α-Synucleinopathies display pronounced heterogeneity in the spatial distribution of α-synuclein (αSyn) pathology and clinical progression. Although distinct αSyn assemblies-from monomers and soluble oligomers to fibrils-exert non-equivalent biological effects, in vivo studies have predominantly focused on preformed fibrils (PFFs), leaving the pathogenic potential of soluble oligomers insufficiently explored. Here, we investigated the spatiotemporal, molecular, and behavioral consequences of striatal delivery of structurally validated αSyn oligomers in adult mice. Three-month-old male C57BL/6 J mice received bilateral injections of αSyn oligomers into the dorsal caudate-putamen and were analyzed at 30, 90, and 180 days post-injection (dpi) using molecular, histological, and behavioral approaches. αSyn oligomers induced a highly dynamic and region-specific pathological cascade. At 30 dpi, widespread inclusions were evident in cortical and limbic regions projecting to the striatum, followed by a progressive redistribution of pathology toward the striatum at later stages, while inclusions were consistently absent from the substantia nigra pars compacta. In parallel, αSyn oligomers elicited distinct spatiotemporal patterns of inflammatory and oxidative responses across brain regions, characterized by an immediate pro-inflammatory cytokine surge in the striatum, early but transient oxidative response in the cortex and delayed, sustained oxidative stress in the midbrain. Despite modest nigrostriatal degeneration and preserved gross motor performance, sensitive behavioral measures revealed early and persistent motor weakness, suggesting synaptic and axonal dysfunction rather than neuronal loss. Collectively, our findings provide the first in vivo evidence that soluble αSyn oligomers act as potent yet transient drivers of a distributed and partially reversible neuropathological program fundamentally distinct from canonical PFF-based models. By uncovering an oligomer-specific mode of αSyn-induced neurodegeneration, this work demonstrates that disease-relevant pathology can arise from dynamic, regionally uncoupled processes rather than stable fibrillar inclusions, redefining early α-synucleinopathy as a state of selective circuit vulnerability and revealing a previously unrecognized therapeutic window for intervention.
Disease reactivation following cessation of sphingosine 1-phosphate receptor modulators (S1PRM) occurs in ~ 10% of multiple sclerosis (MS) patients. The biological factors underlying this phenomenon remain incompletely understood, including the potential contribution of sex-specific differences. We performed a systematic review on published literature and adverse event registries (FAERS, EudraVigilance), as of January 2024, focusing exclusively on fingolimod (FTY) withdrawal in individuals with MS. Disease severity after FTY withdrawal was assessed in the experimental autoimmune encephalomyelitis (EAE) mouse model, using untreated EAE mice as controls. S1P receptor expression was analyzed by immunofluorescence in spinal cord tissue from EAE mice and in brain biopsies from MS patients with disease reactivation after FTY withdrawal and MS controls (no prior FTY or S1PRM treatment). Analysis of eight studies (n = 2579) demonstrated an association between female sex and disease reactivation after FTY cessation (odds ratios: 1.09-7.20), corroborated by pharmacovigilance data (FAERS: OR = 2.00, p < 0.0001; EudraVigilance: OR = 2.42, p < 0.0001). Female EAE mice exhibited greater post-FTY treatment disease severity (2.5-fold increase, p < 0.0001) with increased S1PR1 expression on CD3+T cells (p < 0.001). Human brain biopsies showed elevated S1PR1 expression on CD3+T cells in active demyelinating lesions during disease reactivation compared to inactive demyelinated lesions (p < 0.0001) and controls (p < 0.05). Across clinical, experimental, and neuropathological analyses, female sex was associated with more pronounced disease activity following FTY withdrawal. Increased S1PR1 expression in T cells represents a potential cellular correlate of this sex-associated vulnerability and warrants further mechanistic investigation.
