Shi SM, Suh RJ, Shon DJ, Garcia FJ, Buff JK, Atkins M, Li L, Lu N, Sun B, Luo J, To NS, Cheung TH, McNerney MW, Heiman M, Bertozzi CR, Wyss-Coray T. Glycocalyx dysregulation impairs blood-brain barrier in ageing and disease. Nature. 2025 Feb 26; Epub 2025 Feb 26 PubMed.
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University of Arizona College of Medicine and Pharmacy
This is an outstanding manuscript. In the blood-brain barrier field, we now appreciate that the BBB is under constant “attack” by neurodegenerative disease states. Across most CNS diseases, it is now recognized that loss or dysregulation of BBB integrity at the level of the tight junction proteins, such as claudin 5, occludin, zona occludens-1, leads it to “leak.” That these authors demonstrate loss of BBB integrity, and then reverse that loss via restored and improved BBB integrity, is very exciting and a watershed publication for the BBB field.
In the paper, the authors write that, “In addition to contributing to the structural integrity of the glycocalyx layer, mucin-domain glycoproteins have diverse biological roles, including in signaling, cell–cell interactions and regulation of membrane morphology.” This demonstrates their deep understanding of the BBB, a series of tight-knit endothelial cells with a unique, very high electrical resistance. The glycocalyx is much more than just an ultrastructural element. It signals, interacts, and regulates BBB morphology.
I fully agree with the authors when they write “removal of mucin-domain glycoproteins from brain endothelial cells broadly compromises BBB integrity, including by modulating tight junctions, increasing oxidative stress, and disrupting other crucial vascular homeostatic pathways.” I also agree with this statement: “These results demonstrate that restoring the brain endothelial glycocalyx may be an effective therapeutic route to combat BBB breakdown in age-related CNS diseases.” I base my opinion on 45 years of much-appreciated NIH funding for our research on the BBB in stroke, pain, AD, hypoxia and drugs of abuse challenges.
View all comments by Tom DavisWashington University in St Louis
University of Auckland
This study offers compelling insight into the role of mucin-domain glycoproteins in the glycocalyx—a dense layer that coats the luminal surface of endothelial cells and serves as a critical interface between the bloodstream and the vasculature. Composed of glycoproteins, proteoglycans, and glycolipids, the glycocalyx provides mechanical stability, regulates vascular permeability, and modulates immune interactions at the blood-brain barrier. Shi et al. provide evidence that brain endothelial glycocalyx dysregulation is a central feature of aging and neurodegenerative disease.
Their findings suggest that loss of mucin-type O-glycosylation contributes to BBB dysfunction and leakiness, key characteristics of neurodegenerative diseases such as Alzheimer's and Huntington’s, and that restoring these modifications can improve both vascular and cognitive function in aged mice. Importantly, taking advantage of previously published single cell-RNA sequencing datasets, they further revealed that key enzymes involved in mucin-type O-glycosylation are disrupted in Alzheimer's and Huntington's patients, highlighting the translational relevance of these findings.
Using a combination of mucin-selective proteomics, in vivo glycocalyx degradation, and endothelial-specific genetic manipulations, the authors demonstrated that loss of mucin-domain glycoproteins compromises the BBB, allowing blood-derived factors to infiltrate the brain. This disruption was associated with oxidative stress, tight junction instability, and increased neuroinflammation, key contributors to neurodegeneration. Notably, AAV-mediated overexpression of the glycosyltransferases C1GALT1 and B3GNT3—key enzymes in mucin-type O-glycan biosynthesis—restored core mucin-type O-glycosylation. This restoration ultimately rescued BBB function, reduced microglial activation, and improved cognitive performance in aged mice. These findings open up possibilities for therapeutic strategies targeting the BBB glycocalyx for central nervous system rejuvenation in aging and age-related neurological diseases.
While therapeutic restoration of mucin-domains in aging was beneficial in promoting vascular repair and associated neurological improvements, the specific mechanisms through which this is achieved remain unclear. This study raises questions about the broader role of glycosylation in neurodegeneration. While glycosaminoglycans like heparan sulfate have been widely studied, the contribution of mucin-domain glycoproteins to BBB stability is a novel and significant finding. It would be particularly interesting in this context to explore whether the reduced glycocalyx during aging contributes to elevated peripheral immune cell infiltration observed in the aged brain and whether this contributes to the observed BBB leakage, neuroinflammation, and cognitive decline—particularly given the group’s (and others’) prior work on T cell infiltration in the aged, and diseased, brain. It would also be interesting to explore additional factors that could contribute to mucin-type O-glycosylation disturbances beyond the reduction in glycosyltransferases, particularly whether various mucinases or proteases could be implicated in the age-related loss.
