Endothelial expression of human amyloid precursor protein leads to amyloid β in the blood and induces cerebral amyloid angiopathy in knock-in mice

Abstract

The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770+ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770+ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (AppNL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770+:AppNL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies. The deposition of amyloid β (Aβ) in blood vessels of the brain, known as cerebral amyloid angiopathy (CAA), is observed in most patients with Alzheimer’s disease (AD). Compared with the pathology of CAA in humans, the pathology in most mouse models of AD is not as evident, making it difficult to examine the contribution of CAA to the pathogenesis of AD. On the basis of biochemical analyses that showed blood levels of soluble amyloid precursor protein (APP) in rats and mice were markedly lower than those measured in human samples, we hypothesized that endothelial APP expression would be markedly lower in rodents and subsequently generated mice that specifically express human WT APP (APP770) in endothelial cells (ECs). The resulting EC-APP770+ mice exhibited increased levels of serum Aβ and soluble APP, indicating that endothelial APP makes a critical contribution to blood Aβ levels. Even though aged EC-APP770+ mice did not exhibit Aβ deposition in the cortical blood vessels, crossing these animals with APP knock-in mice (AppNL-F/NL-F) led to an expanded CAA pathology, as evidenced by increased amounts of amyloid accumulated in the cortical blood vessels. These results highlight an overlooked interplay between neuronal and endothelial APP in brain vascular Aβ deposition. We propose that these EC-APP770+:AppNL-F/NL-F mice may be useful to study the basic molecular mechanisms behind the possible breakdown of the blood–brain barrier upon administration of anti-Aβ antibodies. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder in humans and the leading cause of dementia. The pathology of AD is characterized by the deposition of amyloid β (Aβ) peptide in the brain parenchyma and blood vessels. Aβ is generated by the two-step proteolytic cleavage of amyloid precursor protein (APP), β-site APP cleaving enzyme-1 (1Yan R. Vassar R. Targeting the β secretase BACE1 for Alzheimer's disease therapy.Lancet Neurol. 2014; 13: 319-329Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar), and γ-secretase (2Wertkin A.M. Turner R.S. Pleasure S.J. Golde T.E. Younkin S.G. Trojanowski J.Q. Lee V.M. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9513-9517Crossref PubMed Scopus (195) Google Scholar), the last of which is composed of four transmembrane proteins (presenilin, nicastrin, Pen2, and Aph1). When APP is alternatively cleaved at the α-site within the Aβ sequence, subsequent γ-secretase cleavage cannot produce neurotoxic Aβ peptide. Alternative mRNA splicing generates three major APP isoforms, APP695, APP751, and APP770, each of which exhibits cell-specific expression. Neurons express APP695 (2Wertkin A.M. Turner R.S. Pleasure S.J. Golde T.E. Younkin S.G. Trojanowski J.Q. Lee V.M. Human neurons derived from a teratocarcinoma cell line express solely the 695-amino acid amyloid precursor protein and produce intracellular beta-amyloid or A4 peptides.Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9513-9517Crossref PubMed Scopus (195) Google Scholar), whereas brain vascular endothelial cells (ECs) and platelets express APP770 (3Kitazume S. Tachida Y. Kato M. Yamaguchi Y. Honda T. Hashimoto Y. Wada Y. Saito T. Iwata N. Saido T. Taniguchi N. Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.J. Biol. Chem. 2010; 285: 40097-40103Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 4Miura S. Yoshihisa A. Misaka T. Yamaki T. Kojima T. Toyokawa M. Ogawa K. Shimura H. Yamamoto N. Kasahara K. Takeishi Y. Kitazume S. Amyloid precursor protein 770 is specifically expressed and released from platelets.J. Biol. Chem. 2020; 295: 13194-13201Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 5Tamaoka A. Kalaria R.N. Lieberburg I. Selkoe D.J. Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1345-1349Crossref PubMed Scopus (140) Google Scholar, 6Austin S.A. Sens M.A. Combs C.K. Amyloid precursor protein mediates a tyrosine kinase-dependent activation response in endothelial cells.J. Neurosci. 