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Lack of ApoE inhibits ADan amyloidosis in a mouse model of Familial Danish Dementia

  • Anllely Fernandez
    Affiliations
    Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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  • Maria-Teresa Gomez
    Affiliations
    Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA
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  • Ruben Vidal
    Correspondence
    Corresponding author: Ruben Vidal, Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, 635 Barnhill Dr., MSB A136, Indianapolis, IN 46202, USA: Tel: (317) 274-1729;
    Affiliations
    Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA

    Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN46202, USA
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Open AccessPublished:November 24, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102751

      Abstract

      The Apolipoprotein E-ε4 allele (APOE-ε4) is the strongest genetic risk factor for late onset Alzheimer disease (AD). ApoE plays a critical role in amyloid-β (Aβ) accumulation in AD, and genetic deletion of the murine ApoE gene in mouse models results in a decrease or inhibition of Aβ deposition. The association between the presence of ApoE and amyloid in amyloidoses suggests a more general role for ApoE in the fibrillogenesis process. However, whether decreasing levels of ApoE would attenuate amyloid pathology in different amyloidoses has not been directly addressed. Familial Danish dementia (FDD) is an autosomal dominant neurodegenerative disease
      characterized by the presence of widespread parenchymal and vascular Danish amyloid (ADan) deposition and neurofibrillary tangles. A transgenic mouse model for
      FDD (Tg-FDD) is characterized by parenchymal and vascular ADan deposition. To determine the effect of decreasing ApoE levels on ADan accumulation in vivo, we generated a mouse model by crossing Tg-FDD mice with ApoE knock-out mice (Tg-FDD+/-/ApoE-/-). Lack of ApoE results in inhibition of ADan deposition up to 18 months of age. Additionally, our results from a genetic screen of Tg-FDD+/-/ApoE-/- mice emphasizes the significant role for ApoE in neurodegeneration in FDD via glial-mediated mechanisms. Taken together, our findings suggest that the interaction between ApoE and ADan plays a key role in FDD pathogenesis, in addition to the known role for ApoE in amyloid plaque formation in AD.

      Keywords

      Abbreviationsand nomenclature:

      AβPP (Aβ precursor protein), AD (Alzheimer disease), ApoE (Apolipoprotein E), CAA (Cerebral amyloid angiopathy), ITM2B (Integral membrane protein 2B), FBD (Familial British dementia), FCD (Familial Chinese dementia), FDD (Familial Danish dementia), PCs (Pro-protein convertases), Tg-FDD (Transgenic animal model for FDD), Th-T (Thioflavin-T)

      Introduction

      Mutations in the BRI2 gene (also known as ITM2B (

      Deleersnijder, W., Hong, G., Cortvrindt, R., Poirier, C., Tylzanowski, P., Pittois, K., Van Marck, E., Merregaert, J. (1996) Isolation of markers for chondro-osteogenic, differentiation using cDNA library subtraction. Molecular cloning and characterization of a gene belonging to a novel multigene family of integral membrane proteins. J. Biol. Chem. 271, 19475-19482.

      )), located on the long arm of chromosome 13, cause the autosomal dominant neurodegenerative diseases familial British (FBD), Danish (FDD), and Chinese (FCD) dementia (
      • Vidal R.
      • Frangione B.
      • Rostagno A.
      • Mead S.
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      A stop-codon mutation in the BRI gene associated with familial British dementia.
      ,
      • Vidal R.
      • Revesz T.
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      • Holton J.L.
      • Bek T.
      • Bojsen-Moller M.
      • Braendgaard H.
      • Plant G.
      • Ghiso J.
      • Frangione B.
      A decamer duplication in the 3’ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.
      ,
      • Liu X.
      • Chen K.L.
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      • Huang Y.Y.
      • Chen S.D.
      • Dong Q.
      • Cui M.
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      A Novel ITM2B Mutation Associated with Familial Chinese Dementia.
      ). Point mutations in the stop codon of BRI2 cause FBD and FCD (
      • Vidal R.
      • Frangione B.
      • Rostagno A.
      • Mead S.
      • Revesz T.
      • Plant G.
      • Ghiso J.
      A stop-codon mutation in the BRI gene associated with familial British dementia.
      ,
      • Liu X.
      • Chen K.L.
      • Wang Y.
      • Huang Y.Y.
      • Chen S.D.
      • Dong Q.
      • Cui M.
      • Yu J.T.
      A Novel ITM2B Mutation Associated with Familial Chinese Dementia.
      ), while FDD is caused by a 10-nucleotide duplication insertion (BRI2795-796InsTTTAATTTGT) in the 3’-end of the coding region of the BRI2 gene (
      • Vidal R.
      • Revesz T.
      • Rostagno A.
      • Kim E.
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      • Bek T.
      • Bojsen-Moller M.
      • Braendgaard H.
      • Plant G.
      • Ghiso J.
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      A decamer duplication in the 3’ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.
      ). FDD, originally named heredopathia ophthalmo-oto-encephalica because of the presence of cataracts, hearing problems and neurological disease, was first described in members of a single Danish family in the Djursland peninsula (

      Strömgren E. (1981) Heredopathia ophthalmo-oto-encephalica. In: Handbook of Clinical Neurology. PJ Vinken, GWBruyn (eds), pp. 150–152. North-Holland Publishing Company: Amsterdam.

      ,
      • Strömgren E.
      • Dalby A.
      • Dalby M.A.
      • Ranheim B.
      Cataract, deafness, cerebellar ataxia, psychosis and dementia: a new syndrome.
      ). Neuropathologically, FDD is characterized by the presence of cerebral amyloid angiopathy (CAA) in vessels of the retina and leptomeninges as well as in vessels of the gray and white matter of the central nervous system (CNS) (
      • Vidal R.
      • Revesz T.
      • Rostagno A.
      • Kim E.
      • Holton J.L.
      • Bek T.
      • Bojsen-Moller M.
      • Braendgaard H.
      • Plant G.
      • Ghiso J.
      • Frangione B.
      A decamer duplication in the 3’ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.
      ,
      • Holton J.L.
      • Lashley T.
      • Ghiso J.
      • Braendgaard H.
      • Vidal R.
      • Guerin C.J.
      • et al.
      Familial Danish dementia: a novel form of cerebral amyloidosis associated with deposition of both amyloid-Dan and amyloid-beta.
      ). Amyloid plaques are found in the hippocampus, with abnormal neurites in the vicinity of blood vessels with amyloid. Neurofibrillary tangles (NFTs) composed of tau paired helical filaments (PHFs) and straight filaments (SFs) (
      • Vidal R.
      • Revesz T.
      • Rostagno A.
      • Kim E.
      • Holton J.L.
      • Bek T.
      • Bojsen-Moller M.
      • Braendgaard H.
      • Plant G.
      • Ghiso J.
      • Frangione B.
      A decamer duplication in the 3’ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.
      ,
      • Holton J.L.
      • Lashley T.
      • Ghiso J.
      • Braendgaard H.
      • Vidal R.
      • Guerin C.J.
      • et al.
      Familial Danish dementia: a novel form of cerebral amyloidosis associated with deposition of both amyloid-Dan and amyloid-beta.
      ) are also found. Recent cryo-electron microscopy (cryo-EM) work showed that PHFs and SFs in FDD and FBD are structurally identical to those seen in Alzheimer disease (AD) (
      • Shi Y.
      • Zhang W.
      • Yang Y.
      • Murzin A.G.
      • Falcon B.
      • Kotecha A.
      • et al.
      Structure-based classification of tauopathies.
      ).
      The BRI2 protein contains 266 amino acids and belongs to a family of integral type II single trans-membrane domain proteins (

