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WO2022003225A1 - MODÈLE DE GLYCOSYLATION DE SAPPα ET/OU SAPPβ COMME BIOMARQUEUR DE DIAGNOSTIC DE LA MALADIE D'ALZHEIMER, MÉTHODE ET TROUSSE REPOSANT SUR CELUI-CI - Google Patents

MODÈLE DE GLYCOSYLATION DE SAPPα ET/OU SAPPβ COMME BIOMARQUEUR DE DIAGNOSTIC DE LA MALADIE D'ALZHEIMER, MÉTHODE ET TROUSSE REPOSANT SUR CELUI-CI Download PDF

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Publication number
WO2022003225A1
WO2022003225A1 PCT/ES2021/070478 ES2021070478W WO2022003225A1 WO 2022003225 A1 WO2022003225 A1 WO 2022003225A1 ES 2021070478 W ES2021070478 W ES 2021070478W WO 2022003225 A1 WO2022003225 A1 WO 2022003225A1
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Prior art keywords
sappa
earrb
app
disease
glycosylation pattern
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Spanish (es)
Inventor
Javier SÁEZ VALERO
Inmaculada Belén LÓPEZ FONT
Inmaculada CUCHILLO IBÁÑEZ
Claudia Paola BOIX RODRÍGUEZ
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Consejo Superior de Investigaciones Cientificas CSIC
Universidad Miguel Hernandez de Elche UMH
Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED
Original Assignee
Consejo Superior de Investigaciones Cientificas CSIC
Universidad Miguel Hernandez de Elche UMH
Centro de Investigacion Biomedica en Red de Enfermedades Neurodegenerativas CIBERNED
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Publication of WO2022003225A1 publication Critical patent/WO2022003225A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the invention relates to in vitro methods for the diagnosis of Alzheimer's disease in which the pattern of protein glycosylation is determined.
  • the invention relates to an in vitro method in which the glycosylation pattern of sAPPa and / or eARRb, fragments generated in the proteolytic processing of the amyloid protein precursor (APP), is determined.
  • APP amyloid protein precursor
  • AD Alzheimer's disease
  • P-tau abnormally hyperphosphorylated cytoskeletal protein tau
  • AD is the only tauopathy that presents with fibrillar amyloid deposits, consisting mainly of the b-amyloid (Ab) peptide.
  • Ab is a 40-42 amino acid peptide product of the proteolytic processing of the transmembrane protein known as amyloid protein precursor (APP).
  • APP amyloid protein precursor
  • the processing of APP can be carried out by different routes that coexist under normal physiological conditions, the non-amyloidogenic route and the amyloidogenic route. Long N-terminal fragments (NTFs) are produced in both pathways.
  • the non-amyloidogenic pathway In the non-amyloidogenic pathway, with the sequential action of a-secretase (ADAM10) and y-secretase, Ab is not produced. With the action of ⁇ -secretase, the NTF sAPPa fragment and the C-terminal fragment (CTF) APP-CTF83 are produced. With the action of g-secretase, the APP-CTF83 fragment produces the AICD and P3 fragments.
  • the amyloidogenic pathway directed by the sequential action of the enzymes b-secretase (BACE1) and g-secretase, generates Ab. With the action of b-secretase, the NTF eARRb fragment and the CTF APP-CTF99 fragment are produced. With the action of g-secretase, the APP-CTF99 fragment produces the AICD and Ab fragments.
  • AD pathological "effectors" of AD are the oligomers of Ab and P-tau and that an excess of oligomeric forms of Ab (especially the 42 amino acid form: Ab42) is the primary determinant of the neurotoxicity in AD.
  • CSF cerebrospinal fluid
  • Glycosylation is a process of adding carbohydrates to proteins, which can occur as a cotranslational modification (occurs parallel to protein synthesis when the ribosome is associated with the endoplasmic reticulum) or posttranslational (occurs when the protein has already completed its synthesis ).
