A male-specific mechanism of meningeal nociceptor sensitization promoting migraine headache.

BACKGROUND: We wished to explore possible sexual dimorphism in mechanisms sensitizing or activating meningeal nociceptors that can promote the headache phase of migraine. METHODS: Male and female C57BL6J mice received either supradural orexin B and an inflammatory mediator cocktail (IM) with migraine-like pain behaviors and photophobia recorded. Expression of orexin 2 receptor (OX2R) in trigeminal ganglion (TG) and phosphorylated extracellular signal-regulated kinases (ERK) levels in trigeminal nucleus caudalis (TNC) were evaluated. Orexin B-induced excitability of TG cells was assessed with patch-clamp electrophysiology. Intranasal delivery of CRISPR/Cas9 plasmids was used to edit the expression of OX2R in the TG. RESULTS: Supradural orexin B induced migraine-like pain behaviors, photophobia and increased TNC ERK phosphorylation exclusively in males. Blockade of orexin signaling with supradural suvorexant, a dual orexin receptor antagonist, prevented, but did not reverse, migraine-like pain in males induced by supradural IM cocktail. OX2R expression was higher in male TG and orexin B increased TG neuron excitability in males. Intranasal OX2R CRISPR/Cas9 reduced TG receptor expression and orexin B-induced TNC ERK phosphorylation and prevented migraine-like pain induced by supradural orexin B in males. CONCLUSIONS: Our studies reveal a male-specific mechanism of TG nociceptor sensitization and migraine-like pain behavior mediated by orexin B/OX2R signaling. Sexually dimorphic mechanisms of trigeminal nociceptor sensitization and activation offer opportunities to improve patient outcomes by considering patient sex and may influence clinical trial design and interpretation.

Meningeal lymphatic vessel dysfunction driven by CGRP signaling causes migraine-like pain in mice.

Migraines are a type of headache that occur with other neurological symptoms, but the pathophysiology remains unclear. In this issue of the JCI, Nelson-Maney and authors used constitutive and inducible knockouts of the CGRP receptor components, elegantly demonstrating an essential function of CGRP in modulating meningeal lymphatic vessels (MLVs) in migraine. CGRP was shown to induce rearrangement of membrane-bound gap junction proteins in MLVs, resulting in a reduced CSF flux into cervical lymph nodes. The authors also provided evidence of a primary role for CGRP in modulating neuro-immune function. Finally, by showing that blocking CGRP signaling in MLVs attenuated pain behavior associated with acute migraine in rodents, the authors provided a target for pharmacological blockade of CGRP in relation to primary headache disorders.

Unlocking potential of oxytocin: improving intracranial lymphatic drainage for Alzheimer's disease treatment.

Background: The impediment to ß-amyloid (Aß) clearance caused by the invalid intracranial lymphatic drainage in Alzheimer's disease is pivotal to its pathogenesis, and finding reliable clinical available solutions to address this challenge remains elusive. Methods: The potential role and underlying mechanisms of intranasal oxytocin administration, an approved clinical intervention, in improving intracranial lymphatic drainage in middle-old-aged APP/PS1 mice were investigated by live mouse imaging, ASL/CEST-MRI scanning, in vivo two-photon imaging, immunofluorescence staining, ELISA, RT-qPCR, Western blotting, RNA-seq analysis, and cognitive behavioral tests. Results: Benefiting from multifaceted modulation of cerebral hemodynamics, aquaporin-4 polarization, meningeal lymphangiogenesis and transcriptional profiles, oxytocin administration normalized the structure and function of both the glymphatic and meningeal lymphatic systems severely impaired in middle-old-aged APP/PS1 mice. Consequently, this intervention facilitated the efficient drainage of Aß from the brain parenchyma to the cerebrospinal fluid and then to the deep cervical lymph nodes for efficient clearance, as well as improvements in cognitive deficits. Conclusion: This work broadens the underlying neuroprotective mechanisms and clinical applications of oxytocin medication, showcasing its promising therapeutic prospects in central nervous system diseases with intracranial lymphatic dysfunction.