Epileptogenesis transforms a healthy brain into an epileptic network, yet the temporal and cell-type-specific molecular events driving this transition remain poorly defined. Neuron-glia interactions are essential in this process, but no study has systematically charted their transcriptional dynamics from the acute insult to chronic epilepsy. Using the intracortical kainic acid mouse model that recapitulates key hallmarks of mesial temporal lobe epilepsy with hippocampal sclerosis in humans, we performed Fluorescent Activated Nuclear Sorting of NeuN+ (neuronal) and NeuN⁻ (glia) nuclei followed by RNA sequencing at 1 h, 24 h, and 3 months after status epilepticus. Differential expression and integrative GO/KEGG analyses resolved stage-specific molecular programs across cell types. The majority of genes differentially expressed in neurons and glia were exclusive to the respective time point investigated. We also identify a sequential reorganization of cellular gene expression changes during epileptogenesis. The acute phase is dominated by a shared stress response and DNA-repair programs in both neurons and glia. At 24 h, glia undergoes a marked transcriptional pivot involving necroptosis-associated, TNFR1/IFN-linked, and COX-2/chemokine pathways, while neurons display immune- and plasticity-related signatures. By 3 months, transcriptional activity is largely confined to glia and enriched for inflammatory, angiogenic, and gliogenic processes, consistent with long-term neurovascular remodeling. Only a few transcripts, including Parp3 (neurons) and Tlr1 (glia), are dysregulated across all stages. These findings reveal an orderly transition from an acute protective-leaning program to a early latent glial inflammatory/regulated-death state, culminating in chronic gliopathy. Our work provides, to our knowledge, the first cell-type-resolved temporal atlas of epileptogenesis and identifies the early latent phase as a mechanistically tractable window for antiepileptogenic intervention.
Titinopathies are complex neuromuscular disorders with multiple phenotypes. The gene's size, comprising 364 exons, as well as the protein's size of 3.8 MDa and its extensive network of protein interactors, are key factors underlying this complexity. Various phenotypes characterize titinopathies, and this study focuses on two of them: arthrogryposis and myofibrillar myopathies. The protein deregulations associated with these two phenotypes remain unknown or have been minimally explored; however, understanding these consequences is essential for better characterizing the pathophysiological aspects of these titinopathies.The objective was to analyze protein deregulations in two cohorts of French patients with titinopathies exhibiting the arthrogryposis and myofibrillar myopathy phenotypes, and to compare them with control individuals. Protein extracts were obtained from muscle biopsies of patients, and changes in protein levels within these two groups were analyzed by mass spectrometry. The results indicate specific deregulations in each group. The networks analyzed revealed deregulation of proteins involved in fibrosis mechanisms or in the actomyosin complex for the arthrogryposis phenotype. Regulation of the muscle contraction system through deregulation of proteins involved in the cytoskeleton is impacted in patients with myofibrillar myopathy. The proteins that are quantitatively abnormal in these two groups also provide insights into the major signaling networks disrupted in titinopathies. These findings will contribute to a more precise characterization of titinopathies, enabling the identification of phenotype-specific biomarkers and potentially guiding the search for targeted therapies for these neuromuscular disorders.
