Journal of Neuroinflammation

Although the brain was traditionally considered immune-privileged, recent studies show immune factors play key roles in brain function. Dysfunction of these factors is linked to neurodevelopmental disorders, but mechanisms remain unclear. Using a maternal immune activation (MIA) mouse model, we investigated immune-related genes in neurodevelopmental disorder pathogenesis. MIA mice showed increased locomotor activity and disrupted prepulse inhibition. RNA-seq and qPCR analyses revealed persistent increases in major histocompatibility complex class II (MHCII) expression and persistent decreases in GABAergic synapse-related gene expression, particularly glutamate decarboxylase (Gad) expression, in dopaminergic regions. These expressions were negatively correlated, and immunohistochemistry showed MHCII at postsynaptic GABAergic synapses on dopaminergic neurons. Patch-clamp recordings confirmed reduced mIPSC frequency in MIA mice. MHCII knockout mice showed opposite phenotypes, while MHCII overexpression in dopaminergic neurons decreased Gad expression. These results suggest MIA-induced MHCII upregulation enhances pruning of GABAergic synapses on dopaminergic neurons, leading to behavioral deficits.

Introduction: Gliomas integrate into neural circuits and heighten neuronal excitability, engaging in bidirectional communication whereby neuronal activity promotes tumor growth and proliferation. Aging reshapes the brain microenvironment through extracellular matrix changes, altered secretory factors, and immune dysfunction, creating conditions permissive to tumorigenesis and limiting immunotherapy efficacy in glioblastoma. However, its effect on neuronal excitability and signaling in glioblastoma remains poorly understood. Methods: We developed a novel classification system for glioblastoma by leveraging three classes of DNA methylation-based aging biomarkers: chronological, biological, and mitotic clocks. This approach stratified tumors into accelerated and decelerated epigenetic aging subtypes, which we then characterized at the molecular, functional, and clinical levels using multimodal analyses. Guided by these profiles, we evaluated the in vitro effects of the FDA-approved agents levetiracetam and riluzole, alone and in combination with temozolomide, on U87MG and A172 cell lines. Specifically, we assessed changes in cell viability, apoptosis, and the expression of marker genes related to stemness, neuronal hyperexcitability, and immunosuppression. Results: Tumors with decelerated epigenetic aging showed expression modules and CpG hypomethylation associated with neuronal activity and stemness, and carried significantly worse prognosis. Single-cell and spatial multi-omics analyses revealed enrichment for neurons and malignant neural stem-like cells in these tumors. They also displayed enhanced intercellular communication, driven predominantly by glutamate signaling across the malignant, neuronal, and immune compartments of the tumor microenvironment. In vitro pharmacological inhibition of glutamatergic signaling with levetiracetam and riluzole reduced cell viability, induced apoptosis, and suppressed expression of stemness, neuronal hyperexcitability, and immunosuppression markers. Both agents potentiated the cytotoxic and apoptotic effects of temozolomide, supporting glutamatergic inhibition as a strategy for improving chemosensitivity. Conclusion: By establishing a framework for decoding glioblastoma heterogeneity through epigenetic aging, we identified the glutamatergic pathway as a clinically actionable vulnerability. Our findings suggest that combining anti-glutamatergic therapies with temozolomide exerts synergistic antitumor effects while mitigating adverse chemotherapy-induced phenotypes, such as increased stemness, neuronal hyperexcitability, and immunosuppression, thereby laying the groundwork for novel therapeutic strategies.