Meningeal lymphatic supporting cells govern the formation and maintenance of zebrafish mural lymphatic endothelial cells.

The meninges are critical for the brain functions, but the diversity of meningeal cell types and intercellular interactions have yet to be thoroughly examined. Here we identify a population of meningeal lymphatic supporting cells (mLSCs) in the zebrafish leptomeninges, which are specifically labeled by ependymin. Morphologically, mLSCs form membranous structures that enwrap the majority of leptomeningeal blood vessels and all the mural lymphatic endothelial cells (muLECs). Based on its unique cellular morphologies and transcriptional profile, mLSC is characterized as a unique cell type different from all the currently known meningeal cell types. Because of the formation of supportive structures and production of pro-lymphangiogenic factors, mLSCs not only promote muLEC development and maintain the dispersed distributions of muLECs in the leptomeninges, but also are required for muLEC regeneration after ablation. This study characterizes a newly identified cell type in leptomeninges, mLSC, which is required for muLEC development, maintenance, and regeneration.

Heterogeneous fibroblasts contribute to fibrotic scar formation after spinal cord injury in mice and monkeys.

Spinal cord injury (SCI) leads to fibrotic scar formation at the lesion site, yet the heterogeneity of fibrotic scar remains elusive. Here we show the heterogeneity in distribution, origin, and function of fibroblasts within fibrotic scars after SCI in mice and female monkeys. Utilizing lineage tracing and single-cell RNA sequencing (scRNA-seq), we found that perivascular fibroblasts (PFs), and meningeal fibroblasts (MFs), rather than pericytes/vascular smooth cells (vSMCs), primarily contribute to fibrotic scar in both transection and crush SCI. Crabp2 + /Emb+ fibroblasts (CE-F) derived from meninges primarily localize in the central region of fibrotic scars, demonstrating enhanced cholesterol synthesis and secretion of type I collagen and fibronectin. In contrast, perivascular/pial Lama1 + /Lama2+ fibroblasts (LA-F) are predominantly found at the periphery of the lesion, expressing laminin and type IV collagen and functionally involved in angiogenesis and lipid transport. These findings may provide a comprehensive understanding for remodeling heterogeneous fibrotic scars after SCI.

Spatial transcriptomics analysis identifies a tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease.

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. Here, we characterize the tumor microenvironment of LMD and patient-matched extra-cranial metastases using spatial transcriptomics in a small number of clinical specimens (nine tissues from two patients) with extensive in vitro and in vivo validation. The spatial landscape of melanoma LMD is characterized by a lack of immune infiltration and instead exhibits a higher level of stromal involvement. The tumor-stroma interactions at the leptomeninges activate tumor-promoting signaling, mediated through upregulation of SERPINA3. The meningeal stroma is required for melanoma cells to survive in the cerebrospinal fluid (CSF) and promotes MAPK inhibitor resistance. Knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in tumor cell death and re-sensitization to MAPK-targeting therapy. Our data provide a spatial atlas of melanoma LMD, identify the tumor-promoting role of meningeal stroma, and demonstrate a mechanism for overcoming microenvironment-mediated drug resistance in LMD.

[Explorations about the correlation between biological changes of meninges in periodontitis mice and cognitive impairment via single-cell RNA sequencing].

