. In the healthy context, neuronal progenitor cells are highly responsive to neuronal activity during development. We previously reported a proliferative response of patient-derived DHG-H3G34 cultures to active neurons in vitro, yet the key neuronal mechanisms governing the progression of DHG-H3G34 are unknown. Here, we explored the neuronal activity-dependent mechanisms that drive DHG-H3G34 growth. We found that activity-induced DHG-H3G34 proliferation in neuron-glioma co-culture is abrogated in the presence of a voltage-gated sodium channel blocker (tetrodotoxin) that prevents action potentials or an AMPA receptor inhibitor, confirming that activity-dependent mechanisms drive DHG-H3G34 malignant cell proliferation and raising the possibility of AMPAR-mediated synaptic mechanisms. Immuno-electron microscopy in two independent patient-derived models demonstrated multiple types of synaptic structures in DHG-H3G34 xenografts, including neuron-to-glioma synapses. Preliminary electrophysiological recordings of xenografted DHG-H3G34 cultures confirmed the presence of spontaneous and stimulation-evoked inward currents consistent with excitatory postsynaptic currents (EPSCs). Further optogenetic stimulation of both glutamatergic cortical projection neurons and GABAergic neurons promoted the proliferation of DHG-H3G34 xenografted cells within the stimulated circuits in vivo. Activity-regulated conditioned media harvested from optogenetically stimulated acute cortical slices induced DHG-H3G34 proliferation in monoculture, thus confirming paracrine signaling as one key mechanism in driving tumor growth. Subsequent proteomic analysis of active conditioned media indicated multiple novel proteins of interest including neuronal cell adhesion molecule (NrCAM) and neurofascin (NFASC). Both paracrine factors increased proliferation and migration in multiple DHG-H3G34 cell lines. Overall, these findings suggest that DHG-H3G34 gliomas integrate into neural circuits and leverage both conserved and tumor-type-specific activity-regulated mechanisms. Continued investigation into these activity-dependent mechanisms of glioma growth aims to identify potential therapeutic strategies for these lethal brain cancers. Citation Format: Kathryn R. Taylor, Samuel H. Wu, Richard Drexler, Gustavo A. Cruzeiro, Ilon Liu, Alexis E. Ivec, Lijun Ni, Carlos A. Biagi, Pamelyn J. Woo, Minhui Su, Youkyeong Gloria Byun, Mariella Filbin, Michelle Monje. Neuronal activity-regulated mechanisms promoting growth of diffuse hemispheric glioma, H3G34-mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4029.

Effects of CHD2 depletion on the growth of H3K27M DMG xenografts.

CHD2 regulates neuron-induced proliferation of H3.1K27M DMG cells.

Background There is an urgent need for novel targeted therapeutic strategies for pediatric-type diffuse high-grade glioma (PDHGG) to improve patient outcomes, the development of which demands model systems that accurately recapitulate the specific PDHGG subtypes. Characterization, longitudinal monitoring and, ultimately, evaluation of treatment response in these models requires sensitive non-invasive imaging techniques such as magnetic resonance imaging (MRI).Methods Thirty-five patient-derived, site-specific, orthotopic in vivo models of PDHGG, established using implantation of patient tumor material or patient-derived in vitro cultures maintained in stem cell retaining conditions, were characterized using multiparametric MRI.Results Median survival ranged from 54 to 433 days. Tumors identified on T2-weighted (T2w) images varied in appearance from a diffuse hyperintense signal to well-defined high contrast masses, and distribution of human nuclear antigen positive tumor cells corresponded to regions of T2w signal hyperintensity. Apparent diffusion coefficient was significantly higher in brainstem diffuse midline glioma (DMG) models than in diffuse hemispheric glioma (DHG) tumors, mirroring clinical observations. Lack of contrast-agent enhancement indicated an intact blood-brain barrier in most models, with heterogeneous disruption observed in four DHG models. Upon re-implantation, survival was significantly shortened in 3/4 DHG tumors and 1/10 DMG models, while quantitative MRI parameters remained similar. Furthermore, when 3 models grown in 2D and 3D in vitro were implanted in parallel, poorer survival or improved penetrance was associated with 3D cultures.Conclusion We established a comprehensive pre-clinical platform in which to evaluate the efficacy of therapeutic strategies against PDHGG in vivo, enhanced by the use of multiparametric MRI.

