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The Molecular Misreading of APP and UBB Induces a Humoral Immune Response in Alzheimer’s Disease Patients with Diagnostic Ability

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Abstract

Alzheimer’s disease (AD) is the most common cause of dementia worldwide with 10–30% prevalence in aging population and a high socioeconomic impact. Because AD definitive diagnostic requires post-mortem verification, new approaches to study the disease are necessary. Here, we analyze the humoral immune response in AD to survey whether APP+1 or UBB+1 frameshift proteins, produced as a consequence of the “molecular misreading” alteration in AD occurring in the APP (amyloid precursor protein) and UBB (ubiquitin-B protein) proteins’ mRNA, elicit the production of autoantibodies specific of AD. To this end, APP+1 and UBB+1 peptides were expressed in bacteria as 6xHisHalo fusion proteins and after purification to homogeneity their seroreactivity was analyzed using 81 individual sera from AD patients and 43 individual sera from healthy individuals by luminescence beads immunoassay. We found that as a result of the molecular misreading, APP+1 and UBB+1 frameshift peptides produced a humoral immune response in AD patients, whose autoantibody levels are significantly higher in comparison with healthy controls. Their combination with a previously reported panel of four autoantigens specific of AD (ANTXR1, OR8J1, PYGB, and NUPR1) increased their diagnostic ability assessed by receiver operating characteristic (ROC) curves up to an area under the curve (AUC) of 73.5%. Collectively, our results demonstrate that APP+1 and UBB+1 frameshift proteins, non-previously described as AD-specific autoantigens, elicit the production of autoantibodies which might be useful as blood-based biomarkers to aid in the detection of the disease.

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Acknowledgments

We thank the excellent technical support of Maricruz Sánchez.

Funding

This work was supported by the Ramon y Cajal programme of the MINECO, and the financial support of the SAF2014-53209-R and the PI17CIII/00045 grants from the MINECO and the AES-ISCIII program to R.B., respectively. A.M-C. was supported by a predoctoral contract of the Fundación Tatiana Pérez de Guzmán el Bueno. P.SS-A. and A.M-C. are recipients of a FPU fellowship from the Ministerio de Educación, Cultura y Deporte. M.G-A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

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Authors and Affiliations

  1. UFIEC, Chronic Disease Programme, Instituto de Salud Carlos III, E-28220, Majadahonda, Madrid, Spain

    Ana Montero-Calle, Pablo San Segundo-Acosta, María Garranzo-Asensio & Rodrigo Barderas

  2. Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040, Madrid, Spain

    Pablo San Segundo-Acosta

  3. Alzheimer Disease Research Unit, CIEN Foundation, Queen Sofia Foundation Alzheimer Center, E-28031, Madrid, Spain

    Alberto Rábano

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  1. Ana Montero-Calle
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  2. Pablo San Segundo-Acosta
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  3. María Garranzo-Asensio
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  4. Alberto Rábano
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Contributions

Conception and design: R.B. Development of methodology: A.M-C., P.SS-A., M.G-A., and R. B. Perform Research: A.M-C., P.SS-A., M.G-A. Analysis and interpretation of data: A.M-C., and R.B. Writing, review, and/or revision of the manuscript: A. M-C., P.SS-A., M. G-A., and R.B. Technical, obtaining and processing of samples, or material support: A.M-C., P.SS-A., M. G-A., A.R., and R.B.

Corresponding author

Correspondence to Rodrigo Barderas.

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Written informed consent was obtained from all patients. The Institutional Ethical Review Board of the Spanish Research Center for Neurological Diseases Foundation (CIEN), the Complutense University of Madrid, and the ISCIII approved this study (CEI PI 49) on biomarker discovery of Alzheimer’s disease.

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Supplementary Fig. S1

Analysis of the expression and purification of the 6xHis-Halo fusion proteins by Coomassie blue staining (left) and WB (right) of soluble (SF) and insoluble (P) fractions of E. coli cultures expressing the indicated 6xHis-Halo tagged peptides. Proteins were found in both fractions at similar levels. Fusion proteins were purified from the soluble fraction using the Ni-NTA Agarose Resin. After the purification process, proteins were purified to homogeneity (E1 and E2 lines). NR: non-retained proteins; E1: elute 1; E2: elute 2. Nitrocellulose membranes were incubated with the anti-6xHis mAb. (PPTX 321 kb)

Supplementary Fig. S2

3D representation of the seroreactivity of both fusion proteins using 81 individual AD sera and 43 healthy control individual sera. a AD sera. b Healthy control sera. Both peptides were differently recognized by the individual sera, demonstrating the specific response against both AD-related frameshifts. *, AD patients younger than 80 years. The Braak stage of AD patients post-mortem assessed is also depicted. (PPTX 791 kb)

Supplementary Fig. S3

Analysis of the seroreactivity to APP+1 or UBB+1 frameshift peptides according to the age of the individuals. The humoral response exhibited by AD patients was similar independently on the age of the individuals for APP+1, whereas for UBB+1 signals were higher for AD patients of the 60-80 group than older patients. Mean and SEM are depicted as bar graphs. SEM: Standard error of the mean; R.L.U.: Relative luminescence units. (PPTX 333 kb)

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Montero-Calle, A., San Segundo-Acosta, P., Garranzo-Asensio, M. et al. The Molecular Misreading of APP and UBB Induces a Humoral Immune Response in Alzheimer’s Disease Patients with Diagnostic Ability. Mol Neurobiol 57, 1009–1020 (2020). https://doi.org/10.1007/s12035-019-01809-0

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