      Deleersnijder, W., Hong, G., Cortvrindt, R., Poirier, C., Tylzanowski, P., Pittois, K., Van Marck, E., Merregaert, J. (1996) Isolation of markers for chondro-osteogenic, differentiation using cDNA library subtraction. Molecular cloning and characterization of a gene belonging to a novel multigene family of integral membrane proteins. J. Biol. Chem. 271, 19475-19482.

      ,
      • Garringer H.J.
      • Sammeta N.
      • Oblak A.
      • Ghetti B.
      • Vidal R.
      Amyloid and intracellular accumulation of BRI2.
      ). BRI2 is produced as a pro-protein with a pro-peptide sequence that is cleaved by pro-protein convertases (PCs) between the BRI2 ectodomain that releases the ADan amyloid in FDD (
      • Garringer H.J.
      • Sammeta N.
      • Oblak A.
      • Ghetti B.
      • Vidal R.
      Amyloid and intracellular accumulation of BRI2.
      ) (Fig. 1). A transgenic animal model for FDD (Tg-FDD) expressing the Danish mutant form of human BRI2 under the control of the mouse prion protein promoter shows significant vascular and parenchymal ADan deposition, amyloid associated gliosis, intracellular and extracellular deposition of oligomeric forms of ADan as well as tau immunoreactive deposits in the neuropil (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ). A double transgenic mouse model generated by crossing Tg-FDD mice with mice expressing human 4-repeat P301S mutant tau shows significant enhancement of tau deposition and decrease in synaptophysin levels, suggesting that ADan and amyloid-β (Aβ) may share similar pathogenic pathway(s) (
      • Garringer H.J.
      • Murrell J.
      • Sammeta N.
      • Gnezda A.
      • Ghetti B.
      • Vidal R.
      Increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant BRI2/tau transgenic mice.
      ).
      Figure thumbnail gr1
      Figure 1Proteolytic processing the type-II single trans-membrane (TM) domain BRI2 protein. A) Processing by ADAM10 in the ectodomain of BRI2 releases the BRICHOS domain (∼100 amino acid (aa)) and an N-terminal fragment which the subject of additional proteolysis by SPPL2. Disulfide-bonded loops in the BRICHOS domain and in the carboxy-terminus of BRI2 (aa 5 and 22 of the Bri2-23 peptide) are indicated as well as the single N-glycosylation site (Glyc) at position 170. B). Cleavage of the pro-protein (or immature protein, im-BRI2) by pro-protein convertases (PCs) generates the 23 aa peptide (Bri2-23) and a mature form of BRI2 (m-BRI2). Processing of the FBD, FDD and FCD forms of BRI2 by PCs releases the 34 aa amyloid peptides (ABri, ADan and AChi) in FBD, FDD and FCD, respectively.
      Genetic analyses have shown that the apolipoprotein E (ApoE) gene is the strongest genetic risk factor for developing late-onset AD (
      • Corder E.H.
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      Strittmatter W.J., Saunders A.M., Schmechel D., Pericak-Vance M., Enghild J., Salvesen G.S., Roses A.D. (1993) Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci U S A. 90(5), 1977-1981.

      ). Three alleles of ApoE (ε2, ε3, and ε4) exist, which generate three different ApoE proteins (ApoE2, ApoE3, and ApoE4) that differ by only one or two amino-acids at positions 112 and 158, which seem to modify the structure and function of ApoE (
      • Mahley R.W.
      • Huang Y.
      Apolipoprotein (apo) E4 and Alzheimer's disease: unique conformational and biophysical properties of apoE4 can modulate neuropathology.
      ). The influence of ApoE on AD risk occurs in an isoform-dependent manner with the ε4 allele as the strongest genetic risk factor for AD (
      • Bertram L.
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      ,
      • Farrer L.A.
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      Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium.
      ). Using antibodies against ApoE, it was observed that ApoE immunoreactivity was associated with amyloid deposits in AD and Creutzfeldt-Jakob disease (

      Namba Y., Tomonawa M., Kawasaki H., Otomo E., Ikeda K. (1991) Apolipoprotein E immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in Alzheimer's disease and kuru plaque amyloid in Creutzfeld-Jacob disease. Brain Res 541,163-166.

      ), as well as in other cerebral and systemic amyloid diseases (
      • Wisniewski T.
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      Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid.
      ,
      • Gallo G.
      • Wisniewski T.
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      ), suggesting a more general role for ApoE in amyloid diseases as an amyloid catalyst or “pathological chaperone” (
      • Wisniewski T.
      • Frangione B.
      Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid.
      ). Genetic deletion of the murine ApoE gene (which is expressed as a single isoform) in mouse models overexpressing mutant forms of the human Aβ precursor protein (AβPP) results in inhibition of Aβ deposition or in a decrease in amyloid plaques (
      • Bales K.R.
      • Verina T.
      • Dodel R.C.
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      • Cummins D.J.
      • Piccardo P.
      • Ghetti B.
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      Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition.
      ,
      • Holtzman D.M.
      • Bales K.R.
      • Wu S.
      • Bhat P.
      • Parsadanian M.
      • Fagan A.M.
      • Chang L.K.
      • Sun Y.
      • Paul S.M.
      Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease.
      ,

      Holtzman D.M., Bales K.R., Tenkova T., Fagan A.M., Parsadanian M., Sartorius L.J., Mackey B., Olney J., Mckeel D., Wozniak D., Paul S.M. (2000) Apolipoprotein Eisoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci U S A 97, 2892-2897.