Malignant pediatric brain tumors remain the leading cause of cancer-related mortality in children. Current diagnostics and monitoring rely on imaging and invasive biopsy, which may not capture tumor heterogeneity. Liquid biopsy-based biomarkers offer a novel, minimally invasive option. Among these, tumor-educated platelets have shown diagnostic value in adult cancers, but their utility in pediatric brain tumors has not been investigated. We analyzed platelet transcriptomes of 73 blood samples from 23 pediatric brain tumor patients, classified as high-grade or low-grade tumor patients, and 25 cancer-free controls. Platelets were isolated, CD45+ depleted, and subjected to RNA sequencing. CD45+ depletion efficiency was assessed using xCell-based leukocyte enrichment scores. Differential gene expression was assessed with DESeq2 and Gene Ontology over-representation analysis. Gene-level discrimination between groups was evaluated by receiver operating characteristic analysis, and a logistic regression model with patient-grouped 5-fold cross-validation was trained to classify high-grade tumor patients versus controls. Platelets from brain tumor patients showed transcriptional remodeling compared to controls, especially pronounced in high-grade tumor patients. We identified 315 and 338 differentially expressed genes in the brain tumor group versus controls and high-grade tumor patients versus control comparisons, respectively, and 9 genes in high-grade tumor patients versus low-grade tumor patients. In low-grade tumor patients versus controls, 30 genes met the significance threshold. Platelet gene expression of high-grade tumor patients showed consistent dysregulation of cancer-associated genes. Gene enrichment analyses highlighted pathways related to cytoskeleton dynamics, angiogenesis, and extracellular matrix organization. Multiple genes demonstrated encouraging classification performance, and logistic regression classifier based on selected transcripts achieved an area under the curve of 0.91, sensitivity of 85%, and a specificity of 92% in identifying high-grade tumor patients. This study provides the first evidence that platelets exhibit distinct transcriptomic signatures in pediatric brain tumor patients. Platelet RNA profiles separated high-grade tumor patients from controls, possibly reflecting tumor presence. These findings suggest that platelet transcriptomic profiling may warrant further investigation as a potential minimally invasive biomarker for pediatric brain tumors. The observed transcriptomic alterations and enriched pathways also raise the possibility that platelets participate in tumor-associated biological processes. Larger multicenter studies are needed to validate clinical applicability.
Synapse loss is the best correlate of cognitive decline in neurodegenerative diseases (NDDs). In Alzheimer's disease complement dysregulation, triggered by amyloid-β accumulation, plays a major role in synapse loss, but its contribution in other NDDs where amyloid is absent, most notably tauopathies, is elusive. Aggregation of tau is a prominent co-pathology in many NDDs and is characteristic of classical tauopathies in which amyloid pathology is lacking. Here we explore the effect of tau accumulation on complement dysregulation and its contribution to neuronal damage and synapse loss in a mouse tauopathy model harbouring the familial P301S tau mutation and post-mortem brain samples from human tauopathies. Complement gene and protein expression were analysed in P301S mice at 2, 4 and 6 M of age. Complement dysregulation was evident from qPCR analysis showing increased classical pathway (C4, C2), and complement receptor (C3ar1, Cd11b, Cd11c) gene expression in P301S mice compared to wildtype (WT). C1q protein levels were markedly increased in brain homogenates from P301S mice compared to WT, accompanied by C1q deposition on tau aggregates. Synapse loss was evident for both excitatory and inhibitory synapses and was accompanied by an increased percentage of C1q positive excitatory synapses, unaffected by proximity to tau aggregates. The classic pathway regulator CSMD1 was present on synapses and decreased on C1q positive synapses in P301S mice, implying a loss of protection from complement attack. Observations in human tauopathy brains demonstrated decreased CSMD1-labelled excitatory synapses, consistent with findings in P301S mice. These findings demonstrate that complement dysregulation occurs in areas of tau pathology and may contribute to synapse loss in tauopathies.
Although adamantinomatous craniopharyngioma (ACP) is a histologically benign intracranial tumor, its invasive growth pattern, characterized by finger-like protrusions, leads to a poor prognosis. In-depth analysis of the heterogeneity of the pathological structure of ACP and the development and evolution of tumor cells is crucial for expanding precision treatment strategies. However, a detailed understanding of the complex pathological structural characteristics of ACP is lacking. Here, we used laser microdissection to obtain 40 samples from 6 different pathological structures for Smart-Seq2 sequencing and conducted in-depth analysis of 6 single-nucleus RNA sequencing samples and 4 spatial transcriptome samples. We also performed comprehensive validation using 3 external single-cell databases. Through histopathological observation, multiomic analysis, and immunofluorescence staining, we resolved the single-cell expression profiles of two distinct functional subpopulations of the whorl-like cell clusters, providing insights into their potential biological roles in tumor progression. Additionally, we discovered the epithelial-mesenchymal transition (EMT) or partial EMT development direction of the stellate reticulum and different cell fates related to terminal keratinization. Moreover, we reported the presence of previously neglected cyst-wall epithelium and the constitutive epithelium within ACP. The latter was found to possess basal cell characteristics and act as a potential progenitor cell pool for tumor maintenance and evolution. Through pseudotime analysis combined with co-expression network analysis, we constructed a differentiation regulatory network based on the constitutive epithelium. Our research results complement existing knowledge and provide a comprehensive analysis of the pathological features of ACP, providing new concepts and targets for precision targeted therapy.
A repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet existing mouse models incompletely engage spinal regions implicated in disease. Here, an adeno-associated virus encoding (G4C2)149 repeats was delivered via neonatal intrathecal injection, achieving widespread CNS expression with robust spinal cord targeting. This approach was applied to mice with graded loss of endogenous C9orf72 to interrogate both gain- and loss-of-function mechanisms. Longitudinal motor, behavioral, and pathological analyses revealed that repeat expression primarily drives mild, progressive muscle weakness, whereas coordination deficits were largely genotype dependent. Subtle gait abnormalities and hyperactivity were also observed. Within spinal motor regions, repeat-expressing mice exhibited dipeptide repeat protein accumulation, reduced NeuN-positive area, fewer motor neurons, glial activation, sparse phosphorylated TDP-43 pathology, and increased cryptic TDP-43 splicing. Cross-domain correlations further linked repeat expression, spinal pathology, and motor dysfunction. Collectively, these findings establish that CNS-wide repeat expression combined with reduced C9orf72 produces a coherent, mild ALS/FTD model.
Intraneuronal α-synuclein (αS) accumulation is a central event in the pathogenesis of Parkinson's disease and dementia with Lewy bodies. The spread of αS pathology throughout the central nervous system contributes to disease progression via seed-dependent propagation, yet disease-modifying therapies remain unavailable. We previously reported that porphyrin compounds inhibit αS aggregation in vitro; however, because of their high molecular weight, porphyrins poorly penetrate the blood-brain barrier (BBB). Heme, a porphyrin derivative, is synthesized from 5-aminolevulinic acid (5-ALA) via the heme biosynthetic pathway. In this study, we investigated whether 5-ALA modulates αS accumulation and propagation. Although porphyrin compounds inhibited αS seeding in vitro, 5-ALA itself did not. However, treatment of mouse primary neurons with 5-ALA enhanced heme synthesis and suppressed seed-dependent αS aggregation, suggesting a potential role for intracellular heme in modulating αS propagation. We further evaluated the effects of 5-ALA in an in vivo αS propagation model. Preformed αS fibrils were unilaterally injected into the striatum of wild-type mice, followed by oral administration of 5-ALA at three doses (0, 1.7, and 20 mg/kg/day) for 4 weeks. Biochemical analyses demonstrated that 5-ALA significantly reduced the propagation of sarkosyl-insoluble αS to the contralateral hemisphere. Immunohistochemical analyses revealed a marked reduction in phosphorylated αS pathology in the amygdala and substantia nigra in the 5-ALA-treated groups. Collectively, these findings indicate that oral administration of 5-ALA suppresses αS propagation in vivo, potentially through enhancement of intracellular heme synthesis. Modulation of the heme biosynthetic pathway may represent a novel therapeutic strategy for synucleinopathies.
