Conformationally distinct "strains" of α-synuclein aggregates are believed to contribute to the clinical and pathological diversity observed among synucleinopathies such as multiple system atrophy (MSA) and Parkinson's disease. Cases of MSA can be classified into two distinct subtypes: the cerebellar variant, MSA-C, and the parkinsonian variant, MSA-P, as determined by differences in their clinical and/or neuropathological presentations. To assess whether distinct α-synuclein strains may be present in individuals with MSA-C versus MSA-P, we characterized the conformational and seeding properties of α-synuclein aggregates in various brain regions from MSA-C and MSA-P patients and performed propagation studies in M83 transgenic mice. Biochemical fingerprinting of α-synuclein aggregates using limited proteolysis and a conformational stability assay failed to reveal differences between MSA-C and MSA-P either before or after propagation in mice. Similarly, using brain extracts from either MSA patients or MSA-inoculated mice, MSA-C and MSA-P α-synuclein aggregates exhibited indistinguishable seeding attributes in a seed amplification assay. Finally, no differences were observed in either the kinetics of disease progression or the extent of cerebral α-synuclein deposition in M83 mice inoculated with either MSA-C or MSA-P, regardless of the brain region from which the injected α-synuclein aggregates were derived. These results suggest that MSA subtypes are unlikely to arise because of distinct α-synuclein strains. Instead, our findings support a model in which the same α-synuclein strain initially forms in different brain regions, leading to differences in disease manifestation.

TAR DNA-binding protein 43 (TDP-43) inclusions are often associated with hyperphosphorylated tau, thus neurofibrillary tangles as the hallmark of Alzheimer's disease (AD) and primary age-related tauopathy (PART). TDP-43 in AD is associated with cognitive impairment, and while staging is known, the localization, cellular and inclusion characteristics of TDP-43 are yet to be elucidated. We investigate relationships between TDP-43 inclusions and the tangle maturation continuum in AD, PART, and co-pathologies by multiplex immunostaining combined with artificial intelligence (AI)-based segmentation via object recognition, reconstruction, and quantification. We performed anti-phosphorylated TDP-43 immunofluorescence with phosphorylated tau labeling different stages and modifications of tangles (AT8, pS396, TauC3, MN423, GT38) in three controls, three cases with PART and TDP-43 (PART-TDP), five cases with high likelihood AD and TDP-43 (AD-TDP), and four cases of high likelihood AD with TDP-43 and Lewy Body disease (AD-TDP-LBD). Confocal imaging was taken from eight regions: amygdala (amygdala-BL and amygdala-CM) and hippocampus (Cornu Ammonis (CA)-1, CA2/3, CA4, dentate gyrus (DG), subiculum (SUB)), and entorhinal cortex (ERC) and quantified with AI segmentation to identify 3D spatial relations, thus the maturity of neurofibrillary tangle associated TDP-43 (TAT) inclusions. TATs, which were either identified by pTDP-43 and AT8 or pTDP-43 and pS396 double positivity, were also investigated by Thioflavin S (ThioS) histochemistry. We found pS396 labeled mature TATs predominated in PART and AD in every region. Basolateral and centromedial amygdala displayed overall greatest number of pre-TATs and mature TATs. Mature TATs were homogenously distributed among hippocampal subfields whereas CA4 and DG had the greatest mature TAT composition. ERC revealed closer numbers of pre-TATs and mature TATs yet mature TATs predominated all groups. Unbiased AI-based object identification, reconstruction, and TAT maturation analysis pipeline in conjunction with TDP-43, tau, and ThioS multiplex immunostaining demonstrated unique aggregation and maturation patterns, highlighting region-specific dynamics in the neurodegenerative processes of PART and AD.