Viral infections have long been proposed as environmental contributors to neurodegenerative diseases, including Parkinson's disease (PD), yet the molecular mechanisms linking infection and neurodegeneration are not well defined. Neuroinflammation and disruption of central nervous system (CNS) homeostasis have emerged as potential mediators. In this study, we used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, as a model pathogen to investigate convergent molecular pathways between viral infection and PD. Single-nucleus RNA sequencing (snRNA-seq) was performed on post-mortem striatal tissue from 14 individuals stratified into four groups: COVID-19 only (COVID-19), PD only (PD), comorbid PD with COVID-19 (PD/COVID-19), and controls (Control). The PD/COVID-19 group exhibited an expanded astrocytic population and a pronounced interferon-associated molecular signature characterized by increased expression of canonical interferon-stimulated genes, including IFI44L (average log2FC= 3.9; adjusted p=2.3 x 10-373), IFI44 (average log2FC=2.9; adjusted p=8.0 x 10-266), ISG15 (average log2FC=3.1; adjusted p=1.2 x 10-197), and RSAD2 (average log2FC= 3.5; adjusted p=8.6 x 10-111). Pathway analyses demonstrated activation of innate immune and antiviral signaling pathways, particularly within microglia and astrocytes, including interferon signaling, pattern-recognition receptor pathways, and complement-associated responses. In parallel, genes involved in lipid metabolism, cholesterol homeostasis, synaptic maintenance, and neuronal signaling were reduced across disease groups. Proteomic analyses independently confirmed enrichment of antiviral and interferon-associated pathways and identified convergent suppression of sterol, cholesterol, and lipid metabolic processes. Our findings identify a convergent molecular signature linking PD and COVID-19, pronounced in comorbid individuals and characterized by interferon-driven innate immune activation, glial inflammatory responses, and dysregulation of lipid metabolic homeostasis. Collectively, the data support a model in which severe viral infection amplifies biological pathways already implicated in PD pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive dysfunction and loss of upper and lower motor neurons. Although motor neuron degeneration ultimately drives paralysis, neuronal dysfunction may precede cell death by a prolonged interval, suggesting that vulnerable neurons engage stress-adaptive programs that permit survival despite impaired function. Cellular senescence represents one such persistent stress response and has increasingly been implicated in neurodegenerative disease, including disorders associated with TDP-43 pathology. Here, we investigated whether senescence-associated molecular states are present in vulnerable motor neurons in ALS and whether they differ according to anatomical region and phosphorylated TDP-43 (pTDP-43) pathology. Postmortem primary motor cortex, cervical spinal cord, and lumbar spinal cord were obtained from the Department of Veterans Affairs Biorepository Brain Bank from individuals with ALS classified as pTDP-43-positive or pTDP-43-negative, together with non-ALS controls. Targeted bulk transcriptomic profiling was combined with GeoMx Digital Spatial Profiling of individual motor neurons to characterize disease-, region-, and pathology-associated molecular phenotypes while preserving anatomical context. Across ALS cases, we identified alterations in pathways related to cell-cycle regulation, RNA processing, mitochondrial function, proteostasis, inflammation, and synaptic signaling. These signatures varied by anatomical region and pTDP-43 status, indicating substantial heterogeneity in the molecular response to ALS pathology. Despite these differences, both ALS groups exhibited convergent proteomic and transcriptomic features associated with cellular senescence. These findings identify senescence-associated molecular states within vulnerable neuronal populations in ALS and support a model in which persistent stress adaptation may permit neuronal survival while contributing to progressive cellular dysfunction. This spatially resolved analysis links neuronal phenotype to anatomical and pathological context and supports further evaluation of senescence-associated pathways as therapeutic vulnerabilities in ALS.

Cholesterol efflux is an important determinant of cellular lipid homeostasis. However, how microglial excessive cholesterol accumulation affects neuronal synaptic integrity remains poorly understood, particularly in the context of Alzheimer's disease. Here, we utilized a conditional knockout mouse model targeting the cholesterol transporters ABCA1 and ABCG1 in microglia. The microglia-specific ABCA1/ABCG1 deficiency triggered marked cholesterol accumulation, microglial hypertrophy, downregulation of the homeostatic marker P2ry12, and upregulation of the reactivity-associated marker CD11b, indicating shift toward a reactive phenotype. This phenotype was accompanied by increased reactive oxygen species, consistent with enhanced oxidative stress in ABCA1/ABCG1-deficient microglia compared with control. Using organotypic hippocampal slice cultures, we investigated the downstream neuronal outcomes of microglial ABCA1/ABCG1 deficiency. Under basal conditions, microglial ABCA1/ABCG1 knockdown did not significantly alter dendritic spine density in CA1 pyramidal neurons. However, upon exposure to amyloid-beta (A{beta}) stress, microglial ABCA1/ABCG1 deficiency markedly exacerbated dendritic spine loss in CA1 pyramidal neurons. Taken together, our findings highlight an important role for ABCA1/ABCG1-dependent cholesterol efflux in maintaining microglial homeostasis and limiting neuronal synaptic vulnerability to A{beta}-associated stress. These results support further investigation of microglial cholesterol transport as a potential target for preserving synaptic resilience in Alzheimer's disease.

