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• W2008884445 abstract "Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aβ) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aβ in islet amyloid in type 2 diabetic patients and Aβ deposits in brains of patients with Alzheimer disease. Aβ reactivity was not detected in islet amyloid although islet β cells express AβPP and convertases necessary for Aβ production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aβ deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aβ shown here may represent a molecular link between type 2 diabetes and Alzheimer disease. Several proteins have been identified as amyloid forming in humans, and independent of protein origin, the fibrils are morphologically similar. Therefore, there is a potential for structures with amyloid seeding ability to induce both homologous and heterologous fibril growth; thus, molecular interaction can constitute a link between different amyloid forms. Intravenous injection with preformed fibrils from islet amyloid polypeptide (IAPP), proIAPP, or amyloid-beta (Aβ) into human IAPP transgenic mice triggered IAPP amyloid formation in pancreas in 5 of 7 mice in each group, demonstrating that IAPP amyloid could be enhanced through homologous and heterologous seeding with higher efficiency for the former mechanism. Proximity ligation assay was used for colocalization studies of IAPP and Aβ in islet amyloid in type 2 diabetic patients and Aβ deposits in brains of patients with Alzheimer disease. Aβ reactivity was not detected in islet amyloid although islet β cells express AβPP and convertases necessary for Aβ production. By contrast, IAPP and proIAPP were detected in cerebral and vascular Aβ deposits, and presence of proximity ligation signal at both locations showed that the peptides were <40 nm apart. It is not clear whether IAPP present in brain originates from pancreas or is locally produced. Heterologous seeding between IAPP and Aβ shown here may represent a molecular link between type 2 diabetes and Alzheimer disease. Amyloid fibril formation is a self-propagating process that implicates a prion-like mechanism for amyloid growth and spreading of amyloid in tissues. Alzheimer disease (AD) and type 2 diabetes (T2D) are multifactorial diseases, but both have local amyloid depositions in their pathogenesis. In T2D, islet amyloid is made up by islet amyloid polypeptide (IAPP; amylin)1Westermark P. Wernstedt C. Wilander E. Hayden D.W. O'Brien T.D. Johnson K.H. Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells.Proc Natl Acad Sci U S A. 1987; 84: 3881-3885Crossref PubMed Scopus (879) Google Scholar (reviewed in Westermark et al2Westermark P. Andersson A. Westermark G.T. Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.Physiol Rev. 2011; 91: 795-826Crossref PubMed Scopus (714) Google Scholar) that arises through posttranslational processing of its precursor, proIAPP.3Marzban L. Soukhatcheva G. Verchere C.B. Role of carboxypeptidase E in processing of pro-islet amyloid polypeptide in β-cells.Endocrinology. 2005; 146: 1808-1817Crossref PubMed Scopus (49) Google Scholar Accumulation of IAPP amyloid leads to β-cell death4Huang C.J. Lin C.Y. Haataja L. Gurlo T. Butler A.E. Rizza R.A. Butler P.C. High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes.Diabetes. 2007; 56: 2016-2027Crossref PubMed Scopus (335) Google Scholar, 5Hull R.L. Zraika S. Udayasankar J. Aston-Mourney K. Subramanian S.L. Kahn S.E. Amyloid formation in human IAPP transgenic mouse islets and pancreas, and human pancreas, is not associated with endoplasmic reticulum stress.Diabetologia. 