PolySia is associated with GFAP-positive cells

BackgroundNeuronal injury in autoimmune encephalitis (AE) may involve antibodies or T cells, with or without complement activation. Cell-free DNA (cf-DNA), released during cell death, and the complement split product C4d may reflect underlying tissue damage and immune activation. This study examines cf-DNA and C4d levels in the CSF and plasma of AE patients, focusing on differences between paraneoplastic and non-paraneoplastic subtypes.MethodsThirty patients with AE (including paraneoplastic and non-paraneoplastic cases) and 18 healthy and disease controls were included. Total cf-DNA and cf-DNA integrity (cfDI), defined as the ALU-247/ALU-115 ratio, were measured in the CSF and plasma using quantitative polymerase chain reaction (qPCR). Interleukins IL-6 and IL-17A and the complement split product C4d were measured by ELISA. Clinical and radiological parameters were recorded.ResultsCSF cf-DNA levels were significantly elevated in AE patients compared to controls (p < 0.01). Patients with paraneoplastic AE showed higher cfDI values (p < 0.05), indicating a predominance of necrotic cell death. CSF C4d levels were markedly increased in AE patients, particularly those with tumors (p < 0.001). CSF C4d showed the highest diagnostic accuracy for detecting underlying tumors at AE diagnosis (AUC = 0.818). Elevated CSF ALU-115 levels (p = 0.025) were significantly associated with MRI-confirmed encephalitic lesions, while increased cfDI correlated with electroencephalogram abnormalities indicative of epileptiform activity, underscoring their potential as biomarkers of disease severity.ConclusionsElevated CSF levels of necrotic cf-DNA and the complement split product C4d reflect heightened CNS tissue injury and inflammatory activity in AE, particularly in paraneoplastic cases. These biomarkers may serve as useful tools for early diagnosis, disease monitoring, and subtype differentiation in AE.

As tissue-resident macrophages of the central nervous system parenchyma, microglia perform diverse essential functions during homeostasis and perturbations1. They primarily interact with neurons by means of synaptic engulfment and through the rapid elimination of apoptotic cells and non-functional synapses2. Here, by combining unbiased lipidomics and high-resolution spatial lipid imaging, deep single-cell transcriptome analysis and novel cell-type-specific mutants, we identified a previously unknown mode of microglial interaction with neurons. During homeostasis, microglia deliver the lysosomal enzyme β-hexosaminidase to neurons for the degradation of the ganglioside GM2 that is integral to maintaining cell membrane organization and function. Absence of Hexb, encoding the β subunit of β-hexosaminidase, in both mice and patients with neurodegenerative Sandhoff disease leads to a massive accumulation of GM2 derivatives in a characteristic spatiotemporal manner3. In mice, neuronal GM2 gangliosides subsequently engage the macrophage galactose-type lectin 2 receptor on microglia through N-acetylgalactosamine residues, leading to lethal neurodegeneration. Notably, replacement of microglia with peripherally derived microglia-like cells is able to break this degenerative cycle and fully restore central nervous system homeostasis. Our results reveal a mode of bidirectional microglia-neuron communication centred around GM2 ganglioside turnover, identify a microgliopathy and offer therapeutic avenues for these maladies.

The transcriptomic classification of primary colorectal cancer (CRC) into distinct consensus molecular subtypes (CMSs) is a well‐described strategy for patient stratification. However, the molecular nature of CRC metastases remains poorly investigated. To this end, this study aimed to identify and compare organotropic CMS frequencies in CRC liver and brain metastases. Compared to reported CMS frequencies in primary CRC, liver metastases from CRC patients were CMS4‐enriched and CMS3‐depleted, whereas brain metastases mainly clustered as CMS3 and rarely as CMS4. Regarding overall survival rates, CMS4 was the most favorable subtype for patients with hepatic lesions, followed by CMS1 and CMS2. The survival of patients with brain metastases did not correlate with CMS. However, we identified a CMS3‐related metabolic gene signature, specifically upregulated in central nervous system (CNS)‐infiltrating CRC, as a negative prognostic marker and potential tumor progressor. In summary, subtyping of CRC metastases revealed an organotropic CMS distribution in liver and brain with impact on patient survival. CNS‐infiltrating CRC samples were enriched for CMS3 and predictive metabolic biomarkers, suggesting metabolic dysregulation of CRC cells as a prerequisite for metastatic colonization of the brain.

