Blanchard JW, Bula M, Davila-Velderrain J, Akay LA, Zhu L, Frank A, Victor MB, Bonner JM, Mathys H, Lin YT, Ko T, Bennett DA, Cam HP, Kellis M, Tsai LH. Reconstruction of the human blood-brain barrier in vitro reveals a pathogenic mechanism of APOE4 in pericytes. Nat Med. 2020 Jun;26(6):952-963. Epub 2020 Jun 8 PubMed. Correction.

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  1. The Tsai lab provides some remarkable insight into the generation of cerebral amyloid angiopathy, which may identify potential therapeutics for Alzheimer's disease. A large percentage of people with AD develop amyloid plaques along their blood vessels. This cerebral amyloid angiopathy can lead to severe pathology and may play a critical role in cognitive decline. There has long been speculation about why amyloid builds up around the vessels, with two main models: 1) vascular amyloid builds up due to overproduction by vascular cells including endothelial cells, pericytes, and vascular smooth muscle cells, 2) vascular amyloid buildup is due to a dysfunction of vascular clearance pathways including efflux across the blood-brain barrier and by the glymphatic system.

    The Tsai lab examined vascular amyloid deposition with a three-dimensional model of the blood-brain barrier using endothelial cells, mural cells (pericytes and vascular smooth muscle cells), and astrocytes generated from human iPSC cells, and came up with a really novel finding. They were able to show that vascular amyloid builds up through unique neural-pericyte interactions. They found significant vascular amyloid buildup only when exogenous amyloid-β was given to the BBB models, and this buildup was much greater when the vascular cells were derived from APOE4/4 iPSCs than from APOE3/3 iPSCs. By mixing and matching the genotypes of the different cells, they were able to identify that the critical vascular cells were the mural cells, as amyloid deposition occurred when these cells were APOE4/4 regardless of the genotype of the other cells, and that this genotype led to overproduction of APOE4 by the mural cells. Thus, vascular amyloid deposition occurred through the interaction of neural derived Aβ with pericyte-derived APOE.

    This is very interesting, as it is currently not known how neurons and pericytes may interact in the healthy brain. The finding of an interaction between neural-derived Aβ and pericyte-derived APOE is incredibly novel and it will be interesting to understand whether these interactions take place in the normal healthy brain (for instance to regulate blood flow) or whether this interaction is specific to Alzheimer's pathology. It is also not clear how excess mural cell ApoE leads to vascular amyloid deposition, whether this drives precipitation of the peptides or alters vascular amyloid clearance. This also is very interesting in the context of the recent finding that APOE4 carriers have pericyte damage and early blood-brain barrier breakdown that correlate with cognitive decline, because it suggests that neural-pericyte interactions may drive the pathology observed in the APOE4 carriers. The Tsai lab also shows that inhibition of calcineurin-NFAT signaling with approved drugs was able to limit APOE production by APOE4/4 pericytes, and thus limit the vascular buildup of amyloid. This is a truly remarkable finding as it suggests a novel potential treatment for AD.

    View all comments by Richard Daneman

  2. Joel Blanchard and Li-Huei Tsai have developed an interesting in vitro BBB model made of human induced pluripotent stem cells (iPSCs) differentiated into endothelial cells, pericytes, and astrocytes, that they named iBBB. First, they found that iPSC-derived pericytes from isogenic APOE4 carrier cells significantly increased their expression of ApoE as compared with iPSC-derived pericytes from isogenic APOE3 carrier cells. Then, they uncovered that elevated ApoE levels trigger more Aβ to accumulate through calcineurin–nuclear factor of activated T cells (NFAT) signaling. Interestingly, the authors used FDA-approved drugs targeting this pathway, including cyclosporine A (CsA), and were able to reduce the levels of ApoE proteins and the buildup of Aβ after administering the drugs in their in vitro iBBB model and also in vivo in mice. Even more interesting, there are known publications following those individuals who received organ transplants under medication with this drug and these people turned out to have a reduced risk of developing dementia.

    Another interesting point is that CsA is a known inhibitor of Cyclophilin A (CypA). Long story short, we have recently shown that the pericytic pro-inflammatory CypA-matrix metalloproteinase 9 (MMP9) pathway is activated in individuals carrying the APOE4 gene (not APOE3) which leads to basement membrane disruption and tight junction loss, ultimately increasing the BBB permeability (Montagne et al., 2020). 

    The vascular contribution to dementia is increasingly recognized. Having a drug that can protect BBB function as well as reduce amyloidosis is very exciting for the field.

    References:

    Montagne A, Nation DA, Sagare AP, Barisano G, Sweeney MD, Chakhoyan A, Pachicano M, Joe E, Nelson AR, D'Orazio LM, Buennagel DP, Harrington MG, Benzinger TL, Fagan AM, Ringman JM, Schneider LS, Morris JC, Reiman EM, Caselli RJ, Chui HC, Tcw J, Chen Y, Pa J, Conti PS, Law M, Toga AW, Zlokovic BV. APOE4 leads to blood-brain barrier dysfunction predicting cognitive decline. Nature. 2020 May;581(7806):71-76. Epub 2020 Apr 29 PubMed.