Objective: To clarify the potential correlation between biological changes of meninges in periodontitis mice and cognitive impairment by analyzing the biological changes of meninges in periodontitis mice using single-cell RNA sequencing. Methods: Thirty C57BL/6 mice were divided into two groups by using random number table method (15 mice in each group). Mice in the control group were locally administered 2% carboxyl methyl cellulose (CMC) without Porphyromonas gingivalis (Pg) on both buccal sides. A mixture of Pg W83 and 2% CMC was applied on both buccal sides in the experimental group mice three times a week, lasting for 16 weeks in total. The absorption of alveolar bone, locomotor activity and cognitive function, the activation of microglia and astrocytes in the cortex were observed and assessed. The mRNA expression levels of Occludin in meninges and brain were detected in two groups. Single-cell RNA sequencing data of meninges were processed by uniform manifold approximation and projection (UMAP). Differential genes expressions of endothelial cells were processed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. In addition, real-time fluorescence quantitative PCR (RT-qPCR) was used to verify the expressions of transcription activating factor 3 (Atf3) and apolpoprotein L domain-containing 1 (Apold 1). Results: Methylene blue staining found the distances of buccal and palatal cement-enamel junction-alveolar bone crest in experimental mice [(185.60±17.60), (206.90±13.37) µm] increased significantly compared with the control group [(135.33±9.57), (163.05±14.98) µm] (t=5.02, P=0.002; t=4.37, P=0.005). Open field experiment showed the total distance and average speed of mice in the experimental group [(971.88±164.57) cm, (3.25±0.55) cm/s] were not statistically significant compared with the control group [(914.24±278.81) cm, (3.05±0.93) cm/s] (t=0.65, P=0.525; t=0.65, P=0.520). The recognition index of the experimental group [(48.02±16.92) %] was lower than the control group [(66.27±17.90) %] (t=2.40, P=0.027) by novel object recognition tests. Compared with the control group [(63.56±11.88) %], the alternation of experimental group [(50.99±14.17) %] was significantly decreased in Y maze tests (t=2.33, P=0.030). Immunohistochemistry results showed microglia and astrocytes were activated in the cortex of experimental mice. Compared with the control group (1.02±0.25, 1.04±0.31), the relative mRNA expressions of Occludin decreased significantly in the meninges and brain of periodontitis mice, respectively (0.61±0.10, 0.64±0.20) (t=3.47, P=0.010; t=2.66, P=0.024). By single-cell RNA sequencing, meninges cells were divided into 11 types, such as endothelial cells, fibroblasts, immune cells and so on. Endothelial cells were the main cell types in meninges [the control group: 26.47% (1 589/6 004), the experimental group: 26.26% (807/3 073)]. Compared with the control group [5.56% (334/6 004)], the percentage of granulocytes increased in the periodontitis mice [11.65% (358/3 073)]. Using clustering analysis to further focus on endothelial cells, GO enrichment analysis revealed differential genes were mainly related to angiogenesis, cell adhesion, apoptosis and so on. KEGG enrichment analysis revealed that differential genes were related to signaling pathways of interleukin-17, relaxin and so on. The relative mRNA expressions of Atf3 and Apold1 in meninges of periodontitis mice (0.42±0.24, 0.54±0.27) were significantly lower than the control group (1.03±0.26, 1.02±0.23) (t=3.88, P=0.005; t=3.02, P=0.017). Conclusions: The mice chronically infected with Pg W83 occurred memory impairment, neuroinflammation and changes of barrier function. In the meninges of periodontitis mice, there were infiltration of immune cells and down-regulation expressions of Atf3 and Apold1 by single-cell RNA sequencing. Meningeal immunity and changes of barrier function may play an important role in the cognitive impairment caused by periodontitis.

Impaired Meningeal Lymphatics and Glymphatic Pathway in Patients with White Matter Hyperintensity.

White matter hyperintensity (WMH) represents a critical global medical concern linked to cognitive decline and dementia, yet its underlying mechanisms remain poorly understood. Here, humans are directly demonstrated that high WMH burden correlates with delayed drainage of meningeal lymphatic vessels (mLVs) and glymphatic pathway. Additionally, a longitudinal cohort study reveals that glymphatic dysfunction predicts WMH progression. Next, in a rat model of WMH, the presence of impaired lymphangiogenesis and glymphatic drainage is confirmed, followed by elevated microglial activation and white matter demyelination. Notably, enhancing meningeal lymphangiogenesis through adeno-associated virus delivery of vascular endothelial growth factor-C (VEGF-C) mitigates microglial gliosis and white matter demyelination. Conversely, blocking the growth of mLVs with a VEGF-C trap strategy exacerbates these changes. The findings highlight the role of mLVs and glymphatic pathway dysfunction in aggravating brain white matter injury, providing a potential novel strategy for WMH prevention and treatment.

