Age-related meningeal extracellular matrix remodeling compromises CNS lymphatic function

Abstract

Efficient clearance of central nervous system (CNS) waste proteins and appropriate immune surveillance is essential for brain health. These processes are facilitated by lymphatic networks present in the meninges that drain cerebrospinal fluid (CSF). Age-related impairments to meningeal lymphatic drainage contribute to CNS waste accumulation and immune dysfunction, yet the underlying mechanisms remain poorly understood. Here, we identify extracellular matrix (ECM) remodeling in the aged dura as a key driver of CSF clearance deficits, demonstrating that peri-lymphatic collagen accumulation disrupts lymphatic function. Exploring immune-derived factors contributing to this ECM remodeling, we identify transforming growth factor beta 1 (TGFβ1) as a major regulator using primary human dural fibroblasts. Using a novel mouse model with constitutively active TGFβ receptor 1 (TGFβR1) signaling in dural fibroblasts, we show that excessive peri-lymphatic collagen deposition impairs meningeal lymphatic drainage and alters meningeal immunity. Mechanistically, we reveal that ECM-associated matrix stiffness disrupts lymphatic junction integrity and impairs lymphangiogenesis in human lymphatic endothelial cells. These findings establish dural immune cell and fibroblast-mediated ECM remodeling as a critical regulator of CSF clearance and highlight it as a potential therapeutic target for restoring brain waste clearance in aging.

Keywords: Aging; Extracellular matrix; Fibrosis; Lymphatics; Meningeal immunity; Meninges; Traumatic brain injury.

Fig. 1

Collagen alterations in the aged dura. (A) Dural whole mount stained with Fast Green dye indicating collagen-dense regions. Insets represent enlarged regions of non-sinus and transverse sinus regions. (B) Sirius Red staining of a dural sinus cross-section, with collagen type I/III visualized by polarized light. (C) Transmission electron microscopy (TEM) demonstrating composition of mouse dural sinus including endothelial cells (ECs) lining the lumen and collagen bundles. (D) Unlabeled Liquid chromatography-mass spectrometry (LC-MS) analysis for collagen subtype expression in human and mice dural homogenates, n = 4 dural samples per species. (E) Pie chart demonstrating the abundance of extracellular matrix (ECM) proteins that make up the mouse dura from mass spectrometry analysis. (F) Principal component analysis plot for dural protein expression determined by Tandem Mass Tag (TMT)-labeled LC-MS analysis from young (2-3 months) and old (20-24 months) mice showing relative changes in collagen proteins, n = 5 dural samples per age. (G) Volcano plot of average log₂ fold change and -log₂ adjusted p value for alterations in old and young dural proteins. Collagen proteins are coloured in magenta. Data points represent the average of n = 5 mice per age. (H) Immunohistochemistry of Col1a1 coverage in dural whole mounts and the peri-lymphatic regions of the transverse sinus regions of young (2-3 months) and old (20-24 months) mice. An intra-cisterna magna injection of OVA-A594 (2.5 μL OVA-A594 1 µg/mL) was performed one hour prior to euthanasia, indicating sites of CSF access in the transverse sinus region. Arrowheads highlight peri-lymphatic collagen. (I) Quantification of Col1a1 coverage in the peri-lymphatic regions of the transverse sinus. *, P < 0.05 (Student’s t test), n = 6 young mice, n = 7 old mice. (J) qPCR analysis of Col1a1 expression in the dura of young and old mice. **, P < 0.01 (Student’s t test), n = 6 per age. (K) Analysis of significant differentially expressed genes (DEGs) relating to fibrosis pathways, using the curated gene list from Fibroatlas, in old mouse dura compared to young mouse dura by cell type. Data are from scRNA-seq analysis of young and old whole dura from [16]

Fig. 2

TGFβ1 promotes a fibrotic response in vitro in human dural fibroblasts. (A) Schematic for the growth of human dural fibroblasts obtained from tissue biopsies. (B) Phenotypic characterization of dural fibroblast cultures demonstrating expression of Col1a1, α-SMA, vimentin, TMEM119, PDGFRβ, CD13 and PDGFRα and absence of CD45 and CD31. Images are representative of n = 3 independent cases. (C) Dot plot showing scaled expression and percentage of cells expressing these genes in mouse dural stroma populations from reanalysis of scRNA-seq data in [16]. (D) tSNE visualization of scRNA-seq data from young and old whole dura samples by [16] showing the Cd3e T cell cluster, and presence of Th1, Th2, Th17 and Treg cells determined by expression of Ifng, Il4, Il17a, and Tgfb1 respectively, n = 5 individual young and old dura per experiment, n = 2 independent experiments, 10 dura samples per age total.(E) Schematic of the cytokine cocktails corresponding to different secretion patterns of T cell polarized states, namely Th1, Th2, Th17, and Treg. (F-J) Immunocytochemistry and quantification of Col1a1, Sirius Red, and fibronectin expression in human dural fibroblast cultures after treatment with Veh, Th1, Th2, Th17 or Treg cytokine cocktail at 10 ng/mL for 48 hours. *, P < 0.05; **, P < 0.01; ***, P < 0.001 One-way ANOVA with Kruskal-Wallis post-hoc test, n = 6 independent human dural cultures for Col1a1, n = 8 independent human dural cultures for Sirius Red, n = 9 independent human dural cultures for fibronectin