  • Carbohydrates can be linked to proteins by N-glycosylation, in which they bind to the nitrogen of the side chains of the amino acids asparagine or arginine, or by O-glycosylation, in which they bind to the oxygen of the hydroxyl group of the side chains of the amino acids serine, threonine or tyrosine.
  • Glycosylation is a specific process of the cell type and the moment of cell development, and it shows alterations associated with some pathologies. Glycosylation also determines the interaction of proteins, their functionality and further processing. The glycosylation pattern of proteins and / or peptides as biomarkers has been described for the diagnosis of AD.
  • US2002022242A1 describes the glycosylation pattern of the enzyme butyrylcholinesterase as a biomarker for the diagnosis of AD.
  • Said document describes the detection of the glycosylation pattern of butyrylcholinesterase by binding to lectins.
  • Lectins are proteins that recognize terminal sugars exposed in glycoproteins with very high specificity.
  • W02012056008A1 describes the O-glycosylation of an amino acid of Ab as a biomarker for the diagnosis of AD and describes, among others, the following techniques to detect the O-glycosylated amino acid in Ab: ELISA, mass spectrometry, emission tomography of positrons (PET), magnetic resonance imaging, radioimmunoassays, lectin-binding assays, immunohistochemistry, Western blot, and flow cytometry.
  • ELISA EPT
  • PET emission tomography of positrons
  • PET emission tomography of positrons
  • magnetic resonance imaging radioimmunoassays
  • lectin-binding assays immunohistochemistry
  • Western blot and flow cytometry.
  • the term "subject” refers to a human. More preferably, said subject has Alzheimer's disease or is suspected of having, or is at risk of having, said disease. Subjects who are affected by said disease can be identified by the symptoms accompanying the disease, which are known in the state of the art. However, a subject suspected of being affected by the aforementioned disease may also be an apparently healthy subject, for example, investigated by a clinical examination of routine, or it may be a subject at risk of developing the aforementioned disease.
  • sample refers to a sample of a body fluid, a sample of cells, a sample of a tissue, or a sample of wash / rinse fluid obtained from an external body surface or internal.
  • samples are samples of cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
  • glycosylation pattern of sAPPa and / or eARRb refers, generally, to the set of carbohydrates linked to sAPPa and / or eARRb.
  • any pattern recognition method known in the state of the art can be used, so as to detect differences between the subject's sAPPa and / or eARRb glycosylation pattern relative to a reference glycosylation pattern.
  • a numerical value or a range of numerical values depending on the glycosylation pattern can be obtained, allowing the comparison between the glycosylation pattern of sAPPa and / or eARRb of the subject with respect to the glycosylation pattern of sAPPa and / or eARRb of reference.
  • lectin binding assays can be used to determine the glycosylation pattern of sAPPa and / or eARRb.
  • a percentage or fraction of sAPPa and / or eARRb bound to a lectin can be obtained, said percentage or fraction of sAPPa and / or eARRb depends on the glycosylation pattern of sAPPa and / or eARRb and allows the comparison between the glycosylation pattern of sAPPa and / or eARRb in the subject versus the glycosylation pattern of sAPPa and / or eARRb in reference.
  • the term "compare” refers to contrasting the glycosylation pattern of sAPPa and / or eARRb in the sample to be analyzed with the glycosylation pattern in a suitable reference sample, as specified. later in the present description.
  • the comparison refers to that of the parameters or values corresponding to said glycosylation patterns, for example, a percentage or fraction of sAPPa and / or eARRb bound to lectins is compared with a percentage or reference fraction of sAPPa and / or eARRb bound to lectins; an intensity signal obtained from the glycosylation pattern of sAPPa and / or eARRb linked to lectins in a sample is compared with the same type of intensity signal from said glycosylation pattern of sAPPa and / or eARRb linked to lectins in a reference sample .
  • the Referred comparison can be carried out manually or computer-aided.
  • the value of the determined quantity can be compared with the values corresponding to the appropriate references that are stored in a database by means of a computer program. Consequently, the identification result referred to in this document may be automatically provided in a suitable output format.