2009; 52: 1102-1111Crossref PubMed Scopus (48) Google Scholar and development of diabetes.6Guardado-Mendoza R. Davalli A.M. Chavez A.O. Hubbard G.B. Dick E.J. Majluf-Cruz A. Tene-Perez C.E. Goldschmidt L. Hart J. Perego C. Comuzzie A.G. Tejero M.E. Finzi G. Placidi C. La Rosa S. Capella C. Halff G. Gastaldelli A. DeFronzo R.A. Folli F. Pancreatic islet amyloidosis, beta-cell apoptosis, and alpha-cell proliferation are determinants of islet remodeling in type-2 diabetic baboons.Proc Natl Acad Sci U S A. 2009; 106: 13992-13997Crossref PubMed Scopus (116) Google Scholar, 7Rivera J.F. Costes S. Gurlo T. Glabe C.G. Butler P.C. Autophagy defends pancreatic beta cells from human islet amyloid polypeptide-induced toxicity.J Clin Invest. 2014; 124: 3489-3500Crossref PubMed Scopus (162) Google Scholar, 8Kim J. Cheon H. Jeong Y.T. Quan W. Kim K.H. Cho J.M. Lim Y.M. Oh S.H. Jin S.M. Kim J.H. Lee M.K. Kim S. Komatsu M. Kang S.W. Lee M.S. Amyloidogenic peptide oligomer accumulation in autophagy-deficient beta cells induces diabetes.J Clin Invest. 2014; 124: 3311-3324Crossref PubMed Scopus (118) Google Scholar We have shown that incomplete enzymatic cleavage of proIAPP into IAPP triggers amyloid formation9Paulsson J.F. Westermark G.T. Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation.Diabetes. 2005; 54: 2117-2125Crossref PubMed Scopus (96) Google Scholar and that the first intracellular amyloid in part consists of proIAPP.10Paulsson J.F. Andersson A. Westermark P. Westermark G.T. Intracellular amyloid-like deposits contain unprocessed pro-islet amyloid polypeptide (proIAPP) in beta cells of transgenic mice overexpressing the gene for human IAPP and transplanted human islets.Diabetologia. 2006; 49: 1237-1246Crossref PubMed Scopus (90) Google Scholar It is conceivable that amyloid that escapes elimination during cell debris clearance acts as seed and promotes extracellular amyloid growth. AD is the most common neurodegenerative disease, with a prevalence of 10% at age 70 years,11Plassman B.L. Langa K.M. Fisher G.G. Heeringa S.G. Weir D.R. Ofstedal M.B. Burke J.R. Hurd M.D. Potter G.G. Rodgers W.L. Steffens D.C. Willis R.J. Wallace R.B. Prevalence of dementia in the United States: the aging, demographics, and memory study.Neuroepidemiology. 2007; 29: 125-132Crossref PubMed Scopus (1368) Google Scholar and local deposits of Aβ amyloid are present in cortex and vessels in the brain.12Glenner G.G. Wong C.W. Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein.Biochem Biophys Res Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1254) Google Scholar, 13Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Amyloid plaque core protein in Alzheimer disease and Down syndrome.Proc Natl Acad Sci U S A. 1985; 82: 4245-4249Crossref PubMed Scopus (3654) Google Scholar Aβ peptide is generated by enzymatic cleavage of the precursor AβPP14Walter J. Kaether C. Steiner H. Haass C. The cell biology of Alzheimer's disease: uncovering the secrets of secretases.Curr Opin Neurobiol. 2001; 11: 585-590Crossref PubMed Scopus (158) Google Scholar (often referred to as APP), and accumulated data point to a partially intracellular route for Aβ plaque formation.15Friedrich R.P. Tepper K. Ronicke R. Soom M. Westermann M. Reymann K. Kaether C. Fandrich M. Mechanism of amyloid plaque formation suggests an intracellular basis of Abeta pathogenicity.Proc Natl Acad Sci U S A. 2010; 107: 1942-1947Crossref PubMed Scopus (215) Google Scholar, 16Takahashi R.H. Milner T.A. Li F. Nam E.E. Edgar M.A. Yamaguchi H. Beal M.F. Xu H. Greengard P. Gouras G.K. Intraneuronal Alzheimer abeta42 accumulates in multivesicular bodies and is associated with synaptic pathology.Am J Pathol. 