Primary meningeal melanocytic tumors (PMMTs) are rare neoplasms currently stratified by the World Health Organization (WHO) into three grades of malignancy based exclusively on histopathological criteria, while the prognostic significance of molecular alterations remains poorly defined. This study aimed to investigate the prognostic relevance of histopathological and molecular features in PMMTs and to assess the diagnostic utility of immunohistochemical and molecular markers for distinguishing PMMTs from malignant melanotic nerve sheath tumors (MMNSTs). Thirty-six primary central nervous system (CNS) melanocytic tumors, including 24 PMMTs (10 grade 1, 12 grade 2, and 2 grade 3) and 12 MMNSTs, were analyzed through integrated histopathological, immunohistochemical, genetic, and epigenetic characterization. Among grade 2 PMMTs, descriptively defined CNS-invasive otherwise benign melanocytomas defined a clinically favorable subgroup, whereas SF3B1 mutations and chromosome 8q gains were associated with higher recurrence rates and shorter recurrence-free survival. Losses of chromosome 17 or 21q were observed exclusively in MMNSTs, supporting their potential diagnostic utility in distinguishing MMNSTs from PMMTs. These findings demonstrate biological heterogeneity among intermediate-grade PMMTs and identify molecular markers associated with adverse clinical outcomes. Integrated histopathological and molecular characterization may enhance prognostic stratification and diagnostic accuracy in primary CNS melanocytic tumors.

Brain metastases (BrM) are a frequent and life-threatening complication of solid tumours, with lung cancer representing their most common source. While cancer-associated fibroblasts (CAFs) are well-established contributors to tumour progression in many extracranial malignancies, their presence and function within the brain tumour microenvironment, where fibroblasts have long been considered scarce or absent, remain poorly understood. Here, we isolated and characterised fibroblast-like cells from 13 human BrMs of diverse origins. These BrM-associated CAFs (BrM-CAFs) expressed canonical CAF markers and showed reduced proliferation and increased senescence compared to normal fibroblasts. Their transcriptome was enriched for extracellular matrix (ECM)-related genes, including multiple collagens, fibronectin, and matrix-remodelling enzymes. In vitro, BrM-CAFs produced a fibrillar ECM, and in BrM tissues, their abundance was associated with collagen I and fibronectin deposition. Transcriptomic, proteomic, and secretome analyses further revealed that BrM-CAFs produce multiple cytokines, chemokines, and growth factors that promote cell motility. BrM-CAF conditioned medium promoted both monocyte migration and the migration of cancer cells, including established cell lines and patient-derived lung cancer BrM cells; monocyte migration was partially reduced by inhibition of CCL2/CCR2 signalling, whereas blocking CXCL12, CXCL16, or CX3CL1 attenuated BrM-CAF-induced cancer cell migration. Beyond these effects on migration, exposure to BrM-CAFs increased cancer cell invasion in transwell and heterotypic 3D spheroid assays. In contrast, their effects on cancer cell proliferation were limited and did not indicate a growth-promoting role. Exposure to BrM-CAFs was also associated with increased expression of interferon-stimulated genes in cancer cells. Together, our findings support a role for BrM-CAFs in shaping the brain metastatic microenvironment through ECM remodelling and the secretion of pro-migratory mediators, promoting monocyte and cancer cell migration and enhancing cancer cell invasion. These data identify BrM-CAFs as active stromal participants in BrM biology and support further investigation of their biological, diagnostic, and therapeutic relevance.

Patients suffering from aquaporin-4 antibody-positive neuromyelitis spectrum disorder may present with radically different types of lesions in the central nervous system. They may show large tissue destructive lesions, as typically seen in medulla, spinal cord, and optic nerves, and lesions with much better tissue preservation, as seen in the area postrema. The causes for the resilience of the area postrema to the action of aquaporin 4-antibodies are currently unclear. We performed detailed immunohistochemical analysis combined with spatial transcriptomics and functional annotation clustering, and identified key differences in the immunological landscape between the area postrema and the medulla. In the area postrema, only few chemokines were upregulated in response to AQP4 antibodies. Therefore, the immune response was dominated by local microglial/macrophages which displayed a low grade of microgliosis and an overall low level of activation. Il-1β expression was below our cut-off for detection. This was in marked contrast to lesions in the medulla, which were characterized by the expression of many chemokines underlying the recruitment of macrophages and neutrophils from the periphery to this site, and by Il-1β expression. Aldehyde dehydrogenase 1 family member A2 (Aldh1a2), the key molecule for retinoic acid synthesis, was strongly expressed in the area postrema, but not in the medulla. Because retinoic acid can prevent the activation of astrocytes and microglia, can reduce the production of cytokine/chemokine transcripts, can alleviate the expression of IL-1β, and can promote an M1- to M2-transition in macrophages, our data implicate a protective role of retinoic acid at this site.