      ,
      • Katsouri L.
      • Georgopoulos S.
      Lack of LDL receptor enhances amyloid deposition and decreases glial response in an Alzheimer's disease mouse model.
      ,
      • Irizarry M.C.
      • Cheung B.S.
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      • Bales K.R.
      • Hyman B.T.
      Apolipoprotein E afects the amount, form, and anatomical distribution of amyloid β-peptide deposition in homozygous APP V717F transgenic mice.
      ,
      • Fagan A.M.
      • Watson M.
      • Parsadanian M.
      • Bales K.R.
      • Paul S.M.
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      Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease.
      ,
      • Lewandowski C.T.
      • Maldonado Weng J.
      • LaDu M.J.
      Alzheimer's disease pathology in APOE transgenic mouse models: The Who, What, When, Where, Why, and How.
      ,
      • Balu D.
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      • York J.M.
      • Valencia-Olvera A.C.
      • LaDu M.J.
      The role of APOE in transgenic mouse models of AD.
      ). The effect of ApoE on amyloid deposition seems to be dose dependent since a significantly lower number of amyloid plaques is seen in mice hemizygous for ApoE (
      • Bales K.R.
      • Verina T.
      • Dodel R.C.
      • Du Y.
      • Altstiel L.
      • Bender M.
      • Hyslop P.
      • Johnstone E.M.
      • Little S.P.
      • Cummins D.J.
      • Piccardo P.
      • Ghetti B.
      • Paul S.M.
      Lack of apolipoprotein E dramatically reduces amyloid beta-peptide deposition.
      ,
      • Holtzman D.M.
      • Bales K.R.
      • Wu S.
      • Bhat P.
      • Parsadanian M.
      • Fagan A.M.
      • Chang L.K.
      • Sun Y.
      • Paul S.M.
      Expression of human apolipoprotein E reduces amyloid-beta deposition in a mouse model of Alzheimer's disease.
      ,
      • Irizarry M.C.
      • Cheung B.S.
      • Rebeck G.W.
      • Paul S.M.
      • Bales K.R.
      • Hyman B.T.
      Apolipoprotein E afects the amount, form, and anatomical distribution of amyloid β-peptide deposition in homozygous APP V717F transgenic mice.
      ,

      Kim J., Jiang H., Park S., Eltorai A.E.M., Stewart F.R., Yoon H., et al. (2011) Haploinsufficiency of human APOE reduces amyloid deposition in a mouse model of amyloid-β amyloidosis. J Neurosci. 31, 18007-18012.

      ,

      Bien-Ly N., Gillespie A.K., Walker D., Yoon S.Y., Huang Y. (2012) Reducing human apolipoprotein E levels attenuates age-dependent Aβ accumulation in mutant human amyloid precursor protein transgenic mice. J Neurosci. 32, 4803-4811.

      ,
      • Huynh T.-P.V.
      • Liao F.
      • Francis C.M.
      • Robinson G.O.
      • Serrano J.R.
      • Jiang H.
      • et al.
      Age-dependent effects of apoE reduction using antisense oligonucleotides in a model of β-amyloidosis.
      ), with astrocytic ApoE3 and ApoE4 having a major role on the deposition and accumulation of Aβ in amyloid plaques (
      • Mahan T.E.
      • Wang C.
      • Bao X.
      • Choudhury A.
      • Ulrich J.D.
      • Holtzman D.M.
      Selective reduction of astrocyte apoE3 and apoE4 strongly reduces Aβ accumulation and plaque-related pathology in a mouse model of amyloidosis.
      ). A similar effect of ApoE in other cerebral or systemic amyloidoses has not been experimentally demonstrated.
      Analysis of FDD knock-in mice (FDDKI mice), which do not develop amyloid deposits, crossed with human ApoE-ε3 and ApoE-ε4 targeted replacement mice suggest that the FDD mutation may differentially affect learning and memory in ApoE-ε4 carriers and non-carriers (
      • Biundo F.
      • Ishiwari K.
      • Del Prete D.
      • D'Adamio L.
      Interaction of ApoE3 and ApoE4 isoforms with an ITM2b/BRI2 mutation linked to the Alzheimer disease-like Danish dementia: Effects on learning and memory.
      ). Herein, we characterized the impact that ApoE has on amyloid pathology in a FDD transgenic mouse model, a model of cerebral ADan amyloidosis. We found that Tg-FDD mice on the murine ApoE knockout background (Tg-FDD+/-/ApoE-/-) do not deposit ADan amyloid. We also observed accumulation of mature and immature forms of the BRI2 precursor protein in the hippocampus of Tg-FDD+/-/ApoE-/- mice, which is not seen in sex/age matched Tg-FDD mice carrying the murine ApoE allele, and a significant change in the expression profile of genes related to autophagy, vesicle trafficking, angiogenesis and activation of microglia. Our results suggest that apart from influencing amyloid plaque formation in AD, the interaction between ApoE and ADan plays a key role in FDD pathogenesis.

      Results

      ADan aggregation in the presence of different ApoE isoforms.

      The interaction between ADan and ApoE variants was assessed by monitoring ADan aggregation in the presence of 300 nM recombinant ApoE2, ApoE3, or ApoE4 using a Thioflavin-T (Th-T) binding assay. As a control, the wild-type Bri2-23 peptide was also analyzed. ADan aggregation kinetics at pH 7.2 displayed a sigmoideal curve shape. After 6 hours, a peak in ADan aggregation was observed. The addition of the three different ApoE isoforms led to a pronounced decrease of Th-T incorporation, leading to lower plateau levels, without modifying lag-phases (Fig. 2A). No aggregation of the Bri2-23 peptide was observed under the same conditions over the entire time course of the experiment (Fig. 2B). Presence of the different ApoE isoforms had no effect on the aggregation of the Bri2-23 peptide.
      Figure thumbnail gr2
      Figure 2Thioflavin-T (Th-T) binding assay. A) Th-T binding of ADan (30 μM) alone and in the presence of ApoE2, ApoE3 and ApoE4 (300 μM). B) Th-T binding of Bri2-23 (30 μM) alone and in the presence of ApoE2, ApoE3 and ApoE4 (300 μM). Fluorescence is expressed in arbitrary units (A.U.). All data are expressed as mean ± SD. Experiments were repeated three times.

      ApoE knockout and ADan deposition in vivo.