Background: Spatial organization is increasingly recognized as a key determinant of tumor-immune interactions in head and neck squamous cell carcinoma (HNSCC). The GSE300147 Xenium spatial transcriptomic resource generated by McCord and colleagues established a framework for mapping spatially coordinated T-cell states in HNSCC. However, how tumor-enriched epithelial immune states relate to metabolic, redox, and stress-adaptive transcript programs remains incompletely defined. Methods: A secondary, data-driven reanalysis of GSE300147 was performed, focusing on 17 confirmed HNSCC Xenium sections after exclusion of a non-HNSCC ameloblastoma specimen. A total of 1,148,244 cells were analyzed, including 558,867 EpCAM+ tumor-enriched epithelial cells. Tumor-enriched epithelial cells were classified into Hot, Intermediate, and Cold states using a Composite Hotness framework integrating T-cell inflammatory signature score, checkpoint-associated signaling, CD274 expression, IFN/antigen-presentation signature score (IFN/AP), and tumor-immune proximity. Six metabolic ecosystem states, neighborhood profiling, spatial permutation testing, and an integrated Immune-Metabolic-Redox Ecosystem Score (IMRES) were then applied. Results: Immune activation was spatially heterogeneous across HNSCC sections. Immune-hot tumor-enriched epithelial regions showed not only inflammatory, checkpoint-associated, and antigen-presentation signature scores, but also coordinated metabolic, oxidative-redox, and stress-response transcript programs. IMRES, derived from available immune, metabolic, redox, and stress-response transcript components represented in the Xenium panel, increased progressively from Cold to Intermediate to Hot tumor-enriched epithelial states and was associated with NFE2L2, GDF15, HLA-DRA, CD274, KEAP1, and MDM2. Integrating IMRES with Composite Hotness identified a distinct Hot+IMREShigh ecosystem comprising 106,874 tumor-enriched epithelial cells. This state showed the strongest immune-active and stress-adaptive features and was positioned closer to immune populations than expected by random assignment. An alternative rank-based robustness analysis reproduced the IMRES-associated ecosystem axis and correlated with the original module-based score (Spearman r = 0.597). Conclusions: This secondary reanalysis extends the original spatial T-cell framework by defining a complementary tumor-centered immune-metabolic-redox ecosystem in HNSCC. IMRES provides a transcript-derived framework for identifying Hot+IMREShigh neighborhoods where immune activation, checkpoint signaling, metabolic remodeling, and stress adaptation converge, providing a hypothesis-generating framework for studying immune resistance and therapeutic vulnerability.

Tauopathies are characterized by the accumulation and spread of pathogenic tau aggregates throughout the brain, a process that is increasingly recognized to involve not only neurons but also microglia. However, whether pathogenic MAPT directly alters microglial degradative capacity remains poorly understood. Here, using isogenic human induced pluripotent stem cell-derived microglia carrying the pathogenic MAPT IVS10+16 mutation, we identify tau as a regulator of microglial lysosomal function. MAPT IVS10+16 microglia exhibited coordinated suppression of lysosomal and autophagic pathways, reduced lysosomal protease abundance and activity, and impaired autophagosome-lysosome fusion. Mutant microglia also showed reduced uptake of extracellular tau aggregates, reduced tau accumulation in acidic compartments, and a blunted lysosomal response to proteopathic stress. Conversely, genetic loss of MAPT increased lysosomal degradative capacity and accumulation of extracellular tau aggregates within acidic compartments, supporting a cell-intrinsic role for endogenous tau in regulating microglial degradative function. Pharmacologic enhancement of the autophagy lysosome pathway in MAPT IVS10+16 microglia increased proteolytic activity and improved tau handling. Together, these findings reveal a reciprocal relationship between tau and microglial lysosome function and identify degradative capacity as a modifiable component of the microglial response to tau pathology.

Background: Multiple sclerosis (MS) is a chronic neurodegenerative disorder in which inflammatory demyelinating lesions affect the brain, optic nerve and spinal cord. MS lesion formation is accompanied by profound changes to blood vessels, including the density of PDGFR{beta}+ mural cells, historically identified as pericytes. Intriguingly, in recent years, single-cell and lineage tracing studies have shown that the PDGFR{beta}+ cell population is heterogeneous, comprising both pericytes and perivascular fibroblasts. Yet, due to their overlapping expression profiles, the spatial distribution of these cell populations in MS lesions remains poorly understood. Methods: We employed multiplex immunohistochemistry for endothelial cells (CD31), basement membrane (laminin), fibroblasts (PDGFR{beta}, COL1A1, SMA), pericytes (PDGFR{beta}, SLC6A12) and immune cells (CD45, CD68) to characterize the spatial localization of fibroblasts and pericytes in MS lesions, and how this relates to perivascular space enlargement and immune cell presence. Results: We analysed 17633 individual vessels across 5 control white matter, 5 normal-appearing white matter, 4 active and 4 chronic active MS lesions. By carefully delineating endothelium and perivascular compartments, we find that perivascular space area but not number of vessels is increased in MS lesions. Through mining of publicly available sequencing datasets, we confirm COL1A1 and SLC6A12 as fibroblast and pericyte markers, respectively, in the human brain. COL1A1+ and SLCA12+ vessels were largely distinct of one another. Unsupervised clustering of the expression profile of PDGFR{beta}, COL1A1 and SLC6A12 in individual vessels distinguished three partially overlapping vessel clusters. Of these, the fibroblast-associated vessel type (COL1A1 high, SLC6A12 low) was increased in chronic active lesion rim and center. Importantly, fibroblast-associated vessels were related to increased perivascular space enlargement and more accumulation of immune cells. Conclusion: We identify distinct fibroblast- and pericyte-associated vascular phenotypes in human white matter. Notably, fibroblast-associated vessels are increased in chronic active lesions, where they are related to immune cell cuffs. These findings provide a spatial link between perivascular fibroblasts and chronic inflammation in MS.