Mild Malformation of Cortical Development with Oligodendroglial Hyperplasia in Epilepsy (MOGHE) is a recently described disease entity primarily affecting young children with drug-resistant epilepsy, mainly affecting the frontal lobe. The condition is histopathologically defined by focal lesions with patchy areas of increased oligodendroglial cell density at the grey-white matter boundary and heterotopic neurons in the white matter. Approximately half of the individuals with MOGHE carry brain somatic variants in the SLC35A2 gene, which affects the UDP-galactose transporter and thus sphingolipid glycosylation. To investigate the impact of SLC35A2 variants on protein expression, we analysed MOGHE brain tissue with and without SLC35A2 mosaicism, distinguishing missense from nonsense variants. We developed an antibody targeting the N-terminus of the SLC35A2 galactose transporter and applied it for immunofluorescence (IF) analyses in a MOGHE cohort comprising 59 genetically tested individuals selected from three centres in Germany. The cohort included 13 individuals with SLC35A2 missense variants and 15 with SLC35A2 nonsense variants. Our findings confirm the localisation of the SLC35A2 protein in the Golgi apparatus of all neuroepithelial cell types as well as within Golgi outposts along oligodendroglial processes. The protein distribution was altered in MOGHE samples dependent on the SLC35A2 variant and its allelic frequency. Western blot and IF analyses revealed a significant SLC35A2 reduction in MOGHE tissues carrying nonsense variants. Ultrastructural analyses from three MOGHE samples demonstrated hypomyelination in regions with increased oligodendroglial cell densities, regardless of the harbouring of SLC35A2 variants. Notably, this hypomyelination pattern decreased with age. These results suggested a role for the SLC35A2 protein in the pathogenesis of MOGHE and indicated the presence of additional myelin-associated pathomechanisms in those individuals who do not carry a pathogenic SLC35A2 variant.

Background and PurposeFGF, VEGFR-2 and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG35-55-induced experimental autoimmune encephalomyelitis (EAE) prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSFR and VEGFR-2 by fexagratinib (formerly known as AZD4547) may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model.Experimental ApproachFemale C57BL/6J mice were treated with fexagratinib (6.25 or 12.5 mgkg-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (Day 18/20 post immunization) and the chronic phase of EAE (Day 41/42).Key ResultsIn the prevention experiment, treatment with 6.25 or 12.5 mgkg-1 fexagratinib prevented severe first clinical episodes by 66.7% or 84.6% respectively. Mice treated with 12.5 mgkg-1 fexagratinib hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, fexagratinib resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased by both experimental approaches.Conclusion and ImplicationsMulti-kinase inhibition by fexagratinib in a well-tolerated dose of 1 mgkg-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

Background:Radiation necrosis is a recognized complication following radiotherapy for primary brain tumors, presenting diagnostic and therapeutic challenges, and potentially masquerading as tumor recurrence. This study aims to delineate the clinical trajectory, management strategies, and outcomes of histologically confirmed radiation necrosis in patients treated for primary brain tumors. Methods:We conducted a retrospective review of patients who underwent surgical intervention for suspected tumor recurrence at our institution between 2010 and 2022, following adjuvant radiotherapy. Cases with histopathologically confirmed radiation necrosis were identified and analyzed for onset, clinical symptoms, radiological features, correlation with radio- and chemotherapy, management approaches, and disease progression. Results:Out of 276 patients operated for suspected recurrent brain tumors, 14 (5%) were histopathologically diagnosed with radiation necrosis. The latency period from radiotherapy to diagnosis ranged from 3 to 40 months. Notably, patients with oligodendrogliomas exhibited a significantly higher incidence of radiation necrosis (26%), underscoring a substantial risk association (P < 0.001). Conversely, the rates of radiation necrosis in patients with glioblastoma and astrocytoma (WHO grade II and III) were lower, at 2% and 0%, respectively, suggesting a lower risk association (P < 0.001 and P = 0.036, respectively). The majority (79%) of these patients were asymptomatic and exhibited a favorable clinical course, with most cases showing no progression of necrosis. During the follow-up period, tumor recurrence was verified in 2 patients. Conclusion:Radiation necrosis post-radiotherapy for primary brain tumors occurs infrequently but predominantly in patients with oligodendrogliomas, often following a benign course. The study underscores the importance of close monitoring for this condition, given the potential for sampling errors and the critical need for histopathological confirmation to guide appropriate management.