FOSL1 colocalizes with CHD2 to regulate cell viability and gene expression.

Validation of hits from CRISPR/Cas9 screens.

CHD2 and FOSL1 regulate H3K27me3 and H3K27ac in H3.1K27M DMG cells.

Analysis of CRISPR screens for hits in H3.1K27M DMG cells.

Analysis for DEGs of CHD2-depleted H3.1K27M DMG cells.

CHD2 binds to the gene regulatory elements of axon-guidance and synaptic genes.

Calcium imaging in brain slices with SU-DIPG36 xenografts in hippocampal CA1.

Analysis for shared DEGs between CHD2 and FOSL1 depletion in H3.1K27M DMG cells.

High-grade gliomas (HGGs) are the leading cause of brain cancer-related death. HGGs include clinically, anatomically and molecularly distinct subtypes that stratify into diffuse midline gliomas (DMGs), such as H3K27M-altered diffuse intrinsic pontine glioma, and hemispheric HGGs, such as IDH wild-type glioblastoma. Neuronal activity drives glioma progression through paracrine signalling1,2 and neuron-to-glioma synapses3-6. Glutamatergic AMPA receptor-dependent synapses between neurons and glioma cells have been demonstrated in paediatric3 and adult4 high-grade gliomas, and early work has suggested heterogeneous glioma GABAergic responses7. However, neuron-to-glioma synapses mediated by neurotransmitters other than glutamate remain understudied. Using whole-cell patch-clamp electrophysiology, in vivo optogenetics and patient-derived orthotopic xenograft models, we identified functional, tumour-promoting GABAergic neuron-to-glioma synapses mediated by GABAA receptors in DMGs. GABAergic input has a depolarizing effect on DMG cells due to NKCC1 chloride transporter function and consequently elevated intracellular chloride concentration in DMG malignant cells. As membrane depolarization increases glioma proliferation3,6, we found that the activity of GABAergic interneurons promotes DMG proliferation in vivo. The benzodiazepine lorazepam enhances GABA-mediated signalling, increases glioma proliferation and growth, and shortens survival in DMG patient-derived orthotopic xenograft models. By contrast, only minimal depolarizing GABAergic currents were found in hemispheric HGGs and lorazepam did not influence the growth rate of hemispheric glioblastoma xenografts. Together, these findings uncover growth-promoting GABAergic synaptic communication between GABAergic neurons and H3K27M-altered DMG cells, underscoring a tumour subtype-specific mechanism of brain cancer neurophysiology.

Pediatric high-grade gliomas (HGGs) are the leading cause of brain cancer-related death in children. HGGs include distinct subtypes defined by anatomical location as well as molecular characteristics that stratify into H3K27M-altered diffuse midline gliomas (DMG) and hemispheric HGGs such as isocitrate dehydrogenase-wild type (IDH-WT) glioblastoma. Neuronal activity drives HGG progression both through paracrine signaling and direct neuron-to-glioma synapses. Using whole-cell patch clamp electrophysiology, in vivo optogenetics, and patient-derived glioma xenograft mouse models, we identify functional, tumor-promoting GABAergic neuron-to-glioma synapses mediated by GABAA receptors in DMGs. GABAergic input has a depolarizing effect on DMG cells due to chloride uptake by the Na-K-2Cl cotransporter NKCC1 and consequent elevated intracellular chloride concentration. In contrast, depolarizing GABAergic currents were not detected in IDH-WT HGG. Membrane depolarization is known to increase glioma proliferation and accordingly, we find that the activity of GABAergic interneurons promotes DMG proliferation in vivo. Increasing GABA signaling with the benzodiazepine lorazepam increases glioma proliferation and reduces survival in xenograft models of DMG but not IDH-WT HGG. Conversely, we find that the anti-seizure medication levetiracetam attenuates low-frequency depolarizing GABAergic synaptic currents in a glioma-specific manner, reducing those in DMG but not in healthy neurons. The effect in DMG is independent of action on synaptic vesicle glycoprotein 2A (SV2A), the chief mechanism by which levetiracetam suppresses seizures. Levetiracetam reduces glioma proliferation and extends survival of mice bearing DMG xenografts, but not IDH-WT HGG xenografts. Retrospective real-world clinical data demonstrate longer overall survival for children with DMG who were taking levetiracetam, which was not evident in pediatric hemispheric HGG. These findings uncover growth-promoting GABAergic synaptic communication between GABAergic neurons and DMG cells, underscoring a tumor subtype-specific mechanism of brain cancer neurophysiology with potentially important implications for commonly used drugs in this disease context, which should be further studied in future prospective clinical studies.