Astrocyte border formation is a conserved neuroprotective response to neural tissue disruption, yet astrocyte border states at implanted biomaterials remain less well characterized than injury responses. Here, we developed the Astrocyte Border Characterization (ABC) Tool, which leverages a shear-thinning, injectable biomaterial to locally deliver astrocyte-specific RiboTag AAVs and small molecule regulators in the mouse striatum, enabling molecular profiling and phenotypic modulation of astrocyte border (AB) cells. Spatially precise delivery of AAV using the ABC Tool yielded enhanced specificity and robust RiboTag expression in AB cells from 7-70 days post injection. Temporal transcriptomic profiling of AB cells revealed predominantly acute, transient changes in genes governing dedifferentiation, proliferation, metabolic reprogramming, and inflammation regulation. Persistent changes accounted for only 14% of regulated genes but involved critical gain of functions in immune regulation and host defense that mirrored astrocyte border responses at chronic CNS injuries. Local delivery of indiscriminate or astrocyte-selective ablation molecules delayed, rather than prevented, border formation, ultimately yielding thicker astrocytes borders with increased inflammation and fibrosis at the biomaterial-tissue interface. Conversely, local delivery of {beta}-hydroxybutyrate (BHB) from the ABC Tool altered key aspects of the transcriptional reprogramming to attenuate chronic astrocyte reactivity and prevent biomaterial contraction without exacerbating inflammation or fibrosis. Our findings establish the ABC Tool as a bioassay for studying and manipulating astrocyte borders at implanted biomaterials and identify focal metabolic regulation as a strategy to modulate AB cell phenotypes and enhance the CNS biocompatibility of biomaterials.

Microglia are central mediators of Alzheimers disease (AD) pathogenesis, yet the mechanisms driving disease-associated microglial states and their therapeutic modulation remain poorly understood. Here, we integrated single-nucleus transcriptomic datasets across the AD spectrum and identified disease- and lipid-associated microglia (DLaM) as a major AD-enriched population linked to genetic risk, neuropathology and cognitive decline. To model this state experimentally, we screened AD-relevant perturbations in human induced pluripotent stem cell (hiPSC)-derived microglia and found that ferric ammonium citrate (FAC) reproducibly induced a DLaM-like state characterized by lipid accumulation, lysosomal dysfunction and impaired A{beta} phagocytosis. Using a transcriptomics-based state-reversion screen, we identified LY2090314 as a potent modulator that restored microglial function and induced a distinct lysosomal-metabolic state. These findings establish a framework for transcriptomic disease-state-guided therapeutic discovery in AD.

Amyotrophic lateral sclerosis (ALS) is a motor neuron disease, leading to progressive muscle weakness and motor impairment. Growing evidence indicates that cerebellar Purkinje cells, which play a central role in motor coordination, are also affected in ALS. However, it is unclear whether the molecular events that initiate neurodegeneration in these ALS-relevant motor-controlling neurons are shared or distinct. Here, we used a MATR3 S85C knock-in (KI) mouse model of early-stage ALS with stage-specific motor phenotypes and selective vulnerability of motor neurons and Purkinje cells to decipher the molecular events underlying neurodegeneration in these two neuronal populations. We found that a profound reduction in detectable MATR3 S85C immunoreactivity (hereafter referred to as MATR3 loss) in both motor neurons and Purkinje cells precedes the onset of motor dysfunction and neuropathology, implicating MATR3 loss as the earliest detectable molecular event. Our bulk cerebellar RNA profiling and motor neuron-specific RNA profiling data at the onset of MATR3 loss revealed distinct molecular signatures. In the cerebellum, Ngfr expression emerged in Purkinje cells before the onset of neuronal loss and remained elevated throughout the disease course. This increase was accompanied by activation of the JNK-mediated cell death pathway. In the motor neurons, elevated Fgf21 and integrated stress response (ISR) gene expression were the first to be observed and persisted throughout disease progression, consistent with previous findings in SOD1 mouse models. Our findings provide mechanistic insights into the initiation of neurodegeneration in ALS-relevant motor-controlling neurons and implicate potential neuron type-specific targets for future therapeutics.