This study leverages x-ray phase-contrast tomography (XPCT) for detailed analysis of neurodegenerative diseases, focusing on the three-dimensional (3D) visualization and quantification of neuropathological features within fixed human postmortem tissue. XPCT with synchrotron radiation offers micrometer and even sub-micron resolution, enabling us to examine intra- and extraneuronal aggregates and inclusions such as Lewy bodies (LBs), granulovacuolar degeneration (GvD), Hirano bodies (HBs), neurofibrillary tangles (NFTs), β-amyloid plaques, and vascular amyloid deposits in three dimensions. In the reconstructions, we identified the highest electron densities in Hirano and LBs, while NFTs exhibited no significant increase in XPCT contrast. Using cutting-edge high-resolution x-ray synchrotron beamlines, we were now able to detect even detect subcellular differences in electron densities found in GvD. Small-scale inhomogeneities of the electron density were also detected in LBs, potentially relating to inclusions of organelles. Additionally, we reveal here a peculiar 3D geometry of HBs and demonstrate the co-occurrence with GvD in the same neuron. These findings underscore the potential of XPCT as a powerful, label-free tool for spatially resolved neuropathological investigations, opening new avenues for the systematic 3D characterization of inclusions and aggregates in neurodegeneration.

Progressive neurological decline in multiple sclerosis is associated with axonal loss and synaptic dysfunction in the non-demyelinated normal appearing gray matter (NAGM) and prominently in the cerebellum. In contrast to early disease stages, where synaptic and neuro-axonal pathology correlates with the extent of T cell infiltration, a prominent role of the innate immune system has been proposed for progressive MS. However, the specific contribution of microglia and astrocytes to synaptic cerebellar pathology in the NAGM- independent of an adaptive T cell response - remains largely unexplored. In the present study, we quantified synaptic changes in the cerebellar NAGM distant from demyelinated lesions in a mouse model of toxic demyelination. Proteomic analysis of the cerebellar cortex revealed differential regulation of synaptic and glutamate transport proteins in the absence of evident structural synaptic pathology or local gray matter demyelination. At the functional level, synaptic changes manifested as a reduction in frequency-dependent facilitation at the parallel fiber- Purkinje cell synapse. Further, deficiency of MyD88, an adaptor protein of the innate immune response, associated with a functional recovery in facilitation, reduced changes in the differential expression of synaptic and glutamate transport proteins, and reduced transcription levels of inflammatory cytokines. Nevertheless, the characteristics of demyelinating lesions and their associated cellular response were similar to wild type animals. Our work brings forward an experimental paradigm mimicking the diffuse synaptic pathology independent of demyelination in late stage MS and highlights the complex regulation of synaptic pathology in the cerebellar NAGM. Moreover, our findings suggest a role of astrocytes, in particular Bergmann glia, as key cellular determinants of cerebellar synaptic dysfunction.

Supplementary Table S1.Demographic and clinical parameters of GB patient cohorts.Supplementary Table S2.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for the presence of a functional SIGLEC16 allele (A) or for the occurrence of polySia on tumor cells (B).Supplementary Table S3.Cox proportional hazards regression analysis of covariates for SIGLEC16 and polySia status in relation to OS (MHH and UMG cohorts combined).Supplementary Table S4.Median age of GB cases stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S5.OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined).Supplementary Table S6.OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for SIGLEC16 genotype and polySia on tumor cells (MHH cohort only).Supplementary Table S7.OS analysis of GB cases receiving gross total resection and chemotherapy stratified for SIGLEC16 genotype and polySia on tumor cells (MHH and UMG cohorts combined, n=101).Supplementary Table S8.OS analysis of IDH-wildtype GB cases (UMG cohort) stratified for SIGLEC16 genotype and polySia on tumor cells.Supplementary Table S9.OS analysis of UMG cohort stratified for MGMT gene promoter methylation and SIGLEC16 genotype, polySia on tumor cells, CD163 and CD74 status.Supplementary Table S10.Characteristics of the two cohorts of n = 70 (MHH) and n = 100 (UMG) GB patients stratified for CD74low and CD74high.Supplementary Table S11. Median age at resection of GB cases stratified for CD163 to CD68 (A) or CD74 to IBA1 ratios (B) alone or in combination with the SIGLEC16 genotype.Supplementary Table S12. OS analysis of GB cases receiving (A) or not receiving gross total resection (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined).Supplementary Table S13. OS analysis of GB cases receiving (A) or not receiving chemotherapy (B) stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH cohort only).Supplementary Table S14.OS analysis of GB cases receiving both gross total resection and chemotherapy stratified for CD163 to CD68 or CD74 to IBA1 ratios alone or in combination with the SIGLEC16 genotype (MHH and UMG cohorts combined, n=101).

