The central nervous system is surrounded by three interconnected membranes referred to as the meninges, which host a diverse immune network1-3. Within the skull-interfacing dura mater are venous sinuses, large veins that are traditionally viewed as passive blood drains for the brain and skull4,5. However, these structures also constitute an important neuroimmune interface6-8. Here we used intravital microscopy to gain mechanistic insight into this interface and reveal that dural sinuses and their endothelial cells form a highly dynamic surface that continually restructures to regulate blood flow, fluid movement and immune surveillance. We show that sinuses are not passive conduits, but instead undergo RAMP1-dependent constriction and dilation mediated by smooth muscle, resembling arterial behaviour. Moreover, the superior sagittal sinus in mice is bifurcated into upper and lower chambers that contribute to intracranial pressure regulation. Both chambers are lined by specialized, highly fenestrated sinus endothelial cells (SECs) that permit movement of fluids, macromolecules and microorganisms between the sinus lumen and leukocyte-rich perisinus space. To safeguard this permeable interface, SECs dynamically open and close intercellular boundaries in a RAMP2-dependent manner. Transcranial RAMP2 antagonism impaired SEC boundary dynamics and reduced immune cell trafficking along the sinus wall during homeostasis and systemic viral infection. Disruption of SEC dynamics during infection compromised local antiviral immunity and promoted pathogen entry into the meninges. Together, these findings establish dural sinuses as dynamic venous structures that regulate fluid exchange and support immune surveillance and antiviral defence.

The autosomal dominant p.Ala165Val mutation in LIM Domain Binding Protein 3 (LDB3) causes myofibrillar myopathy marked by Z-disc disruption, accumulation of filamin-C (FLNc) and chaperone proteins, and progressive muscle weakness. We previously showed that this mutation interferes with the LDB3-protein kinase C alpha (PKCα)-FLNc mechanosensing axis and impairs chaperone-assisted selective autophagy (CASA), establishing a gain-of-function mechanism. In this study, we examined whether mutant allele-specific knockdown could reverse the disease or mitigate disease progression in-vivo. A single intramuscular-injection of an AAV9-delivered microRNA-based shRNA produced substantial knockdown of mutant Ldb3 transcripts and protein in Ldb3 Ala165Val/+ knock-in mice treated either before or after the onset of pathology. Treatment after disease onset reduced filamin-C and CASA protein aggregates and improved muscle strength, whereas early intervention prevented development of molecular and histological features of myopathy. Phosphoproteomic profiling further showed broad remodeling of dysregulated phosphorylation networks, including restoration of PKCα-responsive sites and normalization of altered sarcomeric and cytoskeletal signaling observed in Ldb3 Ala165Val/+ mice. These findings identify disruption of the LDB3-PKCα-FLNc mechanosensing pathway as a central disease driver and suggest that restoring this signaling axis may complement mutant allele-specific RNA interference (RNAi). Overall, our results support RNAi as a promising therapeutic strategy for dominant LDB3-related myofibrillar myopathy.

