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Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

Rebecca Mancusi, Eva Tatlock, Kiarash Shamardani, Lehi Acosta-Alvarez, Richard Drexler, Vrunda Trivedi, Avishai Gavish, Kouta Niizuma, Neeraj Soni, Pamelyn Woo, Sara Mulinyawe, Samin Jahan, Natalie Logan, Karen Malacon, Youkyeong Gloria Byun, Anna Geraghty, Tara Barron, Kathryn Taylor, Michelle Monje

Posted on: 13 August 2026

Preprint posted on 25 December 2025

Bad company: how microglial function is corrupted in diffuse midline glioma, to promote pathogenic neuronal hyperexcitability.

Selected by CRM UoE Journal Club

This preLight was prepared by Alsadeg Bilal, Mary Lorino, and Sophie Brumm, members of the Centre for Regenerative Medicine (CRM) Journal Club at The University of Edinburgh

Created with BioRender.com

Background

Diffuse midline glioma (DMG) is a lethal form of paediatric brain cancer, arising from the midline structures such as the pons, the brainstem and the thalamus. The median survival is under 12 months, and this has remained largely unchanged for decades (Jovanovich et al., 2023). Patients typically present with progressive neurological deficits related to tumour location, such as cranial nerve palsy, ataxia, and seizures. These features contribute substantially to morbidity, loss of independence and a reduced quality of life.

DMG is understood as a neurodevelopmental circuit integrated tumour. It transcriptionally resembles oligodendrocyte precursor cells, a population whose proliferation and differentiation are regulated by neuronal activity. This developmental context is important because neuronal activity also promotes glioma progression. Activity-regulated release of paracrine factors, including neuroligin-3, and the formation of bona fide neuron-to-glioma synapses create a feed-forward loop in which active neural circuits promote tumour growth, while tumour cells increase neuronal excitability (Venkatesh et al., 2015).

The observed hyperexcitability reflects a disrupted excitatory-to-inhibitory (E:I) balance in the brain. Previous work has shown that glioma cells receive functional glutamatergic synaptic inputs, and more recent work has identified tumour-promoting GABAergic neuron-to-glioma synapses in DMG (Venkataramani et al., 2019; Barron et al., 2025). Recent work has therefore focused on a self-amplifying feedback loop, in which neuronal activity promotes glioma growth through secreted synaptogenic or mitogenic factors and direct synaptic integration, while glioma cells further increase neuronal excitability (Venkatesh et al., 2015; Venkataramani et al., 2019; Taylor et al., 2023).

Mancusi et al. highlight that the contribution of microglia in the regulation of neuron-to-glioma synapses is understudied. In the healthy brain, microglia can refine neural circuits by surveying synapses and engulfing complement-tagged synaptic material (Schafer et al., 2012; Soteros et al., 2021). This process, often referred to as synaptic pruning, helps remove inappropriate or excessive connections. Microglia engage with synapses in an activity-dependent manner and therefore play a vital role in maintaining circuit homeostasis (Schafer et al., 2012).

The authors set out to study whether microglial function is altered in a DMG context, which may contribute to the skewed E:I balance. Their observations suggest that healthy microglial pruning mechanisms are reversed in DMG, to promote excitatory over inhibitory synapses in an activity-dependent manner.

Key findings

DMG shifts microglial engulfment away from excitatory synapses and towards inhibitory synaptic material

The authors first sought to compare microglia engulfment of excitatory and inhibitory synapses in xenograft mouse models. In this experiment, patient-derived DMG cells or buffered saline (nontumor control) were delivered to the frontal cortex of immunocompromised NSG mice. Their analysis indicated that in the DMG model, microglia exhibited increased engulfment of both inhibitory and excitatory synapses, compared to the nontumor control. The authors further observed that when the tumour burden was increased, microglia began to engulf fewer excitatory synapses, whereas engulfment of inhibitory synapses remained unchanged. These observations suggest that microglia modulate the E:I balance in DMG.