The vascular heterogeneity observed in this study is particularly intriguing, as the authors report pronounced BBB dysfunction in the ventricles and brain borders such as the meninges, suggesting that certain regions of the vasculature may be more vulnerable to disruption. Notably, the study observed no similar reduction in luminal mucin-domain glycoprotein coverage in the heart or liver with aging, implying that the mechanisms altering BBB and brain border vascular function may be unique to the brain’s vasculature—a factor that could be highly advantageous for targeted therapies.
However, it remains unclear why some areas of the brain’s vasculature and border regions are more affected than others. Further studies are needed to determine whether differences in endothelial composition, local immune environment, or regional metabolic demands contribute to this selective vulnerability. Understanding these mechanisms could provide crucial insight into how the glycocalyx functions across different segments of the brain’s vasculature, and identify specific pathways that drive age-related BBB dysfunction.
Interestingly, no drugs have been found that specifically target components of the glycocalyx. This remains an intriguing avenue for therapeutic development. Given the challenges associated with gene therapy for humans, such as delivery and long-term expression, exploring small molecules or biologics that could modulate the glycocalyx or its key components may offer a less-invasive alternative for treating age-related BBB dysfunction and neurodegenerative diseases. Interventions aimed at restoring the glycocalyx could represent a promising new strategy for mitigating BBB breakdown in aging and disease.
In conclusion, this study opens exciting possibilities for therapeutic intervention targeting the glycocalyx in neurodegenerative diseases. Future investigation into the underlying mechanisms involved will be crucial for determining whether these findings can be translated into effective treatments for aging-related vascular dysfunction and cognitive decline.
View all comments by Taylor StevensonOHSU
In this impressive and data-rich study, Shi et al. assessed the role of dysregulation of the endothelial glycocalyx in the function of the blood-brain-barrier in aging and neurodegenerative conditions. They revealed that, in wild-type mice, perturbation in O-glycosylated proteins (mucin-domain glycoproteins) results in dysregulation of the BBB and, in severe cases, in brain hemorrhaging. In addition, restoring core 1 mucin-type O-glycans to the brain endothelium using adeno-associated viruses (AAV-B3GNT3 and AAV-C1GALT1) improved BBB function and reduced neuroinflammation.
However, treatment of 17-month-old mice for eight weeks with AAV-B3GNT3, but not with AAV-C1GALT1, improved hippocampus-dependent cognitive performance in the Y maze (percent spontaneous alternation) and hippocampus-dependent contextual fear memory, but not hippocampus-independent cued fear memory, in aged wild-type mice, as compared to age-matched AAV-EGFP controls. While similar beneficial effects of AAV-B3GNT3 and AAV-C1GALT1 were seen on all BBB-related markers, quantification of the area occupied by immunoreactivity of IBA1, a microglial marker, in the cortex was lower in AAV-B3GNT3-, but not in AAV-C1GALT1-treated mice, as compared to age-matched AAV-EGFP controls. These data suggest that neuroinflammation in combination with BBB injury might be driving cognitive injury, and that improving BBB function by itself might not be sufficient to improve cognitive function.
The authors also conducted differential glycosylation-related gene analysis on previously published single-nucleus RNA-Seq (snRNA-Seq) datasets of Alzheimer’s and Huntington’s diseases; brain endothelial mucin-type O-glycan biosynthesis was revealed as a shared downregulated pathway enriched in both neurodegenerative conditions and was affected in the aged wild-type mice in this study as well, suggesting the potential for treating healthy age-related cognitive decline in the absence of frank dementia as well as for treating neurodegenerative conditions with dementia.
An important role of the integrity of the BBB for cognitive health is consistent with studies in human apolipoprotein E3 and E4 targeted replacement mice on a standard and Western diet (Rhea et al., 2020; Rhea et al., 2021) and with the ability of viruses, including the non-replicating SARS-CoV-2 and viral proteins like the S1 protein of SARS-CoV-2, to cross the BBB and negatively affect brain function in mice (Rhea et al., 2020; Erickson et al., 2023; Raber et al., 2023). The results of the current study are also consistent with the decline in BBB function in aging and neurodegeneration (Andjelkovica et al., 2023) and efforts to protect or restore BBB function (Knox et al., 2022). The age-related changes in BBB function are more profound in individuals with E4 (Banks et al., 2021).
The upregulation of genes involved in heparan sulfate metabolism in brain endothelial cells reported in this study is consistent with the role of heparan sulfate in neuronal loss in fly models of AD (Schultheis et al., 2024) and the altered expression of heparan sulfate and its role in metabolism of Aβ in Alzheimer’s disease (Ozsan et al., 2023).