2009; 29: 14451-14462Crossref PubMed Scopus (28) Google Scholar), and APP751 shows a relatively ubiquitous expression pattern. Cerebral amyloid angiopathy (CAA) is defined as the deposition of Aβ on the vascular walls of the meninges and brain, giving rise to intracerebral hemorrhage and cognitive impairment in the elderly (7Charidimou A. Gang Q. Werring D.J. Sporadic cerebral amyloid angiopathy revisited: Recent insights into pathophysiology and clinical spectrum.J. Neurol. Neurosurg. Psychiatry. 2012; 83: 124-137Crossref PubMed Scopus (376) Google Scholar). However, epidemiological and clinicopathological data alone do not fully describe the pathological association between CAA and AD. Impaired clearance and/or overproduction of Aβ lead to its accumulation in the brains of patients with AD (8Mawuenyega K.G. Sigurdson W. Ovod V. Munsell L. Kasten T. Morris J.C. Yarasheski K.E. Bateman R.J. Decreased clearance of CNS beta-amyloid in Alzheimer's disease.Science. 2010; 330: 1774Crossref PubMed Scopus (1427) Google Scholar). Most neuronal Aβ is transported across the blood–brain barrier into the blood (9Tarasoff-Conway J.M. Carare R.O. Osorio R.S. Glodzik L. Butler T. Fieremans E. Axel L. Rusinek H. Nicholson C. Zlokovic B.V. Frangione B. Blennow K. Menard J. Zetterberg H. Wisniewski T. et al.Clearance systems in the brain-implications for Alzheimer disease.Nat. Rev. Neurol. 2015; 11: 457-470Crossref PubMed Scopus (750) Google Scholar), and as such, the blood Aβ can derive from a mixture of APP sources including from neurons, smooth muscle cells, ECs, and pericytes, all of which can contribute to vascular Aβ deposition (10Burgermeister P. Calhoun M.E. Winkler D.T. Jucker M. Mechanisms of cerebrovascular amyloid deposition. Lessons from mouse models.Ann. N. Y. Acad. Sci. 2000; 903: 307-316Crossref PubMed Scopus (56) Google Scholar). Accumulating evidence shows that several anti-Aβ immunotherapies reduce the amyloid burden in brain parenchyma, while augmenting that of vascular Aβ accumulation (11Nicoll J.A. Wilkinson D. Holmes C. Steart P. Markham H. Weller R.O. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: A case report.Nat. Med. 2003; 9: 448-452Crossref PubMed Scopus (1271) Google Scholar, 12Pfeifer M. Boncristiano S. Bondolfi L. Stalder A. Deller T. Staufenbiel M. Mathews P.M. Jucker M. Cerebral hemorrhage after passive anti-Abeta immunotherapy.Science. 2002; 298: 1379Crossref PubMed Scopus (451) Google Scholar), indicating that Aβ accumulation in the brain parenchyma and vessels is regulated in different ways. A major therapeutic concern with some anti-Aβ antibodies is the possible breakdown of the blood–brain barrier associated with brain edema and cerebral microhemorrhages, which are observed under magnetic resonance imaging as amyloid-related imaging abnormalities (ARIAs) (13Sperling R. Salloway S. Brooks D.J. Tampieri D. Barakos J. Fox N.C. Raskind M. Sabbagh M. Honig L.S. Porsteinsson A.P. Lieberburg I. Arrighi H.M. Morris K.A. Lu Y. Liu E. et al.Amyloid-related imaging abnormalities in patients with Alzheimer's disease treated with bapineuzumab: A retrospective analysis.Lancet Neurol. 2012; 11: 241-249Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). It is thus important to clarify how brain Aβ accumulates in the vascular walls. Unfortunately, most AD model mice with parenchymal Aβ deposition exhibit only moderate Aβ deposition in the brain blood vessels (14Sasaguri H. Nilsson P. Hashimoto S. Nagata K. Saito T. De Strooper B. Hardy J. Vassar R. Winblad B. Saido T.C. APP mouse models for Alzheimer's disease preclinical studies.EMBO J. 2017; 36: 2473-2487Crossref PubMed Scopus (327) Google Scholar). Based on our initial study showing that human vascular ECs express a significantly high level of APP770 (3Kitazume S. Tachida Y. Kato M. Yamaguchi Y. Honda T. Hashimoto Y. Wada Y. Saito T. Iwata N. Saido T. Taniguchi N. Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.J. Biol. Chem. 2010; 285: 40097-40103Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and that the level of serum soluble amyloid precursor protein (sAPP) in rodents is less than 1% of that in human serum, we explored the possibility that limited endothelial APP expression could result in a markedly reduced CAA pathology in rodent models. we a mouse model that human APP770 in We that the mice exhibited significantly high levels of blood and whereas the accumulation of Aβ in blood vessels However, crossing with APP knock-in mice in an expanded CAA These results that neuronal and endothelial APP contribute to vascular Aβ deposition. We measured blood levels in and Human serum and were to and The for is that platelets in and it into the serum upon activation S. Yoshihisa A. Misaka T. Yamaki T. Kojima T. Toyokawa M. Ogawa K. Shimura H. Yamamoto N. Kasahara K. Takeishi Y. Kitazume S. Amyloid precursor protein 770 is specifically expressed and released from platelets.J. Biol. Chem. 2020; 295: 13194-13201Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, R.N. B. R. S. E. J. D. A. P. The amyloid precursor protein of Alzheimer's disease is released by human platelets.J. Biol. Chem. Full Text PDF PubMed Google Scholar). We measured blood which for and our in human significantly lower than that in serum the case of and between and levels in and serum were and serum levels in rats were and which were markedly lower than those measured in human blood levels were in some AD model showed a level in serum, less than of the level in human Even though APP is to be expressed H. of APP Med. 2012; PubMed Scopus Google Scholar), we hypothesized that endothelial APP expression be limited in which could result in markedly lower levels of blood and CAA pathology in most AD mouse models. It is that a Aβ is for amyloid in models T. Y. N. J. Nilsson P. S. Iwata N. Saido T.C. knock-in mouse models of Alzheimer's disease.Nat. Neurosci. 2014; PubMed Scopus Google Scholar). our we a mouse model that specifically with the M. T. C. L. P. C. Lieberburg I. Selkoe D.J. of the beta-amyloid precursor protein in Alzheimer's disease beta-protein 1992; PubMed Scopus Google Scholar), which Aβ in vascular We generated mice under the and and these mice with which specifically express in the ECs by J. J.A. M. A model for endothelial in Biol. PubMed Scopus Google Scholar), to mice We the brains from EC-APP770+ and mice to ECs, which were the cells for endothelial cell and for the neuronal in The results showed that an increased APP not in brain from of neurons and cells, which are major cell in do not express and ECs are a cell in the brain H. J. S. M. L. M. et of Alzheimer's PubMed Scopus Google Scholar). we could an increased APP in the of brain ECs from EC-APP770+ an the of APP that is to mouse and we the of and APP in the ECs from EC-APP770+ and mice and the expression level of endothelial in EC-APP770+ mice to be three than that in We ECs from the of of mice and observed an increased APP in EC-APP770+ in the ECs derived from brains and of EC-APP770+ mice showing that endothelial expression is not limited to the these that is specifically expressed in the ECs of EC-APP770+ A of the of endothelial APP770 is cleaved for from ECs, and the cells secrete and in the (3Kitazume S. Tachida Y. Kato M. Yamaguchi Y. Honda T. Hashimoto Y. Wada Y. Saito T. Iwata N. Saido T. Taniguchi N. Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.J. Biol. Chem. 2010; 285: 40097-40103Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We thus that human and Aβ would be in the serum of EC-APP770+ serum human levels in and EC-APP770+ mice were to whereas were in mice We to and measured levels in EC-APP770+ mice to be whereas not in The serum level in EC-APP770+ mice is less than of which with the case in humans, in which the level is the level B. J.M. amyloid precursor in of a and Scopus Google Scholar). for serum or and we serum levels in and We that serum and levels were markedly in EC-APP770+ mice than in mice our these data for the that endothelial APP to blood Aβ levels. We did not an in serum and levels between and We to EC-APP770+ mice increased CAA pathology with We that levels of brain were between and EC-APP770+ mice We ECs from the brains of EC-APP770+ and the accumulation of in EC-APP770+ mice analyses to in cells for the endothelial in the brains of EC-APP770+ mice These results that endothelial APP expression not lead to Aβ deposition in brain blood vessels. We endothelial APP expression AD and/or CAA pathology and EC-APP770+ mice with AD model of the brain of EC-APP770+:AppNL-F/NL-F mice the amyloid in the cerebral vessels, a pathological of CAA Winkler D.T. P. M. E. S. D. C. K. S.G. Staufenbiel M. Mathews P.M. Jucker M. is to the in a mouse model of cerebral hemorrhage with Neurosci. PubMed Scopus Google Scholar), in to parenchymal amyloid examine in we a on of and mouse brains M. Iwata N. Y. K. K. Saido T.C. and of amyloid in Neurosci. PubMed Scopus Google Scholar). EC-APP770+ and mouse brains exhibit whereas mouse brains exhibit parenchymal in and as T. Y. N. J. Nilsson P. S. Iwata N. Saido T.C. knock-in mouse models of Alzheimer's disease.Nat. Neurosci. 