Glioblastoma (GBM) is the most common malignant primary brain tumor in adults. Cystic GBM is a phenotypic subset requiring distinct clinical management. This study aimed to comprehensively assess the genomic landscape of cystic GBM. GBM, IDH wild-type patients with available next-generation sequencing and presurgical brain MRI data were evaluated for cysts by neuroimaging specialists and a senior neuroradiologist. Patients with incomplete radiological or histopathological data, treatment-related cysts, or indeterminate cyst status were excluded. We used logistic regression to assess the association of cystic GBM with demographic variables and the top 24 altered genes. We employed Cox regression to analyze the association of cystic GBM with survival. Among 374 eligible patients, we identified 111 (30%) with cystic GBM and 263 (70%) with non-cystic GBM. Multivariable logistic regression revealed the association of cystic GBM with alterations of the KDR (OR 8.75 [1.77-43.3], p = 0.038), RB1 (OR 5.12 [2.58-10.1], p = 0.008), TP53 (OR 2.72 [1.67-4.43], p = 0.008), CDK4 (OR 2.36 [1.27-4.37], p = 0.036) genes, as well as wild type EGFR (OR 0.33 [0.20-0.53], p = 0.008). Cystic GBM was not associated with overall survival (HR 0.82 [0.61-1.11], p = 0.205). Cystic GBM is marked by alterations of the Rb and p53 pathways as well as KDR alterations. This provides new insights into the pathogenesis of cystic GBM. Several alterations associated with cystic GBM, including CDK4 and KDR, are targets of small-molecule inhibitors. Further research is required to explore the diagnostic and therapeutic implications of this association.
The amygdala is highly vulnerable to protein aggregation and heavily affected in Lewy body diseases (LBDs). However, vulnerability might vary per amygdalar nucleus and it is unclear if the pattern of vulnerability across the nuclei differs between types of protein aggregation and between LBDs. In this study, we aimed to assess the vulnerability of amygdalar nuclei to multiple types of protein aggregation across LBDs. Post-mortem amygdala tissue of donors with incidental LBD (iLBD, n = 6), Parkinson's disease (PD; n = 18), dementia with Lewy bodies (DLB; n = 9) and Alzheimer's disease with Lewy bodies (AD + LB; n = 15) was immunostained with antibodies against alpha-synuclein (aSyn; EP1536Y and 5G4), amyloid beta (Aβ; 4G8), phosphorylated tau (p-tau; AT8) and phosphorylated TDP-43 (p-TDP-43; 11-9), and quantitatively analyzed using QuPath. Neuronal and astrocytic aSyn pathology were most pronounced in the parahippocampal-amygdaloid transition area (PHA) and the basal nucleus, a pattern shared by all disease groups. Vulnerability to Aβ pathology varied per group but was highest in the PHA in AD + LB, whereas diffuse plaques were most common in the accessory basal nucleus. The PHA of DLB and both the basal and accessory basal nucleus of AD + LB cases were most susceptible to p-tau pathology, with fine granular cytoplasmic neuronal tau inclusions being mostly observed in the basal nucleus and neurofibrillary tangles in the accessory basal nucleus. The nuclei in the ventromedial part of the amygdala (PHA, ventral part of the basal nucleus, and cortical nucleus) were found to be hotspots for protein aggregation across LBDs. aSyn pathology in these nuclei predominantly correlated with dementia, hallucinations and anxiety. Our results show that amygdalar nuclei vulnerability differs per protein aggregate and disease entity, although the PHA, basal nucleus and cortical nucleus are generally more vulnerable. Together, our study provides a deeper insight into the selective vulnerability of amygdalar nuclei to protein aggregates and their relation to clinical characteristics in LBDs.