Mature astrocytes become activated upon non-specific tissue damage and contribute to glial scar formation. Proliferation and migration of adult reactive astrocytes after injury is considered very limited. However, the regenerative behavior of individual astrocytes following selective astroglial loss, as seen in astrocytopathies, such as neuromyelitis optica spectrum disorder, remains unexplored. Here, we performed longitudinal in vivo imaging of cortical astrocytes after focal astrocyte ablation in mice. We discovered that perilesional astrocytes develop a remarkable plasticity for efficient lesion repopulation. A subset of mature astrocytes transforms into reactive progenitor-like (REPL) astrocytes that not only undergo multiple asymmetric divisions but also remain in a multinucleated interstage. This regenerative response facilitates efficient migration of newly formed daughter cell nuclei towards unoccupied astrocyte territories. Our findings define the cellular principles of astrocyte plasticity upon focal lesion, unravelling the REPL phenotype as a fundamental regenerative strategy of mature astrocytes to restore astrocytic networks in the adult mammalian brain. Promoting this regenerative phenotype bears therapeutic potential for neurological conditions involving glial dysfunction.

Background Ultra-high-resolution magnetic resonance imaging of the ex vivo brain is increasingly becoming an indispensable tool for studying the morphology and potential pathology of the brain. Despite the important role of the cerebellum in nervous system functions and motor control, as well as its potential damage in neurological diseases, it remains relatively understudied compared to other brain regions. One major reason is the even finer structures.Methods A balanced steady state free precession approach with receiver bandwidths as low as 50Hz/pixel and long repetition times of 36ms is suggested and optimized, called “LoBa-bSSFP”, which enhances the signal-to-noise ratio and alleviates strain on the gradient system for the ultra-high spatial resolutions. A radiofrequency phase cycle scheme is used to reduce potential artifacts. Only 3T MRI standard equipment is utilized for acquisition and basic image reconstruction of the ex vivo brain immersed in perfluoropolyether.Results The presented LoBa-bSSFP approach provides images with very good soft tissue contrast and a detailed visualization of cerebellar morphology. It enables isotropic resolutions of 98-microns for the entire cerebellum, a further refinement allows even up to 77-microns isotropic on a purely clinical MR system. The acquisitions preserved the integrity of the ex vivo cerebellum, so maintaining its connection to the cerebrum and brainstem.Conclusions Our findings demonstrate the feasibility of employing 3T based LoBa-bSSFP for true ultra-high-resolution ex vivo imaging of the cerebellum, reaching resolutions up to 77-microns isotropic and the potential to reveal subtle microscopic abnormalities of the cerebellar cortex. LoBa-bSSFP may be superior to conventional FLASH sequences in terms of acquisition efficiency and - in some cases - even contrast.### Competing Interest StatementThe authors have declared no competing interest.* bSSFP : balanced SSFP FLASH : fast low-angle shot GM : gray matter LoBa : Low-Bandwidth URI : ultra-high-resolution imaging WM : white matter

Age-related myelin damage induces inflammatory responses, yet its involvement in Alzheimer's disease remains uncertain, despite age being a major risk factor. Using a mouse model of Alzheimer's disease, we found that amyloidosis itself triggers age-related oligodendrocyte and myelin damage. Mechanistically, CD8+ T cells promote the progressive accumulation of abnormally interferon-activated microglia that display myelin-damaging activity. Thus, our data suggest that immune responses against myelinating oligodendrocytes may contribute to neurodegenerative diseases with amyloidosis. The involvement of age-related myelin damage in Alzheimer's disease is unclear. Kedia et al. show that T cell-mediated microglia activation triggers a response against myelinating oligodendrocytes contributing to neurodegenerative diseases with amyloidosis.