    View all comments by Axel Montagne

  3. This is a very elegant study, both technically in establishing a model of blood-brain barrier from induced brain endothelial, astrocyte, and pericyte-like cells, and then applying it to study the effects of ApoE4 on amyloid deposition and calcineurin-NFAT-dependent upregulation of APOE expression.

    ApoE4 has been known to associate with increased vascular and plaque Aβ accumulation for more than 25 years, but somehow the precise mechanisms haven’t been fully established. The effect appears to be even stronger for vascular than for plaque amyloid, possibly the reason for increased ARIA among APOE4 carriers in anti-amyloid immunotherapy trials. E4-induced increases in APOE expression seems like a plausible—and potentially treatable—mechanism.

    View all comments by Steven Greenberg

  4. The authors developed methods to generate a BBB-like structure by putting together brain endothelial cells (BEC), mural cells (iMC), and astrocytes differentiated from human iPSCs. Because each of the BBB components can be derived individually, the authors were able to investigate the contribution of each cell type to cerebral amyloid angiopathy. Importantly, they discovered that the major cell type in which ApoE4 contributes to CAA is mural cells. They showed that Aβ accumulation became prominent in the BBB-like structure when mural cells were derived from iPSCs with the APOE4 genotype.

    By performing gene expression analysis, the authors found that NFAT signaling was highly upregulated in APOE4 mural cells. The data leads to the important hypothesis that inhibition of NFAT signaling, via inhibitors such as Cyclosporine A (CsA) or FK506, can rescue the ApoE4-mediated accumulation of Aβ. Indeed, these inhibitors rescued the phenotypes.

    It is very interesting that in previous clinical studies, patients chronically treated with CsA or FK506 showed significantly less incidence of dementia. Optimization of CsA or FK506, or related compounds that inhibit NFAT pathway but have a limited side effect, could be a potential drug for CAA.

    View all comments by In-Hyun Park

  5. Tsai and colleagues present several converging lines of evidence to bring new insight to the role of ApoE and calcineurin–nuclear factor of activated T cells (NFAT) signaling in cerebral amyloid angiopathy (CAA) and Alzheimer’s disease (AD). Evidence to support a pathogenic mechanism for ApoE4 is drawn from multiple sources including in vitro, in vivo, and ex vivo blood-brain barrier (BBB) models and supported by human postmortem single-nucleus transcriptomic data. The authors developed a novel, 3D, induced pluripotent stem cell (iPSC)-derived BBB model (iBBB) to demonstrate greater amyloid accumulation in the APOE4 iBBB. Furthermore, they demonstrated dysregulation of calcineurin–NFAT signaling and APOE in pericyte-like mural cells leading to APOE4-associated CAA pathology.

    The novel iBBB is elegantly formed by spontaneous reorganization of iPSC-derived cell types into a model that recapitulates both anatomical and physiological properties of the human BBB. There is clear evidence of mural cells (MC) migrating to positions proximal to the brain endothelial cells (BEC) forming vessel-like structures. Additionally, aquaporin-4 positive astrocytes surround the vessels and extend GFAP+ projections into the perivascular space. Definition of pericyte markers has improved with the availability of single-cell transcriptomic datasets (Vanlandewijck et al. 2018), and the authors add to this body of literature showing their iMCs to be highly similar to hippocampal pericytes, identifying pericyte markers useful for future investigations.

    Populating the iBBB with iPSC-derived cell types allowed the authors to leverage genetically engineered isogenic pairs of APOE3/3 and APOE4/4 lines and additional APOE3/E4 donors to investigate the effects of APOE genotype on amyloid deposition. APOE4 is a prominent risk factor for capillary CAA (Thal et al., 2002), which is characterized by deposition of amyloid in the BBB basement membrane. The authors demonstrate that iBBB cultures remodel the extracellular matrix, acquiring proteins found in the BBB basement membrane—the site of amyloid deposition in capillary CAA. It would be interesting to investigate if the amyloid deposition observed in the APOE4 iBBB, much of which appears non-vascular or within the BEC and MCs, localizes to the basement membrane generated by the iBBB.

    Vessels in other self-assembly BBB models (Campisi et al., 2018) have perfusable lumens enabling studies of amyloid clearance. Does Tsai’s iBBB model have the ability to study clearance? We acknowledge that not all BBB models are able to answer multiple biological questions and strongly support the use of complementary models to supplement functional analyses as the authors have done with their transwell model to explore questions of barrier permeability and efflux transporters.

