The full complexity of the blood-brain barrier, with its different cell types acting together, has been difficult to model in vitro. In the June 8 Nature Medicine, researchers led by Li-Huei Tsai at the Massachusetts Institute of Technology, Cambridge, debuted a model that combined endothelial cells, astrocytes, and pericyte-like cells in a three-dimensional gel. The endothelial cells formed capillaries that had high electrical resistance and held back the escape of solutes, suggesting the presence of tightly sealed junctions like those in the brain-blood barrier.
- Blood-brain barrier model combines human astrocytes, pericytes, and endothelial cells.
- APOE4 in pericytes, but not the other cells, promoted cerebral amyloid angiopathy.
- Blocking NFAT signaling protected pericytes.
Other researchers called the model an important advance. “This is a very innovative, versatile blood-brain barrier model, with human relevance,” said Guojun Bu at the Mayo Clinic in Jacksonville, Florida. Costantino Iadecola of Weill Cornell Medical College in New York noted, “This is a step forward in terms of the ability to reproduce what we see in vivo.”
Using this model, Tsai and colleagues found that APOE4, a risk factor for cerebral amyloid angiopathy, worked its mischief through pericytes, but not the other cell types. When pericytes in the model expressed APOE4, they ramped up a transcriptional pathway that boosted ApoE secretion and led to amyloid deposition on capillary walls. Blocking this pathway prevented deposition. “Emerging evidence indicates an important role for pericytes in the physiology and pathology of blood vessels in the brain,” Tsai told Alzforum.
Capillary in a Dish. Two weeks after combining brain endothelial cells, mural cells, and astrocytes in hydrogel, mural cells (middle, green) line blood vessels (red), while astrocyte end-feet (right, green) touch vessels (red). Nuclei are blue. [Courtesy of Blanchard et al., Nature Medicine.]
A paper in the June 15 Nature Cell Biology highlights this. Researchers led by Asifa Akhtar at the Max Planck Institute of Immunobiology and Epigenetics in Freiburg, Germany, generated mice with a defective chromatin-regulating complex. Their neuronal metabolism went haywire, and this in turn inflamed pericytes, leading to a disrupted vasculature. The finding may help explain why people who carry a mutation in the same complex are at elevated risk for neurodegeneration.
Researchers have established numerous in vitro models that capture various aspects of the blood-brain barrier. Cheryl Wellington at the University of British Columbia in Vancouver, Canada, used human cells to build artificial blood vessels on a tube-like support. The tubes transported fluid and mimicked CAA when Aβ was added (Oct 2017 news). Don Ingber and Kit Parker at Harvard study interactions between brain cells and blood vessels on polymer chips (Aug 2018 news). Doo Yeon Kim and Rudolph Tanzi at Harvard Medical School juxtaposed an endothelial cell layer with a three-dimensional neuronal culture to model the BBB (Oct 2014 news). In-Hyun Park and colleagues at Yale School of Medicine in New Haven, Connecticut, grew cerebral organoids with a functioning vasculature (Oct 2019 news).
Few of these models focus on the interactions of astrocytes, pericytes, and endothelial cells, however. To generate these cells, Tsai and colleagues separately differentiated human induced pluripotent stem cells into either endothelial cells, astrocytes, or mural cells. Mural cells come in different varieties, including the smooth muscle cells that hug larger vessels and the pericytes that line capillaries. Researchers disagree on where the dividing line is between them (Jun 2015 news; Feb 2017 news; May 2017 news). Because no specific markers have been identified for mural cell types, the authors characterized them by RNA-Seq, finding that they had an expression profile very similar to in vivo pericytes, and distinct from that of smooth muscle cells.
First author Joel Blanchard combined all three cell types in a Matrigel matrix and allowed them to grow for two weeks in the presence of platelet-derived growth factor BB and vascular endothelial growth factor A. At the end of that time, the endothelial cells had assembled into capillary-like structures surrounded by basement membrane. Pericytes had migrated to perch on these tubes, as they do in vivo, while astrocytes extended end-feet to touch the vessels (see image above). The capillaries expressed tight junction proteins, retained solutes, and exhibited a high electrical resistance, suggesting the presence of a blood-brain barrier.
The authors then used this system to examine how APOE4 boosts the risk for cerebral amyloid angiopathy (Premkumar et al., 1996; Rannikmäe et al., 2014). They generated endothelial cells, pericytes, and astrocytes from people who were either APOE3/3 or APOE3/4, then used different combinations to model the BBB. To promote CAA, the authors exposed their models to conditioned media from neuronal cultures carrying an APP duplication.