Central nervous system (CNS) tumors originate from neuroectodermal cells and cause a significant burden of neurological and systemic disease in humans. CNS tumors exhibit diverse biological behaviors, ranging from benign, slow-growing masses to highly aggressive, infiltrative malignancies. We found activation of integrated stress response (ISR) in tumor cells. ISR is the final common pathway activated in response to a range of cellular stressors. ISR activation suppresses global protein translation except ATF4, leading to apoptosis in normal cells. Tumors can evade ISR activation to support proliferation, making ISR targetable. The relevance of ISR activation in the context of CNS tumors is unknown. ISR activation was analyzed with single cell transcriptomes (scRNAseq) and single cell ATAC seq (scATACseq) of pituitary adenomas (PA), schwannomas (SCH), and ependymomas (EPN) tumors from our patients. Analysis was supplemented with publicly available datasets including scRNAseq from medulloblastomas (MB) and meningiomas (MG), and corresponding human normal tissue datasets. ISR activity scores were significantly (p<0.05) higher in PA, EPN and MG tumor cells compared to their normal counterparts. Global functional enrichment analysis between tumor and normal cells revealed upregulation of translation, protein folding, and ER stress-related pathways. ATF4 target genes ATF6, and XBP1 were upregulated in tumor cells. These genes mediate ER stress resolution and drive the expression of HSPA5 (BiP), a molecular chaperone. HSPA5 itself was upregulated in tumor cells as well. scATACseq revealed increased promoter accesibility in several ISR-regulated genes including HSPA5, XBP1, and DDIT3 suggesting broad epigenetic reprogramming that allows ISR escape. Our findings suggests that ISR activation in some CNS tumors (PA, EPN, and MG) may play a critical role in tumor adaptation by enhancing protein homeostasis and stress resilience. These findings highlight the ISR escape as a tumor adaptation mechanism and potential therapeutic target in CNS tumors like PA, EPN, and MG.

Chronic neuroinflammation and neurodegeneration are critical but unresolved drivers of disability accumulation in progressive multiple sclerosis (MS). Chronic active white matter lesions, identifiable radiologically as paramagnetic rim lesions (PRL), indicate progression-relevant chronic neuroinflammation. Using single-cell transcriptomics (scRNAseq) and T-cell receptor sequencing (scTCR-seq), we profiled cerebrospinal fluid (CSF) and blood immune cells of 34 radiologically characterized adults with MS (17 untreated, 6 treated with B-cell-depletion) and 5 healthy controls. Coupled with proteomics, we found PRL-associated enrichment of interferon (IFN) signaling and upregulation of TCR signaling in CSF and blood. This was accompanied by clonal expansion of CD8+ T effector memory (TEM) cells, with the highly expanded clonal cells exhibiting T helper type 1 (TH1)-like and cytotoxic profiles. Validating the cytotoxic immune profile in blood using flow cytometry, we identified a cellular correlate of PRL exhibiting features of CD8+ TEMRA cells. Despite chronic B-cell depletion, PRL-associated neuroinflammation, driven by myeloid activation and CD8+ T-cell cytotoxicity, persisted. Serum and CSF proteomic networks showed PRL-pertinent signatures, including networks unaffected by B-cell depletion. CSF:serum protein ratios revealed compartmentalized myeloid activation in cases with PRL. Using in silico perturbation, we nominated therapeutic targets, including MYD88, TNF, MYC, TYK2, JAK2, and BTK, for alleviating chronic neuroinflammation in MS. Our findings highlight mechanisms of chronic neuroinflammation in MS and point to potential biomarkers for monitoring disease progression.

Neuronal heterogeneity is a defining feature of the developing mammalian brain, but the mechanisms regulating the diversification of closely related cell types remain elusive. Here, we investigated granule cell (GC) subtype composition in the dentate gyrus (DG) and the influence of a psychosis-associated V321L mutation in Neuregulin1 (Nrg1). Using morphoelectric characterization, single-nucleus gene expression, and chromatin accessibility profiling, we identified distinctions between typical GCs and a rare subtype known as semilunar granule cells (SGCs). We found that the V321L mutation, which disrupts Nrg1 nuclear back-signaling, results in overabundance of SGC-like cells. Pseudotime analyses suggest a GC-to-SGC transition potential, supported by the accessibility of SGC-enriched genes in non-SGCs. In WT mice, SGC-like gene expression increases during adolescence, coinciding with reduced Nrg1 back-signaling capacity. These results suggest that intact Nrg1 nuclear signaling represses SGC-like fate and that its developmental or pathological loss may permit acquisition of this fate. Our findings reveal a novel role of Nrg1 in maintaining DG cell-type composition and suggest that disrupted subtype regulation may contribute to disease-associated changes in the DG.