      To assess the effect of ApoE on ADan deposition in an in vivo model of amyloidosis, we generated mice in which the Danish mutant form of human BRI2 is expressed under the control of the mouse prion protein promoter (Tg-FDD) (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ) on an ApoE knockout (ApoE-/-) background (Tg-FDD+/-/ApoE-/-). Mice were analyzed at 3, 6, 9, 12, and 18 months of age. In Tg-FDD mice, ADan amyloid is detectable in animals older than 7-8 month of age. At ∼7 months of age, transgenic animals consistently begin to exhibit CAA primarily in pial (leptomeningeal) cerebellar vessels. Immunohistochemical analysis of Tg-FDD+/-/ApoE-/- mice using polyclonal and monoclonal antibodies specific for the ADan amyloid peptide (SupFig 1) showed that knockout of the murine ApoE gene led to a complete inhibition of ADan deposition up to the last age analyzed (18 months) (Fig. 3). Thioflavin S-positive ADan deposition in leptomeningeal cerebellar vessels can be seen in age-matched Tg-FDD+/- control mice (Fig. 3) (SupFig 2). No parenchymal amyloid deposition was observed in Tg-FDD+/-/ApoE-/- mice. Immunohistochemical analysis using an antibody that recognizes the BRI2 precursor protein shows a different pattern of immunoreactivity in the hippocampus of Tg-FDD+/-/ApoE-/- mice, compared to Tg-FDD+/- mice (Fig. 4). The antibody against the N-terminus of the BRI2 precursor protein labeled neuronal cell bodies in the CA3 region of the hippocampus of Tg-FDD+/-/ApoE-/- mice but not in Tg-FDD+/- mice (Fig. 4A). Western blot analyses of protein extracts from the hippocampus showed a statistically significant increase in the levels of the im-BRI2 and m-BRI2 precursor protein in Tg-FDD+/-/ApoE-/- mice compared to Tg-FDD+/- mice (Fig. 4B).
      Figure thumbnail gr3
      Figure 3Amyloid deposition in Tg-FDD mice at 18 months of age. Typical cerebellar amyloid deposition in leptomeniges in Tg-FDD mice can be seen using the monoclonal antibody 10A1 and polyclonal antibody 1700 in Tg-FDD+/+ and Tg-FDD+/- mice. The deposits are typically Th-S-positive, indicating the presence of fibrillar ADan. Amyloid deposits are not observed in Tg-FDD+/- mice on the ApoE-/- background (Tg-FDD+/-/ApoE-/-) by immunohistochemistry and by Th-S. Scale bar: 100 μm.
      Figure thumbnail gr4
      Figure 4Accumulation of mutant BRI2 in Tg-FDD+/-/ApoE-/- mice. A) Intracellular accumulation of BRI2 in neuronal cell bodies of the CA3 region of the hippocampus of Tg-FDD+/-/ApoE-/- mice but not Tg-FDD+/- mice can be observed by immunohistochemistry using an anti-BRI2 (anti-ITM2B) ab. Sections were from the hippocampus of 18-month old mice. Scale bar: 200μm. B) Western blot analysis of brain homogenates from the hippocampus using the anti-BRI2 ab shows a significant accumulation of immature (im-BRI2) and mature (m-BRI2) forms of BRI2 in Tg-FDD+/-/ApoE-/- mice. Samples were run in triplicates. Representative blots are shown. C) Quantification of im-BRI2 by densitometry corrected with the densitometry of tubulin (** p=0.0051. D) Quantification of m-BRI2 by densitometry corrected with the densitometry of tubulin (** p=0.0018). All data are expressed as mean ± SD and a t-test was performed for quantification.

      Gene co-expression analysis defines modules associated with ADan deposition and ApoE.

      To profile the molecular changes associated with the lack of amyloid deposition in Tg-FDD+/-/ApoE-/- mice, we performed a targeted neuropathological transcriptome analysis using a customized Nanostring nCounter panel containing 770 genes specific for neurodegeneration. Analysis of hippocampal samples from three Tg-FDD+/- and three Tg-FDD+/-/ApoE-/- mice at 12 months of age show forty-two significantly mis-regulated genes, fourteen downregulated genes and twenty-eight upregulated genes (Fig. 5, STable 1) (SupFig 3). Hierarchical clustering analysis showed distinct cluster separation between Tg-FDD+/-/ApoE-/- and Tg-FDD+/- mice (Fig. 5b). Network analysis using STRING software to visualize protein-protein interactions of most upregulated and downregulated genes identified four regulated pathways with a high significance score (Fig. 6). The top gene networks identified by genes mis-regulated in Tg-FDD+/-/ApoE-/- mice compared to Tg-FDD+/- mice include Autophagy: Hexb (1.3-fold, p=0.03), Cd68 (1.3-fold, p=0.03); Activated microglia: Ptgs2 (1.5-fold, p=0.03), C1qb (1.4-fold, p=0.01), C1qc (1.4-fold, p=0.02), C1qa (1.4-fold, p=0.01), Cd68 (1.3-fold, p=0.03); Angiogenesis: Ptgs2 (1.5-fold, p=0.03), C1qb (1.4-fold, p=0.01), C1qc (1.4-fold, p=0.02), C1qa (1.4-fold, p=0.01); Vesicle trafficking: Ptgs2 (1.5-fold, p=0.03), Cntn4 (-1.4-fold, p=0.04) (Fig. 6).
      Figure thumbnail gr5
      Figure 5Transcriptomics analysis of aged Tg-FDD+/- mice vs. Tg-FDD+/-/ApoE-/- mice. A) Volcano plot of Nanostring results comparing mRNA isolated from hippocampi of 12 month old mice, done in triplicate. Knock-out of ApoE expression resulted in the upregulation of 28 genes (red), and down-regulation of 14 genes (green). Genes that are below the cut-off (FDR False Discovery Rate  ≤ 1.3.) are marked in gray. Some of the highly regulated genes are indicated in the plot. B) Heatmap of log2 fold changes of genes in Tg-FDD+/- mice vs. Tg-FDD+/-/ApoE-/- mice. Vertical lanes represent biological replicates. Genes are color coded as stated above.
      Figure thumbnail gr6
      Figure 6Prediction of the protein-protein interaction network affected by ApoE knock-out in Tg-FDD mice. Network showing the interaction of the top 24 differentially expressed genes, with bold lines indicating the highest confidence score interaction.