When the pericyte-like cells were APOE3/3, little amyloid deposited, no matter what genotype the astrocytes and endothelial cells were. When the pericyte-like cells expressed APOE4, however, extensive CAA occurred (see image above).
What did APOE4 do to pericytes? By RNA-Seq, the authors found about 2,300 genes elevated and almost 2,000 repressed in APOE3/4 pericyte-like cells compared with APOE3/3. They looked for possible master regulators, and noticed upregulation of nuclear factor of activated T cells (NFAT). The phosphatase calcineurin activates NFAT, allowing it to move into the nucleus and initiate transcription. Calcineurin and NFAT are elevated in Alzheimer’s brain, and NFAT interacts with the APOE promoter (Maloney et al., 2007; Reese and Taglialatela, 2011). APOE3/4 pericytes, but not the other cell types, had elevated calcineurin, nuclear NFATc1, and APOE expression. The data hinted at a positive feedback loop, with APOE4 in pericyte-like cells stimulating NFAT signaling to boost its own expression. It is unclear why APOE4 could have such different effects in distinct cell types.
Elevated APOE expression was the culprit behind CAA, the authors found. Overexpressing ApoE3 in the blood-brain barrier model mimicked the APOE4 genotype, causing more CAA, while knocking out the gene prevented it. Blocking NFAT signaling was equally effective. Treating the BBB model with calcineurin inhibitors for two weeks lowered ApoE, normalized the expression profile of APOE3/4 pericytes to that of APOE3/3 cells, and prevented CAA after exposure to Aβ. The same strategy worked in vivo. The authors crossed APOE4 knock-in mice with 5xFAD mice, and treated the offspring with calcineurin inhibitors for three weeks. Treated mice expressed less ApoE in the brain and had less CAA compared with untreated littermates. Likely, ApoE seeds amyloid aggregation in blood vessels as it does in brain, Tsai believes.
Does the mechanism hold in people? In postmortem brain samples from APOE3 and APOE4 carriers, the authors found higher levels of NFAT and ApoE in pericytes in the latter. However, commenters questioned how well findings from this model will translate. Iadecola noted that most CAA in the brain occurs in larger vessels, not capillaries. Bu noted that APOE4 carriers produce less ApoE in parenchyma than do noncarriers, the opposite of what was seen in the pericytes in this model. Because ApoE is secreted, the parenchymal protein might reach blood vessels, complicating the picture. “Is the vasculature exposed to a lower or higher amount of ApoE in ApoE4 carriers? We don’t know,” Bu said.
Nonetheless, researchers found the data exciting. “The interaction between neural-derived Aβ and pericyte-derived ApoE is incredibly novel … it suggests that neural-pericyte interactions may drive the pathology observed in APOE4 carriers,” Richard Daneman at the University of California, San Diego, wrote to Alzforum (full comment below). Steven Greenberg at Massachusetts General Hospital, Boston, wondered if the potent effect of ApoE4 on vascular amyloid drives the increased ARIA seen in APOE4 carriers on anti-amyloid immunotherapy (comment below).
Researchers were curious about therapeutic implications. Calcineurin inhibitors such as cyclosporin A and FK506 are approved by the Food and Drug Administration for use as immunosuppressants, typically to prevent rejection of transplanted organs. Intriguingly, people taking these drugs have a lower risk of dementia (Taglialatela et al., 2015). Christopher Norris of the University of Kentucky, Lexington, noted that calcineurin inhibitors exert beneficial effects on brain cells, calming reactive astrocytes and promoting synaptic plasticity in neurons. “Calcineurin is a major mediator of cellular dysfunction in numerous neurodegenerative diseases,” he said. Norris collaborates with Elizabeth Head at UC Irvine to test calcineurin inhibitors in a dog model of amyloidosis. In preliminary data, they see benefits to neuronal connectivity at drug levels low enough not to suppress the immune system, suggesting the therapy could be worth testing in people.
Tsai’s findings train a spotlight on pericytes, a cell type that has been obscure in Alzheimer’s disease until recently. ApoE4 has previously been implicated in boosting the pro-inflammatory cytokine cyclophilin A in pericytes, causing them to produce proteinases that damage the blood-brain barrier and make it leak (May 2012 news). Recently, researchers led by Axel Montagne and Berislav Zlokovic at the University of Southern California in Los Angeles tied this pericyte-mediated damage to cognitive decline (May 2020 news).
