Neuron-Glial Interactions: Implications for Plasticity, Behavior, and Cognition.

The traditional view of glial cells as mere supportive tissue has shifted, due to advances in technology and theoretical conceptualization, to include a diversity of other functions, such as regulation of complex behaviors. Astrocytes, the most abundant glial cells in the central nervous system (CNS), have been shown to modulate synaptic functions through gliotransmitter-mediated neurotransmitter reuptake, influencing neuronal signaling and behavioral functions. Contemporary studies further highlight astrocytes' involvement in complex cognitive functions. For instance, inhibiting astrocytes in the hippocampus can lead to memory deficits, suggesting their integral role in memory processes. Moreover, astrocytic calcium activity and astrocyte-neuron metabolic coupling have been linked to changes in synaptic strength and learning. Microglia, another type of glial cell, also extend beyond their supportive roles, contributing to learning and memory processes, with microglial reductions impacting these functions in a developmentally dependent manner. Oligodendrocytes, traditionally thought to have limited roles postdevelopment, are now recognized for their activity-dependent modulation of myelination and plasticity, thus influencing behavioral responses. Recent advancements in technology and computational modeling have expanded our understanding of glial functions, particularly how astrocytes influence neuronal circuits and behaviors. This review underscores the importance of glial cells in CNS functions and the need for further research to unravel the complexities of neuron-glia interactions, the impact of these interactions on brain functions, and potential implications for neurological diseases.

Integrated electrophysiological and genomic profiles of single cells reveal spiking tumor cells in human glioma.

Prior studies have described the complex interplay that exists between glioma cells and neurons; however, the electrophysiological properties endogenous to glioma cells remain obscure. To address this, we employed Patch-sequencing (Patch-seq) on human glioma specimens and found that one-third of patched cells in IDH mutant (IDHmut) tumors demonstrate properties of both neurons and glia. To define these hybrid cells (HCs), which fire single, short action potentials, and discern if they are of tumoral origin, we developed the single cell rule association mining (SCRAM) computational tool to annotate each cell individually. SCRAM revealed that HCs possess select features of GABAergic neurons and oligodendrocyte precursor cells, and include both tumor and non-tumor cells. These studies characterize the combined electrophysiological and molecular properties of human glioma cells and describe a cell type in human glioma with unique electrophysiological and transcriptomic properties that may also exist in the non-tumor brain.

Dominant Malignant Clones Leverage Lineage Restricted Epigenomic Programs to Drive Ependymoma Development.

ZFTA-RELA is the most recurrent genetic alteration seen in pediatric supratentorial ependymoma (EPN) and is sufficient to initiate tumors in mice. Despite ZFTA-RELA's potent oncogenic potential, ZFTA-RELA gene fusions are observed exclusively in childhood EPN, with tumors located distinctly in the supratentorial region of the central nervous system (CNS). We hypothesized that specific chromatin modules accessible during brain development would render distinct cell lineage programs at direct risk of transformation by ZFTA-RELA. To this end, we performed combined single cell ATAC and RNA-seq analysis (scMultiome) of the developing mouse forebrain as compared to ZR-driven mouse and human EPN. We demonstrate that specific developmental lineage programs present in radial glial cells and regulated by Plagl family transcription factors are at risk of neoplastic transformation. Binding of this chromatin network by ZFTA-RELA or other PLAGL family motif targeting fusion proteins leads to persistent chromatin accessibility at oncogenic loci and oncogene expression. Cross-species analysis of mouse and human EPN reveals significant cell type heterogeneity mirroring incomplete neurogenic and gliogenic differentiation, with a small percentage of cycling intermediate progenitor-like cells that establish a putative tumor cell hierarchy. In vivo lineage tracing studies reveal single neoplastic clones that aggressively dominate tumor growth and establish the entire EPN cellular hierarchy. These findings unravel developmental epigenomic states critical for fusion oncoprotein driven transformation and elucidate how these states continue to shape tumor progression. HIGHLIGHTS: 1. Specific chromatin modules accessible during brain development render distinct cell lineage programs at risk of transformation by pediatric fusion oncoproteins.2. Cross-species single cell ATAC and RNA (scMultiome) of mouse and human ependymoma (EPN) reveals diverse patterns of lineage differentiation programs that restrain oncogenic transformation.3. Early intermediate progenitor-like EPN cells establish a tumor cell hierarchy that mirrors neural differentiation programs.4. ZFTA-RELA transformation is compatible with distinct developmental epigenetic states requiring precise 'goldilocks' levels of fusion oncoprotein expression.5. Dominant tumor clones establish the entire EPN cellular hierarchy that reflects normal gliogenic and neurogenic differentiation programs.