NOTCH2NLC-related neuronal intranuclear inclusion disease (NIID) is a progressive neurodegenerative disorder with marked clinical heterogeneity and an increasingly recognized phenotypic spectrum. Although hydrocephalus-like presentations, i.e. ventriculomegaly with concomitant with cognition, motor, and/or bladder dysfunctions, have been observed in NIID sporadically, their prevalence, clinicopathological characteristics, and potential clinical implications remain poorly understood. This retrospective cohort study systematically characterized this newly recognized hydrocephalus-like phenotype. We conducted an MRI-based screening to identify previously undiagnosed NIID cases from a cohort of 498 adults with imaging diagnosis of communicating hydrocephalus. Four such cases were identified and incorporated into our NIID cohort, with genetic and pathological confirmation. A total of 68 NIID patients were analyzed, of whom 19 (27.9%) exhibited marked ventriculomegaly (Evans index > 0.34) disproportionate to parenchymal atrophy and were grouped as ventriculomegalic NIID. Compared with those without ventriculomegaly, these patients were associated with male predominance, more frequent walking difficulty, and less frequent tremor. Nearly half of ventriculomegalic cases (9/19) fulfilled the idiopathic normal pressure hydrocephalus-like clinical triad. Radiological features included markedly enlarged ventricles, smaller callosal angle, less frequent diffusion-weighted imaging hyperintensities, and overrepresented periventricular-dominant white matter lesion pattern. A subset of ventriculomegalic NIID patients (5/8) showed improvement in gait, cognition, and/or urinary function following cerebrospinal fluid drainage, and two patients who underwent ventriculoperitoneal shunt surgery experienced sustained clinical benefit. Brain autopsy was performed in one ventriculomegalic NIID patient, revealing widespread intranuclear inclusions, particularly in ependymal and choroid plexus epithelial cells and astrocytes, alongside dysmorphic astrocytes and loss of aquaporin-4 polarization. Collectively, our findings suggest that a hydrocephalus-like phenotype with distinctive clinical and radiological features is relatively common in NIID and frequently misdiagnosed as idiopathic normal pressure hydrocephalus. Pathological alterations in the ependyma, choroid plexus, and astrocytes and subsequently disturbed cerebrospinal fluid dynamics may contribute to its pathogenesis. Some patients might benefit from shunt surgery, highlighting a possible symptomatic and partially reversible component and broadening therapeutic opportunities in this otherwise progressive neurodegenerative disorder.
The glymphatic system plays a key role in clearing waste products from the brain and is essential for maintaining brain homeostasis. When dysfunctional, it appears to contribute to pathological changes that exacerbate brain disorders, including neurodegenerative diseases. Additionally, wasteosomes, also known as corpora amylacea, are structures that function as waste containers and are thought to increase in response to chronic glymphatic insufficiency. Hence, in this study, we evaluated whether the accumulation and distribution of wasteosomes are compatible with both the potential role of wasteosomes as a hallmark of the chronic glymphatic insufficiency and the presence of this insufficiency in certain neurodegenerative diseases. Accordingly, brain tissue from 185 donors was analysed, including cases of Alzheimer's disease, amyotrophic lateral sclerosis with TDP-43 proteinopathy, frontotemporal lobar degeneration with TDP-43 or tau proteinopathy, and non-diseased controls. Wasteosomes were examined across 28 brain regions comprised within 5 major brain areas, using region-specific scoring systems. Analysis was conducted through variance and covariance analyses, along with decision tree procedures. The findings reveal that wasteosomes are consistently found in specific critical regions, with a higher burden in donors with neurodegenerative diseases compared with controls. These regions are independent of the regional distribution of the underlying proteinopathy, and are potentially associated with glymphatic drainage pathways. From an integrated perspective, although further studies are required, the increased presence of wasteosomes in these critical regions across all diseased groups is consistent with the potential presence of chronic glymphatic insufficiency in these diseases.