    Tsai and colleagues convincingly demonstrate that APOE4 pericytes produce more ApoE than do APOE3 pericytes in their iBBB and in mouse and human brain sections. From conditioned media experiments, they conclude that ApoE secreted from APOE4 pericytes is sufficient to result in increased amyloid accumulation in the iBBB. Bu and colleagues (Yamazaki et al., 2020) also recently reported BBB dysfunction resulting from APOE4 pericytes, where they observed impaired basement membrane formation. However, noncontact co-culture—a similar paradigm to conditioned media—was not sufficient to induce changes in endothelial expression of basement-membrane components in Bu’s study, whereas conditioned media was sufficient to increase amyloid accumulation in the iBBB. This may suggest that APOE4 pericytes cause BBB dysfunction by multiple mechanisms, both via secreted factors and via direct contact with endothelial cells. Notably, Bu and colleagues did not see the same trends in ApoE protein levels that Tsai and colleagues have reported with APOE transcripts, a relative decrease in astrocyte ApoE in APOE4, and a relative increase in pericyte ApoE in APOE4.

    The lipidation status of the ApoE secreted by APOE4 pericytes in this iBBB model is of interest given the 2018 findings of Holtzman and colleagues that an antibody specifically targeting nonlipidated, aggregated ApoE could inhibit amyloid accumulation in ApoE4-knockin, APP/PS1 mice (Liao et al., 2018). Whether the neutralized ApoE in the Holtzman study came primarily from pericytes and whether the pericyte ApoE in the study by Tsai is nonlipidated and aggregated are therefore interesting questions for future investigation.

    With regards to novel therapeutic targets arising from this paper, this group demonstrated that inhibiting the protein phosphatase calcineurin in the NFAT signaling pathway was sufficient to reduce the pathogenic APOE expression, and Aβ deposition. These results are enticing because calcineurin inhibitors are already commercially available, and the authors cite a paper in the discussion that reported decreased incidence of AD/dementia in patients treated with calcineurin inhibitors (Taglialatela et al., 2015), but there are considerable risks of long-term treatment with an immunosuppressant.

    A more recent paper found that long-term use of calcineurin inhibitors in patients who received liver transplants is associated with impaired cognitive function, and white-matter hyperintensities (Pflugrad et al., 2018), and there is emerging evidence of other potentially neurotoxic effects. Especially given the current climate with COVID-19, it is unlikely that an immunosuppressant would be widely accepted as a preventative treatment strategy. Calcineurin inhibitors may not be the way forward, but if we can find targets in this pathway more specific to the AD pathology, this may be a viable preventative treatment avenue.

    Overall, the authors bring together observations from multiple different BBB models and complementary transcriptomic analyses for a thorough investigation of pathogenic mechanism for APOE4 in pericytes and present new targets for intervention.

    References:

    Campisi M, Shin Y, Osaki T, Hajal C, Chiono V, Kamm RD. 3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes. Biomaterials. 2018 Oct;180:117-129. Epub 2018 Jul 12 PubMed.

    Liao F, Li A, Xiong M, Bien-Ly N, Jiang H, Zhang Y, Finn MB, Hoyle R, Keyser J, Lefton KB, Robinson GO, Serrano JR, Silverman AP, Guo JL, Getz J, Henne K, Leyns CE, Gallardo G, Ulrich JD, Sullivan PM, Lerner EP, Hudry E, Sweeney ZK, Dennis MS, Hyman BT, Watts RJ, Holtzman DM. Targeting of nonlipidated, aggregated apoE with antibodies inhibits amyloid accumulation. J Clin Invest. 2018 May 1;128(5):2144-2155. Epub 2018 Mar 30 PubMed.

    Pflugrad H, Schrader AK, Tryc AB, Ding X, Lanfermann H, Jäckel E, Schrem H, Beneke J, Barg-Hock H, Klempnauer J, Weissenborn K. Longterm calcineurin inhibitor therapy and brain function in patients after liver transplantation. Liver Transpl. 2018 Jan;24(1):56-66. 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.

    Thal DR, Ghebremedhin E, Rüb U, Yamaguchi H, Del Tredici K, Braak H. Two types of sporadic cerebral amyloid angiopathy. J Neuropathol Exp Neurol. 2002 Mar;61(3):282-93. PubMed.

    Vanlandewijck M, He L, Mäe MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Laviña B, Gouveia L, Sun Y, Raschperger E, Räsänen M, Zarb Y, Mochizuki N, Keller A, Lendahl U, Betsholtz C. Author Correction: A molecular atlas of cell types and zonation in the brain vasculature. Nature. 2018 Aug;560(7716):E3. PubMed.

    Yamazaki Y, Shinohara M, Yamazaki A, Ren Y, Asmann YW, Kanekiyo T, Bu G. ApoE (Apolipoprotein E) in Brain Pericytes Regulates Endothelial Function in an Isoform-Dependent Manner by Modulating Basement Membrane Components. Arterioscler Thromb Vasc Biol. 2020 Jan;40(1):128-144. Epub 2019 Oct 31 PubMed.

    View all comments by Cheryl Wellington

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  1. Human Blood-Brain Barrier Model Blames Pericytes for CAA

Mutations

  1. APOE C130R (ApoE4)