Changes to cellular lipids accompany shifts in microglial cell state, but the functional significance of these metabolic changes remains poorly understood. In human induced pluripotent stem cell-derived microglia, we observed that both extrinsic activation (by lipopolysaccharide treatment) and intrinsic triggers (the Alzheimer's disease-associated APOE4 genotype) result in accumulation of triglyceride-rich lipid droplets. We demonstrate that lipid droplet accumulation is not simply concomitant with changes in the cell state. In fact, both triglyceride biosynthesis and catabolism are critical for the activation-induced transcription and secretion of inflammatory cytokines and chemokines, as well as changes in phagocytosis. In microglia harboring the Alzheimer's disease risk APOE4 genotype, inhibiting triglyceride biosynthesis attenuates disease-associated transcriptional states. Triglyceride biosynthesis inhibition also rescues microglial surveillance defects observed in slices from APOE4 humanized transgenic mice. Together, our findings establish that modulating triglyceride metabolism can tune microglial immune activity in response to extrinsic activation and in APOE4-associated disease.

Neurodegenerative diseases activate innate and adaptive immune responses that can either slow or accelerate disease progression. Here, we sought to define beneficial immune pressures that emerge during tauopathy development in mice and humans. Using mice that express mutant human tau in neurons, we observed that microglia slowed tauopathy development by controlling the spread of phosphorylated tau (pTau) in the central nervous system and blood. However, over time microglia converted into distressed antigen-presenting cells, acquired neuronal transcripts and were targeted by resident, clonally expanded CD8+ T cells. These cells did not express traditional effector molecules, such as IFNγ, TNF or granzymes a/b/c, but instead deposited granzyme K (GZMK) onto microglia and were regulated by immune checkpoint proteins (TIGIT, PD-1), as blockade of TIGIT and PD-1 enhanced disease progression. GZMK+CD8+ T cells also targeted microglia in pTau-rich human brain lesions resulting from age, Alzheimer’s disease or chronic traumatic encephalopathy. Deletion of CD8+ T cells in mice promoted the emergence of distressed microglia containing neuronal transcripts, markedly enhanced pTau spread and accelerated neurological decline. These data demonstrate that GZMK+CD8+ T cells are a signature of tauopathy development and could potentially be harnessed to slow disease progression. McGavern and colleagues identified a CD8+ T cell population that expresses GZMK in ʽtau-richʼ brains, where it may play a protective role against neurodegeneration.

ABSTRACT One of the most important properties of human embryonic stem cells (hESCs) is related to their primed and naïve pluripotent states. Our previous meta-analysis indicates the existence of heterogeneous pluripotent states derived from diverse naïve protocols. In this study, we have characterized a commercial medium (RSeT)-based pluripotent state under various growth conditions. Notably, RSeT hESCs can circumvent hypoxic growth conditions as required by naïve hESCs, in which some RSeT cells (e.g., H1 cells) exhibit much lower single cell plating efficiency, having altered or much retarded cell growth under both normoxia and hypoxia. Evidently, hPSCs lack many transcriptomic hallmarks of naïve and formative pluripotency (a phase between naive and primed states). Integrative transcriptome analysis suggests our primed and RSeT hESCs are close to the early stage of post-implantation embryos, similar to the previously reported primary hESCs and early hESC cultures. Moreover, RSeT hESCs did not express naïve surface markers such as CD75, SUSD2, and CD130 at a significant level. Biochemically, RSeT hESCs exhibit a differential dependency of FGF2 and co-independency of both Janus kinase (JAK) and TGFβ signaling in a cell-line-specific manner. Thus, RSeT hESCs represent a previously unrecognized pluripotent state downstream of formative pluripotency. Our data suggest that human naïve pluripotent potentials may be restricted in RSeT medium. Hence, this study provides new insights into pluripotent state transitions in vitro .