The upregulation of hyaluronan in isolated microvessels of aged mice as compared to the young wild-type mice is consistent with the positive correlation of HA with neuropathology in Alzheimer’s disease (Reed et al., 2019) and the association of age-related accumulation of HA in the hippocampus with reduced neurogenesis and cognitive injury (Su et al., 2017)
To assess the molecular changes in brain endothelial cells induced by mucin degradation, the authors performed bulk RNA-Seq on bEnd.3 cells after 16h of StcE treatment. Differential gene expression analysis revealed downregulation of genes involved in vascular development and integrity, TGF signaling, and in the regulation of reactive oxygen species, including the glutathione peroxidase 1 gene. These results are consistent with the role of ROS in aging and neurodegeneration and the recovery of spatial memory and reduced neuronal loss in the hippocampus following traumatic cortical brain injury to the developing brain in mice with transgenic overexpression of glutathione peroxidase (Tsuru-Aoyagi et al., 2009).
For the mouse studies, young (3-month-old) and aged (17- to 19-month-old) wild-type mice were used. It would be good to consider including middle-aged mice in follow-up studies, as age-related brain changes in wild-type often start at middle age (Benice et al., 2006). Also, the age-related changes in hippocampus-dependent cognition (Benice et al., 2006) and in BBB function (Dion-Albert et al., 2022) are sex-dependent. Therefore, it will be important to compare these outcome measures in female and male mice (in the current study, it was not clear to me whether female and/or male mice were used). Also, it would be good to include wild-type mice without viral EGFP expression to exclude potential effects of EGFP on any of the outcome measures.
It is very encouraging that the age-related dysregulation of the endothelial glycocalyx in BBB function seen in wild-type mice resembles that seen in patients with Alzheimer’s and Huntington’s. It will be important to consider follow-up studies using mouse models of Alzheimer’s, Huntington’s, and other neurodegenerative conditions.
View all comments by Jacob RaberWashington University in St. Louis
Washington University in St. Louis, School of Medicine
The microenvironment of the brain is controlled by a series of border tissues that regulate the entry and exit of molecules from the periphery. Chief amongst these is the blood-brain barrier (BBB), which refers to a series of properties in the brain endothelium: tight junctions, suppressed bulk transcytosis, and a range of import and efflux pumps. Less well-studied is the glycocalyx of the brain vasculature, which is very thick compared to the endothelial glycocalyx of other organs and forms a non-adhesive coating to blood vessels (Ando et al., 2018).
Here, Shi et al. show that the glycocalyx of the brain vasculature is dramatically altered in aging mice. It loses much of its coating, and changes composition. Most notably, a particular modification, mucin-type O-glycosylation, was found to be reduced. Further work suggested that this was related to changes in the glycosylation biosynthetic pathway, driven by C1galt1, an enzyme that adds glycans to mucins.
Using an adenovirus under a promoter specific to endothelial cells (Cldn5) to target the BBB, Shi et al. then perturbed C1galt1 in mice. When its expression was reduced, there was dramatic BBB opening, and even the presence of bleeds. On the other hand, in old mice, rescue of another glycocalyx-modifying enzyme, B3gnt3, functionally restored BBB integrity and was sufficient to rescue cognitive dysfunction.
This work sheds light on an understudied player in BBB biology, and there are many questions. What drives changes in the glycocalyx during aging and diseases? This could be triggered by brain dysfunction or circulating challenges, and indeed the glycocalyx is highly sensitive to inflammation (Ando et al., 2018).
Most importantly, how does the glycocalyx help maintain the BBB? The negatively charged glycocalyx may protect brain endothelial cells from the actions of circulating cytokines, which are predominantly negatively charged (Messina et al., 2024). Alternatively, the glycocalyx is a major impediment that restricts the interactions of immune cells with the BBB, so its loss may enable circulating immune cells to interact with the BBB, to trigger its breakdown (Schmidt et al., 2012). This may, in turn, contribute to capillary stalling as has been reported previously in mouse models of Alzheimer’s disease (Cruz Hernandez et al., 2019).
Finally, the ability to target the glycocalyx with viral vectors suggests that this work could be translated, either through viral vectors or drugs that target the enzymes that form the glycocalyx, to restore BBB function during aging or neurological disease.
References:
Ando Y, Okada H, Takemura G, Suzuki K, Takada C, Tomita H, Zaikokuji R, Hotta Y, Miyazaki N, Yano H, Muraki I, Kuroda A, Fukuda H, Kawasaki Y, Okamoto H, Kawaguchi T, Watanabe T, Doi T, Yoshida T, Ushikoshi H, Yoshida S, Ogura S. Brain-Specific Ultrastructure of Capillary Endothelial Glycocalyx and Its Possible Contribution for Blood Brain Barrier. Sci Rep. 2018 Nov 30;8(1):17523. PubMed.