2002; 161: 1869-1879Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar IAPP and Aβ exhibit an overall 25% amino acid (aa) sequence identity, a property that increases in regions important for fibril assembly.17O'Nuallain B. Williams A.D. Westermark P. Wetzel R. Seeding specificity in amyloid growth induced by heterologous fibrils.J Biol Chem. 2004; 279: 17490-17499Crossref PubMed Scopus (338) Google Scholar Two regions of Aβ (aa 11 to 21 and aa 23 to 37) with high binding affinity for IAPP, and two analogous regions on IAPP (aa 8 to 20 and aa 21 to 37) with corresponding affinity for Aβ have been identified.18Andreetto E. Yan L.M. Tatarek-Nossol M. Velkova A. Frank R. Kapurniotu A. Identification of hot regions of the Abeta-IAPP interaction interface as high-affinity binding sites in both cross- and self-association.Angew Chem Int Ed Engl. 2010; 49: 3081-3085Crossref PubMed Scopus (150) Google Scholar These binding zones include residues that are important for self-assembly. Several clinical studies link together AD and T2D, and individuals with T2D have almost a twofold greater risk of developing AD,19Leibson C.L. Rocca W.A. Hanson V.A. Cha R. Kokmen E. O'Brien P.C. Palumbo P.J. Risk of dementia among persons with diabetes mellitus: a population-based cohort study.Am J Epidemiol. 1997; 145: 301-308Crossref PubMed Scopus (557) Google Scholar, 20Ott A. Stolk R.P. van Harskamp F. Pols H.A. Hofman A. Breteler M.M. Diabetes mellitus and the risk of dementia: the Rotterdam Study.Neurology. 1999; 53: 1937-1942Crossref PubMed Google Scholar, 21Peila R. Rodriguez B.L. Launer L.J. Honolulu-Asia Aging StudyType 2 diabetes, APOE gene, and the risk for dementia and related pathologies: the Honolulu-Asia Aging Study.Diabetes. 2002; 51: 1256-1262Crossref PubMed Scopus (1010) Google Scholar whereas other studies suggest an inverse relationship.22Janson J. Laedtke T. Parisi J.E. O'Brien P. Petersen R.C. Butler P.C. Increased risk of type 2 diabetes in Alzheimer disease.Diabetes. 2004; 53: 474-481Crossref PubMed Scopus (700) Google Scholar We here show that intravenous injection of preformed fibrils of synthetic IAPP, proIAPP, or Aβ in transgenic mice expressing human IAPP can act as a seed for IAPP amyloid in the islet of Langerhans. Morphological analysis with the highly sensitive and specific proximity ligation assay (PLA) of pancreases from T2D subjects and brain from AD patients revealed colocalization of IAPP and Aβ peptide in Aβ cerebral plaques, whereas no Aβ was detected in islet amyloid deposited in the endocrine pancreas. Recombinant human proIAPP1-67 (hproIAPP), human IAPP1-37 (hIAPP), and human des-31,32 proinsulin (CA) produced as described,23Paulsson J.F. Schultz S.W. Kohler M. Leibiger I. Berggren P.O. Westermark G.T. Real-time monitoring of apoptosis by caspase-3-like protease induced FRET reduction triggered by amyloid aggregation.Exp Diabetes Res. 2008; 2008: 865850Crossref PubMed Scopus (17) Google Scholar and Aβ1-42 (Aβ; Bachem, Bubendorf, Switzerland) (Figure 1A) were solubilized in dimethyl sulfoxide (DMSO) and diluted with distilled water to a final concentration of 100 μmol/L peptide in 1% DMSO, and allowed to form fibrils overnight at room temperature. Male mice (FVB; 4- to 6-week-old) expressing human IAPP behind the rat insulin-1 promotor, but deficient of mouse IAPP (hIAPP+/+mIAPP−/−),24Westermark