      Discussion

      ApoE has been found present in amyloid deposits in AD and other cerebral and systemic amyloidoses, suggesting a general role for ApoE in amyloid diseases as a pathological chaperone (
      • Wisniewski T.
      • Frangione B.
      Apolipoprotein E: a pathological chaperone protein in patients with cerebral and systemic amyloid.
      ,
      • Gallo G.
      • Wisniewski T.
      • Choi-Miura N.H.
      • Ghiso J.
      • Frangione B.
      Potential role of apolipoprotein-E in fibrillogenesis.
      ). Numerous studies have focused on the direct interaction of Aβ with ApoE and the formation of Aβ and ApoE complexes (
      • Wisniewski T.
      • Castano E.M.
      • Golabek A.
      • Vogel T.
      • Frangione B.
      Acceleration of Alzheimer’s fibril formation by apolipoprotein E in vitro.
      ,
      • Sadowski M.J.
      • Pankiewicz J.
      • Scholtzova H.
      • Mehta P.D.
      • Prelli F.
      • Quartermain D.
      • Wisniewski T.
      Blocking the apolipoprotein E/amyloid-β interaction as a potential therapeutic approach for Alzheimer’s disease.
      ); however, whether similar mechanism(s) may be at play in other amyloids remains to be determined. Like what has been seen in AD, ApoE has been found closely associated with ABri and ADan parenchymal and vascular deposits in FBD and FDD, respectively (
      • Lashley T.
      • Holton J.L.
      • Verbeek M.M.
      • Rostagno A.
      • Bojsen-Møller M.
      • David G.
      • van Horssen J.
      • Braendgaard H.
      • Plant G.
      • Frangione B.
      • Ghiso J.
      • Revesz T.
      Molecular chaperons, amyloid and preamyloid lesions in the BRI2 gene-related dementias: a morphological study.
      ), with Aβ immunoreactivity, mainly in a perivascular position, also present in patients with FDD (
      • Vidal R.
      • Revesz T.
      • Rostagno A.
      • Kim E.
      • Holton J.L.
      • Bek T.
      • Bojsen-Moller M.
      • Braendgaard H.
      • Plant G.
      • Ghiso J.
      • Frangione B.
      A decamer duplication in the 3’ region of the BRI gene originates an amyloid peptide that is associated with dementia in a Danish kindred.
      ,
      • Holton J.L.
      • Lashley T.
      • Ghiso J.
      • Braendgaard H.
      • Vidal R.
      • Guerin C.J.
      • et al.
      Familial Danish dementia: a novel form of cerebral amyloidosis associated with deposition of both amyloid-Dan and amyloid-beta.
      ).
      In the present study, we determined whether the presence of different ApoE isoforms influence the aggregation of synthetic ADan in vitro. ADan-amyloid formation kinetics showed a classical sigmoidal curve, with a lag phase, an elongation phase, and a plateau (saturation) phase. The addition of the different ApoE isoforms to ADan led to a pronounced decrease of Th-T incorporation, with lower plateau levels, but without modifying lag-phases. Thus, in our in vitro system, ApoE did not modify exponential fibril growth (elongation phase), but rather modified the level of the plateau, which may be caused by a shift to non-fibrillar structures. Future studies will determine whether the same result is obtained using lipidated ApoE instead of recombinant ApoE. No effect was observed when assessing the Bri2-23 peptide in the same assay. The role of ApoE in ADan aggregation in vivo was assessed using a cohort of mice expressing the Danish mutant form of human BRI2 on an ApoE knockout background. Lack of ApoE in Tg-FDD+/-/ApoE-/- mice, examined up to 18 months of age, prevented completely the age-dependent deposition of ADan previously observed in Tg-FDD mice (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ). Knock-out of ApoE in Tg-FDD+/-/ApoE-/- mice seems to block the conversion of soluble ADan to fibrillar ADan in both the brain parenchyma and blood vessels. We did not observe a delay in ADan deposition, although we cannot discard amyloid deposition occurring in animals older than 18 months of age. In some transgenic Aβ models, genetic deletion of the murine ApoE gene has been shown to delay Aβ accumulation and amyloid deposition (
      • Balu D.
      • Karstens A.J.
      • Loukenas E.
      • Maldonado Weng J.
      • York J.M.
      • Valencia-Olvera A.C.
      • LaDu M.J.
      The role of APOE in transgenic mouse models of AD.
      ). During the neuropathologic examination of mice, we unexpectedly found BRI2 accumulation in hippocampal neurons of Tg-FDD+/-/ApoE-/- mice. We have previously reported accumulation of mutant BRI2 in FBD, FDD and the Tg-FDD model (
      • Garringer H.J.
      • Sammeta N.
      • Oblak A.
      • Ghetti B.
      • Vidal R.
      Amyloid and intracellular accumulation of BRI2.
      ) and speculated that the accumulation of the BRI2 precursor may be due to the mutant sequences interfering with the correct folding of BRI2 and the enzymatic activity of pro-protein convertases. Alternatively, the mutant sequence may disrupt information required for efficient Golgi exit. Further work may clarify whether knockout of ApoE may affect protein processing or protein sorting, leading to intracellular accumulation of BRI2.
      Total RNA sequencing studies from brains of AD individuals showed the involvement of the classical complement pathway (CCP) and the phosphorylation of tau to be associated with AD in an APOE genotype-specific manner (
      • Panitch R.
      • Hu J.
      • Chung J.
      • Zhu C.
      • Meng G.
      • Xia W.
      • et al.
      Integrative brain transcriptome analysis links complement component 4 and HSPA2 to the APOE ε2 protective effect in Alzheimer disease.
      ,
      • Jun G.
      • You Y.
      • Zhu C.
      • Meng G.
      • Chung J.
      • Panitch R.
      • et al.
      Protein phosphatase 2A, complement component 4, and APOE genotype linked to Alzheimer disease using a systems biology approach. medRxiv.
      ). Molecular profiling of the Tg-FDD+/-/ApoE-/- model revealed changes in autophagy, activated microglia, angiogenesis and vesicle trafficking pathways, with C1q, the first subcomponent of the C1 complex of the CCP of complement activation, as one of the most upregulated genes and Ppp2r5c (the protein phosphatase 2 (PP2) regulatory subunit B'gamma), involved in the dephosphorylation of Tau, as one of the most downregulated genes. A total of 14 significantly downregulated genes and 28 significantly upregulated genes were observed. Among these 28 genes, 5 of them are highly expressed in microglia (Brain RNA seq database; http://www.brainrnaseq.org/). The top five most significantly upregulated genes detected were Smyd1, Entpd4, Fcrls, Bcas1 and C1q (composed of C1qa, C1qb, and C1qc). Smyd1, a histone methyltransferase, is a transcription factor characterized extensively in hematopoietic cells and cardiac/skeletal muscle (
      • Mayfield R.D.
      • Zhu L.
      • Smith T.A.
      • Tiwari G.R.
      • Tucker H.O.
      The SMYD1 and skNAC transcription factors contribute to neurodegenerative diseases.