  • the term "reference sAPPa and / or eARRb glycosylation pattern" is derived from samples of healthy subjects known to be free from Alzheimer's disease.
  • a suitable reference sAPPa and / or eARRb glycosylation pattern can be determined, by the methods of the present invention, from a reference sample to be analyzed together, that is, simultaneously, or subsequently, with the test sample.
  • a cut-off value can be used as a reference value associated with the reference sAPPa and / or eARRb glycosylation pattern.
  • the term "difference in comparison" may correspond to a decrease or an increase in a numerical value or range of values dependent on the glycosylation pattern of sAPPa and / or eARRb in the sample to be analyzed. , relative to a numerical value or range of values dependent on the glycosylation pattern of sAPPa and / or eARRb, in a suitable reference sample, as specified above.
  • said difference is statistically significant, that is, a statistically significant decrease, or a statistically significant increase.
  • Whether a difference is statistically significant can be determined, using various statistical evaluation tools, well known in the art, for example, determination of confidence intervals and determination of the p-value, for example, through binomial tests.
  • Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%.
  • the significance levels of the statistical tests are preferably 0.1, 0.05, 0.01, 0.005 or 0.0001.
  • the term "indicative" refers to the fact that a difference between the glycosylation pattern of sAPPa and / or eARRb in the sample to be analyzed, with respect to The glycosylation pattern of sAPPa and / or eARRb in a suitable reference sample makes it possible to diagnose whether a subject has Alzheimer's disease.
  • “Western blot”, also called “immunoblot” or “electroblot”, refers to an analytical technique used in cellular and molecular biology to identify specific proteins in a complex mixture of proteins, such as the one described above. Presented in cellular or tissue extracts. The technique uses the following three steps: size separation, transfer to a solid support, and finally visualization by protein binding to appropriate primary or secondary antibodies.
  • enzyme-linked immunosorbent assay refers to an immunoassay technique in which an immobilized antigen is detected by an antibody bound to an enzyme (peroxidase, alkaline phosphatase, etc.) capable of to generate a detectable product from a substrate, by means of a color change or some other type of change, caused by the enzymatic action on said substrate.
  • an enzyme peroxidase, alkaline phosphatase, etc.
  • a primary antibody that recognizes the antigen and which in turn is recognized by a secondary antibody linked to said enzyme.
  • Antigen can be indirectly detected in the sample by spectrophotometrically measured color changes.
  • alternative splicing refers to a process in which, from a primary transcript of mRNA or pre-mRNA, different isoforms of mRNA and proteins are obtained, which may have different functions. This process occurs mainly in eukaryotes.
  • pan-specific antibodies refers to antibodies with specificity against a specific domain present only on a target, for example, the exclusive domain that differentiates various forms or variants of a protein.
  • the technical problem to be solved consists of the development of an in vitro diagnostic method for the diagnosis of AD based on new biomarkers.
  • the present invention as defined in the claims, provides a solution to said technical problem.
  • the present invention provides an in vitro method of diagnosing Alzheimer's disease in a subject, comprising:
  • said biomarker is sAPPa.
  • the biological sample is selected from the group consisting of: cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
  • said glycosylation pattern is determined by a technique selected from the group consisting of: ELISA, mass spectrometry, positron emission tomography (PET), nuclear magnetic resonance (NMR), radioimmunoassays, assays binding to lectins, immunohistochemistry, Western blot and flow cytometry.
  • said glycosylation pattern is determined by lectin binding assays.
  • said lectins are Concanavalin A from Canavalia ensiformis (Con A) and / or PHA from Phaseolus vulgaris.
  • the amount or concentration of sAPPa and / or eARRb is detected by discriminating between bound or not bound to lectins.
  • the detection of the amount or concentration of sAPPa and / or eARRb is carried out by ELISA or Western blot.
  • sAPPa and / or eARRb are derived from any of the variants of the amyloid protein precursor (APP).
  • APP variants are selected from the group consisting of: APP695, APP751 and APP770.
