Abstract
Impairments of the blood–brain barrier (BBB) and vascular dysfunction contribute to Alzheimer’s disease (AD) from the earliest stages. However, the influence of AD-affected astrocytes on the BBB remain largely unexplored. In the present study, we created an in vitro BBB using human-immortalized endothelial cells in combination with immortalized astroglial cell lines from the hippocampus of 3xTG-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively). We found that co-culturing endothelial monolayers with WT-iAstro upregulates expression of endothelial tight junction proteins (claudin-5, occludin, ZO-1) and increases the trans-endothelial electrical resistance (TEER). In contrast, co-culturing with 3Tg-iAstro does not affect expression of tight junction proteins and does not change the TEER of endothelial monolayers. The same in vitro model has been used to evaluate the effects of extracellular vesicles (EVs) derived from the WT-iAstro and 3Tg-iAstro. The EVs derived from WT-iAstro increased TEER and upregulated expression of tight junction proteins, whereas EVs from 3Tg-iAstro were ineffective. In conclusion, we show for the first time that immortalized hippocampal astrocytes from 3xTG-AD mice exhibit impaired capacity to support BBB integrity in vitro through paracrine mechanisms and may represent an important factor underlying vascular abnormalities during development of AD.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
Data available within the article or its supplementary materials.
Code Availability
Not applicable.
References
Alvarez JI, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, Terouz S, Sabbagh M, Wosik K, Bourbonniere L, Bernard M, van Horssen J, de Vries HE, Charron F, Prat A (2011) The hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 334:1727–1731. https://doi.org/10.1126/science.1206936
Alvarez JI, Katayama T, Prat A (2013) Glial influence on the blood brain barrier. Glia 61:1939–1958
Basun H, Bogdanovic N, Ingelsson M, Almkvist O, Näslund J, Axelman K, Bird TD, Nochlin D, Schellenberg GD, Wahlund LO, Lannfelt L (2008) Clinical and neuropathological features of the arctic APP gene mutation causing early-onset Alzheimer disease. Arch Neurol. https://doi.org/10.1001/archneur.65.4.499
Bell RD, Winkler EA, Singh I, Sagare AP, Deane R, Wu Z, Holtzman DM, Betsholtz C, Armulik A, Sallstrom J, Berk BC, Zlokovic BV (2012) Apolipoprotein e controls cerebrovascular integrity via cyclophilin A. Nature. https://doi.org/10.1038/nature11087
Bogorad MI, DeStefano J, Karlsson J, Wong AD, Gerecht S, Searson PC (2015) Review: in vitro microvessel models. Lab Chip 15:4242–4255
Bourasset F, Ouellet M, Tremblay C, Julien C, Do TM, Oddo S, LaFerla F, Calon F (2009) Reduction of the cerebrovascular volume in a transgenic mouse model of Alzheimer’s disease. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2009.01.006
Campisi M, Shin Y, Osaki T, Hajal C, Chiono V, Kamm RD (2018) 3D self-organized microvascular model of the human blood-brain barrier with endothelial cells, pericytes and astrocytes. Biomaterials. https://doi.org/10.1016/j.biomaterials.2018.07.014
Chiarini A, Armato U, Gardenal E, Gui L, Dal Prà I (2017) Amyloid β-exposed human astrocytes overproduce phospho-tau and overrelease it within exosomes, effects suppressed by calcilytic NPS 2143-further implications for Alzheimer’s therapy. Front Neurosci. https://doi.org/10.3389/fnins.2017.00217
Delpech JC, Herron S, Botros MB, Ikezu T (2019) Neuroimmune crosstalk through extracellular vesicles in health and disease. Trends Neurosci. 42:361–372
Dermaut B (2001) Cerebral amyloid angiopathy is a pathogenic lesion in Alzheimer’s disease due to a novel presenilin 1 mutation. Brain. https://doi.org/10.1093/brain/124.12.2383
