15 Nov 2019

Whether in the wake of a stroke, seizure, massive neuroinflammation, or a blow to the head, the endothelial cells of the blood-brain barrier respond with remarkable similarity, according to a study published in the November issue of Nature Neuroscience. Researchers led by Richard Daneman at the University of California, San Diego, reported that the endothelial cells that make up the BBB normally express a suite of genes that distinguishes them from the endothelia of other organs. However, BBB cells damaged in various ways lost this specialized signature, changing over to an expression profile more akin to endothelial cells in other parts of the body. The findings suggest that common mechanisms of BBB dysfunction underlie different brain injuries and diseases.

Endothelial cells that form the BBB have a distinct gene-expression profile.

It responded similarly to stroke, seizure, head trauma, neuroinflammation.

This “BBB dysfunction signature” mirrored endothelial gene expression in other organs.

David Attwell of University College London called the study exciting, noting that the similarities in gene-expression changes evoked by different insults was surprising. “This raises the possibility that successfully preventing (or increasing) endothelial cell gene-expression changes that occur in one disease may lead to a potential therapy for other types of CNS disorders,” he wrote.

“This exciting study provides a comprehensive data resource on the molecular underpinnings of brain endothelial cell dysfunction,” wrote Tony Wyss-Coray and Andrew Yang of Stanford University in a joint comment to Alzforum. “It was not known a priori whether such a core dysfunction module existed, and its identification opens a variety of fascinating questions.”

Tasked with shielding the precious brain from toxic insults while allowing crucial nutrients to cross, the blood-brain barrier is highly selective. Ergo, the endothelial cells that line the brain’s vessels are highly specialized, forming ultra-tight junctions and mobilizing molecular transporters not typically found in vessels supplying other organs. Disruption of the barrier is thought to play a hand in the pathogenesis of multiple injuries and diseases—including traumatic brain injury, stroke, seizures, and neurodegenerative disease such as Alzheimer’s (Mar 2008 news; Feb 2015 webinar; Jan 2019 news).

What molecular shenanigans compromise the barrier? And are they similar across neurological diseases? Co-first authors Roeben Munji and Allison Soung and colleagues addressed these questions by comparing the transcriptomes of endothelial cells. Before diving into injury or disease models, the researchers compared the gene-expression profiles of healthy endothelial cells taken from the brain and other organs of mice. Compared with their counterparts from the kidney, lung, and liver, endothelial cells from the brain expressed a unique set of genes that encode metabolic enzymes and components of tight junctions, transporters, and the extracellular matrix, as well as units of the Wnt-β-catenin pathway known to support the BBB’s formation and maintenance. In contrast, endothelial cells in other organs of the body expressed a plethora of genes involved in immune functions, including receptors that latch onto leukocytes and whisk them across vessel walls.

How do brain endothelial cells change in the face of injury or disease? The researchers tracked gene-expression changes following four different insults known to disrupt the barrier: seizures, stroke, trauma, and experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. They induced seizures by injecting kainic acid, triggered stroke by occluding the middle cerebral artery, caused traumatic brain injury by dropping weights onto the heads of anesthetized mice, and set off EAE by injecting the 35-55 amino acid fragment of myelin oligodendrocyte glycoprotein, which elicited an inflammatory autoimmune response.

They then tracked the permeability of the barrier at three time points. In the earliest, or “acute” phase of each injury, the researchers found only minimal disruption of the BBB. However, in the so-called “subacute phase,” which was one or two days later depending on the model, the leakiness of the BBB reached its peak. About a month later, in the “chronic phase,” the barrier had partially or fully regained its integrity.

Each injury induced a bevy of gene-expression changes in brain endothelial cells. They varied substantially among injuries in the acute phase, but shared striking commonalities in the subacute phase, when the barrier was leakiest. By the chronic phase, gene expression had largely returned to normal in the stroke, seizure, and TBI models, but remained highly altered in the EAE model. Interestingly, the researchers found that the genetic signature of the healthy BBB endothelium was most downregulated in the acute phase of TBI, and the subacute phase of stroke, seizure, and EAE. Conversely, genes expressed predominantly in endothelial cells outside of the brain were turned up in the brain endothelium at these time points.