G.T. Gebre-Medhin S. Steiner D.F. Westermark P. Islet amyloid development in a mouse strain lacking endogenous islet amyloid polypeptide (IAPP) but expressing human IAPP.Mol Med. 2000; 6: 998-1007PubMed Google Scholar and where islet amyloid starts to occur after 10 months on high-fat feeding were used for seeding experiments. Preformed fibrils of hproIAPP, hIAPP, Aβ, and CA equivalent to 20 μg of peptide in 100 μL of 0.15 mol/L NaCl were sonicated 10 seconds (Vibra Cell; Sonic & Materials, Danbury, CT) and injected in the tail vein of four groups of seven hIAPP transgenic mice.24Westermark G.T. Gebre-Medhin S. Steiner D.F. Westermark P. Islet amyloid development in a mouse strain lacking endogenous islet amyloid polypeptide (IAPP) but expressing human IAPP.Mol Med. 2000; 6: 998-1007PubMed Google Scholar A fifth group of seven mice received an i.v. injection of 100 μL of 0.15 mol/L NaCl. Mice were caged individually, maintained on a 12 hours light–dark cycle, and fed standard chow with free access to water and lard (Ellco Food, Gothenburg, Sweden) for 10 months. In addition, four groups of three mice each were injected with hproIAPP, hIAPP, Aβ, or CA fibrils and sacrificed 24 hours later. The study was approved by the animal ethics committee at Linköping University, Sweden. At sacrifice, pancreas, spleen, liver, lung, heart, and kidney were collected, fixed in 10% neutral buffered formalin, and the presence of amyloid was investigated after Congo Red staining. Antibodies and dilutions used in the present study are listed in Table 1.Table 1Description of Antibodies Used for Proximity Ligation Assay, Immunohistochemistry, Immunofluorescence, and Western BlotAntibodyAntigenSpeciesDilutionSupplier4G8Aβ 17–24Mouse1:500 (IF)1:500 (PLA)1:1000 (WB)Signet6E10Aβ 1–16Mouse1:500 (PLA)SignetSM1341IAPP 7–17Mouse1:500 (PLA)AcrisSc1616β-ActinGoat1:1000 (WB)Santa CruzAntiserum A110mIAPP1–37Rabbit1:2000 (PLA)Authors25Betsholtz C. Christmansson L. Engstrom U. Rorsman F. Svensson V. Johnson K.H. Westermark P. Sequence divergence in a specific region of islet amyloid polypeptide (IAPP) explains differences in islet amyloid formation between species.FEBS Lett. 1989; 251: 261-264Crossref PubMed Scopus (155) Google Scholar A114hCGRP 1–37Rabbit1:500 (PLA)Authors∗See Supplemental Figure S2A. A133hIAPP 20–29Rabbit1:250 (IHC)1:2000 (PLA)1:1000 (WB)Authors9Paulsson J.F. Westermark G.T. Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation.Diabetes. 2005; 54: 2117-2125Crossref PubMed Scopus (96) Google Scholar A169hproIAPP 6–13Rabbit1:50 (IHC)Authors9Paulsson J.F. Westermark G.T. Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation.Diabetes. 2005; 54: 2117-2125Crossref PubMed Scopus (96) Google Scholar A165hproIAPP 46–55Rabbit1:200 (IHC)Authors9Paulsson J.F. Westermark G.T. Aberrant processing of human proislet amyloid polypeptide results in increased amyloid formation.Diabetes. 2005; 54: 2117-2125Crossref PubMed Scopus (96) Google Scholar A142hproIAPP 52–67Rabbit1:2000 (PLA)Authors10Paulsson J.F. Andersson A. Westermark P. Westermark G.T. Intracellular amyloid-like deposits contain unprocessed pro-islet amyloid polypeptide (proIAPP) in beta cells of transgenic mice overexpressing the gene for human IAPP and transplanted human islets.Diabetologia. 2006; 49: 1237-1246Crossref PubMed Scopus (90) Google Scholar A159ApoAI 9–20Rabbit1:2000 (PLA)Authors26Mucchiano G.I. Haggqvist B. Sletten K. Westermark P. Apolipoprotein A-1-derived amyloid in atherosclerotic plaques of the human aorta.J Pathol. 