TDP-43 mislocalization and pathology occurs across a range of neurodegenerative diseases, but the pathways that modulate TDP-43 in neurons are not well understood. We generated a Halo-TDP-43 knock-in human induced pluripotent stem cell (iPSC) line and performed a genome-wide CRISPR interference FACS-based screen to identify modifiers of TDP-43 levels in neurons. A meta-analysis of our screen and publicly available screens identified both specific hits and pathways present across multiple screens, the latter likely responsible for generic protein level maintenance. We identified BORC, a complex required for anterograde lysosome transport, as a specific modifier of TDP-43 protein, but not mRNA, levels in neurons. BORC loss led to longer half-life of TDP-43 and other proteins, suggesting lysosome location is required for proper protein turnover. As such, lysosome location and function are crucial for maintaining TDP-43 protein levels in neurons.

Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NF-κB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling are not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN, associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it plays a role in the sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, and OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 plays a role in STING-mediated innate immune signaling, acting as a negative regulator of IRF3-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.

The CNS is covered by three interconnected membranes referred to as the meninges that host a diverse immune network. Situated within the skull-interfacing dura mater are venous sinuses, which are large veins thought to serve predominantly as passive blood drains for the brain and skull. Here, using intravital imaging, we revealed that the superior sagittal sinus and its associated endothelial cells are highly dynamic and constantly restructure to modulate fluid movement and immune surveillance. Specifically, we observed that the sagittal sinus is not a passive drain but instead constricts and dilates under smooth muscle control and Ramp1 signaling. In addition, the sinus wall is comprised of specialized endothelial cells that are highly fenestrated, allowing movement of fluids, macromolecules, and microbes between the sinus lumen and leukocyte-rich parasinus space. To protect this permeable interface, sinus endothelial cells (SECs) continually open and close their boundaries in a Ramp2 dependent manner. Transcranial Ramp2 antagonism impeded SEC boundary dynamics and immune cell movement along the sinus wall during steady state and after systemic viral infection. Interference with SEC dynamics during viral infection disrupted local antiviral immunity and facilitated pathogen entry into the meninges. These findings demonstrate that dural sinuses are dynamic venous drains with specialized SECs designed to support both steady state immune surveillance and antiviral immunity. This research was supported by the intramural program of the National Institute of Neurological Disorders and Stroke, NIH. Neuroimmunology (NEUR)

Neurodegenerative diseases trigger innate and adaptive immune responses that can either slow or accelerate disease progression. Here, we sought to define beneficial immune pressure that emerged during development of tauopathies in mice and humans. Using mice that express mutant human tau in neurons, we observed that microglia slowed tauopathy development by controlling the spread of tau throughout the CNS and into the blood. Single cell TCR sequencing revealed clonally expanded CD8+ T cells in the CNS and draining lymph nodes of tauopathy mice that expressed granzyme K, but not traditional effector molecules (e.g., IFNg, TNF, granzymes a/b/c), which was deposited onto the microglia they targeted. Engagement of microglia by granzyme K expressing CD8+ T cells was a signature of tauopathy development in mice as well as humans with tau rich brain lesions linked to age, Alzheimer’s disease, or chronic traumatic encephalopathy. Deletion of CD8+ T cells in mice promoted the appearance of distressed microglia containing neuronal transcripts, markedly enhanced tau spread, and accelerated neurological decline. These data highlight a beneficial immune reaction involving microglia and granzyme K expressing CD8+ T cells that can slow tauopathy progression. Enhancement of this coordinated response offers the potential to improve outcomes in tauopathy patients. This work was supported by the intramural program at the National Institute of Neurological Disorders & Stroke and the National Institute of Allergy & Infectious Diseases, NIH, as well as The Defense Health Agency (310286-1.00-65642 and 311661-5.00-66323). Neuroimmunology (NEUR)