Fig. 3

Modeling dural ECM deposition using a constitutively active TGFβR1. (A) Schematic depicting our fibrosis model via the delivery of an AAV9 carrying a constitutively active TGFβR1 (muT) to the CSF-filled cisterna magna. (B) Whole mount mouse dura depicting GFP coverage surrounding CSF access points (CAPs) one month after CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL). Insets represent dotted box regions. (C) Quantification of GFP coverage in CAPs of GFP and muT mice. ***, P < 0.001 (Student’s t test), n = 7 mice per AAV. (D) Dot plot demonstrating scaled gene expression and percentage of cells expressing these genes for cell-specific markers in populations from the whole dura or (E) in dura stromal populations from scRNA-seq analysis by [16]. (F) Confocal images of non-sinus and sinus region showing GFP expression in IL33⁺ fibroblasts around the transverse sinus but not non sinus regions one month after CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL).(G) Flow cytometry gating strategy for the identification of GFP expressing cells in the whole dura, one month after delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL). (H) Quantification of the proportions of fibroblasts, CD45⁺ cells or double negative (DNs) cells within the GFP⁺ gate. (I) Quantification for GFP expression within CD45^-CD31^-PDPN⁺ fibroblasts, CD45⁺CD11b⁺Ly6g^-Ly6c^-F4/80⁺ macrophages, or CD45^-CD31⁺ endothelial cells. (J, K) Immunocytochemistry and quantification for Col1a1 expression one week following AAV9 transduction with 1x10¹⁰ GC/mL AAV9-GFP or AAV9-muT. **, P < 0.01 (Student’s t test), n = 6 independent mouse dura cultures per group. (L) Whole mount mouse dura depicting Col1a1 coverage surrounding CAPs one month after CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL). Insets represent regions outlined by dotted box. (M, N) Quantification of Col1a1 and Lyve1 coverage in CAPs of GFP and muT mice. ***, P < 0.001 (Student’s t test), n = 7 mice per AAV

Fig. 4

Dural ECM deposition impairs CSF clearance. (A) Schematic representing the proposed model to promote dural fibrosis via CSF-delivery of AAVs and assess CSF clearance one month later (lymphatic drainage, glymphatic influx, and CSF efflux). (B) Confocal imaging of the transverse sinus region following CSF delivery of OVA-A594 (2.5 µL of 1 mg/mL) one month after CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL).(C) Flow cytometry gating strategy for the identification of dural immune populations. (D-F) Flow cytometry and quantification of CSF-delivered OVA-A594 (2.5 µL of 1 mg/mL) uptake one hour post injection in dural macrophages and dendritic cells (DCs) one month after CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL. ***, P<0.001 (Two-way ANOVA with Sidak’s post hoc test), n = 5 mice per AAV. (G) Quantification of immune cell frequency (% of all CD45⁺ cells) in the dura one month post CSF delivery of AA9-CMV-GFP or muT (3 μL of 1x10¹³ GC/mL). *, P < 0.05; ***, P < 0.001 (Two-way ANOVA with Sidak’s post hoc test), n = 5 mice per AAV. (H-J) Immunohistochemistry and quantification for Lyve1⁺ lymphatics and OVA-A594 drainage (2.5 µL of 1 mg/mL stock) one hour post injection one month after CSF delivery of AAV9-GFP or AAV9-muT (3 µL of 1x10¹³ GC/mL). **, P < 0.01; NS,  P >0.05 (Student’s t-test),n = 10 mice per AAV

Fig. 5

Tissue stiffness impairs lymphatic function. (A) Immunostaining of dural whole mounts showing abundant IL33⁺ fibroblasts surrounding bridging veins around dural sinuses but limited presence around arachnoid cuff exit (ACE) points. (B, C) Immunostaining and quantification for GFP coverage around dural sinuses or DPP4⁺ ACE points following CSF delivered AAV9-GFP expression (3 µL of 1x10¹³ GC/mL of GFP) one month post, n = 5 mice. (D) Summary schematic for the development of induced pluripotent stem cell (iPSC)-derived lymphatic endothelial cells (iLECS). iLECs express key lymphatic markers including PROX1 and VE-Cadherin. (E-G) Flow cytometry and quantification for surface staining of VE-cadherin and VEGFR3 72 hours after culturing on specially-formatted stiffness plates (0.2 kPA and 8 kPA). *,  P < 0.05 Wilcoxon matched-pairs signed rank test, n = 6 independent iLEC differentiations from n = 3 iPSC lines. (H) Immunocytochemistry of VE-cadherin in iLECS 72 hours after culturing on stiffness plates (0.2 kPA and 8 kPA). White arrows indicate regions of VE-Cadherin disruption in stiff plates. (I) iLECs labelled with ActinGreen sprouting on identX microfluidic chips in low stiffness (5 mg/mL fibrinogen to 4 U/mL thrombin), medium stiffness (25 mg/mL fibrinogen to 10 U/mL thrombin) and stiff stiffness (50 mg/mL fibrinogen to 20 U/mL thrombin) fibroin/thrombin gels towards lymphangiogenic factors (VEGF-C, angiopoietin 1 (Ang1) and hepatocyte growth factor (HGF), 100 ng/mL each) for 6 days. Z-depth colour coding shows vessels sprouting in 3-D. (J, K) Quantification of ActinGreen coverage of iLEC sprouting in variable stiffness fibrin/thrombin or Collagen 1 (low stiffness 1 mg/mL, medium stiffness 2 mg/mL or stiff stiffness 4 mg/mL) gels. **, P < 0.01 One way ANOVA with Dunnett’s post hoc test, n = 3/4 independent iLEC differentiations from n = 3 independent iPSC lines