The non-canonical isocitrate dehydrogenase 1 (IDH1) R132C mutation is rare in diffuse gliomas but appears to be enriched in tumors associated with Li-Fraumeni syndrome (LFS), which is caused by germline tumor protein p53 (TP53) mutations. However, the molecular relationship between TP53 alterations and IDH1 R132C, as well as the prevalence of LFS among patients with R132C-mutant gliomas, remains unclear. We analyzed 93 consecutive patients retrospectively with IDH1-mutant diffuse gliomas (Central Nervous System World Health Organization (CNS WHO) Grades 2 and 3) treated at our institution between 2005 and 2025. IDH, TP53, and ATRX status were assessed by immunohistochemistry, Sanger sequencing, multiplex ligation-dependent probe amplification, and targeted next-generation sequencing. Germline TP53 mutations were assessed using peripheral blood samples. DNA methylation profiling was performed using the Methylscape platform and the DKFZ methylation classifier. Three of 90 IDH1-mutant gliomas (3%) harbored the R132C variant. All three tumors were astrocytomas with concurrent TP53 mutation, ATRX alteration, and 1p/19q non-codeletion. Exploratory cancer cell fraction analysis suggested that both IDH1 R132C and TP53 mutations were present in the major tumor cell population, supporting a biological association between TP53 alterations and the occurrence of the IDH1 R132C variant. One patient harbored a pathogenic germline TP53 mutation despite lacking a family history or fulfilling the established clinical diagnostic criteria for LFS. DNA methylation analysis demonstrated partial overlap with conventional IDH-mutant astrocytoma and also revealed atypical clustering patterns. IDH1 R132C-mutant gliomas are characterized by concurrent TP53 mutation, ATRX alteration, and 1p/19q non-codeletion. Although based on a small cohort, our findings support a biological association between TP53 alterations and the rare IDH1 R132C variant and suggest that germline TP53 testing may be considered in selected patients, particularly younger individuals, to identify previously unrecognized Li-Fraumeni syndrome.
WHO grade 2 meningiomas exhibit highly heterogeneous clinical courses. While the Ki67 proliferation index is a standard biomarker, its prognostic utility remains limited by methodological inconsistency and potential time-dependent dynamics. We evaluated an automated, artifact-adjusted Ki67 assessment and its integration with molecular risk profiling. 98 WHO grade 2 meningiomas (WHO 2021) were analyzed using an automated QuPath-based pipeline with HistoART for artifact exclusion. Molecular risk was defined by methylation and copy number profiling to calculate the integrated molecular-morphologic risk score by Maas et al. We employed extended Cox models to account for proportional hazards violations. Automated Ki67 values were significantly lower than routine pathological estimates (median 2.91% vs. 10%; p < 0.001) and correlated modestly with integrated risk scores (ρ = 0.26, p = 0.009). We identified a biphasic risk pattern: within the first 38 postoperative months, an automated Ki67 > 3.62% was a strong independent predictor for local recurrence (HR 5.06, p < 0.001) and progression-free survival (HR 4.15, p = 0.002), remaining significant alongside subtotal resection and the integrated risk group. Beyond 38 months, prognostic impact attenuated. Ki67 and the integrated molecular risk score contributed independently in multivariable models, suggesting complementary biological dimensions. Automated, artifact-adjusted Ki67 quantification provides time-dependent, independent prognostic information in WHO grade 2 meningioma, complementary to molecular risk stratification. It may serve as a cost-effective surveillance marker-both as an adjunct to molecular profiling and as a standalone tool where molecular testing is unavailable.
Glioblastoma (GBM) is a highly heterogeneous, invasive brain tumor with profound metabolic plasticity. Patient-derived cultures provide valuable preclinical models, yet the stability and clinical relevance of their metabolic phenotypes during ex vivo maintenance remain unclear. Here, we performed longitudinal functional profiling of mitochondrial respiration, substrate utilization, and migration in primary patient-derived GBM cultures at one and five weeks ex vivo using Seahorse Bioanalyzer assays and wound-healing assays, complemented by exploratory integration with routine clinical parameters (MGMT promoter methylation, p53 expression, sex, and age). Early profiling identified two mitochondrial subgroups distinguished by spare and maximal respiratory capacity, with MGMT promoter methylation observed exclusively in the high-respiration cluster. Over time, most cultures retained their initial phenotype, whereas a subset transitioned from low to high respiration, indicating dynamic adaptation under standardized conditions. Extended culture was accompanied by patient-specific shifts in fuel utilization, including increased glutamine dependency and reduced inter-sample variability in glucose and fatty-acid parameters. Exploratory analyses suggested an association between higher age and lower fatty-acid oxidation capacity and indicated constrained fatty-acid dependency in p53-positive cultures. Migration assays revealed marked inter-sample heterogeneity and a 2-6 times faster gap closure in male-derived than female-derived cultures. Notably, early migratory behavior showed no strong association with OCR-defined metabolic state, and early fuel-flex parameters did not recapitulate OCR cluster structure. Together, these data provide a standardized longitudinal functional benchmark showing that patient-derived GBM cultures preserve intrinsic metabolic heterogeneity while undergoing time-dependent adaptation, and that migration represents an additional, partially independent functional axis. These findings are hypothesis-generating and support stratified, time-sensitive follow-up studies of metabolic and invasive phenotypes in GBM.