Diffuse midline glioma (DMG) is a near-universally lethal form of pediatric high-grade glioma, driven by neuronal activity-regulated paracrine signaling and synaptic integration of malignant cells into neural circuits. In turn, DMG increases neuronal excitability, augmenting neuron-to-glioma signaling. In the healthy brain, microglia, the resident immune cells of the central nervous system (CNS), regulate neuronal excitability and synaptic connectivity. However, the role of microglia in promoting tumor-associated hyperexcitable neural networks in glioma remains unknown. Here, we investigate the activity-regulated engulfment of neuronal synapses by microglia in both healthy and glioma-bearing mice, and further explore how glioma cells alter microglia-mediated circuit refinement, contributing to pathogenic neuronal hyperexcitability. Microglia-mediated circuit refinement in the glioma microenvironment was characterized through synaptic engulfment analysis of both excitatory and inhibitory synapses by microglia in healthy mice and patient-derived DMG xenograft models, paired with optogenetic stimulation in the neocortex. We found that glutamatergic neuronal activity in the healthy brain increased excitatory synaptic engulfment by microglia in a previously unappreciated negative feedback mechanism that may guard against hyperexcitability. In contrast, this activity-regulated increase in excitatory synaptic engulfment was abrogated in DMG-infiltrated brains. Instead, inhibitory synaptic engulfment was significantly increased in DMG in response to glutamatergic neuronal activity. Together, these dysregulated synaptic engulfment mechanisms may create imbalance in the excitatory to inhibitory (E:I) synapse ratio predicted to increase neuronal excitability. Complementary single-nuclei sequencing studies revealed concordant tumor-specific, activity-regulated changes in microglia-neuron signaling showing reduced expression of excitatory synaptic refinement gene programs in microglia, potentially mediating the aberrant synaptic engulfment observed in DMG. These findings reveal novel cancer-neuron-immune interactions in DMG and provide an opportunity to potentially modulate tumor-associated neuronal hyperexcitability by targeting aberrant microglial synaptic engulfment.

Glutamatergic neuronal activity promotes proliferation of both oligodendrocyte precursor cells (OPCs) and gliomas, including diffuse midline glioma (DMG). However, the role of neuromodulatory brainstem neurons projecting to midline structures where DMGs arise remains unexplored. Here, we demonstrate that midbrain cholinergic neuronal activity modulates OPC and DMG proliferation in a circuit-dependent manner. Optogenetic stimulation of the cholinergic pedunculopontine nucleus (PPN) promotes glioma growth in pons, while stimulation of the laterodorsal tegmentum nucleus (LDT) drives proliferation in thalamus. DMG-bearing mice exhibit higher acetylcholine release and increased cholinergic neuronal activity over the disease course. In co-culture, cholinergic neurons enhance DMG proliferation, and acetylcholine directly acts on DMG cells. Single-cell RNA sequencing revealed high CHRM1 and CHRM3 expression in primary DMG samples. Pharmacological or genetic blockade of M1/M3 receptors abolished cholinergic activity-driven DMG proliferation. Taken together, these findings demonstrate that midbrain cholinergic long-range projections promote activity-dependent DMG growth, mirroring a parallel proliferative effect on healthy OPCs.