  • a decrease in a subject, relative to a reference value derived from samples of healthy subjects, of the levels of sAPPa derived from APP695 and derived from the combination of the variants APP-KPI, APP751 and APP770, not bound to Con A or PHA lectins is indicative of a positive diagnosis of Alzheimer's disease in said subject;
  • a decrease in a subject, relative to a reference value derived from samples from healthy subjects, of the level of eARRb derived from APP695, not bound to Con A or PHA lectins is indicative of a positive diagnosis of Alzheimer's disease; and in which an increase in a subject, with respect to a reference value derived from samples of healthy subjects, of the level of eARRb derived from the combination of the APP-KPI variants, APP751 and APP770, not bound to the lectins Con A or PHA is indicative of a positive diagnosis of Alzheimer's disease in said subject.
  • O-glycosylation amino acids 633, 651, 652, 659, 663, 667, 681.
  • Amino acids 1-17 of APP correspond to the signal peptide.
  • the eARRb fragment corresponds to amino acids 18-671 of APP.
  • the sAPPa fragment corresponds to amino acids 18-687. This information is also accessible in the protein database UniProtKB (Accession No. P05067).
  • the present invention also provides the sAPPa and / or eARRb biomarkers for use in an in vitro method of diagnosing Alzheimer's disease in which the glycosylation pattern of sAPPa and / or eARRb is determined in a biological sample.
  • the present invention also provides the use of the sAPPa and / or eARRb biomarkers in the in vitro diagnosis of Alzheimer's disease, in which the glycosylation pattern of sAPPa and / or eARRb is determined in a biological sample.
  • said biomarker is sAPPa.
  • the biological sample is selected from the group consisting of: cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
  • the biological sample has been obtained from a subject.
  • the present invention also provides an Alzheimer's disease diagnostic kit, comprising reagents for determining the glycosylation pattern of sAPPa and / or eARRb in a biological sample, wherein said reagents comprise Con A and / or PHA lectin and specific antibodies against sAPPa and / or eARRb.
  • said specific antibodies are the polyclonal antibody IBL-a, specific against sAPPa and the monoclonal antibody IBL-b, specific against eARRb.
  • the kit of the invention further comprises at least one buffer solution.
  • buffers are: phosphate buffer, phosphate saline buffer, acetate buffer, borate-chloride buffer, carbonate buffer, glycine buffer, and Tris buffer.
  • FIG. 1 Schematic representation and biochemical characterization of NTF and CTF fragments of APP.
  • A Schematic representation of the proteolytic fragments sAPPa, eARRb, CTFa and OTRb generated by a-secretase (non-amyloidogenic pathway) and b-secretase (amyloidogenic pathway) (not drawn to scale). The location of the KPI domain present in the APP751 and APP770 variants, but not in APP695, is indicated. Epitopes for the antibodies used in this study are also shown.
  • the immunoblots of CHO-PS70 extracts were detected simultaneously with two different antibodies: the C-terminal antibody generated in rabbit and recognizing a domain common to CTFa and (IPTb; and the antibody generated in rat 2D8 and recognizing the N- domain). Terminal Ab which therefore only detects (IPTb (the band that accumulates after treatment with DAPT is indicated by arrow). Positions corresponding to a molecular mass of 6.5, 10 and 14 kDa are indicated.
  • FIG. 3 The sAPPa and eARRb variants remain unchanged in AD brain tissue.
  • FIG. 4 CTFa and OTRb remain unchanged in AD brain tissue.
  • B Densitometric quantification of the ⁇ TRb (B) and CTFa (C) species, using GAPDH as a loading control to ensure that equivalent amounts of protein were loaded in each lane. The calculations were carried out in duplicate.
  • D Graph of the relationship obtained for each sample dividing the immunoreactivity of ⁇ TRb by that of CTFa. There were no statistically significant differences evident.
  • Figure 5. Comparison of APP695 and APP-KPI glycosylation in brain tissue from control and AD subjects.