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DNA methylation profiling has become crucial for accurate classification of CNS tumors, yet Illumina EPIC arrays are restricted by long turnaround times, batching requirements, and limited accessibility in non-specialized centers. Rapid, affordable alternatives are urgently needed, particularly in resource-constrained settings. We aimed to evaluate nanopore sequencing as a real-world diagnostic tool for resolving diagnostically challenging CNS tumors. We applied the ROBIN pipeline to 34 consecutive CNS tumors that remained NOS/NEC after routine histopathological and molecular workup. Samples comprised 16 routine FFPE blocks and 18 frozen tissues (9 fresh-frozen, 9 pre-extracted DNA) from three pathology centers. Nanopore sequencing was performed on PromethION R10.4.1 flow cells, targeting definitive methylation-based classification report within 24 h. A confident methylation class was assigned in 28/34 cases (82.4%), including 14/16 (87.5%) methylation calls of FFPE samples with median target coverage of 0.03×. Integration of simultaneously generated CNV profiles rescued an integrated glioblastoma diagnosis in two methylation-unclassifiable cases via 7+/10- signatures. The nanopore-based classifications also achieved 84.6% (11/13) concordance with paired result from Illumina Methylation EPIC arrays. In high-coverage samples, diagnostic-relevant SNVs (BRAF V600E, PTEN, NF1/TP53) were recovered from the same run. SNV detection was strongly coverage-dependent, however: it was not achievable in the low-coverage FFPE and fresh-frozen specimens. Nanopore sequencing enables rapid, accurate, multidimensional molecular classification of diagnostically intractable CNS tumors using routine FFPE and frozen tissue, achieving high diagnostic resolution in comparison with reference methods while drastically reducing turnaround time and cost, making precision neuropathology feasible beyond quaternary centers.
Approximately 30% of group 4 medulloblastomas (G4-MBs) present with metastasis at diagnosis, yet the molecular mechanisms remain unclear. To elucidate differences of genes' expression between primary tumors of metastatic (M+) and non-metastatic (M0) G4-MB, we performed multi-omics profiling, including RNA sequencing, proteomics, single-nucleus RNA sequencing (snRNA-seq), and spatial transcriptomics on tumor samples. Integrative analyses identified VCAN, a chondroitin sulfate proteoglycan, as the most significantly up-regulated gene in primary tumors of M+ G4-MB. High expression of this gene was correlated with poor patient prognosis. Functional assays demonstrated that VCAN promotes proliferation and invasion while inhibiting apoptosis. In addition, NR3C1 was predicted as the key activator of VCAN by Single-Cell Regulatory Network Inference and Clustering (SCENIC). High-definition spatial transcriptomics revealed that NR3C1 and VCAN are highly co-expressed within the same spatial domains. Multiplex immunofluorescence confirmed the co-localization, providing spatial evidence of their regulatory interaction. ChIP-qPCR subsequently confirmed direct binding of NR3C1 to the VCAN promoter. Knockdown of NR3C1 or VCAN suppressed invasion and proliferation and induced apoptosis of the tumor cells, which were partially reversed by VCAN overexpression. Together, these findings revealed that the NR3C1-VCAN axis played a pivotal role in the metastatic progression of G4-MB, highlighting a potential therapeutic target for high-risk patients.
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