Cruz Hernández JC, Bracko O, Kersbergen CJ, Muse V, Haft-Javaherian M, Berg M, Park L, Vinarcsik LK, Ivasyk I, Rivera DA, Kang Y, Cortes-Canteli M, Peyrounette M, Doyeux V, Smith A, Zhou J, Otte G, Beverly JD, Davenport E, Davit Y, Lin CP, Strickland S, Iadecola C, Lorthois S, Nishimura N, Schaffer CB. Neutrophil adhesion in brain capillaries reduces cortical blood flow and impairs memory function in Alzheimer's disease mouse models. Nat Neurosci. 2019 Mar;22(3):413-420. Epub 2019 Feb 11 PubMed.
Messina JM, Luo M, Hossan MS, Gadelrab HA, Yang X, John A, Wilmore JR, Luo J. Unveiling cytokine charge disparity as a potential mechanism for immune regulation. Cytokine Growth Factor Rev. 2024 Jun;77:1-14. Epub 2023 Dec 26 PubMed.
Schmidt EP, Yang Y, Janssen WJ, Gandjeva A, Perez MJ, Barthel L, Zemans RL, Bowman JC, Koyanagi DE, Yunt ZX, Smith LP, Cheng SS, Overdier KH, Thompson KR, Geraci MW, Douglas IS, Pearse DB, Tuder RM. The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nat Med. 2012 Aug;18(8):1217-23. Epub 2012 Jul 22 PubMed.
View all comments by Jonathan KipnisYale University
This manuscript provides a comprehensive picture of changes in glycoprotein in the blood-brain barrier, offering an impressive view of the glycoprotein landscape along the lifespan of the mouse. The discovery that mucin-domain glycoproteins are lost during aging is critical in developing therapeutics for a large number of neurodegenerative diseases or even for normal, aging-mediated breakdown of the BBB. While this manuscript focuses more on the physical barrier glycoproteins provide for endothelial cells in the BBB, their role in signaling with other cellular components, including astrocytes and pericytes, but also even with neurons, will be highly important future directions of study that could reveal ways to delay or reverse neurodegeneration.
View all comments by In-Hyun ParkGifu University Graduate School of Medicine
I am impressed by this paper, which echoes the words of Dr. William Osler, who said, “A man is as old as his arteries." It is a very novel study, suggesting that with aging, the glycocalyx thins, and that its restoration improves vascular permeability, decreases neuroinflammation, and leads to improvement of dementia. It emphasizes that we cannot talk about aging without considering the vascular endothelium.
It is known that the vascular endothelial glycocalyx is composed of a complex of various proteoglycans and glycoproteins, the structure and components of which vary from organ to organ. Glycosaminoglycans, which are negatively charged, prevent blood proteins, which are also negatively charged, from leaking out of blood vessels. They mask adhesion factors such as integrins that exist on the vascular endothelium, and they prevent inflammatory cells and platelets from adhering to vascular endothelial cells, thereby helping to maintain microcirculation.
The glycocalyx in the vascular endothelium of the brain is thicker than in other organs. It is thought that this thick glycocalyx contributes to the maintenance of the blood-brain barrier, a structure that prevents unwanted substances from entering the blood vessels. On the other hand, since glycocalyx is known to thin out due to various diseases, it is thought that aging and disease are caused by the thinning of glycocalyx.
When various signaling molecules travel throughout the body, they pass through blood vessels. They must pass the vascular wall to migrate to the tissues, and the glycocalyx on the surface of the wall is the front line of defense on the tissue side, so to speak. It is very reasonable to assume that abnormalities in this area allow various substances to enter the tissues, which is why it is so important for aging and a variety of diseases. In this sense, I think the involvement of mucin domain glycoproteins, which make up glycocalyx, is significant, and I think there is a good chance that this repair will improve brain homeostasis.
The structure of blood vessels and glycocalyx differs not only by organ but also by organ site, suggesting that the phenotype of the brain as a whole, as well as differences by site, are important to study. Since blood vessels are present in almost all tissues, all organs are potential targets. One of the most interesting of these organs is the kidney, whose function declines with aging. An interesting disease is cancer, depending on type. Cancer is interesting because it is covered with bulky glycocalyx, involves blood vessels when it metastasizes, and is more common in the elderly.
I am interested in glycocalyx as a therapeutic target because I believe aging affects not only the brain but other organs and diseases. This work inspires us to explore these relationships further.
View all comments by Hideshi OkadaMake a Comment
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