  • Figure 6 Comparison of glycosylation of sAPPa and eARRb in brain tissue of control subjects and subjects with AD.
  • the p-values are indicated in the figure.
  • Figure 7 Comparison of cerebral glycosylation of sAPP between control subjects and subjects with AD. Graphical representation of the percentage of the sAPPa fragment derived from APP695 (A) and APP-KPI (B) in the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA and Con A. Graphical representation of the percentage of the eARRb fragment derived from APP695 (C) and APP-KPI (D) in the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA and Con A. The p-values are indicated in the figure (ns, not significant).
  • NINCDS-ADRDA clinical criteria for probable AD
  • the patients with AD corresponded to sporadic cases that were selected based on their clinical history and neuropathological diagnosis based on the CERAD diagnostic criteria, from the English Consortium to Establish a Registry for Alzheimer's Disease.
  • the cases were categorized as stages V-VI during the neuropathological analysis following the Braak and Braak scale (Braak and Braak, 1991).
  • the mean postmortem interval of the tissue was between 1, 5 and 6 hours, without significant differences in both groups.
  • the control subjects were negative in their histopathological analysis, and had no history of neurological or psychiatric symptoms or memory impairment.
  • the brain tissue samples collected in the tissue banks were kept at -80 ° C at all times. Subsequently, they were slowly thawed on ice and homogenized in an extraction buffer (10% w / v) Tris-HCl 50 mM; pH 7.4; 500 mM NaCl; 5 mM EDTA; 1% (w / v) Nonidet P-40; 0.5% (w / v) Triton X-100, supplemented with a protease inhibitor cocktail (Sigma P834). The homogenate was sonicated using a Misonix Microson ultrasonic cell disruptor sonicator in 3 series of 10 pulses.
  • BSA bovine serum albumin
  • DMEM middle Eagle Modified by Dulbecco
  • GlutaMAX TM Gibco® Life Technologies, Paisley, UK
  • FBS fetal calf serum
  • Gibco fetal calf serum
  • the cells were treated with the y-secretase inhibitor DAPT (5 mM, LY-374973 t-butyl ester of (N- [N- (3,5-difluorophenacetyl) -l-alanyl] -S-phenylglycine; Calbiochem®, Merck KGaA).
  • Control cells were treated with only the same volume of dimethylsulfoxide (DMSO).
  • DMSO dimethylsulfoxide
  • the cells were washed twice with cold phosphate buffered saline (PBS) and They were resuspended in 100 ⁇ l of ice-cold extraction buffer (described above) supplemented with a cocktail of protease inhibitors (described above).
  • Cell lysates were sonicated and centrifuged for 1 hour at 70,000 xg and 4 ° C, and the extracts were frozen at -80 ° C for future analysis.
  • RNA was isolated from brain tissue samples using TRIzol reagent and the PureLink TM Micro-to-Midi Total RNA Purification System (Invitrogen), according to the kit manufacturer's instructions and performed an analysis of messenger RNA molecules (mRNA) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR).
  • mRNA messenger RNA molecules
  • qRT-PCR quantitative reverse transcriptase polymerase chain reaction
  • cDNA First strand complementary DNA was obtained by reverse transcription of total RNA (1.5 pg), using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, United Kingdom), according to with the kit manufacturer's instructions.
  • PCR polymerase chain reaction
  • Brain tissue samples (30 pg per well) were boiled at 95 ° C for 5 minutes, separated by electrophoresis on 7.5% sodium dodecyl sulfate (SDS-PAGE) polyacrylamide gels in Tris-Tricine and subsequently they were transferred to 0.45 pm nitrocellulose membranes (Schleicher & Schuell Bioscience, GmbH, Dassel, Germany).
  • the immunoreactive signal of the APP bands was quantified in the Western blots.
  • the APP species in the samples were detected using: a rabbit C-terminal anti-APP polyclonal antiserum (1: 1000; Sigma Aldrich, St.