2001; 193: 270-275Crossref PubMed Scopus (85) Google Scholar A179MedinRabbit1:2000 (PLA)Authors27Larsson A. Peng S. Persson H. Rosenbloom J. Abrams W.R. Wassberg E. Thelin S. Sletten K. Gerwins P. Westermark P. Lactadherin binds to elastin–a starting point for medin amyloid formation?.Amyloid. 2006; 13: 78-85Crossref PubMed Scopus (38) Google ScholarApoAI, apolipoprotein AI; hIAPP, human IAPP; hproIAPP, human proIAPP; IHC, immunohistochemistry; IF, immunofluorescence; mIAPP, mouse IAPP; PLA, proximity ligation assay; WB, Western blot.∗ See Supplemental Figure S2A. Open table in a new tab ApoAI, apolipoprotein AI; hIAPP, human IAPP; hproIAPP, human proIAPP; IHC, immunohistochemistry; IF, immunofluorescence; mIAPP, mouse IAPP; PLA, proximity ligation assay; WB, Western blot. Reactivity patterns for antibodies produced against Aβ 17 to 24 (4G8), hIAPP 20 to 29 (A133), mIAPP 1 to 37 (A110), and hproIAPP 52 to 67 (A142) were scrutinized with slot blot analysis. Aβ 1 to 42 (Bachem), hIAPP (recombinant expressed),23Paulsson J.F. Schultz S.W. Kohler M. Leibiger I. Berggren P.O. Westermark G.T. Real-time monitoring of apoptosis by caspase-3-like protease induced FRET reduction triggered by amyloid aggregation.Exp Diabetes Res. 2008; 2008: 865850Crossref PubMed Scopus (17) Google Scholar hproIAPP residues 52 to 67,28Ma Z. Westermark G.T. Li Z.C. Engstrom U. Westermark P. Altered immunoreactivity of islet amyloid polypeptide (IAPP) may reflect major modifications of the IAPP molecule in amyloidogenesis.Diabetologia. 1997; 40: 793-801Crossref PubMed Scopus (16) Google Scholar and human αCGRP 1 to 37 (hαCGRP; Sigma-Aldrich, Stockholm, Sweden), were dissolved in DMSO, diluted in 50 mmol/L sodium carbonate buffer (pH 9.6), and applied (27 pmol) to nitrocellulose membranes (Hybond-ECL; GE Healthcare, Uppsala, Sweden). The membranes were boiled to expose epitopes and thus increase antibody binding in 4.3 mmol/L Na2HPO4, 1.4 mmol/L KH2PO4, 2.7 mmol/L KCl, and 137 mmol/L NaCl (PBS), pH 7.4, for 5 minutes in a microwave oven, blocked in a solution of 0.1% Tween-20, 50 mmol/L Tris-buffer with 150 mmol/L NaCl (pH 7.5) with 5% (w/v) nonfat dry milk for 1 hour followed by incubation with A110, A114, A133, A142, or 4G8 overnight at 4°C. Reactivity was visualized with horseradish peroxidase–conjugated secondary antibodies (Dako, Stockholm, Sweden) and chemiluminescence detection (Immobilon; Merck Millipore, Stockholm, Sweden). Ten sections (20 μm thick) from formalin-fixed, paraffin-embedded pancreas from patients with T2D (n = 4) (Table 2) were collected in Eppendorf tubes and then deparaffinized and rehydrated. Tissue was incubated in 70% formic acid (FA) for 60 minutes followed by centrifugation at 16,000 × g for 10 minutes. Supernatant was lyophilized and redissolved in DMSO.Table 2Clinical Information on PatientsPatient no.SexADDuration of disease (years)DiagnosisAD1M64UnknownADAD2M619ADAD3F843AD+Lewy bodyAD4F9212ADAD5M85>10ADAD6M88>10ADNon-AD1M88MDCFTD1M70FTDFTD2F61FTDPSPM70PSP+mild degenerative changes of AD typeNon-AD2M74Familial TTR amyloidosis without AD diagnosis, but with diffuse plaquesT2D1M8216T2DT2D2F785T2DT2D3F7415T2DT2D4M6810T2DF, female; M, male; AD, Alzheimer disease; FTD, frontotemporal dementia; MDC, mild degenerative changes; PSP, progressive supranuclear palsy; T2D, type 2 diabetes; TTR, transthyretin amyloid. Open table in a new tab F, female; M, male; AD, Alzheimer disease; FTD, frontotemporal dementia; MDC, mild degenerative changes; PSP, progressive supranuclear palsy; T2D, type 2 diabetes; TTR, transthyretin amyloid. Frozen temporal lobe brain samples (approximately 150 mg) from autopsy-confirmed AD patients (n = 4) and non-AD patients (n = 4) (Table 2) were recovered from the brain bank at Uppsala University. The tissues were homogenized with a mechanical homogenizer (Polytron PT-MR2100; Kinematica, Littau-Luzern, Switzerland) in 20 mmol/L Tris buffer, 137 mmol/L NaCl, and 2 mmol/L EDTA (pH 7.6) with protease inhibitors; 50 μg/mL phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, and 2 μg/mL aprotinin (10 μL/mg brain tissue; Sigma-Aldrich) at 4°C. The homogenate was centrifuged at 100,000 × g for 1 hour at 4°C (TLA110; Beckman Coulter, Bromma, Sweden), and supernatant was collected (hereafter referred to as TB fraction). Remaining pellet was rehomogenized in 70% FA (10 μL/mg brain tissue), incubated 45 minutes at room temperature, sonicated, and then centrifuged at 100,000 × g for 1 hour at 4°C. The supernatant was lyophilized and redissolved in DMSO to generate the FA fraction. Frozen kidney tissue was extracted in 150 mmol/L Tris-HCl (pH 6.8), 6% mercaptoethanol, 12% SDS, and 30% glycerol (SDS sample buffer) with cycles of mechanical homogenization and heating at 95°C. The supernatant was recovered and used as negative control in Western blot. Anonymized fresh brain tissue was from the brain bank, and formalin-fixed tissues were recovered from the amyloid archive, Uppsala University, and approved for research by the local ethical committee, Uppsala University. Proteins from tissue extracts were solubilized with SDS sample buffer, separated by 6% to 12.5% tricine-SDS-PAGE, and then transferred onto a nitrocellulose membrane (Hybond-ECL) and boiled in PBS for 5 minutes. Membrane was blocked in Tween-Tris–buffered saline with 5% (w/v) nonfat dry milk for 1 hour and then incubated with primary antibodies A133 or 4G8 diluted in Tween-Tris–buffered saline (Table 1) overnight at 4°C. Reactivity was visualized with horseradish peroxidase–conjugated secondary antibody (Dako) and chemiluminescence detection (Immobilon). Total protein quantification was performed in TB and FA fractions with Pierce BCA protein assay (Fischer Scientific, Gothenburg, Sweden). Digitalized images of Western blot bands were used to quantify protein levels based on optical density of all reactivity for each antibody (ImageJ software version 1.48, NIH, Bethesda, MD). In situ PLA can be used for colocalization studies of proteins in tissue sections. The high specificity of PLA derives from a dual binding event of two primary antibodies with different species origin, to target proteins. The primary antibodies are detected with oligonucleotide-conjugated secondary antibodies (PLA probes) that generate an amplifiable circular DNA only if both target proteins are in close proximity (<40 nm).29Soderberg O. Gullberg M. Jarvius M. Ridderstrale K. Leuchowius K.J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.G. Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat Methods. 2006; 3: 995-1000Crossref PubMed Scopus (1754) Google Scholar The sensitivity derives from the up to 1300-fold amplification of the circular DNA strand,30Baner J. Nilsson M. Mendel-Hartvig M. Landegren U. Signal amplification of padlock probes by rolling circle replication.Nucleic Acids Res. 1998; 26: 5073-5078Crossref PubMed Scopus (379) Google Scholar which is visualized after hybridization with fluorescence-labeled oligonucleotides. Formalin-fixed and paraffin-embedded sections (10 μm) from pancreas and brain were rehydrated and pretreated with 95% FA for 3 minutes at room temperature to expose antigens. After