Meningeal lymphatic CGRP signaling governs pain via cerebrospinal fluid efflux and neuroinflammation in migraine models.

Recently developed antimigraine therapeutics targeting calcitonin gene-related peptide (CGRP) signaling are effective, though their sites of activity remain elusive. Notably, the lymphatic vasculature is responsive to CGRP signaling, but whether meningeal lymphatic vessels (MLVs) contribute to migraine pathophysiology is unknown. Mice with lymphatic vasculature deficient in the CGRP receptor (CalcrliLEC mice) treated with nitroglycerin-mediated (NTG-mediated) chronic migraine exhibit reduced pain and light avoidance compared with NTG-treated littermate controls. Gene expression profiles of lymphatic endothelial cells (LECs) isolated from the meninges of Rpl22HA/+;Lyve1Cre RiboTag mice treated with NTG revealed increased MLV-immune interactions compared with cells from untreated mice. Interestingly, the relative abundance of mucosal vascular addressin cell adhesion molecule 1-interacting (MAdCAM1-interacting) CD4+ T cells was increased in the deep cervical lymph nodes of NTG-treated control mice but not in NTG-treated CalcrliLEC mice. Treatment of cultured hLECs with CGRP peptide in vitro induced vascular endothelial-cadherin (VE-cadherin) rearrangement and reduced functional permeability. Likewise, intra cisterna magna injection of CGRP caused rearrangement of VE-cadherin, decreased MLV uptake of cerebrospinal fluid (CSF), and impaired CSF drainage in control mice but not in CalcrliLEC mice. Collectively, these findings reveal a previously unrecognized role for lymphatics in chronic migraine, whereby CGRP signaling primes MLV-immune interactions and reduces CSF efflux.

A brain-specific angiogenic mechanism enabled by tip cell specialization.

Vertebrate organs require locally adapted blood vessels1,2. The gain of such organotypic vessel specializations is often deemed to be molecularly unrelated to the process of organ vascularization. Here, opposing this model, we reveal a molecular mechanism for brain-specific angiogenesis that operates under the control of Wnt7a/b ligands-well-known blood-brain barrier maturation signals3-5. The control mechanism relies on Wnt7a/b-dependent expression of Mmp25, which we find is enriched in brain endothelial cells. CRISPR-Cas9 mutagenesis in zebrafish reveals that this poorly characterized glycosylphosphatidylinositol-anchored matrix metalloproteinase is selectively required in endothelial tip cells to enable their initial migration across the pial basement membrane lining the brain surface. Mechanistically, Mmp25 confers brain invasive competence by cleaving meningeal fibroblast-derived collagen IV α5/6 chains within a short non-collagenous region of the central helical part of the heterotrimer. After genetic interference with the pial basement membrane composition, the Wnt-ß-catenin-dependent organotypic control of brain angiogenesis is lost, resulting in properly patterned, yet blood-brain-barrier-defective cerebrovasculatures. We reveal an organ-specific angiogenesis mechanism, shed light on tip cell mechanistic angiodiversity and thereby illustrate how organs, by imposing local constraints on angiogenic tip cells, can select vessels matching their distinctive physiological requirements.

Involvement of the glymphatic/meningeal lymphatic system in Alzheimer's disease: insights into proteostasis and future directions.

BACKGROUND:  Alzheimer's disease (AD) is pathologically characterized by the abnormal accumulation of Aß and tau proteins. There has long been a keen interest among researchers in understanding how Aß and tau are ultimately cleared in the brain. The discovery of this glymphatic system introduced a novel perspective on protein clearance and it gained recognition as one of the major brain clearance pathways for clearing these pathogenic proteins in AD. This finding has sparked interest in exploring the potential contribution of the glymphatic/meningeal lymphatic system in AD. Furthermore, there is a growing emphasis and discussion regarding the possibility that activating the glymphatic/meningeal lymphatic system could serve as a novel therapeutic strategy against AD. OBJECTIVES:  Given this current research trend, the primary focus of this comprehensive review is to highlight the role of the glymphatic/meningeal lymphatic system in the pathogenesis of AD. The discussion will encompass future research directions and prospects for treatment in relation to the glymphatic/meningeal lymphatic system.