Autophagic vacuoles in muscle fibers are a characteristic finding in several muscle diseases and usually indicate perturbed lysosomal protein degradation. Some of these are associated with defects in proteins directly involved in autophagy and lysosomal degradation. The gene CLN8 encodes an endoplasmic reticulum transmembrane protein, previously associated with childhood-onset neuronal ceroid lipofuscinosis (NCL), a group of lysosomal storage diseases. We describe the clinical features and results from pathology, genetic, and proteomic investigations in an adult-onset myopathy with autophagic vacuoles associated with biallelic variants in CLN8. A 40-year-old woman presented with seizures followed by transient muscle weakness and myalgia. Creatine kinase and myoglobin levels were moderately elevated. Over time, she developed progressive muscle weakness and cognitive fatigue. Muscle biopsy showed an autophagic vacuolar myopathy with fat tissue replacement and increased interstitial connective tissue. There was a marked immunohistochemical increase of markers of autophagy such as lysosomal-associated membrane protein 2 (LAMP2), microtubule-associated protein 1A/1B-light chain 3 (LC3), and sequestosome1/p62, as well as lysosomal deposition of curvilinear-like, autofluorescent material containing subunit c of mitochondrial adenosine triphosphate (ATP) synthase (mitochondrial ATP synthase membrane subunit c locus 3 [ATP5MC3/SCMAS]), typical for some forms of NCLs, including CLN8. Blood lymphocytes showed typical fingerprint inclusions. Genetic analysis revealed biallelic CLN8 variants, c.511C>T; p.P171S and c.536T>A; p.L179H. Proteomic analysis demonstrated upregulation of proteins involved in autophagy, muscle regeneration, and protein turnover. Proteins associated with oxidative phosphorylation were downregulated, except for ATP5MC3/SCMAS, which showed accumulation. In conclusion, we describe a novel myopathy with autophagic vacuoles and characteristic features of ceroid lipofuscinosis, including autophagosomal/lysosomal deposition of curvilinear-like, autofluorescent material containing ATP5MC3/SCMAS. This disease appears to be an unusual adult-onset form of CLN8.

Patient-derived tumor organoids are widely used in cancer research, yet the biological impact of tissue processing during model generation remains unclear. Fragment-based and dissociation-based (DIS) approaches are commonly assumed to trade fidelity for uniformity, but their molecular consequences remain incompletely defined. To address this gap, we performed a proteome-wide comparison of fragment-based (cut-and-culture [CUT]) and DIS glioblastoma organoid protocols using quantitative mass spectrometry. Organoids from multiple patient tumors were cultured under growth factor-free or growth factor-supplemented conditions and compared with matched primary tissue. Results show that both protocols produced technically robust glioblastoma organoids when maintained in their native media. However, CUT organoids matched the reproducibility of DIS cultures while preserving a broader extracellular matrix (ECM) repertoire and networks linked to collagen assembly, vascular support, and cell-matrix signaling. DIS cultures were biased toward exogenous basement membrane components and proliferative, growth factor-responsive states. Across tumors, CUT organoids consistently showed greater proteomic similarity to matched primary tissue, retaining neural, glial, stromal, and extracellular features largely absent from DIS models. Taken together, fragment-based glioblastoma organoids can be both reproducible and biologically faithful. Tissue dissociation acts as a major perturbation that reshapes ECM organization, cellular states, and tumor identity, making protocol choice a critical determinant of model fidelity and translational relevance.

A meaningful subset of IDH-mutant astrocytoma harbors ATRX missense variants that retain ATRX IHC immunoreactivity yet exhibit functional ALT activation, supporting the biologic relevance of these missense mutations.