      ). Recent work suggests that Smyd1 functions also in neuronal cells as regulators of genes disrupted in different neurodegenerative diseases (
      • Mayfield R.D.
      • Zhu L.
      • Smith T.A.
      • Tiwari G.R.
      • Tucker H.O.
      The SMYD1 and skNAC transcription factors contribute to neurodegenerative diseases.
      ). Smyd1 is expressed in microglia cells and astrocytes and was also observed in endothelial cells (ECs) where it may have a role in inflammation-triggered signaling in ECs (
      • Shamloul A.
      • Steinemann G.
      • Roos K.
      • Liem C.H.
      • Bernd J.
      • Braun T.
      • Zakrzewicz A.
      • Berkholz J.
      The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells.
      ). Entpd4, the ectonucleoside triphosphate diphosphohydrolase 4 (or NTPDase4), catalyzes the hydrolysis of nucleoside triphosphates and diphosphates in a calcium- or magnesium-dependent manner. The gene is expressed in many different organs, including the brain, and may play a role in the development of gastric cancer (
      • Gorelik A.
      • Labriola J.M.
      • Illes K.
      • Nagar B.
      Crystal structure of the nucleotide-metabolizing enzyme NTPDase4.
      ). Fcrls, which encodes the Fc Receptor-like S, is a scavenger receptor expressed by microglia in mice (
      • Butovsky O.
      • Jedrychowski M.P.
      • Moore C.S.
      • Cialic R.
      • Lanser A.J.
      • Gabriely G.
      • et al.
      Identification of a unique TGF-β-dependent molecular and functional signature in microglia.
      ). Fcrls are expressed in multiple brain macrophage subsets (
      • Brioschi S.
      • Zhou Y.
      • Colonna M.
      Brain Parenchymal and Extraparenchymal Macrophages in Development, Homeostasis, and Disease.
      ). Fcrls binds ligands that are non-self or self-altered molecules and remove them by phagocytosis. Bcas1, the breast carcinoma amplified sequence 1, is specifically expressed in immature oligodendrocytes undergoing maturation and myelination. Bcas1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions (
      • Fard M.K.
      • van der Meer F.
      • Sánchez P.
      • Cantuti-Castelvetri L.
      • Mandad S.
      • Jäkel S.
      • et al.
      BCAS1 expression defines a population of early myelinating oligodendrocytes in multiple sclerosis lesions.
      ) and Bcas1-positive immature oligodendrocytes seem to be affected by α-synuclein-induced pathology in multiple system atrophy (
      • Kaji S.
      • Maki T.
      • Ueda J.
      • Ishimoto T.
      • Inoue Y.
      • Yasuda K.
      • Sawamura M.
      • Hikawa R.
      • Ayaki T.
      • Yamakado H.
      • Takahashi R.
      BCAS1-positive immature oligodendrocytes are affected by the α-synuclein-induced pathology of multiple system atrophy.
      ). C1q is the recognition molecule complex of the innate immune system that initiates the CCP. C1q is secreted by macrophages and may play a role in synapse elimination in healthy brain and in neurodegenerative diseases (
      • Stevens B.
      • Allen N.J.
      • Vazquez L.E.
      • Howell G.R.
      • Christopherson K.S.
      • Nouri N.
      • et al.
      The classical complement cascade mediates CNS synapse elimination.
      ,
      • Vadászi H.
      • Kiss B.
      • Micsonai A.
      • Schlosser G.
      • Szaniszló T.
      • Kovács R.Á.
      • et al.
      Competitive inhibition of the classical complement pathway using exogenous single-chain C1q recognition proteins.
      ). Recently, it has been reported that in ApoE-deficient mice, oxidized lipids activate the CCP and that all three ApoE isoforms and serum-derived ApoE3 bind C1 and C1q (
      • Yin C.
      • Ackermann S.
      • Ma Z.
      • Mohanta S.K.
      • Zhang C.
      • Li Y.
      • Nietzsche S.
      • et al.
      ApoE attenuates unresolvable inflammation by complex formation with activated C1q.
      ). Additional important upregulated genes are Hexb, Cd68, Ptgs2 and Cntn4. Hexb encodes the beta subunit of β-hexosaminidase. Mutations in Hexb lead to developmental problems with seizures, and childhood death (
      • Bley A.E.
      • Giannikopoulos O.A.
      • Hayden D.
      • Kubilus K.
      • Tifft C.J.
      • Eichler F.S.
      Natural history of infantile G(M2) gangliosidosis.
      ,
      • Smith N.J.
      • Winstone A.M.
      • Stellitano L.
      • Cox T.M.
      • Verity C.M.
      GM2 gangliosidosis in a UK study of children with progressive neurodegeneration: 73 cases reviewed.
      ), and may be associated with AD and cerebral amyloid angiopathy (
      • Sierksma A.
      • Lu A.
      • Mancuso R.
      • Fattorelli N.
      • Thrupp N.
      • Salta E.
      • Zoco J.
      • Blum D.
      • et al.
      Novel Alzheimer risk genes determine the microglia response to amyloid-beta but not to TAU pathology.
      ,
      • Mufson E.J.
      • He B.
      • Ginsberg S.D.
      • Carper B.A.
      • Bieler G.S.
      • Crawford F.
      • Alvarez V.E.
      • Huber B.R.
      • Stein T.D.
      • McKee A.C.
      • Perez S.E.
      Gene Profiling of Nucleus Basalis Tau Containing Neurons in Chronic Traumatic Encephalopathy: A Chronic Effects of Neurotrauma Consortium Study.
      ). Recent work in mouse models demonstrated that Hexb heterozygosity leads to neuropathologic changes, consistent with previous reports that described a biochemical relationship between Hexb and AD (
      • Brioschi S.
      • Zhou Y.
      • Colonna M.
      Brain Parenchymal and Extraparenchymal Macrophages in Development, Homeostasis, and Disease.
      ,
      • Whyte L.S.
      • Fourrier C.
      • Hassiotis S.
      • Lau A.A.
      • Trim P.J.
      • Hein L.K.
      • Hattersley K.J.
      • et al.
      Lysosomal gene Hexb displays haploinsufficiency in a knock-in mouse model of Alzheimer's disease.
      ). Cd68 is a marker for macrophage lineage cells, primarily localized to microglia within the brain parenchyma, and perivascular macrophages in cerebral blood vessels (
      • Hopperton K.E.
      • Mohammad D.
      • Trépanier M.O.
      • Giuliano V.
      • Bazinet R.P.
      Markers of microglia in post-mortem brain samples from patients with Alzheimer's disease: a systematic review.
      ). Cd68 labels lysosomal and endosomal transmembrane glycoprotein of microglia, indicating phagocytic activity, with presence of CD68, MSR-A and HLA-DR being related to dementia and scores of poor cognitive function in AD (
      • Minett T.
      • Classey J.
      • Matthews F.E.
      • Fahrenhold M.
      • Taga M.
      • Brayne C.
      • Ince P.G.
      • Nicoll J.A.
      • Boche D.
      Microglial immunophenotype in dementia with Alzheimer's pathology; MRC CFAS.
      ). Ptgs2 encodes the cyclooxygenase-2 (Cox-2), which is involved in the synthesis of prostanoids. Cox-2 seems to be expressed under pathological conditions and to have detrimental effects in AD pathophysiology and neurodegeneration (
      • Tyagi A.
      • Kamal M.A.
      • Poddar N.K.
      Integrated Pathways of COX-2 and mTOR: Roles in Cell Sensing and Alzheimer's Disease.