2014; PubMed Scopus Google Scholar). A observed in EC-APP770+:AppNL-F/NL-F mouse brain, amyloid deposition in cortical and showed that the of in the in EC-APP770+:AppNL-F/NL-F mice of the with the for a vascular smooth muscle cell whereas most were in EC-APP770+:AppNL-F/NL-F mice indicating that crossing EC-APP770+ mice with mice results in the deposition of amyloid in brain blood vessels. of blood vessels showed that were in the of vessels. the amyloid in we in which amyloid were and on the basis of amyloid to of were in EC-APP770+:AppNL-F/NL-F mice not in mice that most parenchymal were less than and were vascular of EC-APP770+ mice with mice exhibit Aβ deposition in brain blood vessels. of of mouse brains is The cortical and from and mice were with to parenchymal The of brain is as the with cortical from and mice were with and The and parenchymal and vascular amyloid in cortical and from and mice were and by and are in with the of parenchymal in and mouse brain were and are as the were by amyloid APP, amyloid precursor endothelial to on parenchymal a and we were to these in and The of parenchymal in cortical of EC-APP770+:AppNL-F/NL-F mice than that in mice However, as a observed in we that could be to an from of the We to the of the by and that it within the between and of the in The by and the The a of the of which shows expression in the and B. 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Saido T.C. knock-in mouse models of Alzheimer's disease.Nat. Neurosci. 2014; PubMed Scopus Google and mice were with mice J. J.A. M. A model for endothelial in Biol. PubMed Scopus Google Scholar). The by for The by EC-APP770+ and EC-APP770+ mice were with WT EC-APP770+ mice were with AD model (AppNL-F/NL-F) mice T. Y. N. J. Nilsson P. S. Iwata N. Saido T.C. knock-in mouse models of Alzheimer's disease.Nat. Neurosci. 2014; PubMed Scopus Google Scholar). The of of the The to the S. R. L. R. M. A. V. S. of models of with a 90: PubMed Scopus Google Scholar) is at analyses were to the and brain mice were with and with and with were and in the for on brain brains were in for at or of were the were with in 1% serum for and with for The were with and antibodies and of the and antibodies are in the of M. Iwata N. Y. K. K. Saido T.C. and of amyloid in Neurosci. PubMed Scopus Google Scholar). The were with 1% containing for by with in 1% for E. W. preferentially cerebrovascular as imaging for cerebral amyloid PubMed Scopus Google Scholar). The were observed an We imaging as T. Y. N. J. Nilsson P. S. Iwata N. Saido T.C. knock-in mouse models of Alzheimer's disease.Nat. Neurosci. 2014; PubMed Scopus Google Scholar). reduce the data were from at four with were and with in with the ECs were with or with (3Kitazume S. Tachida Y. Kato M. Yamaguchi Y. Honda T. Hashimoto Y. Wada Y. Saito T. Iwata N. Saido T. Taniguchi N. Brain endothelial cells produce amyloid {beta} from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form.J. Biol. Chem. 2010; 285: 40097-40103Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). from mouse brain or were with containing were with acid protein of were to and to with in the were with antibodies and antibodies. were with an of the protein were and in or serum, a Human a Human APP770 an a P. S. M. J. R. V. M. H. T. The vascular in 2009; PubMed Scopus (56) Google Scholar) and a S. R. L. R. M. A. V. S. of models of with a 90: PubMed Scopus Google Scholar) were a A mouse to serum in from mice were to were with a in with the The of of or the with and the in with the The and for are in and those for were and The for with the at its and the at its The for with at its and the at its The of each expression the data by the PubMed Scopus Google of and for and in a data are as between analyses were by were a three or by a or The data were and in a and data are from the upon The that of with the of We M. of for I. Saito of The of for the and at the for APP770 by the for of the of The of S. K. Y. T. Y. S. Y. H. and Y. M. N. S. and Y. K. N. A. and T. S. data Y. T. H. T. and T. S. S. K. N. T. by the for and S. the S. and S. and to

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