United in Dysfunction. A core group of 54 genes were upregulated in the subacute phase of all four disease models, while 136 (red numbers) were turned up in at least three. [Courtesy of Munji et al., Nature Neuroscience, 2019.]

At the subacute time point—when the barrier was leakiest across all models—54 genes were upregulated in common across all four models, and 136 were turned up in at least three models (see image at right). The researchers defined these 136 genes as a common BBB dysfunction module. It contained 68 genes normally expressed by endothelial cells outside of the brain, including many that belong to pathways involved in angiogenesis and inflammation.

Together, the findings suggest that while brain endothelial cells may initially respond differently to unique insults, they soon converge on a gene-expression profile that resembles those of endothelial cells in other organs of the body, said Daneman. Among other functions, these gene-expression changes likely ramp up interactions between the endothelium and circulating immune cells, he added.

The findings jibe with a recent study led by Wyss-Coray, which reported the increased expression of the leukocyte adhesion receptor VCAM1 on brain endothelial cells with age (Jul 2018 conference news; May 2019 news). Aging also weakens the BBB.

Important unanswered questions were whether the gene-expression changes Mungi and colleagues identified alter the integrity of the blood-brain barrier, and whether they are helpful or harmful to the brain, Daneman said. The full genetic dataset will be available to researchers at NCBI’s Gene Expression Omnibus.

Will these findings extend to neurodegenerative diseases such as AD? Dementias typically have a much longer prodromal phase than the acute injuries modeled here, and the blood-brain barrier likely erodes more slowly. Some studies have been unable to detect overt breaches in the BBB in AD (Oct 2015 news). This important question is challenging to address, Daneman said, not only because the BBB is more intact in these diseases, but also because mouse models of AD poorly recapitulate the cerebrovascular pathologies associated with the disease. His lab is developing techniques to isolate endothelial cells from postmortem brain tissue of AD and PD patients. Daneman is also looking for serum biomarkers of BBB disruption.

Axel Montagne of the University of Southern California, Los Angeles, wrote that the paper could spur an investigation of how different cells in the brain’s vasculature, such as pericytes and smooth muscle cells, change when the BBB is compromised, and how gene-expression profiles of cells compare throughout the cerebrovascular tree, and in different regions of the brain (Vanlandewijck et al., 2018). Montagne envisions doing cerebrovascular-profiling studies in mouse models of AD.

“We also wonder whether this [BBB dysfunction] module will be found in chronic settings of neurodegeneration and aging,” wrote Yang and Wyss-Coray. “A hint arises in the module’s de-enrichment of BBB-specific genes, suggesting a common perturbation in surrounding mural-cell signals, such as from a loss of pericytes,” they added. “As pericyte loss has been reported in Alzheimer’s disease, this module may indeed be an even more generalizable hallmark of BBB dysfunction.”

Giuseppe Faraco of Weill Cornell Medical College in New York wondered whether early changes to brain endothelial function that precede overt BBB disruption might play a role in the pathogenesis of neurodegenerative diseases. Faraco recently reported that in response to a high-salt diet, dysfunctional brain endothelial cells trigger tau aggregation within the parenchyma that might wreak havoc on the brain (Oct 2019 news). Others have reported that BBB weakness itself correlated with dementia, regardless of ongoing tau or Aβ pathology (Jan 2019 news).

The study underscores the emerging view that the BBB is a transcriptionally dynamic sensor of a variety of environmental stimuli, and that its gene products may functionally affect overall brain health, noted Yang and Wyss-Coray. “Whether these ‘sense-and-response’ capabilities decline with normal aging and neurodegenerative disease may be an important area of study,” they wrote.—Jessica Shugart