Glioblastoma Neurovascular Progenitor Orchestrates Tumor Cell Type Diversity.

Glioblastoma (GBM) is the deadliest form of primary brain tumor with limited treatment options. Recent studies have profiled GBM tumor heterogeneity, revealing numerous axes of variation that explain the molecular and spatial features of the tumor. Here, we seek to bridge descriptive characterization of GBM cell type heterogeneity with the functional role of individual populations within the tumor. Our lens leverages a gene program-centric meta-atlas of published transcriptomic studies to identify commonalities between diverse tumors and cell types in order to decipher the mechanisms that drive them. This approach led to the discovery of a tumor-derived stem cell population with mixed vascular and neural stem cell features, termed a neurovascular progenitor (NVP). Following in situ validation and molecular characterization of NVP cells in GBM patient samples, we characterized their function in vivo. Genetic depletion of NVP cells resulted in altered tumor cell composition, fewer cycling cells, and extended survival, underscoring their critical functional role. Clonal analysis of primary patient tumors in a human organoid tumor transplantation system demonstrated that the NVP has dual potency, generating both neuronal and vascular tumor cells. Although NVP cells comprise a small fraction of the tumor, these clonal analyses demonstrated that they strongly contribute to the total number of cycling cells in the tumor and generate a defined subset of the whole tumor. This study represents a paradigm by which cell type-specific interrogation of tumor populations can be used to study functional heterogeneity and therapeutically targetable vulnerabilities of GBM.

Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

Astrocytes remember inflammation.

Astrocytes respond to all forms of central nervous system maladies. In a recent issue of Nature, Lee et al. demonstrate that astrocytes encode inflammatory stimuli as epigenetic memory, which strengthens responses to subsequent stimuli and exacerbates pathology in disease models.

Glioblastoma disrupts cortical network activity at multiple spatial and temporal scales.

The emergence of glioblastoma in cortical tissue initiates early and persistent neural hyperexcitability with signs ranging from mild cognitive impairment to convulsive seizures. The influence of peritumoral synaptic density, expansion dynamics, and spatial contours of excess glutamate upon higher order neuronal network modularity is unknown. We combined cellular and widefield imaging of calcium and glutamate fluorescent reporters in two glioblastoma mouse models with distinct synaptic microenvironments and infiltration profiles. Functional metrics of neural ensembles are dysregulated during tumor invasion depending on the stage of malignant progression and tumor cell proximity. Neural activity is differentially modulated during periods of accelerated and inhibited tumor expansion. Abnormal glutamate accumulation precedes and outpaces the spatial extent of baseline neuronal calcium signaling, indicating these processes are uncoupled in tumor cortex. Distinctive excitability homeostasis patterns and functional connectivity of local and remote neuronal populations support the promise of precision genetic diagnosis and management of this devastating brain disease.

Next Directions in the Neuroscience of Cancers Arising outside the CNS.

SUMMARY: The field of cancer neuroscience has begun to define the contributions of nerves to cancer initiation and progression; here, we highlight the future directions of basic and translational cancer neuroscience for malignancies arising outside of the central nervous system.

Integrated electrophysiological and genomic profiles of single cells reveal spiking tumor cells in human glioma.

Prior studies have described the complex interplay that exists between glioma cells and neurons, however, the electrophysiological properties endogenous to tumor cells remain obscure. To address this, we employed Patch-sequencing on human glioma specimens and found that one third of patched cells in IDH mutant (IDH mut ) tumors demonstrate properties of both neurons and glia by firing single, short action potentials. To define these hybrid cells (HCs) and discern if they are tumor in origin, we developed a computational tool, Single Cell Rule Association Mining (SCRAM), to annotate each cell individually. SCRAM revealed that HCs represent tumor and non-tumor cells that feature GABAergic neuron and oligodendrocyte precursor cell signatures. These studies are the first to characterize the combined electrophysiological and molecular properties of human glioma cells and describe a new cell type in human glioma with unique electrophysiological and transcriptomic properties that are likely also present in the non-tumor mammalian brain.

The immune system and metabolic products in epilepsy and glioma-associated epilepsy: emerging therapeutic directions.