  • Vinculin (1: 2000, mouse anti-vinculin monoclonal antibody, sc-73614 Santa Cruz; 1: 1000 rabbit anti-vinculin antiserum, Sigma V4139) and GAPDH (1: 10000 mouse anti-GAPDH monoclonal antibody; Proteintech 60004-1) were used as loading controls.
  • Band intensities were analyzed using Image Studio Lite (LI-COR) software The antibodies used were cross-reactive between the fragments sAPPa and eARRb less than 1.5%, and none of the antibodies cross-react with full-length APP.
  • the solubilized brain glycoproteins were treated with a ProZyme enzymatic de-glycosylation kit (GK80110), according to the kit manufacturer's instructions, and then subjected to SDS-PAGE and Western blot analysis.
  • Frontal cortex extracts were denatured and de-glycosylated by incubation with N-Glycanase, O-Glycanase, and Sialidase A. This treatment removes all N-linked glycans and O-linked simple glycans (including polysialylated) from the glycoproteins. Binding to lectins
  • the cerebral cortex samples were incubated at 4 ° C overnight with specific lectins for terminal sugars: the lectin from Canavalia ensiformis Concanavalina A (Con A) (Sigma; Catalog number C9017), with high specificity for terminal mannose residues. , or the lectin from Phaseolus vulgaris PHA (Vector; Catalog number AL113), with high specificity for terminal galactose residues, immobilized on sepharose (Con A) or agarose (PHA).
  • Con A Canavalia ensiformis Concanavalina A
  • PHA Phaseolus vulgaris PHA
  • the glycoprotein fraction not bound to lectins was separated by centrifugation and analyzed in Western blot using antibodies against sAPPa and sAPPp.
  • the proportion of APP not bound to lectin was calculated as the ratio between the immunoreactivity of APP not bound to lectin and the total immunoreactivity, obtained from an aliquot kept under the same conditions, but not incubated with a lectin. All analyzes were done in duplicate.
  • Example 1 Increased expression of APP in the brain of subjects with AD
  • APP messenger RNA mRNA
  • EA APP messenger RNA
  • primers that corresponded to sequences in APP exons 10-11 and that are common to the main brain variants. Therefore, the total mRNA levels of the APP695, APP751 and APP770 variants were determined. Consequently, the levels of APP transcripts as a whole were significantly higher in brain tissue from AD subjects than in brain tissue from control subjects (C) (p ⁇ 0.001; Figure 1).
  • Example 2 Characterization of sAPPa, sAPPp, CTFa and CTFp in the brain tissue of subjects with AD
  • sAPPa or sAPPp were characterized in Western blot of the brain tissue of subjects with AD, a method that allowed discriminating different species of APP, especially those with different molecular masses.
  • sAPPa and sAPPp are distinguished relative to each other by only 16 amino acids (representing 1-2 kDa in molecular mass), and sAPPa or eARRb has been predicted to be only -5-10 kDa smaller than full-length APP, and therefore, no distinguished by electrophoretic separation.
  • small differences in electrophoretic migration can also be attributed to differences in glycosylation, or even reflect immature forms of the protein.
  • sAPPa and eARRb there are 3 alternative splicing variants in the brain, the 695 amino acid, APP695, mostly expressed in neurons, and the APP751 and APP770 variants, expressed in glial cells, which present the serine protease type inhibitor domain.
  • Kunitz KPI from English Kunitz-type serine protease ⁇ h ⁇ ⁇ o ⁇ .
  • NTFs of APP sAPPa and eARRb are distinguished electrophoretically and by Western blotting with pan-specific antibodies against the exclusive C-terminal end of sAPPa and eARRb. Brain tissue, the different APP species and their long N-terminal fragments were detected in several bands that migrated in the range 100-130 kDa.
  • FIG. 2A shows a schematic representation of the full length of APP and the N- and C-terminal fragments determined in this example, as well as the epitopes recognized by the different antibodies used.