rinsing in 50 mmol/L Tris-HCl buffer with 150 mmol/L NaCl (pH 7.4), pancreas sections were incubated with primary antibody pairs, IAPP antiserum (A133), and mouse anti-Aβ antibody 4G8 or A133 and mouse anti-IAPP antibody (SM1341) (Table 1). Brain sections were incubated with mouse anti-Aβ antibodies 4G8 or 6E10 mixed with anti-IAPP antisera (A110, A133) or anti-proIAPP (A142). Mouse anti-Aβ antibodies 4G8 were also used in combination with anti-αCGRP antiserum (A114), anti-apoA1 antiserum (A159), or anti-medin antiserum (A179) (Table 1). Primary antibody incubations were performed overnight at room temperature (Table 1). Duolink in situ PLA (Olink Bioscience, Uppsala, Sweden) was performed according to the manufacturer's protocol. Briefly, for detection of primary antibody pairs, sections were incubated for 90 minutes with oligonucleotide-conjugated anti-mouse IgG MINUS and anti-rabbit IgG PLUS (PLA probes) diluted 1:6 in Tris-buffered saline at 37°C. Amplified DNA strands were detected with oligonucleotides conjugated to a fluorophore, and nuclei stained with Hoechst. Formalin-fixed, paraffin-embedded mouse pancreas sections (6 μm thick) from hIAPP transgenic mouse injected with preformed hproIAPP fibrils were rehydrated and incubated with primary antibody A169, A133, or A165 (Table 1) overnight at room temperature. Sections were rinsed in Tris-buffered saline, and reactivity was detected with alkaline phosphatase–labeled secondary antibody (Dako) and Fast red (Sigma-Aldrich). Sections were counterstained with Mayer's hematoxylin. Formalin-fixed, paraffin-embedded human brain sections (10 μm, AD5, AD6, and non-AD2) (Table 2) were rehydrated, treated with 70% FA for 5 minutes, and then incubated with primary antibody 4G8 (Table 1) overnight at room temperature. Sections were rinsed in Tris-buffered saline, and reactivity was visualized with secondary antibody labeled with Alexa 488 nm (Invitrogen, Gothenburg, Sweden). Alkaline Congo Red staining was performed as previously described.31Puchtler H. Sweat F. Congo red as a stain for fluorescence microscopy of amyloid.J Histochem Cytochem. 1965; 13: 693-694Crossref PubMed Scopus (141) Google Scholar This amyloid-specific dye stains amyloid fibrils pink and discloses green birefringence when viewed in cross-polarized light; a red fluorescence signal is detected when the material is analyzed in a fluorescence microscope at 543-nm excitation. PLA and Congo-stained sections were analyzed in a Nikon Eclipse E800 confocal microscope (Nikon, Kawasaki, Japan) equipped with argon 488-nm and HeNe 543-nm lasers. Sections stained with Congo Red and used for quantification of amyloid were analyzed in cross-polarized light (Olympus, Stockholm, Sweden). Fibrils were analyzed at 75 kV, with a Hitachi 7100 transmission electron microscope (Hitachi, Tokyo, Japan), and micrographs were captured with Gatan multiscan camera with Gatan digital micrograph software version 3.6.4 (Gatan, Pleasanton, CA). Islet amyloid frequency in hIAPP+/+mIAPP−/− mice was analyzed with Fischer's exact test, and IAPP concentrations in AD and non-AD patients were analyzed with unpaired, two-tailed Student's t-test (GraphPad Prism 6 Software, San Diego, CA). P < 0.05 was regarded as significant. Amyloid properties of hproIAPP, hIAPP, Aβ, and CA fibrils were confirmed with Congo Red staining, and all preparations exhibited green birefringence when viewed in a polarization microscope (Figure 1, B–E); typical amyloid-like fibrils were identified after negative staining with 2% uranyl acetate in 50% ethanol and