All drugs of abuse induce long-lasting changes in synaptic transmission and neural circuit function that underlie substance use disorders. Here, we demonstrate that dopaminergic neuronal activity-regulated myelin plasticity is a key modulator of dopaminergic circuit function and opioid reward. Oligodendroglial lineage cells respond to dopaminergic neuronal activity evoked by either optogenetic stimulation or by morphine administration specifically within the reward center ventral tegmental area (VTA), but not along the axonal projections in the medial forebrain bundle nor within the target nucleus accumbens (NAc). Genetic blockade of oligodendrogenesis dampens NAc dopamine release dynamics, which is critical for reward learning, and impairs behavioral conditioning to morphine. Our findings identify dopaminergic neuronal activity-regulated myelin plasticity as an important circuit modification that is required for opioid reward. One-Sentence Summary Activity-dependent myelin plasticity in the ventral tegmental area modulates dopaminergic circuit function and opioid reward

Abstract Neuronal activity robustly drives glioma progression, mediated through paracrine and synaptic neuron-to-glioma interactions. Recent research has focused on glutamatergic and GABAergic neurons, while the impact of neuromodulatory neuron subpopulations and their long-range projections remain unexplored. Here, we explore the glioma-promoting effects of serotonergic and cholinergic brainstem neurons, which project to defined regions throughout the brain. By employing optogenetic stimulation of midbrain serotonergic or cholinergic neurons in mice bearing high-grade glioma allografts or xenografts, we observed robust, circuit-specific effects of each neuromodulatory neuronal subtype on proliferation. Concordant with the anatomy of serotonergic projections, we found that serotonergic neurons of the dorsal raphe nucleus promoted glioma proliferation in the neocortex and pons, while those from the median raphe nucleus increased proliferation in the thalamus. Similarly, stimulation of cholinergic neurons in the laterodorsal tegmentum nucleus enhanced thalamic glioma growth, and stimulation of the pedunculopontine nucleus promoted pontine tumor growth. The long-range neuronal activity-regulated effects on glioma cells were mediated by neurotransmitter secretion at axon terminals, and paracrine signalling protein release locally in the brainstem. The activity-regulated paracrine factor profiles are distinct between serotonergic and cholinergic neurons, illustrating neuron subtype-specific mechanisms affecting glioma cells beyond neurotransmitter differences. Co-culture with hESC/hiPSC-derived cholinergic or serotonergic neurons demonstrated synaptic structures between both neuromodulatory subpopulations and glioma cells, indicating a synaptic component to these neuron-glioma interactions. ScRNA-seq data integration revealed distinct receptor abundances between DMGs and IDH-WT glioblastomas correlating with specific cellular states. Inhibition of these targets – such as M1 or M3 cholinergic receptors in DMG – mitigated the proliferation-inducing effects of the relevant neuron subtype on glioma cells. In summary, these findings introduce brainstem cholinergic and serotonergic neuromodulatory neurons as drivers of high-grade glioma growth in a circuit-specific manner in midline structures and in neocortex, adding to the complexity with which gliomas integrate into brain-wide neural circuitry.

Nervous system activity regulates homeostasis, development, and plasticity of the brain. Certain glial cell populations regulate neuronal activity by controlling neurotransmitter availability at the synapse or regulating extracellular ion concentrations. Glial precursor cells can give rise to gliomas, which are the leading cause of brain cancer-related death. Recent studies have demonstrated the influence of neuronal activity on the progression of a range of high-grade gliomas. One process through which neurons influence gliomas is the formation of functional bona fide synapses between presynaptic neurons and postsynaptic glioma cells. Here, we present multiple techniques that can be utilized to study these neuron-to-glioma synapses. We present a coculture method for in vitro studies and layout the process for generating patient-derived xenograft models in mice for in vivo and in situ studies. We describe the use of electron microscopy to observe the structural characteristics of synapses and electrophysiological studies to investigate the electrical properties of such synapses. We also outline two-photon calcium imaging as a powerful tool to study network-level consequences of activity-depended currents in glioma cells. While similar questions can be answered using electrophysiology and calcium imaging, electrophysiology is useful for directly probing synaptic responses and membrane potential changes while calcium imaging is useful for studying network-level changes.