Do TM, Alata W, Dodacki A, Traversy MT, Chacun H, Pradier L, Scherrmann JM, Farinotti R, Calon F, Bourasset F (2014) Altered cerebral vascular volumes and solute transport at the blood-brain barriers of two transgenic mouse models of Alzheimer’s disease. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2014.02.010
Goetzl EJ, Schwartz JB, Abner EL, Jicha GA, Kapogiannis D (2018) High complement levels in astrocyte-derived exosomes of Alzheimer disease. Ann Neurol. https://doi.org/10.1002/ana.25172
Grabowski TJ, Cho HS, Vonsattel JPG, William Rebeck G, Greenberg SM (2001) Novel amyloid precursor protein mutation in an Iowa family with dementia and severe cerebral amyloid angiopathy. Ann Neurol. https://doi.org/10.1002/ana.1009
Hunt A, Schönknecht P, Henze M, Seidl U, Haberkorn U, Schröder J (2007) Reduced cerebral glucose metabolism in patients at risk for Alzheimer’s disease. Psychiatry Res Neuroimaging. https://doi.org/10.1016/j.pscychresns.2006.12.003
Jones VC, Atkinson-Dell R, Verkhratsky A, Mohamet L (2017) Aberrant iPSC-derived human astrocytes in Alzheimer’s disease. Cell Death Dis. https://doi.org/10.1038/cddis.2017.89
Katt ME, Linville RM, Mayo LN, Xu ZS, Searson PC (2018) Functional brain-specific microvessels from iPSC-derived human brain microvascular endothelial cells: the role of matrix composition on monolayer formation. Fluids Barriers CNS. https://doi.org/10.1186/s12987-018-0092-7
Kisler K, Nelson AR, Montagne A, Zlokovic BV (2017) Cerebral blood flow regulation and neurovascular dysfunction in Alzheimer disease. Nat Rev Neurosci 18:419–434
Lemere CA, Lopera F, Kosik KS, Lendon CL, Ossa J, Saido TC, Yamaguchi H, Ruiz A, Martinez A, Madrigal L, Hincapie L, Arango LJC, Anthony DC, Koo EH, Goate AM, Selkoe DJ, Arango VJC (1996) The E280A presenilin 1 Alzheimer mutation produces increased Aβ42 deposition and severe cerebellar pathology. Nat Med. https://doi.org/10.1038/nm1096-1146
Montagne A, Barnes SR, Sweeney MD, Halliday MR, Sagare AP, Zhao Z, Toga AW, Jacobs RE, Liu CY, Amezcua L, Harrington MG, Chui HC, Law M, Zlokovic BV (2015) Blood-brain barrier breakdown in the aging human hippocampus. Neuron. https://doi.org/10.1016/j.neuron.2014.12.032
Motallebnejad P, Thomas A, Swisher SL, Azarin SM (2019) An isogenic hiPSC-derived BBB-on-a-chip. Biomicrofluidics. https://doi.org/10.1063/1.5123476
Olabarria M, Noristani HN, Verkhratsky A, Rodríguez JJ (2010) Concomitant astroglial atrophy and astrogliosis in a triple transgenic animal model of Alzheimer’s disease. Glia. https://doi.org/10.1002/glia.20967
Paul J, Strickland S, Melchor JP (2007) Fibrin deposition accelerates neurovascular damage and neuroinflammation in mouse models of Alzheimer’s disease. J Exp Med. https://doi.org/10.1084/jem.20070304
Phan DTT, Bender RHF, Andrejecsk JW, Sobrino A, Hachey SJ, George SC, Hughes CCW (2017) Blood–brain barrier-on-a-chip: microphysiological systems that capture the complexity of the blood-central nervous system interface. Exp Biol Med 242:1669–1678
Rocchio F, Tapella L, Manfredi M, Chisari M, Ronco F, Ruffinatti FA, Conte E, Canonico PL, Sortino MA, Grilli M, Marengo E, Genazzani AA, Lim D (2019) Gene expression, proteome and calcium signaling alterations in immortalized hippocampal astrocytes from an Alzheimer’s disease mouse model. Cell Death Dis. https://doi.org/10.1038/s41419-018-1264-8
Rombouts SARB, Goekoop R, Stam CJ, Barkhof F, Scheltens P (2005) Delayed rather than decreased BOLD response as a marker for early Alzheimer’s disease. Neuroimage. https://doi.org/10.1016/j.neuroimage.2005.03.022
Sagare AP, Bell RD, Zhao Z, Ma Q, Winkler EA, Ramanathan A, Zlokovic BV (2013) Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat Commun. https://doi.org/10.1038/ncomms3932
Shin Y, Choi SH, Kim E, Bylykbashi E, Kim JA, Chung S, Kim DY, Kamm RD, Tanzi RE (2019) Blood-brain barrier dysfunction in a 3D in vitro model of Alzheimer’s disease. Adv Sci. https://doi.org/10.1002/advs.201900962
Sofroniew MV (2015) Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci 16:249–263
Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ (2015) TEER measurement techniques for in vitro barrier model systems. J Lab Autom 20:107–126
Stamatovic SM, Keep RF, Kunkel SL, Andjelkovic AV (2003) Potential role of MCP-1 in endothelial cell tight junction “opening”: Signaling via Rho and Rho kinase. J Cell Sci 116:4615–4628
Stamatovic SM, Dimitrijevic OB, Keep RF, Andjelkovic AV (2006) Protein kinase Cα-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability. J Biol Chem. https://doi.org/10.1074/jbc.M513122200
Sweeney MD, Sagare AP, Zlokovic BV (2018) Blood-brain barrier breakdown in Alzheimer dsisease and other neurodegenerative disorders. Nat Rev Neurol 14:133–150
Sweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV (2019) Blood-brain barrier: from physiology to disease and back. Physiol Rev 99:21–78
Szpak GM, Lewandowska E, Wierzba-Bobrowicz T, Bertrand E, Pasennik E, Mendel T, Stepień T, Leszczyńska A, Rafałowska J (2007) Small cerebral vessel disease in familial amyloid and non-amyloid angiopathies: FAD-PS-1 (P117L) mutation and CADASIL. Immunohistochemical and ultrastructural studies. Folia Neuropathol 45:192–204
Théry C, Amigorena S, Raposo G, Clayton A (2006) Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. https://doi.org/10.1002/0471143030.cb0322s30
Théry C, Witwer KW, Aikawa E et al (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. https://doi.org/10.1080/20013078.2018.1535750
Ujiie M, Dickstein DL, Carlow DA, Jefferies WA (2003) Blood-brain barrier permeability precedes senile plaque formation in an Alzheimer disease model. Microcirculation. https://doi.org/10.1038/sj.mn.7800212
Verkhratsky A, Marutle A, Rodriguez-Arellano JJ, Nordberg A (2015). Glial asthenia and functional paralysis: a new perspective on neurodegeneration and Alzheimer's sisease. Neuroscientist 21:552–568
Weksler B, Romero IA, Couraud PO (2013) The hCMEC/D3 cell line as a model of the human blood brain barrier. Fluids Barriers CNS 10:16
Yao Y, Tsirka SE (2011) Truncation of monocyte chemoattractant protein 1 by plasmin promotes blood–brain barrier disruption. J Cell Sci. https://doi.org/10.1242/jcs.082834
Zarranz JJ, Fernandez-Martinez M, Rodriguez O, Mateos B, Iglesias S, Baron JC (2016) Iowa APP mutation-related hereditary cerebral amyloid angiopathy (CAA): a new family from Spain. J Neurol Sci 363:55–56
Acknowledgements
We are grateful to Dr. Aistė Jekabsonė for help with cell lines.
Funding
This work was supported by the Global Grant measure (No. 09.3.3-LMTK-712-01-0082)
Author information
Authors and Affiliations
Contributions
AP and AV conceived the idea. AP, AV, KK and VT designed the experiments. DL made immortalised astrocytes, AK performed TEER experiments. KK performed and analysed most of the experiments. JP performed ELISAs. VT performed qPCRs and helped with analysis of confocal microscopy data. AP and AV wrote the paper. All authors reviewed manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics Approval
Not applicable.
Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
This article has been published at the preprint server of Biology BioRxiv: Kriauciunaite K, Kausyle A, Pajarskiene J, Tunaitis V, Lim D, Verkhratsky A, Pivoriunas A (2020) Immortalised hippocampal astrocytes from 3xTG-AD mice fail to support BBB integrity in vitro: Role of extracellular vesicles in glial-endothelial communication. bioRxiv. https://doi.org/10.1101/2020.03.26.009563.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kriaučiūnaitė, K., Kaušylė, A., Pajarskienė, J. et al. Immortalised Hippocampal Astrocytes from 3xTG-AD Mice Fail to Support BBB Integrity In Vitro: Role of Extracellular Vesicles in Glial-Endothelial Communication. Cell Mol Neurobiol 41, 551–562 (2021). https://doi.org/10.1007/s10571-020-00871-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-020-00871-w