Introduction: Parkinson's disease (PD) and aging both disrupt hypothalamic-pituitary-adrenal (HPA) axis function and peripheral immune homeostasis. Whether aging or -synuclein (-syn) pathology alters glucocorticoid (GC) sensitivity of peripheral immune cells has not been investigated. Methods: Using an ex vivo GC sensitivity assay, we assessed the responsiveness of isolated and lipopolysaccharide (LPS)-stimulated splenocytes to the anti-inflammatory effects of increasing doses of corticosterone (CORT) in a wild-type (WT) aging cohort and in a PD -syn transgenic mouse model and respective age-matched controls. Results: Compared with splenocytes from 6-month-old WT mice, splenocytes from 20-month-old WT mice were less sensitive to 0.1 and 0.5 M CORT. Isolated splenocytes from PD vs. control mice were less sensitive to 0.05, 0.1, and 0.5 M CORT specifically at 16 months of age, but not at 6 or 20 months of age. As peripheral immune phenotyping revealed neither differences in HPA axis-related parameters nor in splenic GC receptor expression between PD and age-matched control mice at 6, 16, and 20 months, splenic GC resistance in PD mice at 16 months of age seems to be mediated by downstream GR signaling dysfunction. Conclusion: Together, our results support the hypothesis that -syn pathology accelerates an aging-associated decline in the peripheral sensitivity to anti-inflammatory GCs and may thereby sustain systemic and neuroinflammatory processes in PD.

Background: Serial glial fibrillary acidic protein (GFAP) trajectories have become an important framework for contextualizing evolving secondary-injury pathophysiology after moderate-to-severe traumatic brain injury (msTBI). However, total GFAP pools release and clearance signals that may be less useful for longitudinal bedside decisions than a proteoform-resolved assay. We compared total GFAP with neoGFAP, defined here as calpain-generated GFAP proteoforms intended to index active astroglial proteolysis during the subacute phase. Methods: We analyzed 651 serial serum samples from 95 msTBI patients from a previously described single-site cohort. Total GFAP and neoGFAP were measured on the same MSD platform from 6 to 240 hours after injury. Early (6 to 72 h) and late (96 to 240 h) windows, data-derived tertiles, and serial trajectory summaries were calculated directly from serial samples. Models were benchmarked against age plus admission post-resuscitation Glasgow Coma Scale (GCS) and the admission IMPACT extended risk score using five-fold stratified cross-validation. Outcomes were unfavorable outcome (GOSE 1 to 4), less-than-good recovery (GOSE 1 to 6), Disability Rating Scale (DRS) [≥]15, mortality, and neuroimaging worsening at 6 months. Results: The cohort contributed 95 serial biomarker profiles, with 90 participants evaluable for 6-month GOSE and 89 for DRS. Unfavorable outcome occurred in 57/90 (63.3%), and less-than-good recovery in 79/90 (87.8%). For unfavorable outcome, IMPACT plus early neoGFAP reached AUROC 0.85 versus 0.84 for IMPACT plus early total GFAP and 0.81 for IMPACT alone. For less-than-good recovery, IMPACT plus late neoGFAP achieved AUROC 0.90 versus 0.84 for late total GFAP and 0.82 for IMPACT alone. Secondary analyses for DRS, mortality, and neuroimaging worsening showed smaller differences. Conclusions: In this retrospective analysis, neoGFAP provided clearer incremental value than total GFAP for recovery-oriented monitoring, especially when late-window reassessment of patients who remained at risk for less-than-good recovery was required. Results support prospective testing of neoGFAP as a pathophysiology-informed adjunct to serial bedside decision making, repeat-assessment thresholds, and recovery stratification.