      ). Cox-2 has been recently to be shown to be critical for the propagation of Aβ and reducing the glycosylation of tau in AD (
      • Guan P.P.
      • Liang Y.Y.
      • Cao L.L.
      • Yu X.
      • Wang P.
      Cyclooxygenase-2 Induced the β-Amyloid Protein Deposition and Neuronal Apoptosis Via Upregulating the Synthesis of Prostaglandin E2 and 15-Deoxy-Δ12,14-prostaglandin J2.
      ). Cntn4, Contactin 4, is an Ig cell adhesion molecule (IgCAM) gene, which has been associated with several neuropsychiatric disorders including AD (
      • Oguro-Ando A.
      • Bamford R.A.
      • Sital W.
      • Sprengers J.J.
      • Zuko A.
      • Matser J.M.
      • Oppelaar H.
      • et al.
      Cntn4, a risk gene for neuropsychiatric disorders, modulates hippocampal synaptic plasticity and behavior.
      ). Genome-wide association studies showed a link between Contactin family and dementia and dysregulated expression of Contactin in the post-mortem brain tissue of AD patients (
      • Bamford R.A.
      • Widagdo J.
      • Takamura N.
      • Eve M.
      • Anggono V.
      • Oguro-Ando A.
      The Interaction Between Contactin and Amyloid Precursor Protein and Its Role in Alzheimer's Disease.
      ).
      The top five most significantly downregulated genes detected are Cyp4x1, Chrnb2, 3110043021Rik (C9ORF72), Pik3ca and Ppp2r5c. Cyp4x1, the cytochrome P450 4x1, is expressed at very high levels in human and murine brain but the function of this protein is unknown. Cyp4x1-knock out mice gained significantly more body weight on normal lab chow diet compared to control flox mice on the same genetic background and had significantly greater intra-abdominal fat deposits (
      • Kharkwal H.
      • Batool F.
      • Koentgen F.
      • Bell D.R.
      • Kendall D.A.
      • Ebling F.J.P.
      • Duce I.R.
      Generation and phenotypic characterisation of a cytochrome P450 4x1 knockout mouse.
      ). Chrnb2 is the nicotinic cholinergic receptor subunit β-2. Genetic analyses have shown an association between Chrnb2 and late-onset AD (
      • Olgiati P.
      • Politis A.M.
      • Papadimitriou G.N.
      • De Ronchi D.
      • Serretti A.
      (2011) Genetics of late-onset Alzheimer's disease: update from the alzgene database and analysis of shared pathways.
      ). Recently, Chrnb2 has been found to be downregulated in chronic traumatic encephalopathy stage III compared to stage II, suggesting cholinergic-related deficits post-traumatic brain injury in humans (
      • Mufson E.J.
      • He B.
      • Ginsberg S.D.
      • Carper B.A.
      • Bieler G.S.
      • Crawford F.
      • Alvarez V.E.
      • Huber B.R.
      • Stein T.D.
      • McKee A.C.
      • Perez S.E.
      Gene Profiling of Nucleus Basalis Tau Containing Neurons in Chronic Traumatic Encephalopathy: A Chronic Effects of Neurotrauma Consortium Study.
      ). 3110043021Rik is the murine homologue of human C9ORF72. Hexanucleotide repeat expansions in the C9ORF72 gene are the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (
      • Breevoort S.
      • Gibson S.
      • Figueroa K.
      • Bromberg M.
      • Pulst S.
      Expanding Clinical Spectrum of C9ORF72-Related Disorders and Promising Therapeutic Strategies: A Review.
      ). In humans, C9ORF72 transcripts are detectable in most tissues, all brain regions, and the spinal cord. C9ORF72 plays an important role in immune regulation. The expression is particularly high in myeloid cells (in particular in CD14+ monocytes, eosinophils, and neutrophils) and lower in lymphoid cells and other tissues (
      • Smeyers J.
      • Banchi E.G.
      • Latouche M.
      C9ORF72: What It Is, What It Does, and Why It Matters.
      ). Pik3ca is the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha. PIP3 is the major product of PIK3CA. PIP3, in turn, is required for translocation of protein kinase B (AKT1, PKB) to the cell membrane, where it is phosphorylated and activated by upstream kinases. Mutations in Pik3ca lead to a plethora of disorders, among others oncogenic and vascular and overgrowth syndromes with some diseases having a neurologic phenotype (
      • Chen W.L.
      • Pao E.
      • Owens J.
      • Glass I.
      • Pritchard C.
      • Shirts B.H.
      • Lockwood C.
      • Mirzaa G.M.
      The utility of cerebrospinal fluid-derived cell-free DNA in molecular diagnostics for the PIK3CA-related megalencephaly-capillary malformation (MCAP) syndrome: a case report.
      , https://www.omim.org/entry/171834). Ppp2r5c is the protein phosphatase 2 (PP2) regulatory subunit B'gamma. The PP2A-PPP2R5C holoenzyme may specifically dephosphorylate and activate TP53 and play a role in DNA damage-induced inhibition of cell proliferation. PP2A-PPP2R5C may also regulate the ERK signaling pathway through ERK dephosphorylation. PP2A is the major tau phosphatase involved in the phosphorylation of Tau in AD (
      • Miao J.
      • Shi R.
      • Li L.
      • Chen F.
      • Zhou Y.
      • Tung Y.C.
      • Hu W.
      • Gong C.X.
      • Iqbal K.
      • Liu F.
      Pathological Tau From Alzheimer's Brain Induces Site-Specific Hyperphosphorylation and SDS- and Reducing Agent-Resistant Aggregation of Tau in vivo.
      ).
      In summary, our findings support the hypothesis that ApoE plays a major role in amyloid deposition in various amyloid diseases. It also demonstrates the beneficial effect of decreasing ApoE levels on ADan accumulation. Whether different isoforms of ApoE can modulate ADan aggregation in FDD, a genetic disorder comparable to familial AD, remains to be determined. The effect of ApoE-ɛ4 on onset age of autosomal dominant AD was confirmed in large pedigrees suggesting that an increased ApoE-ε4 gene dosage may promote the development of the familial form of the disease (
      • Pastor P.
      • Roe C.M.
      • Villegas A.
      • Bedoya G.
      • Chakraverty S.
      • García G.
      • Tirado V.
      • Norton J.
      • Ríos S.
      • Martínez M.
      • Kosik K.S.
      • Lopera F.
      • Goate A.M.
      Apolipoprotein Eepsilon4 modifies Alzheimer's disease onset in an E280A PS1 kindred.
      ,
      • Van Cauwenberghe C.
      • Van Broeckhoven C.
      • Sleegers K.
      The genetic landscape of Alzheimer disease: clinical implications and perspectives.
      ). Our results from gene array assays demonstrated that a number of genes related to autophagy, angiogenesis, vesicle trafficking, microglia, the CCP and tau phosphorylation were dysregulated in the brain of Tg-FDD+/-/ApoE-/- mice. The genetic screen emphasizes the significant role for ApoE at the interface of inflammation and neurodegeneration via glial-mediated mechanisms (
      • Parhizkar S.
      • Holtzman D.M.
      APOE mediated neuroinflammation and neurodegeneration in Alzheimer's disease.
      ). Future in vivo studies may provide additional information regarding the effect of human ApoE isoforms on ADan deposition in vivo and the molecular mechanisms and pathways involved.