Piezo1 regulates meningeal lymphatic vessel drainage and alleviates excessive CSF accumulation.

Piezo1 regulates multiple aspects of the vascular system by converting mechanical signals generated by fluid flow into biological processes. Here, we find that Piezo1 is necessary for the proper development and function of meningeal lymphatic vessels and that activating Piezo1 through transgenic overexpression or treatment with the chemical agonist Yoda1 is sufficient to increase cerebrospinal fluid (CSF) outflow by improving lymphatic absorption and transport. The abnormal accumulation of CSF, which often leads to hydrocephalus and ventriculomegaly, currently lacks effective treatments. We discovered that meningeal lymphatics in mouse models of Down syndrome were incompletely developed and abnormally formed. Selective overexpression of Piezo1 in lymphatics or systemic administration of Yoda1 in mice with hydrocephalus or Down syndrome resulted in a notable decrease in pathological CSF accumulation, ventricular enlargement and other associated disease symptoms. Together, our study highlights the importance of Piezo1-mediated lymphatic mechanotransduction in maintaining brain fluid drainage and identifies Piezo1 as a promising therapeutic target for treating excessive CSF accumulation and ventricular enlargement.

Age-related changes in meningeal lymphatic function are closely associated with vascular endothelial growth factor-C expression.

Meningeal lymphatic vessels (MLVs) have crucial roles in removing metabolic waste and toxic proteins from the brain and transporting them to the periphery. Aged mice show impaired meningeal lymphatic function. Nevertheless, as the disease progresses, and significant pathological changes manifest in the brain, treating the condition becomes increasingly challenging. Therefore, investigating the alterations in the structure and function of MLVs in the early stages of aging is critical for preventing age-related central nervous system degenerative diseases. We detected the structure and function of MLVs in young, middle-aged, and aged mice. Middle-aged mice, compared with young and aged mice, showed enhanced meningeal lymphatic function along with MLV expansion and performed better in the Y maze test. Moreover, age-related changes in meningeal lymphatic function were closely associated with vascular endothelial growth factor-C (VEGF-C) expression in the brain cortex. Our data suggested that the cerebral cortex may serve as a target for VEGF-C supplementation to ameliorate meningeal lymphatic dysfunction, thus providing a new strategy for preventing age-related central nervous system diseases.

Functional analysis and transcriptome profile of meninges and skin fibroblasts from human-aged donors.

The central nervous system (CNS) is surrounded by three membranes called meninges. Specialised fibroblasts, originating from the mesoderm and neural crest, primarily populate the meninges and serve as a binding agent. Our goal was to compare fibroblasts from meninges and skin obtained from the same human-aged donors, exploring their molecular and cellular characteristics related to CNS functions. We isolated meningeal fibroblasts (MFs) from brain donors and skin fibroblasts (SFs) from the same subjects. A functional analysis was performed measuring cell appearance, metabolic activity, and cellular orientation. We examined fibronectin, serpin H1, ß-III-tubulin, and nestin through qPCR and immunofluorescence. A whole transcriptome analysis was also performed to characterise the gene expression of MFs and SFs. MFs appeared more rapidly in the post-tissue processing, while SFs showed an elevated cellular metabolism and a well-defined cellular orientation. The four markers were mostly similar between the MFs and SFs, except for nestin, more expressed in MFs. Transcriptome analysis reveals significant differences, particularly in cyclic adenosine monophosphate (cAMP) metabolism and response to forskolin, both of which are upregulated in MFs. This study highlights MFs' unique characteristics, including the timing of appearance, metabolic activity, and gene expression patterns, particularly in cAMP metabolism and response to forskolin. These findings contribute to a deeper understanding of non-neuronal cells' involvement in CNS activities and potentially open avenues for therapeutic exploration.

Characterization of cortico-meningeal translocator protein expression in multiple sclerosis.