transmission electron microscopy analysis (Figure 1, F–I). After injection of preformed fibrils, animals were housed on standard chow and had free access to lard to stimulate islet amyloidogenesis for 10 months. When the animals were sacrificed, pancreas was recovered and analyzed for islet amyloid after Congo Red staining. Islet amyloid developed in 5 of 7 mice from groups injected with hproIAPP, hIAPP, or Aβ fibrils; in 2 of 6 mice injected with CA fibrils; and in 2 of 7 mice injected with sodium chloride (Table 3). One animal from the CA group died during the study. The number of islets with amyloid was 2.7% and 5% in mice injected with sodium chloride or CA, respectively (P = 0.2), and increased to 24.1% in mice injected with hIAPP fibrils, 15.4% in mice injected with hproIAPP fibrils, and 15.2% in mice injected with Aβ fibrils (P < 0.0005) (Table 3). The amyloid consisted of IAPP in all five groups, and was primarily found perivascularly in mice seeded by hproIAPP, hIAPP, or Aβ preformed fibrils (Figure 2, A–C). Amyloid deposits in control groups injected with CA or sodium chloride were smaller and mainly present in or in association with β cells (Figure 2, D and E). These in vivo results strongly support that not only seeding, but also cross-seeding, is possible of local IAPP amyloid via blood.Table 3Intravenous Injection of Preformed Fibrils Produced from hproIAPP, hIAPP, and Aβ Seed IAPP Amyloid in Islets of hIAPP+/+mIAPP−/− Transgenic MiceFibrilNo. of mice/no. of mice with amyloidTotal no. of islets in mice with amyloid/no. of islets with amyloid% Islets with amyloidIslet amyloid score∗Coherent areas with amyloid/multiple spots of amyloid/a single spot of amyloid in an islet. Fisher's exact test was used for comparison.hIAPP7/5518/12524†P < 0.05, hIAPP versus Aβ.‡P < 0.01, hIAPP versus hproIAPP.§P < 0.001, hIAPP, hproIAPP, and Aβ versus both sodium chloride and CA.7/30/88hproIAPP7/5382/5915.4§P < 0.001, hIAPP, hproIAPP, and Aβ versus both sodium chloride and CA.10/17/32Aβ7/5231/3515.2§P < 0.001, hIAPP, hproIAPP, and Aβ versus both sodium chloride and CA.4/8/23CA6/2280/1450/1/13NaCl7/2301/82.60/2/6∗ Coherent areas with amyloid/multiple spots of amyloid/a single spot of amyloid in an islet. Fisher's exact test was used for comparison.† P < 0.05, hIAPP versus Aβ.‡ P < 0.01, hIAPP versus hproIAPP.§ P < 0.001, hIAPP, hproIAPP, and Aβ versus both sodium chloride and CA. Open table in a new tab To investigate whether hproIAPP fibrils seeded proIAPP and/or IAPP, sections from mice injected with hproIAPP fibrils were subjected to immunohistochemistry with proIAPP-specific antisera A169 and A165, and hIAPP-specific antiserum A133 (Table 1 and Figure 2, F–H), in combination with Congo Red staining (Figure 2, I–K). Because only IAPP-immunoreactivity (IR) was found in perivascular amyloid deposits (Figure 2G), these results suggest that IAPP amyloid grows through deposition of locally secreted IAPP. Pancreata from mice injected with preformed hproIAPP (n = 3), hIAPP (n = 3), CA (n = 3), or Aβ (n = 3) fibrils and sacrificed 1 day later were all free of Congo-positive material. This ensures that the amount of fibrils given for seeding were below the detection level. Also, other organs, ie, spleen, kidney, liver, heart, or lung, recovered from mice 1 day and 10 months after fibril injection were free of amyloid, showing that no systemic amyloidosis was induced. Antibody-based methodology was used for colocalization studies, and to ensure the specificity" @default.
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