Acute circuit activity is no longer dampened in a DMG context

The authors next assessed whether the synaptic engulfment by microglia was activity dependent. In nontumor control mice, optogenetic stimulation of deep layer cortical projection neurons resulted in significantly more engulfment of excitatory synapses, i.e. a dampening of the acutely stimulated neuronal circuits. This control experiment thereby revealed a previously underappreciated negative feedback mechanism that maintains homeostatic circuit excitability. In a DMG context, however, acute optogenetic stimulation had the opposite effect, where microglia preferentially engulfed inhibitory synapses, resulting in a skewed E:I balance. These results further support microglia’s role of regulating the E:I balance in DMG, with input from surrounding neuronal circuits.

DMG-carrying mice display signs of neural hyperexcitability

The authors employed a second mouse model, to study the DMG microenvironment in a fully immunocompetent context. In this model, murine glioma cells were introduced into syngeneic hosts, allowing the authors to examine tumour-microglia-neuron interactions without the limitations of severe immunodeficiency. Optogenetic stimulation was used to probe how tumour-bearing circuits respond to increased neural activity.  This allograft model corroborated neuronal hyperexcitability in DMG through higher postsynaptic potentials and increased frequency of spontaneous excitatory postsynaptic currents, while inhibitory postsynaptic currents were unchanged.

Healthy circuitry dampening mechanisms are reversed in a DMG context

To better understand the transcriptomic changes associated with DMG, the authors performed optogenetic stimulation in the allograft model. Optogenic stimulation triggered  glutamatergic cortical activity, to mimic increased neural activity in DMG.

sn-RNAseq revealed an increase in complement-associated microglial subpopulations in response to glutamatergic neuronal activity in nontumor control mice, as is critical for excitatory circuit pruning. This complement pathway activation of microglia was downregulated in a DMG context, suggesting that microglia can no longer respond to the increased neuronal activity in the tumour microenvironment.

Neuronal activity exacerbates the impact of DMG tumour cells on microglia phenotype

Following the analysis of in vivo models, the authors turned to functionally in vitro experiments to distinguish between the impact of DMG tumour cells and neural activity alone on microglial engulfment. The authors exposed co-cultures of healthy human microglia-like cells, GABAergic and glutamatergic neurons to conditioned media from DMG- or healthy mouse brain slice cultures, following exogenous activation or unstimulated control. This experiment elegantly tests for the presence of secreted factors, which modulate microglia activity regardless of cell contact with DMG tumour cells.

The authors first noted that in the nontumor control setting, stimulation of  neuronal activity released secreted factors, which resulted in microglial engulfment of excitatory synapses. This in vitro observation corroborates the in vivo dampening of acute circuit activity observed in their mouse model.

By contrast, unstimulated DMG-conditioned media promoted a tumour-associated microglial phenotype and impaired normal synaptic engulfment function. Conditioned media from stimulated DMG-bearing slices further exacerbated this effect, reducing microglial engulfment of excitatory synapses and shifting microglia away from their homeostatic circuit-dampening role.

Overall, the authors present data supporting a model in which microglia in DMG-impacted brains contribute to a self-amplifying loop of neuronal hyperexcitability. In healthy tissue, activity-dependent microglial pruning helps restrain excessive excitation. In DMG, this mechanism is corrupted: microglia fail to appropriately prune excitatory synaptic material and may instead preferentially remove inhibitory synaptic elements. This shifts the E:I balance towards excitation, strengthening the neuronal activity state that supports glioma growth.

Why we highlight this preprint

Mancusi et al. demonstrate the strength of integrating multiple in vitro and in vivo models to disentangle complex problems, such as presented by the DMG microenvironment. Their work highlights the importance of taking all cellular players into account when studying and treating the tumour microenvironment. The presented paper offers new research avenues into the secreted factors, which modulate microglia function, that have the potential to impact patient quality of life and treatment outcome.

Questions and suggestions for the authors

  • The most pressing issue is the identity of the factors, which are secreted in DMG-diseased brains, which modulate microglial behaviour. Once identified, druggable targets may emerge which offer additional therapeutic angles.
  • Could the authors describe their plans to identify the key modulating factors, or perhaps share which candidate molecules they may suspect?
  • Have the authors considered a brainstem model, as described in https://doi.org/10.1093/noajnl/vdac079, to complement their work?

Tags: brain cancer, cancer biology, diffuse midline glioma (dmg), glioma, immunology, microglia, neuro-oncology, neuroimmunology, neuron-glia interactions, neuroscience, synaptic engulfment, tumour microenvironment

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