Compartmentalized meningeal inflammation is thought to represent one of the key players in the pathogenesis of cortical demyelination in multiple sclerosis. PET targeting the 18 kDa mitochondrial translocator protein (TSPO) is a molecular-specific approach to quantifying immune cell-mediated density in the cortico-meningeal tissue compartment in vivo. This study aimed to characterize cortical and meningeal TSPO expression in a heterogeneous cohort of multiple sclerosis cases using in vivo simultaneous MR-PET with 11C-PBR28, a second-generation TSPO radioligand, and ex vivo immunohistochemistry. Forty-nine multiple sclerosis patients (21 with secondary progressive and 28 with relapsing-remitting multiple sclerosis) with mixed or high affinity binding for 11C-PBR28 underwent 90-min 11C-PBR28 simultaneous MR-PET. Tracer binding was measured using 60-90 min normalized standardized uptake value ratios sampled at mid-cortical depth and ∼3 mm above the pial surface. Data in multiple sclerosis patients were compared to 21 age-matched healthy controls. To characterize the nature of 11C-PBR28 PET uptake, the meningeal and cortical lesion cellular expression of TSPO was further described in post-mortem brain tissue from 20 cases with secondary progressive multiple sclerosis and five age-matched healthy donors. Relative to healthy controls, patients with multiple sclerosis exhibited abnormally increased TSPO signal in the cortex and meningeal tissue, diffusively in progressive disease and more localized in relapsing-remitting multiple sclerosis. In multiple sclerosis, increased meningeal TSPO levels were associated with increased Expanded Disability Status Scale scores (P = 0.007, by linear regression). Immunohistochemistry, validated using in situ sequencing analysis, revealed increased TSPO expression in the meninges and adjacent subpial cortical lesions of post-mortem secondary progressive multiple sclerosis cases relative to control tissue. In these cases, increased TSPO expression was related to meningeal inflammation. Translocator protein immunostaining was detected on meningeal MHC-class II+ macrophages and cortical-activated MHC-class II+ TMEM119+ microglia. In vivo arterial blood data and neuropathology showed that endothelial binding did not significantly account for increased TSPO cortico-meningeal expression in multiple sclerosis. Our findings support the use of TSPO-PET in multiple sclerosis for imaging in vivo inflammation in the cortico-meningeal brain tissue compartment and provide in vivo evidence implicating meningeal inflammation in the pathogenesis of the disease.

Comparative Gene Signature of Nociceptors Innervating Mouse Molar Teeth, Cranial Meninges, and Cornea.

BACKGROUND: The trigeminal ganglion (TG) collects afferent sensory information from various tissues. Recent large-scale RNA sequencing of neurons of the TG and dorsal root ganglion has revealed a variety of functionally distinct neuronal subpopulations, but organ-specific information is lacking. METHODS: To link transcriptomic and tissue-specific information, we labeled small-diameter neurons of 3 specific subpopulations of the TG by local application of lipophilic carbocyanine dyes to their innervation site in the dental pulp, cornea, and meninges (dura mater). We then collected mRNA-sequencing data from fluorescent neurons. Differentially expressed genes (DEGs) were analyzed and subjected to downstream gene set enrichment analysis (GSEA), and ion channel profiling was performed. RESULTS: A total of 10,903 genes were mapped to the mouse genome (>500 reads). DEG analysis revealed 18 and 81 genes with differential expression (log 2 fold change > 2, Padj < .05) in primary afferent neurons innervating the dental pulp (dental primary afferent neurons [DPAN]) compared to those innervating the meninges (meningeal primary afferent neurons [MPAN]) and the cornea (corneal primary afferent neurons [CPAN]). We found 250 and 292 genes differentially expressed in MPAN as compared to DPAN and to CPAN, and 21 and 12 in CPAN as compared to DPAN and MPAN. Scn2b had the highest log 2 fold change when comparing DPAN versus MPAN and Mmp12 was the most prominent DEG when comparing DPAN versus CPAN and, CPAN versus MPAN. GSEA revealed genes of the immune and mitochondrial oxidative phosphorylation system for the DPAN versus MPAN comparison, cilium- and ribosome-related genes for the CPAN versus DPAN comparison, and respirasome, immune cell- and ribosome-related gene sets for the CPAN versus MPAN comparison. DEG analysis for ion channels revealed no significant differences between the neurons set except for the sodium voltage-gated channel beta subunit 2, Scn2b . However, in each tissue a few ion channels turned up with robust number of reads. In DPAN, these were Cacna1b , Trpv2 , Cnga4 , Hcn1 , and Hcn3 , in CPAN Trpa1 , Trpv1 , Cacna1a , and Kcnk13 and in MPAN Trpv2 and Scn11a . CONCLUSIONS: Our study uncovers previously unknown differences in gene expression between sensory neuron subpopulations from the dental pulp, cornea, and dura mater and provides the basis for functional studies, including the investigation of ion channel function and their suitability as targets for tissue-specific analgesia.