Epilepsy has a profound impact on quality of life. Despite the development of new antiseizure medications (ASMs), approximately one-third of affected patients have drug-refractory epilepsy and are nonresponsive to medical treatment. Nearly all currently approved ASMs target neuronal activity through ion channel modulation. Recent human and animal model studies have implicated new immunotherapeutic and metabolomic approaches that may benefit patients with epilepsy. In this Review, we detail the proinflammatory immune landscape of epilepsy and contrast this with the immunosuppressive microenvironment in patients with glioma-related epilepsy. In the tumor setting, excessive neuronal activity facilitates immunosuppression, thereby contributing to subsequent glioma progression. Metabolic modulation of the IDH1-mutant pathway provides a dual pathway for reversing immune suppression and dampening seizure activity. Elucidating the relationship between neurons and immunoreactivity is an area for the prioritization and development of the next era of ASMs.

Comparative Transcriptomic Analysis of Cerebellar Astrocytes across Developmental Stages and Brain Regions.

Astrocytes are the most abundant glial cell type in the central nervous system, and they play a crucial role in normal brain function. While gliogenesis and glial differentiation occur during perinatal cerebellar development, the processes that occur during early postnatal development remain obscure. In this study, we conducted transcriptomic profiling of postnatal cerebellar astrocytes at postnatal days 1, 7, 14, and 28 (P1, P7, P14, and P28), identifying temporal-specific gene signatures at each specific time point. Comparing these profiles with region-specific astrocyte differentially expressed genes (DEGs) published for the cortex, hippocampus, and olfactory bulb revealed cerebellar-specific gene signature across these developmental timepoints. Moreover, we conducted a comparative analysis of cerebellar astrocyte gene signatures with gene lists from pediatric brain tumors of cerebellar origin, including ependymoma and medulloblastoma. Notably, genes downregulated at P14, such as Kif11 and HMGB2, exhibited significant enrichment across all pediatric brain tumor groups, suggesting the importance of astrocytic gene repression during cerebellar development to these tumor subtypes. Collectively, our studies describe gene expression patterns during cerebellar astrocyte development, with potential implications for pediatric tumors originating in the cerebellum.

Understanding the microenvironment and how this controls cell fate.

The microenvironment influences cell fate. In this collection of voices, researchers from the fields of cancer and regeneration highlight approaches to establish the importance of the microenvironment and discuss future directions to understand the complex interaction between cells and their surrounding environment and how this impacts on disease and regeneration.

Subventricular zone cytogenesis provides trophic support for neural repair in a mouse model of stroke.

Stroke enhances proliferation of neural precursor cells within the subventricular zone (SVZ) and induces ectopic migration of newborn cells towards the site of injury. Here, we characterize the identity of cells arising from the SVZ after stroke and uncover a mechanism through which they facilitate neural repair and functional recovery. With genetic lineage tracing, we show that SVZ-derived cells that migrate towards cortical photothrombotic stroke in mice are predominantly undifferentiated precursors. We find that ablation of neural precursor cells or conditional knockout of VEGF impairs neuronal and vascular reparative responses and worsens recovery. Replacement of VEGF is sufficient to induce neural repair and recovery. We also provide evidence that CXCL12 from peri-infarct vasculature signals to CXCR4-expressing cells arising from the SVZ to direct their ectopic migration. These results support a model in which vasculature surrounding the site of injury attracts cells from the SVZ, and these cells subsequently provide trophic support that drives neural repair and recovery.

Prevalence of pathogenic germline variants in adult-type diffuse glioma.

Background: No consensus germline testing guidelines currently exist for glioma patients, so the prevalence of germline pathogenic variants remains unknown. This study aims to determine the prevalence and type of pathogenic germline variants in adult glioma. Methods: A retrospective review at a single institution with paired tumor/normal sequencing from August 2018-April 2022 was performed and corresponding clinical data were collected. Results: We identified 152 glioma patients of which 15 (9.8%) had pathogenic germline variants. Pathogenic germline variants were seen in 11/84 (13.1%) of Glioblastoma, IDH wild type; 3/42 (7.1%) of Astrocytoma, IDH mutant; and 1/26 (3.8%) of Oligodendroglioma, IDH mutant, and 1p/19q co-deleted patients. Pathogenic variants in BRCA2, MUTYH, and CHEK2 were most common (3/15, 20% each). BRCA1 variants occurred in 2/15 (13%) patients, with variants in NF1, ATM, MSH2, and MSH3 occurring in one patient (7%) each. Prior cancer diagnosis was found in 5/15 patients (33%). Second-hit somatic variants were seen in 3/15 patients (20%) in NF1, MUTYH, and MSH2. Referral to genetics was performed in 6/15 (40%) patients with pathogenic germline variants. 14/15 (93%) of patients discovered their pathogenic variant as a result of their paired glioma sequencing. Conclusions: These findings suggest a possible overlooked opportunity for determination of hereditary cancer syndromes with impact on surveillance as well as potential broader treatment options. Further studies that can determine the role of variants in gliomagenesis and confirm the occurrence and types of pathogenic germline variants in patients with IDH wild type compared to IDH mutant tumors are necessary.