This study investigated the clinicopathological characteristics and molecular spectrum of amyloid-beta (Aβ) in unnatural deaths among relatively young individuals with moderate-to-severe Aβ deposition, including cases of early-onset Alzheimer's disease. A total of 856 forensic autopsy cases aged 40-69 years were analyzed. Detailed semiquantitative and quantitative immunohistochemical analyses were performed in the neocortex, striatum (caudate nucleus, putamen, and nucleus accumbens [NAc]), and amygdala using antibodies targeting multiple Aβ species (Aβ38, Aβ39, Aβ40, Aβ42, Aβ43, pyroglutamate-modified Aβ at the third glutamate residue [AβNp3E], and serine 8-phosphorylated Aβ), as well as phosphorylated tau. Ward's hierarchical cluster analysis was also performed. Thirty-three cases (eight females; 3.9%), including six early-onset Alzheimer's disease cases, were identified. Three cases exhibited genetic abnormalities (one with Down syndrome and two with presenilin 1 [PSEN1] mutations). Marked regional heterogeneity in Aβ molecular profiles was observed, particularly within the striatum. Notably, the NAc displayed a distinct pattern of Aβ deposition and occasional NAc-predominant cerebral amyloid angiopathy. Cases with cognitive impairment (CI) demonstrated more advanced Aβ and tau pathology than CI-negative cases; notably, interstitial Aβ42 and Aβ43 burdens were significantly elevated across all regions. Strikingly, siblings carrying the same PSEN1 mutation-one with CI and the other without-exhibited divergent pathological patterns. Although suicide accounted for approximately one-third of cases, a higher neocortical Aβ burden was associated with a lower frequency of suicide, and no significant difference in suicide rates was observed compared with cases lacking substantial Aβ deposition. Cluster analysis identified a subgroup characterized by relatively elevated striatal Aβ deposition, particularly AβNp3E. Collectively, these findings indicate that the severity of Aβ deposition is not directly associated with suicide. However, early involvement of the NAc may contribute to depressive states, and region-specific Aβ species-particularly Aβ42, Aβ43, and AβNp3E-may be associated with increased cognitive vulnerability in this age group.

BeadChip array-based DNA methylation profiling has been recognized by the World Health Organization (WHO) as a key diagnostic tool for brain tumor classification. While its diagnostic utility has been well established, data on technical reproducibility, interlaboratory comparability, and data interpretation under real-world diagnostic conditions remain limited. Bridging this gap, we here report the results of an international proficiency test using the Infinium MethylationEPIC v2.0 platform and the corresponding Brain Tumor Classifier version 12.8. Tissue slides of eight FFPE brain tumor samples, covering a representative range of CNS tumor entities, were distributed among 24 laboratories in 10 different countries. Participants were asked to report methylation classes and copy number variation (CNV) profiles. Pre-array workflows were left to local procedures and results had to be submitted within 15 working days. Technical data reproducibility was high with a median pairwise beta-value correlation of 0.99 (range 0.93-1.0). In general, participating centers generated high-quality data, reflected by consistently low detection p-values (<0.01). Eighteen of the 24 participating centers (75%) successfully passed the test. Of the six centers that failed the test, two laboratories experienced technical issues that led to misclassification of individual cases and contributed to incorrect CNV reporting. Four additional centers showed substantial discrepancies in the interpretation of diagnostically highly relevant CNVs, whereas methylation classification was not impaired. While accurate DNA quantification proved to be an important pre-array step, the use of the DNA restoration kit had only minor influence on overall results. Taken together, our interlaboratory performance testing on EPIC v2.0 CNS tumor profiling confirms high reproducibility of tumor classification but reveals the need for harmonized CNV reporting.