Structural characterization of SLYM-a 4th meningeal membrane.

Traditionally, the meninges are described as 3 distinct layers, dura, arachnoid and pia. Yet, the classification of the connective meningeal membranes surrounding the brain is based on postmortem macroscopic examination. Ultrastructural and single cell transcriptome analyses have documented that the 3 meningeal layers can be subdivided into several distinct layers based on cellular characteristics. We here re-examined the existence of a 4th meningeal membrane, Subarachnoid Lymphatic-like Membrane or SLYM in Prox1-eGFP reporter mice. Imaging of freshly resected whole brains showed that SLYM covers the entire brain and brain stem and forms a roof shielding the subarachnoid cerebrospinal fluid (CSF)-filled cisterns and the pia-adjacent vasculature. Thus, SLYM is strategically positioned to facilitate periarterial influx of freshly produced CSF and thereby support unidirectional glymphatic CSF transport. Histological analysis showed that, in spinal cord and parts of dorsal cortex, SLYM fused with the arachnoid barrier layer, while in the basal brain stem typically formed a 1-3 cell layered membrane subdividing the subarachnoid space into two compartments. However, great care should be taken when interpreting the organization of the delicate leptomeningeal membranes in tissue sections. We show that hyperosmotic fixatives dehydrate the tissue with the risk of shrinkage and dislocation of these fragile membranes in postmortem preparations.

The murine meninges acquire lymphoid tissue properties and harbour autoreactive B cells during chronic Trypanosoma brucei infection.

The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes human African trypanosomiasis (HAT) or sleeping sickness, accumulates in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTß signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders, including neuropsychiatric lupus and multiple sclerosis.

Proteomic interrogation of the meninges reveals the molecular identities of structural components and regional distinctions along the CNS axis.

The meninges surround the brain and spinal cord, affording physical protection while also serving as a niche of neuroimmune activity. Though possessing stromal qualities, its complex cellular and extracellular makeup has yet to be elaborated, and it remains unclear whether the meninges vary along the neuroaxis. Hence, studies were carried-out to elucidate the protein composition and structural organization of brain and spinal cord meninges in normal, adult Biozzi ABH mice. First, shotgun, bottom-up proteomics was carried-out. Prominent proteins at both brain and spinal levels included Type II collagen and Type II keratins, representing extracellular matrix (ECM) and cytoskeletal categories, respectively. While the vast majority of total proteins detected was shared between both meningeal locales, more were uniquely detected in brain than in spine. This pattern was also seen when total proteins were subdivided by cellular compartment, except in the case of the ECM category where brain and spinal meninges each had near equal number of unique proteins, and Type V and type III collagen registered exclusively in the spine. Quantitative analysis revealed differential expression of several collagens and cytoskeletal proteins between brain and spinal meninges. High-resolution immunofluorescence and immunogold-scanning electronmicroscopy on sections from whole brain and spinal cord - still encased within bone -identified major proteins detected by proteomics, and highlighted their association with cellular and extracellular elements of variously shaped arachnoid trabeculae. Western blotting aligned with the proteomic and immunohistological analyses, reinforcing differential appearance of proteins in brain vs spinal meninges. Results could reflect regional distinctions in meninges that govern protective and/or neuroimmune functions.