Montagne noted that cyclosporin A squelches expression of pro-inflammatory cyclophilin A, the culprit behind this damage. “The vascular contribution to dementia is increasingly recognized, and having a drug that can protect blood-brain barrier function as well as reduce amyloidosis is very exciting for the field,” he wrote to Alzforum (full comment below).
The findings dovetail with the report by Akhtar and colleagues. First author Bilal Sheikh generated transgenic mice that lacked one of the three members of the nonspecific lethal (NSL) chromatin complex, but only in neurons. Conditional knockouts of Mof, Kansl2, and Kansl3 all had the same phenotype, developing cerebrovascular problems and brain hemorrhaging. The authors traced the cause to a breakdown of neuronal metabolism, which led to the buildup of long-chain fatty acids. These fatty acids were secreted, triggering the TLR4 receptor in pericytes. In response to TLR4 signaling, pericytes became inflamed, changed shape, and detached from blood vessels. The vessels became leaky or collapsed.
The same thing may happen in people who carry a Kansl1 mutation, the authors suggested. This mutation causes Koolen-de Vries syndrome, marked by enlarged ventricles and a poorly developed corpus callosum. In the postmortem brain of a fetus who died with the mutation, the authors found evidence of brain hemorrhaging, consistent with what they saw in mouse models. Kansl1 lies close to MAPT, the tau gene, and has been linked to progressive supranuclear palsy (Nov 2018 conference news).
Tsai noted that in toto, the data suggest pericytes may be sensitive to the distress of other cell types such as neurons, and respond by ramping up inflammation. “An anti-inflammatory, immunosuppressant [treatment] approach could be beneficial,” Tsai suggested.—Madolyn Bowman Rogers
- Artificial Human Blood Vessels: A Model for Cerebral Amyloid Angiopathy?
- 'Organ on a Chip' Models the Ins and Outs of the Blood-Brain Barrier
- Alzheimer’s in a Dish? Aβ Stokes Tau Pathology in Third Dimension
- It Bleeds! New Mini-Brains Sport Functioning Blood Vessels
- Smooth Muscle Cells, Not Pericytes, Control Brain Blood Flow
- Pericytes Don’t Go With the Flow—They Change It
- Finally, a Dye to Visualize Pericyte Function
- ApoE4 Makes Blood Vessels Leak, Could Kick Off Brain Damage
- Even Without Amyloid, ApoE4 Weakens Blood-Brain Barrier, Cognition
- International Symposium Puts PSP/CBD on the Map
Research Models Citations
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- Rannikmäe K, Kalaria RN, Greenberg SM, Chui HC, Schmitt FA, Samarasekera N, Al-Shahi Salman R, Sudlow CL. APOE associations with severe CAA-associated vasculopathic changes: collaborative meta-analysis. J Neurol Neurosurg Psychiatry. 2014 Mar;85(3):300-5. Epub 2013 Oct 25 PubMed.
- Maloney B, Ge YW, Alley GM, Lahiri DK. Important differences between human and mouse APOE gene promoters: limitation of mouse APOE model in studying Alzheimer's disease. J Neurochem. 2007 Nov;103(3):1237-57. PubMed.
- Reese LC, Taglialatela G. A role for calcineurin in Alzheimer's disease. Curr Neuropharmacol. 2011 Dec;9(4):685-92. PubMed.
- Taglialatela G, Rastellini C, Cicalese L. Reduced Incidence of Dementia in Solid Organ Transplant Patients Treated with Calcineurin Inhibitors. J Alzheimers Dis. 2015;47(2):329-33. PubMed.
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- Sheikh BN, Guhathakurta S, Tsang TH, Schwabenland M, Renschler G, Herquel B, Bhardwaj V, Holz H, Stehle T, Bondareva O, Aizarani N, Mossad O, Kretz O, Reichardt W, Chatterjee A, Braun LJ, Thevenon J, Sartelet H, Blank T, Grün D, von Elverfeldt D, Huber TB, Vestweber D, Avilov S, Prinz M, Buescher JM, Akhtar A. Neural metabolic imbalance induced by MOF dysfunction triggers pericyte activation and breakdown of vasculature. Nat Cell Biol. 2020 Jun 15; PubMed.