Circadian disruption, notably sleep disturbances, serves as an early indicator of Alzheimers disease (AD), preceding cognitive symptoms like memory loss. The suprachiasmatic nucleus (SCN) governs biological rhythms and receives direct retinal input via melanopsin-expressing retinal ganglion cells (mRGCs) to synchronize with environmental light cycles. The anatomical and functional basis for circadian disruption in AD remains unclear. Here, we explored the multi-level relationships between gene expression, the SCN connectome, and regulations of sleep and circadian rhythms in the APP/PS1 mouse model. The sleep architecture of APP/PS1 mice displayed significantly reduced rapid eye movement sleep (REM), associated with a reduced daily core body temperature amplitude and locomotor hyperactivity. Lastly, APP/PS1 mice showed an impaired response to acute light pulse stimulation and present hyperactivity of mRGCs at a young age and hypoactivity of these cells at older ages. These physiological functions are known to be, at least in part, regulated by the SCN, the main target of mRGCs. We noted several modifications in SCN connectomics using serial blockface electron microscopy (SBEM), including a reduction of the dendro-dendritic chemical synapse (DDCS) network that receives a large part of the retinal input and is thought to be crucial for synchronicity between SCN neurons. In addition, we observed multiple signs of dystrophy, including modifications of the shape of dendrites and cell soma, accumulation of aggregated lysosomes, and swelling of axons. At the same time, we investigated the changes in gene expression using spatial transcriptomics. The SCN presents changes in the expression of genes associated with synapse formation, cell adhesion, and neurite growth. These results suggest that, despite the absence of amyloid plaques in the ventral hypothalamus, the SCN of APP/PS1 mice still undergo profound gene expression changes, impacting connectomics and physiological functions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/744599v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@ceedb0org.highwire.dtl.DTLVardef@156cfaaorg.highwire.dtl.DTLVardef@5bc262org.highwire.dtl.DTLVardef@36df4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microglia are the predominant immune cells in multiple sclerosis (MS) demyelinating lesions, where they phagocytose myelin, but whether they destroy myelin or merely scavenge its debris is unknown. Here, we explore whether pathogenic autoantibodies found in MS may induce the phagocytic destruction of myelin by microglia. Applying patient-derived, myelin-targeting antibodies to the mouse cortex, we developed an in vivo model of MS with focal demyelination that depended on epitope specificity and Fc gamma receptor and complement binding. Longitudinal monitoring of microglia-myelin interactions using in vivo two-photon microscopy revealed rapid microglial envelopment of intact myelin driving myelin loss, while single-cell RNA sequencing identified a demyelination-associated microglial signature. Parallel changes were observed in human MS lesions, where microglia enveloped intact myelin and similar genes were upregulated. Inhibition of Brutons tyrosine kinase (BTK) limited microglial transcriptional changes and prevented myelin loss following microglial envelopment. These findings directly implicate microglia in pathological myelin loss and support BTK inhibition as a therapeutic strategy to prevent demyelination by modulating microglia behavior.

Pathophysiological mechanisms underlying the transition from acute to chronic neuropathic pain remain incompletely understood. The somatosensory and insular cortices are key cortical components of the pain matrix. We examined changes in activation of these cortical regions during the transition from acute to chronic neuropathic pain. The right sciatic nerve was ligated in activity reporter TRAP mice using standard procedures. Mechanical allodynia was confirmed after CCI or sham surgery using von Frey monofilaments applied to the hind paws. To label active neurons, 4-hydroxytamoxifen was administered to separate cohorts at 1, 3, and 6 weeks following nerve ligation. Passive tissue clearing of brain sections and confocal imaging was used to assess active neurons. Progressive reduction of ipsilateral hind paw in CCI mice indicated mechanical allodynia development. CCI mice showed robust neuronal activation in the bilateral somatosensory and insular cortices. The somatosensory cortical activation peaked at 3 weeks post-CCI, whereas insular cortical activity increased during the transition from acute to chronic neuropathic pain. These studies revealed that CCI induced progressive mechanical allodynia and distinct temporal patterns of cortical neuronal activation, with transient peak neuronal activity in the somatosensory cortex and sustained, increasing activation in the insular cortex during acute-to-chronic pain transformation.