We recently reported the deposition of Aβ in the frontal cortex of individuals who died of acute coronavirus disease 2019 (COVID-19), or who did not have COVID-19 but had respiratory distress, or infants with severe cardiac malformations. These Aβ deposits were not the senile, neuritic core "plaques" of Alzheimer's disease (AD) and did not stain for Thioflavin-S or with antibodies to phosphorylated tau protein. Here, we examined multiple sections of the brains of such individuals, finding these non-Alzheimer Aβ deposits (NADAs) predominantly in the isocortex, including frontal, temporal, insular, and occipital, but also in subcortical structures such as the diencephalon, cerebellum, brainstem, and spinal cord, albeit many fewer. NADAs also appeared around blood vessels and within blood vessel walls, but did not incite an inflammatory response. We also examined brains of individuals with metabolic and mitochondrial diseases that lead to hypoxic damage and one individual with severe hyperthermia. All had NADAs identical to the ones we previously reported. These Aβ deposits somewhat resemble the amorphous or diffuse plaques or deposits that have been thought to be associated with early stages of AD, but they are morphologically distinguishable and appear in brains of young individuals who have no Alzheimer pathology. We suggest that the deposits are associated with hypoxia or related mechanisms of injury.

Calcium dysregulation is increasingly recognized as a convergent mechanism underlying neuronal vulnerability and glial overactivation in Alzheimer's disease (AD). Transient Receptor Potential Canonical (TRPC) channels are potential key modulators of Ca[2+] signaling in multiple cell types in central nervous system (CNS), mediating different pathophysiological roles. However, their cell type-specific remodeling and cellular origins of these changes in human AD tissue remain poorly defined. This study investigated their expression patterns with main focus on the two closely related members of TRPC3 and TRPC6 across human AD brains and two relevant mouse models. Formalin-fixed paraffin-embedded cortical and hippocampal tissues from AD patients and age-matched controls were examined using immunohistochemistry. Spatial relationships between TRPC3/TRPC6 and glial fibrillary acidic protein (GFAP)-positive astrocytes were assessed in adjacent serial sections. TRPC3 expression was markedly increased in AD cortex and hippocampus whereas TRPC6 was significantly reduced primarily in pyramidal neurons. TRPC3-positive regions showed close spatial correspondence with reactive astrocytes, particularly in the hippocampal and subcortical white matter regions, suggesting a partial astrocytic origin. TRPC6 exhibited negligible overlap with GFAP. These observations were reproduced in brain sections of both 5xFAD and PS19 transgenic (Tg) mice compared to their littermate controls. Our findings reveal a conserved pattern of divergent TRPC remodeling across human and mouse models with AD pathology. In addition, TRPC1 expression was significantly reduced in AD samples while TRPC4 and TRPC5 had no significant change in expression. Taken together, selected TRPC family members may undergo differential remodeling during AD pathogenesis, with TRPC3 and TRPC6 showing the most prominent and consistent alterations.

Lewy bodies (LBs), composed primarily of aggregated α-synuclein (α-syn), are the pathological hallmark of Lewy body diseases, including Parkinson's disease, Parkinson's disease dementia and dementia with Lewy bodies (DLB). Despite their diagnostic significance, the mechanisms underlying LB formation and their contribution to neurodegeneration remain poorly understood. Previous studies have reported that structures labelled with ubiquitin phosphorylated at serine 65 (p-Ub[S65])-a marker of mitophagy-localise both at the periphery of and within LBs. This study aimed to further characterise these structures and clarify their relationship to α-syn pathology in DLB. Immunofluorescence analysis of post-mortem DLB brain tissue revealed intermittent co-localisation of p-Ub[S65+] puncta with conventional mitochondrial markers. Given previous findings that granulovacuolar degeneration bodies (GVBs)-vesicular structures frequently associated with tau pathology, itself a concomitant feature of DLB-are immunopositive for p-Ub[S65], we next examined whether peri-LB p-Ub[S65+] structures are actually GVBs. p-Ub[S65] puncta co-stained with established GVB markers including charged multivesicular body protein 2B (CHMP2B) and casein kinase 1 delta (CK1D), and, more variably with hyperphosphorylated tau (AT8), across multiple brain regions associated with LB pathology. Notably, GVB abundance within the anterior cingulate gyrus was found to positively correlate with p-α-syn[S129] burden. In summary, p-Ub[S65+] GVBs, containing both auto- and endolysosomal material, cluster around LBs in DLB. Frequently immunoreactive for tau, these hybrid waste vesicles provide a tangible mechanistic link between α-syn and tau pathology and may offer new insight into the processes underlying LB formation in DLB.