  • Brain APP-NTF species were characterized in Western blot, using pan-specific antibodies against the specific C-terminal domains of sAPPa or eARRb ( Figure 2B). When an anti-KPI antibody was used, only the higher molecular mass species showed sizes compatible with the immunoreactive KPI bands.
  • CTFa and (IPTb do not differ between APP variants and were characterized using extracts from CHO-PS70 cells that stably over-express wild-type human APP and the catalytic g-secretase subunit, presenilin-1.
  • Extracts from these cells treated with the g-secretase inhibitor, DAPT were assayed with the C-terminal antibody, providing evidence of accumulation of CTFa (also called C83 for its amino acid length) and OTRb (also called C99 for their amino acid length) in cell extracts, and of coincident bands in parallel loaded brain homogenates (Figure 2D).
  • CTFa also called C83 for its amino acid length
  • OTRb also called C99 for their amino acid length
  • Pan-specific antibodies were used to determine what percentage of sAPPa and eARRb was not recognized by each of the lectins (Table 1).
  • the eARRb fragments derived from both APP695 and APP-KPI showed differences in their interaction with both lectins, both in the control group and in that of patients with AD ( Figures 5A-5D). This may be due to the different cellular origin for the isoforms derived from the APP695 species (neuronal) and the APP-KPIs (mostly glial), since each cell type has a particular glycosylation machinery. However, the different sAPPa fragments exhibited a similar binding pattern to Con A or PHA in both groups.

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Abstract

La présente invention concerne une méthode in vitro de diagnostic de la maladie d'Alzheimer dans laquelle on détermine le modèle de glycosylation de sAPPα et/ou sAPPβ dans un échantillon biologique, les biomarqueurs sAPPα et/ou sAPPβ à utiliser dans ladite méthode in vitro, ainsi qu'une trousse de diagnostic de la maladie d'Alzheimer, qui comprend des réactifs pour déterminer le modèle de glycosylation de sAPPα et/ou sAPPβ dans un échantillon biologique.
PCT/ES2021/070478 2020-07-01 2021-06-30 MODÈLE DE GLYCOSYLATION DE SAPPα ET/OU SAPPβ COMME BIOMARQUEUR DE DIAGNOSTIC DE LA MALADIE D'ALZHEIMER, MÉTHODE ET TROUSSE REPOSANT SUR CELUI-CI Ceased WO2022003225A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020022242A1 (en) * 2000-04-07 2002-02-21 Small David Henry Diagnostic test for alzheimer's disease
US20020150878A1 (en) * 2001-01-23 2002-10-17 Small David Henry Method for the diagnosis of Alzheimer's Disease and other prion related disorders
WO2012056008A1 (fr) * 2010-10-28 2012-05-03 Jonas Nilsson Diagnostic et traitement de la maladie d'alzheimer

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020022242A1 (en) * 2000-04-07 2002-02-21 Small David Henry Diagnostic test for alzheimer's disease
US20020150878A1 (en) * 2001-01-23 2002-10-17 Small David Henry Method for the diagnosis of Alzheimer's Disease and other prion related disorders
WO2012056008A1 (fr) * 2010-10-28 2012-05-03 Jonas Nilsson Diagnostic et traitement de la maladie d'alzheimer

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BAUM, N.: "Editorial comment", JOURNAL OF UROLOGY, LIPPINCOTT WILLIAMS & WILKINS, BALTIMORE, MD, US, vol. 176, no. 3, 1 September 2006 (2006-09-01), BALTIMORE, MD, US , pages 1126, XP005589750, ISSN: 0022-5347, DOI: 10.1016/j.juro.2006.04.156 *
HALIM ADNAN, BRINKMALM GUNNAR, RÜETSCHI ULLA, WESTMAN-BRINKMALM ANN, PORTELIUS ERIK, ZETTERBERG HENRIK, BLENNOW KAJ, LARSON GÖRAN,: "Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, vol. 108, no. 29, 19 July 2011 (2011-07-19), pages 11848 - 11853, XP055897504, ISSN: 0027-8424, DOI: 10.1073/pnas.1102664108 *

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