Abstract The majority of patients with Parkinson disease (PD) experience a loss in their sense of smell and accumulate insoluble α-synuclein aggregates in their olfactory bulbs (OB). Subjects affected by a SARS-CoV-2-linked illness (COVID-19) also frequently experience hyposmia. We previously postulated that microglial activation as well as α-synuclein and tau misprocessing can occur during host responses following microbial encounters. Using semiquantitative measurements of immunohistochemical signals, we examined OB and olfactory tract specimens collected serially at autopsies between 2020 and 2023. Deceased subjects comprised 50 adults, which included COVID19 + patients (n = 22), individuals with Lewy body disease (e.g., PD; dementia with Lewy bodies (n = 6)), Alzheimer disease (AD; n = 3), and other neurodegenerative disorders (e.g., progressive supranuclear palsy (n = 2); multisystem atrophy (n = 1)). Further, we included neurologically healthy controls (n = 9), and added subjects with an inflammation-rich brain disorder as neurological controls (NCO; n = 7). When probing for microglial and histiocytic reactivity in the anterior olfactory nuclei (AON) by anti-CD68 immunostaining, scores were consistently elevated in NCO and AD cases. In contrast, microglial signals on average were not significantly altered in COVID19 + patients relative to healthy controls, although anti-CD68 reactivity in their OB and tracts declined with progression in age. Mild-to-moderate increases in phospho-α-synuclein and phospho-tau signals were detected in the AON of tauopathy- and synucleinopathy-afflicted brains, respectively, consistent with mixed pathology, as described by others. Lastly, when both sides were available for comparison in our case series, we saw no asymmetry in the degree of pathology of the left versus right OB and tracts. We concluded from our autopsy series that after a fatal course of COVID-19, microscopic changes in the rostral, intracranial portion of the olfactory circuitry -when present- reflected neurodegenerative processes seen elsewhere in the brain. In general, microglial reactivity correlated best with the degree of Alzheimer’s-linked tauopathy and declined with progression of age in COVID19 + patients.

Background and Purpose: Fibroblast Growth Factor (FGF), VEGFR2, and CSF1R signalling pathways play a key role in the pathogenesis of multiple sclerosis (MS). Selective inhibition of FGFR by infigratinib in MOG35-55-induced EAE prevented severe first clinical episodes by 40%; inflammation and neurodegeneration were reduced, and remyelination was enhanced. Multi-kinase inhibition of FGFR1-3, CSF1R and VEGFR2 by AZD4547 may be more efficient in reducing inflammation, neurodegeneration and regeneration in the disease model. Experimental Approach: Female C57BL/6J mice were treated with AZD4547 (6.25 mg kg-1 or 12.5 mg kg-1) orally or placebo over 10 days either from time of EAE induction (prevention experiment) or onset of symptoms (suppression experiment). Effects on inflammation, neurodegeneration and remyelination were assessed at the peak of the disease (day 18/20 p.i.) and the chronic phase of EAE (day 41/42 p.i.). Key Results: In the prevention experiment, treatment with AZD4547 prevented severe first clinical episodes by 66.7 or 84.6% respectively. Mice treated with 12.5 mg kg-1 of AZD4547 hardly showed any symptoms in the chronic phase of EAE. In the suppression experiment, treatment with AZD4547 resulted in a long-lasting reduction of severe symptoms by 91 or 100%. Inflammation and demyelination were reduced, and axonal density, numbers of oligodendrocytes and their precursor cells, and remyelinated axons were increased in both experimental approaches. Conclusion and Implications: Multi-kinase inhibition by AZD4547 in a well-tolerated dose of 1 mg kg-1 in humans may be a promising approach to reduce inflammation and neurodegeneration, to slow down disease progression and support remyelination in patients.

Neurological symptoms, including cognitive impairment and fatigue, can occur in both the acute infection phase of coronavirus disease 2019 (COVID-19) and at later stages, yet the mechanisms that contribute to this remain unclear. Here we profiled single-nucleus transcriptomes and proteomes of brainstem tissue from deceased individuals at various stages of COVID-19. We detected an inflammatory type I interferon response in acute COVID-19 cases, which resolves in the late disease phase. Integrating single-nucleus RNA sequencing and spatial transcriptomics, we could localize two patterns of reaction to severe systemic inflammation, one neuronal with a direct focus on cranial nerve nuclei and a separate diffuse pattern affecting the whole brainstem. The latter reflects a bystander effect of the respiratory infection that spreads throughout the vascular unit and alters the transcriptional state of mainly oligodendrocytes, microglia and astrocytes, while alterations of the brainstem nuclei could reflect the connection of the immune system and the central nervous system via, for example, the vagus nerve. Our results indicate that even without persistence of severe acute respiratory syndrome coronavirus 2 in the central nervous system, local immune reactions are prevailing, potentially causing functional disturbances that contribute to neurological complications of COVID-19.