Induction of astrocytic Slc22a3 regulates sensory processing through histone serotonylation.

Neuronal activity drives alterations in gene expression within neurons, yet how it directs transcriptional and epigenomic changes in neighboring astrocytes in functioning circuits is unknown. We found that neuronal activity induces widespread transcriptional up-regulation and down-regulation in astrocytes, highlighted by the identification of Slc22a3 as an activity-inducible astrocyte gene that encodes neuromodulator transporter Slc22a3 and regulates sensory processing in the mouse olfactory bulb. Loss of astrocytic Slc22a3 reduced serotonin levels in astrocytes, leading to alterations in histone serotonylation. Inhibition of histone serotonylation in astrocytes reduced the expression of γ-aminobutyric acid (GABA) biosynthetic genes and GABA release, culminating in olfactory deficits. Our study reveals that neuronal activity orchestrates transcriptional and epigenomic responses in astrocytes while illustrating new mechanisms for how astrocytes process neuromodulatory input to gate neurotransmitter release for sensory processing.

5-aminolevulinic acid, fluorescein sodium, and indocyanine green for glioma margin detection: analysis of operating wide-field and confocal microscopy in glioma models of various grades.

Introduction: Surgical resection remains the first-line treatment for gliomas. Several fluorescent dyes are currently in use to augment intraoperative tumor visualization, but information on their comparative effectiveness is lacking. We performed systematic assessment of fluorescein sodium (FNa), 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX), and indocyanine green (ICG) fluorescence in various glioma models using advanced fluorescence imaging techniques. Methods: Four glioma models were used: GL261 (high-grade model), GB3 (low-grade model), and an in utero electroporation model with and without red fluorescence protein (IUE +RFP and IUE -RFP, respectively) (intermediate-to-low-grade model). Animals underwent 5-ALA, FNa, and ICG injections and craniectomy. Brain tissue samples underwent fluorescent imaging using a wide-field operative microscope and a benchtop confocal microscope and were submitted for histologic analysis. Results: Our systematic analysis showed that wide-field imaging of highly malignant gliomas is equally efficient with 5-ALA, FNa, and ICG, although FNa is associated with more false-positive staining of the normal brain. In low-grade gliomas, wide-field imaging cannot detect ICG staining, can detect FNa in only 50% of specimens, and is not sensitive enough for PpIX detection. With confocal imaging of low-intermediate grade glioma models, PpIX outperformed FNa. Discussion: Overall, compared to wide-field imaging, confocal microscopy significantly improved diagnostic accuracy and was better at detecting low concentrations of PpIX and FNa, resulting in improved tumor delineation. Neither PpIX, FNa, nor ICG delineated all tumor boundaries in studied tumor models, which emphasizes the need for novel visualization technologies and molecular probes to guide glioma resection. Simultaneous administration of 5-ALA and FNa with use of cellular-resolution imaging modalities may provide additional information for margin detection and may facilitate maximal glioma resection.

Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Inhibitory input directs astrocyte morphogenesis through glial GABABR.

Communication between neurons and glia has an important role in establishing and maintaining higher-order brain function1. Astrocytes are endowed with complex morphologies, placing their peripheral processes in close proximity to neuronal synapses and directly contributing to their regulation of brain circuits2-4. Recent studies have shown that excitatory neuronal activity promotes oligodendrocyte differentiation5-7; whether inhibitory neurotransmission regulates astrocyte morphogenesis during development is unclear. Here we show that inhibitory neuron activity is necessary and sufficient for astrocyte morphogenesis. We found that input from inhibitory neurons functions through astrocytic GABAB receptor (GABABR) and that its deletion in astrocytes results in a loss of morphological complexity across a host of brain regions and disruption of circuit function. Expression of GABABR in developing astrocytes is regulated in a region-specific manner by SOX9 or NFIA and deletion of these transcription factors results in region-specific defects in astrocyte morphogenesis, which is conferred by interactions with transcription factors exhibiting region-restricted patterns of expression. Together, our studies identify input from inhibitory neurons and astrocytic GABABR as universal regulators of morphogenesis, while further revealing a combinatorial code of region-specific transcriptional dependencies for astrocyte development that is intertwined with activity-dependent processes.