Abstract Mitochondrial dysfunction causes devastating disorders, including mitochondrial myopathy, but how muscle senses and adapts to mitochondrial dysfunction is not well understood. Here, we used diverse mouse models of mitochondrial myopathy to show that the signal for mitochondrial dysfunction originates within mitochondria. The mitochondrial proteins OMA1 and DELE1 sensed disruption of the inner mitochondrial membrane and, in response, activated the mitochondrial integrated stress response (mt-ISR) to increase the building blocks for protein synthesis. In the absence of the mt-ISR, protein synthesis in muscle was dysregulated causing protein misfolding, and mice with early-onset mitochondrial myopathy failed to grow and survive. The mt-ISR was similar following disruptions in mtDNA maintenance (Tfam knockout) and mitochondrial protein misfolding (CHCHD10 G58R and S59L knockin) but heterogenous among mitochondria-rich tissues, with broad gene expression changes observed in heart and skeletal muscle and limited changes observed in liver and brown adipose tissue. Taken together, our findings identify that the DELE1 mt-ISR mediates a similar response to diverse forms of mitochondrial stress and is critical for maintaining growth and survival in early-onset mitochondrial myopathy.

One of the most important properties of human embryonic stem cells (hESCs) is related to their pluripotent states. In our recent study, we identified a previously unrecognized pluripotent state induced by RSeT medium. This state makes primed hESCs resistant to conversion to naïve pluripotent state. In this study, we have further characterized the metabolic features in these RSeT hESCs, including metabolic gene expression, metabolomic analysis, and various functional assays. The commonly reported metabolic modes include glycolysis or both glycolysis and oxidative phosphorylation (i.e., metabolic bivalency) in pluripotent stem cells. However, besides the presence of metabolic bivalency, RSeT hESCs exhibited a unique metabolome with additional fatty acid oxidation and imbalanced nucleotide metabolism. This metabolic quadrivalency is linked to hESC growth independent of oxygen tension and restricted capacity for naïve reprogramming in these cells. Thus, this study provides new insights into pluripotent state transitions and metabolic stress-associated hPSC growth in vitro.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast ( TNNI2 ) and TnI-slow ( TNNI1 ), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca 2+ ] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca 2+ ], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Neuregulin1 (Nrg1) signaling is critical for neuronal development and function from fate specification to synaptic plasticity. Type III Nrg1 is a synaptic protein which engages in bidirectional signaling with its receptor ErbB4. Forward signaling engages ErbB4 phosphorylation, whereas back signaling engages two known mechanisms: (1) local axonal PI3K-AKT signaling and (2) cleavage by γ-secretase resulting in cytosolic release of the intracellular domain (ICD), which can traffic to the nucleus (Bao et al., 2003; Hancock et al., 2008). To dissect the contribution of these alternate signaling strategies to neuronal development, we generated a transgenic mouse with a missense mutation (V 321 L) in the Nrg1 transmembrane domain that disrupts nuclear back signaling with minimal effects on forward signaling or local back signaling and was previously found to be associated with psychosis (Walss-Bass et al., 2006). We combined RNA sequencing, retroviral fate mapping of neural stem cells, behavioral analyses, and various network analyses of transcriptomic data to investigate the effect of disrupting Nrg1 nuclear back signaling in the dentate gyrus (DG) of male and female mice. The V 321 L mutation impairs nuclear translocation of the Nrg1 ICD and alters gene expression in the DG. V 321 L mice show reduced stem cell proliferation, altered cell cycle dynamics, fate specification defects, and dendritic dysmorphogenesis. Orthologs of known schizophrenia (SCZ)-susceptibility genes were dysregulated in the V 321 L DG. These genes coordinated a larger network with other dysregulated genes. Weighted gene correlation network analysis and protein interaction network analyses revealed striking similarity between DG transcriptomes of V 321 L mouse and humans with SCZ.

INTRODUCTION:Variants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization. METHODS:We described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling. RESULTS:ANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways. DISCUSSION:This study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity. HIGHLIGHTS:ANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.