      Experimental procedures

      Reagents and antibodies

      DMEM medium (#12100046), Fetal bovine serum (#26140079), Penicillin/Streptomycin (#15140122), OptiMEM (#31985062), Lipofectamine (#11668030), PBS (#14190), chemiluminescence kit (#32106), and Imperial Protein Stain (#24615) were purchased from Thermo Fisher. Thioflavin-S (T1892) and Thioflavin-T (T3516) were purchased from Sigma. Anti-ITM2B (sc-374362) was purchased from Santa Cruz; Anti-ApoE (66830) from Proteintech; Anti-C1Q (ab182451) from Abcam; Anti-FCRL4 (PA5-87813) from Invitrogen; Anti-GFAP (G3893) from Sigma; and anti-Myc (sc-40) and anti-Tubulin (sc-23948) from Santa Cruz. Anti-ADan (ab 1700) polyclonal antibody has been reported previously (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ).

      Synthetic peptides

      Bri2-23 (EASNCFAIRHFENKFAVETLICS) was synthesized by ThermoFisher Scientific. ADan (EASNCFAIRHFENKFAVETLICFNLFLNSQEKHY) was synthesized by ERI Amyloid Laboratory. The peptides were reconstituted in hexafluoroisopropanol (HFIP) to a final concentration of 1 mg/ml and incubated for 7 days at room temperature. Stock peptide solutions (1 mM) were prepared in 10 mM DMSO, followed by dilution to 100 μM in PBS buffer.

      Aggregation assay

      Peptide solutions were prepared in PBS with 20 μM Thioflavin-T (Th-T) to a final concentration of 30 μM. ApoE isoforms (apoE2, apoE3, or apoE4) were used at a 300 nM concentration. The volume of aggregation was 100 μl for all the samples. All samples were transferred to a black 96-well Nonbinding Surface microplate with clear bottom. The microplate was transferred to a Cytation C10 reader (Agilent). Fluorescence was measured through the top of the plate, recorded every 15 min for 18 hours (excitation 440 nm, emission 495 nm) at 37°C.

      Antibody generation

      Monoclonal antibodies were generated by GenScript USA Inc. using the antigen sequence CFNLFLNSQEKHY. C57BL/6 mice were immunized using a conventional immunization strategy. Mice with high serum titers (more than 1:60,000), as determined by an ELISA test, were selected for fusion of spleen cells with Myeloma cells type SP2/0. Positive cell supernatants were tested by ELISA. The final positive hybridoma cells secreting antibodies against ADan peptide were stored in -80°C. Supernatants were stable at 2‐8°C for up to 1 month but for long term storage, aliquots of the supernatants were stored at ‐20°C. The 10A1 monoclonal antibody was chosen for further characterization.

      Animal model

      Tg-FDD mice expressing an FDD-associated human mutant BRI2 transgene (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ) were crossed with ApoE tm1Unc mice (
      • Piedrahita J.A.
      • Zhang S.H.
      • Hagaman J.R.
      • Oliver P.M.
      • Maeda N.
      Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells.
      ) (The Jackson Laboratory) and obtained hemizygous mice Tg-FDD+/-/ApoE+/- that were subsequently intercrossed to generate Tg-FDD+/-/ApoE-/- mice. All mice were on an C57Bl6/J background. Male and female mice were used for the experiments. Mice were housed at the Indiana University School of Medicine (IUSM) animal care facility and were maintained according to USDA standards (12-h light/dark cycle, food and water ad libitum), in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD). Animals were anesthetized and euthanized according to IUSM Institutional Animal Care and Use Committee-approved procedures.

      Histological and immunohistochemical studies

      Mice were anesthetized and transcardially perfused with cold 0.9% saline. After perfusion, the animals were decapitated; the skulls opened and the brains removed and kept at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2 or immediately frozen at -80°C. Eight-micrometer-thick sections were cut from the fixed tissue and stained with the Hematoxylin-Eosin (H&E) and thioflavin-S (Th-S) methods following published protocols (
      • Vidal R.
      • Barbeito A.G.
      • Miravalle L.
      • Ghetti B.
      Cerebral amyloid angiopathy and parenchymal amyloid deposition in transgenic mice expressing the Danish mutant form of human BRI2.
      ,
      • Garringer H.J.
      • Murrell J.
      • Sammeta N.
      • Gnezda A.
      • Ghetti B.
      • Vidal R.
      Increased tau phosphorylation and tau truncation, and decreased synaptophysin levels in mutant BRI2/tau transgenic mice.
      ). Antibodies were visualized by using horse anti-mouse/rabbit IgG by the peroxidase-antiperoxidase method utilizing 3,3′diaminobenzidine (DAB) as the chromogen. Images were acquired with a Cytation C10 confocal imaging reader (Agilent).

      Cell transfection

      HEK-293T and COS-7 cells were transfected after reaching 75% cell confluence using a 3:1 ratio of DNA: Lipofectamine in Opti-MEM medium with
      wild-type human BRI2 sequence or FDD-associated human mutant BRI2 cloned into the FUGW lentiviral vector (Addgene) with the addition of an N-terminal MYC tag. Cells were transiently transfected for 5-6 hours with 2 μg of supercoiled plasmid DNA and analyzed after 24 hours.

      Immunofluorescence (IF)

      For IF, COS-7 cells were fixed on coverslips with 4% paraformaldehyde in PBS for 10 minutes, washed with PBS and permeabilized with 0.2% Triton X-100. Then, cells were incubated with blocking solution (10% BSA in PBS) for one hour followed by overnight incubation with primary antibody in blocking solution. Finally, cells were incubated with the corresponding secondary antibodies in blocking solution, washed with PBS and mounted with a mounting medium with DAPI. The images were acquired with Cytation C10 confocal imaging reader (Agilent).

      Western blots

      Transfected HEK-293T cells and brain samples were homogenized in Lysis buffer (Cell Signaling Technology) and proteins were quantified using the BCA method (ThermoFisher). Samples (synthetic peptides or protein samples) were boiled with gel loading buffer for 5 minutes at 100°C and separated in 10% Bris-Tris NuPage gels gels under denaturing conditions. Gels were stained with Coomassie blue or transferred to nitrocellulose membranes. The membranes were incubated with blocking solution (5% non-fat milk in PBS) for 1 hour, then incubated for 1 hour with primary antibody. Washings were carried out after each incubation with PBS. Finally, they were incubated with secondary antibody for 45 minutes. Bands were visualized using a chemiluminescence kit (SuperSignal West Pico, ThermoFisher) according to the manufacturer's specifications. Blots were scanned and quantified using Image J software (U.S. National Institutes of Health, Bethesda, MD, USA).

      RNA isolation and Nanostring analysis

      Hippocampal tissue was dissected from Tg-FDD+/- and Tg-FDD+/-/ApoE-/- mice. RNA was extracted using the miRNeasy Kit (Qiagen). RNA concentration was determined using a Nanodrop. RNA and Nanostring reactions were prepared according to the manufacturer’s recommendation (Mouse nCounter, Nanostring Technologies). Briefly, hippocampi from four Tg-FDD+/- and four Tg-FDD+/-/ApoE-/- mice at 12 months of age were used for gene expression analyses. 100 ng total RNA per sample were analyzed using the nCounter mouse Neuropathology Panel. Data were analyzed using the nSolver Analysis Software 3.0 (Nanostring Technologies) and ROSALIND software. An mRNA was considered differentially expressed if the False Discovery Rate (FDR) ≤ 1.3 or p Value < 0.05.

      Statistical analysis.

      Data are shown as mean ± SD and were considered statistically significant at p < 0.05. with GraphPad Prism 7.0 (GraphPad Software Inc.).

      Data availability

      All the data described in the manuscript are contained within the manuscript.
      This article contains supporting information.

      Uncited reference

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      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank D. Lucas and R. M. Richardson for technical support and Dr. G. I. Hallinan for proof-reading the manuscript.

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