INTRODUCTION: Biomarkers for monitoring disease activity and treatment response in peripheral neuropathies remain limited. Big tau, a high-molecular-weight isoform of tau, is predominantly expressed in the peripheral nervous system (PNS). We investigated serum levels of big tau, brain-derived tau (BD-tau), and neurofilament light chain (NfL) in peripheral neuropathies, multiple sclerosis (MS), Alzheimer disease (AD), and healthy controls (HC). METHODS: Ultra-sensitive blood-based assays run on an HD-X Single Molecule Array analyser (Quanterix) were used to measure big tau and BD-tau in serum from patients with Guillain-Barr&eacute syndrome (GBS, n=81), Miller Fisher syndrome (MFS, n=20), Charcot-Marie-Tooth disease (CMT, n=102), chronic inflammatory demyelinating polyneuropathy (CIDP, n=43), MS (n=159), AD (n=20), and HC (n=41). NfL was measured in patients with neuropathies using an SR-X Single Molecule Array analyser (Quanterix). RESULTS: Serum big tau levels were higher in GBS than in AD (11.4 vs 2.4 pg/mL, p<0.0001) and MS (11.4 vs 9.0 pg/mL, p=0.01), and similar to CIDP and CMT. Contrarily, serum BD-tau levels in GBS were higher than in CIDP (3.0 vs 2.3 pg/mL, p=0.006) and MS (3.0 vs 1.7 pg/mL, p<0.0001), but similar to CMT, and lower than in AD (3.0 vs 9.8 pg/mL, p<0.0001). Serum NfL levels were higher in GBS than in CIDP (32.5 vs 13.0 pg/mL, p=0.0002), CMT (32.5 vs 12.3 pg/mL, p<0.0001), and HC (32.5 vs 7.6 pg/mL, p<0.0001). Compared with GBS, MFS patients showed higher BD-tau (12.7 vs 3.0 pg/mL, p=0.003), lower big tau (5.4 vs 11.4 pg/mL, p=0.002), and higher NfL levels, although the latter did not reach statistical significance (118.3 vs 32.5 pg/mL, p=0.16). The NfL/big tau ratio was significantly higher in MFS than in GBS, CIDP, and CMT. In GBS, BD-tau correlated with early clinical severity (MRC at 1 week; I-RODS at 4 weeks; maximum GBS-DS and GBS-DS at 4 weeks), whereas neither tau biomarker showed long-term clinical correlations. Higher BD-tau and big tau levels were associated with the need for mechanical ventilation (BD-tau: 8.6 vs 2.9 pg/mL, p=0.019; big tau: 19.7 vs 10.7 pg/mL, p=0.007), while higher BD-tau levels were associated with mortality (10.9 vs 2.9 pg/mL, p=0.003). CONCLUSIONS: Higher big tau levels in peripheral neuropathies than in CNS diseases support its role as a PNS-specific biomarker. In MFS, increased serum BD-tau, reduced big tau, and an elevated NfL/big tau ratio suggest CNS involvement with relative preservation of the PNS.

Service members with traumatic brain injury are at approximately two- to four-fold higher risk of Alzheimer's disease or related dementias than those without such an injury, with risk increasing with injury severity. The amyloid/tau/neurodegeneration biomarker framework treats amyloid, tau, and neurodegeneration as independent axes but omits astroglial injury, despite evidence that reactive astrogliosis (indexed by glial fibrillary acidic protein, GFAP) must be elevated for cognitive decline to occur in amyloid-positive individuals. Total GFAP immunoassays aggregate intact protein with multiple calpain- and caspase-cleaved proteoforms, blurring the biological signal. We compared a calpain-cleaved GFAP neoepitope, the glial fibrillary acidic protein neoepitope (neoGFAP), against total GFAP across the full traumatic brain injury--mild cognitive impairment--Alzheimer's disease continuum in Veterans using a two-stage plasma-to-cerebrospinal-fluid biomarker approach. A plasma triage gate combining phosphorylated tau 217 and amyloid beta 42 was applied to 367 unique subjects; a cerebrospinal-fluid benchmarking cohort of 57 subjects (controls, chronic blast traumatic brain injury, mild cognitive impairment, and Alzheimer's disease) received head-to-head neoGFAP and total GFAP measurement. In the whole benchmarking cohort, neoGFAP discriminated mild cognitive impairment plus Alzheimer's disease from non-Alzheimer subjects with an area under the receiver-operating-characteristic curve of 0.81 versus 0.73 for total GFAP, a trend-level advantage that did not reach nominal significance. Within the gate-positive, amyloid-committed subset of 23 subjects, neoGFAP dominance became significant by McNemar's exact test (six discordant subjects favored neoGFAP, none the reverse). Across diagnostic contrasts, neoGFAP outperformed total GFAP for Alzheimer's disease versus control and, importantly for Veterans, for mild cognitive impairment versus chronic blast-exposed Veterans without cognitive impairment. In chronic blast injury, neoGFAP was paradoxically depleted relative to controls, consistent with tissue sequestration of aggregated proteoform fragments. Unbiased proteomic profiling confirmed coordinated elevation across astrocytic, neuronal, mitochondrial, and microglial compartments. An exploratory subject-level reclassification improved accuracy from 71.1 percent using plasma alone to 79.5 percent with added cerebrospinal-fluid markers and age. In a same-cohort ProQuantum replication (n=57), CSF neoGFAP preserved its discrimination advantage over total GFAP for MCI+AD versus non-AD (AUROC 0.76 vs 0.72; cross-platform Spearman {rho}=0.84), while plasma neoGFAP achieved AUROC 0.90, comparable to pTau217 (0.92) and exceeding A{beta}42/40 (0.84). In this small sample, neoGFAP is a superior proteoform-resolved diagnostic and prognostic biomarker across the continuum and supports adding an astroglial-proteoform axis to amyloid/tau/neurodegeneration biomarker frameworks in high-risk populations.

