The blood-brain barrier (BBB) is highly specialized to protect the brain from harmful circulating factors in the blood and maintain brain homeostasis1,2. The brain endothelial glycocalyx layer, a carbohydrate-rich meshwork composed primarily of proteoglycans, glycoproteins and glycolipids that coats the BBB lumen, is a key structural component of the BBB3,4. This layer forms the first interface between the blood and brain vasculature, yet little is known about its composition and roles in supporting BBB function in homeostatic and diseased states. Here we find that the brain endothelial glycocalyx is highly dysregulated during ageing and neurodegenerative disease. We identify significant perturbation in an underexplored class of densely O-glycosylated proteins known as mucin-domain glycoproteins. We demonstrate that ageing- and disease-associated aberrations in brain endothelial mucin-domain glycoproteins lead to dysregulated BBB function and, in severe cases, brain haemorrhaging in mice. Finally, we demonstrate that we can improve BBB function and reduce neuroinflammation and cognitive deficits in aged mice by restoring core 1 mucin-type O-glycans to the brain endothelium using adeno-associated viruses. Cumulatively, our findings provide a detailed compositional and structural mapping of the ageing brain endothelial glycocalyx layer and reveal important consequences of ageing- and disease-associated glycocalyx dysregulation on BBB integrity and brain health.
© 2025. The Author(s).

Transferrin receptor (TfR)-mediated transcytosis across the blood-brain barrier (BBB) is a promising strategy to improve delivery of biologics to the central nervous system (CNS). However, it remains unclear whether age and aging-related diseases impact TfR expression and/or BBB transport capacity. Here, we used the TfR-targeted antibody transport vehicle (ATV TfR ) to enhance CNS delivery in healthy mice and in the 5xFAD mouse model of Alzheimer’s disease (AD). Healthy neonates exhibited the highest vascular TfR expression and ATV TfR brain exposure, whereas BBB transport capacity remained stable across adulthood. Additionally, neither TfR expression nor ATV TfR brain uptake changed significantly in 5xFAD mice. Further, vascular TfR expression in AD patient brains was similar to age-matched controls, suggesting that TfR transport may be conserved for AD in humans. The elevated TfR-mediated brain delivery observed in early mouse development suggests the potential of added efficacy in utilizing TfR platforms for the treatment of early childhood diseases. Preservation of ATV TfR transport in adult mice across healthy aging and in an AD model supports continued application of TfR platforms in age-related diseases.

Existing flow cytometry approaches identify immune cells using the whole infected/inflamed cornea, which limits its ability to distinguish the immune cells infiltrating the corneal epithelium from the corneal stroma. Here, we present a protocol to analyze immune cells in the separated epithelium and stroma from naïve and herpes simplex virus-1 (HSV-1)-infected mouse corneas. We describe steps for viral infection, separation of corneal epithelium from stroma, preparation of a single-cell suspension of the individual epithelium and stroma, and flow cytometry assay.
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.

Physical exercise is generally beneficial to all aspects of human and animal health, slowing cognitive ageing and neurodegeneration1. The cognitive benefits of physical exercise are tied to an increased plasticity and reduced inflammation within the hippocampus2-4, yet little is known about the factors and mechanisms that mediate these effects. Here we show that 'runner plasma', collected from voluntarily running mice and infused into sedentary mice, reduces baseline neuroinflammatory gene expression and experimentally induced brain inflammation. Plasma proteomic analysis revealed a concerted increase in complement cascade inhibitors including clusterin (CLU). Intravenously injected CLU binds to brain endothelial cells and reduces neuroinflammatory gene expression in a mouse model of acute brain inflammation and a mouse model of Alzheimer's disease. Patients with cognitive impairment who participated in structured exercise for 6 months had higher plasma levels of CLU. These findings demonstrate the existence of anti-inflammatory exercise factors that are transferrable, target the cerebrovasculature and benefit the brain, and are present in humans who engage in exercise.
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.

Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus-induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue-resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.
© 2020 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.