Background: Acute phase response is an early immunometabolic response to brain injuries, primarily coordinated by the liver via the activation of acute phase proteins. These immune responses can be both beneficial, promoting tissue repair, and detrimental, exacerbating neurological deficits, if not properly controlled. Despite the central role of the liver in immunometabolism, how hepatic metabolism dynamically adapts to traumatic brain injury remains under explored, primarily due to limited liver-specific modalities that can assess metabolic pathways in vivo. 13C MRI utilizing hyperpolarized 13C-pyruvate can assess key regulatory enzyme activities in hepatic metabolism. Methods: Rats with controlled cortical impact were studied in vivo using hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate under fed and fasted conditions 3-4 days after injury. Hyperpolarized 13C products, including [13C]bicarbonate from [1-13C]pyruvate and [5-13C]glutamate, [1-13C]acetyl-L-carnitine, and [2-13C]phosphoenolpyruvate from [2-13C]pyruvate, were evaluated to assess mitochondrial and gluconeogenic metabolism. In parallel, liver tissues were collected following [U-13C3]pyruvate injection for NMR isotopomer analysis of phosphoenolpyruvate, glucose, and glutamate. Results: While no metabolic differences were detected under fed condition, [13C]bicarbonate and [2-13C]phosphoenolpyruvate increased after brain injury under fasted condition, indicating an upregulation of the hepatic gluconeogenic pathway after injury. 13C NMR of liver tissue extracts from injured rats showed an elevated [2,3-13C2]glutamate-to-[4,5-13C2]glutamate ratio and increased 13C-labeling in phosphoenolpyruvate than controls, confirming enhanced hepatic gluconeogenic pathway. Conclusion: This study demonstrates that hepatic acute phase response to brain injuries can be monitored in vivo by hyperpolarized pyruvate, which may be further utilized for longitudinal immunometabolic evaluation of the liver during pathogenesis and therapeutic interventions.

Background. GBA2-associated hereditary spastic paraplegia (SPG46) is a rare autosomal recessive neurodegenerative disorder caused by loss-of-function mutations in GBA2, encoding the non-lysosomal glucocerebrosidase 2. GBA2 deficiency leads to glucosylceramide (GlcCer) accumulation and glucosylated cholesterol (GlcChol) depletion, causing cytoskeletal defects in immature neurons. However, the mechanisms linking lipid dysregulation to neuronal dysfunction remain poorly understood. Methods. We modelled GBA2 loss of function by chronic pharmacological inhibition in mouse cerebellar granule neurons (CGNs) and assessed neuronal morphology, synaptic organization, Ca2+ dynamics, mitochondrial function and actin cytoskeleton during maturation. Proteomic profiling was performed in GBA2-inhibited and GlcChol-supplemented neurons. Findings were validated in a zebrafish gba2 crispant model by evaluating motor behavior, cerebellar development, neuronal organization and mitochondrial function, and in patient-derived fibroblasts carrying a homozygous pathogenic GBA2 variant (NM_020944). The role of RAC1 was studied in both neurons and patients' cultured skin fibroblasts, and upon rac1 pharmacological inhibition in zebrafish crispants. Results. Chronic GBA2 inhibition impaired axonal outgrowth in immature CGNs but not neurite complexity in mature neurons, suggesting morphological compensation. Nevertheless, mature neurons displayed enlarged presynaptic terminals, impaired synaptic vesicle clustering and altered Ca2+ responses to potassium and glutamate, the latter associated with NMDA receptor redistribution without changes in total receptor levels. Mitochondrial alterations were observed in CGNs, patient fibroblasts and zebrafish, consistent with defective architecture of the mitochondrial network. Proteomics revealed convergent alterations in actin cytoskeleton, synaptic pathways and cellular metabolism following both GBA2 inhibition and GlcChol supplementation. GlcChol bidirectionally regulated RAC1 function, likely altering its spatial distribution rather than its global activation. Confocal imaging confirmed abnormal RAC1 and F-actin localization in patient fibroblasts. Zebrafish gba2 crispants recapitulated motor deficits, Purkinje cell loss, motor neuron disorganization and mitochondrial abnormalities. Pharmacological Rac1 inhibition rescued motor behavior and neuronal organization, linking cytoskeletal disorganization to the observed phenotype in the zebrafish model. Conclusions. Our findings identify a pathogenic GlcChol-RAC1-actin signalling axis linking lipid imbalance to synaptic disorganization, NMDA receptor redistribution and mitochondrial dysfunction in SPG46. The selective vulnerability of corticospinal neurons, cerebellar granule neurons and Purkinje cells may reflect their dependence on this pathway. Rac1 inhibition rescues disease phenotypes in vivo, highlighting this pathway as a promising therapeutic target.