WO2022003225A1 - SAPPα AND/OR SAPPβ GLYCOSYLATION PATTERN AS A DIAGNOSTIC BIOMARKER OF ALZHEIMER'S DISEASE, AND METHOD AND KIT BASED ON SAME - Google Patents

SAPPα AND/OR SAPPβ GLYCOSYLATION PATTERN AS A DIAGNOSTIC BIOMARKER OF ALZHEIMER'S DISEASE, AND METHOD AND KIT BASED ON SAME Download PDF

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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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sappa
earrb
app
disease
glycosylation pattern
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PCT/ES2021/070478
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Spanish (es)
French (fr)
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Javier SÁEZ VALERO
Inmaculada Belén LÓPEZ FONT
Inmaculada CUCHILLO IBÁÑEZ
Claudia Paola BOIX RODRÍGUEZ
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Universidad Miguel Hernández De Elche
Ciberned (Centro De Investigación Biomédica En Red Enfermedades Neurodegenerativas)
Consejo Superior De Investigaciones Científicas (Csic)
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Publication of WO2022003225A1 publication Critical patent/WO2022003225A1/en

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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

The invention relates to: an in-vitro method for diagnosing Alzheimer's disease, wherein the glycosylation pattern of sAPPα and/or sAPPβ in a biological sample is determined; the sAPPα and/or sAPPβ biomarkers for use in the in-vitro method; and an Alzheimer's disease diagnosis kit, which comprises reagents for determining the sAPPα and/or sAPPβ glycosylation pattern in a biological sample.

Description

DESCRIPCIÓN DESCRIPTION
PATRÓN DE GLICOSILACIÓN DE sAPPa Y/O sAPPp COMO BIOMARCADOR DIAGNÓSTICO DE LA ENFERMEDAD DE ALZHEIMER, MÉTODO Y KIT BASADOSPATTERN OF GLYCOSILATION OF sAPPa AND / OR sAPPp AS A DIAGNOSTIC BIOMARKER OF ALZHEIMER'S DISEASE, BASED METHOD AND KIT
EN EL MISMOIN THE SAME
SECTOR DE LA TÉCNICA TECHNICAL SECTOR
La invención se relaciona con métodos in vitro de diagnóstico de la enfermedad de Alzheimer en los que se determina el patrón de glicosilación de proteínas. En particular, la invención se relaciona con un método in vitro en el que se determina el patrón de glicosilación de sAPPa y/o eARRb, fragmentos generados en el procesamiento proteolítico del precursor de la proteína amiloide (APP). The invention relates to in vitro methods for the diagnosis of Alzheimer's disease in which the pattern of protein glycosylation is determined. In particular, 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.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
La enfermedad de Alzheimer (EA) es la forma más común de demencia senil y se caracteriza por la presencia de depósitos proteináceos cerebrales, a nivel extracelular, como placas amiloides y, a nivel intracelular, como ovillos neurofibrilares. La EA es una tauopatía en la que ovillos neurofibrilares de la proteína citoesquelética tau anormalmente hiperfosforilada (P-tau) llevan al colapso y muerte neuronal. La EA es la única tauopatía que cursa con depósitos fibrilares amiloides, constituidos principalmente por el péptido b-amiloide (Ab). Alzheimer's disease (AD) is the most common form of senile dementia and is characterized by the presence of proteinaceous deposits in the brain, at the extracellular level, such as amyloid plaques and, at the intracellular level, as neurofibrillary tangles. AD is a tauopathy in which neurofibrillary tangles of the abnormally hyperphosphorylated cytoskeletal protein tau (P-tau) lead to neuronal collapse and death. AD is the only tauopathy that presents with fibrillar amyloid deposits, consisting mainly of the b-amyloid (Ab) peptide.
El Ab es un péptido de 40-42 aminoácidos producto del procesamiento proteolítico de la proteína transmembrana conocida como precursor de la proteína amiloide (APP). El procesamiento de APP puede llevarse a cabo por distintas vías que coexisten en condiciones fisiológicas normales, la vía no amiloidogénica y la vía amiloidogénica. En ambas vías se producen largos fragmentos N-terminales (NTF, del inglés N-terminal fragment). Ab is a 40-42 amino acid peptide product of the proteolytic processing of the transmembrane protein known as amyloid protein precursor (APP). 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.
En la vía no amiloidogénica, con la acción secuencial de a-secretasa (ADAM10) y y- secretasa, no se produce Ab. Con la acción de la a-secretasa, se producen el fragmento NTF sAPPa y el fragmento C-terminal (CTF, del inglés C-terminal fragment) APP- CTF83. Con la acción de la g-secretasa, el fragmento APP-CTF83 produce los fragmentos AICD y P3. La vía amiloidogénica, dirigida por la acción secuencial de las enzimas b-secretasa (BACE1) y g-secretasa, genera el Ab. Con la acción de la b-secretasa, se produce el fragmento NTF eARRb y el fragmento CTF APP-CTF99. Con la acción de la g-secretasa, el fragmento APP-CTF99 produce los fragmentos AICD y Ab. In the non-amyloidogenic pathway, with the sequential action of a-secretase (ADAM10) and y-secretase, Ab is not produced. With the action of α-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.
Hoy en día se acepta que los verdaderos “efectores” patológicos de la EA son los oligómeros de Ab y el P-tau y que un exceso de formas oligoméricas de Ab (especialmente la forma de 42 aminoácidos: Ab42) es el determinante primigenio de la neurotoxicidad en la EA. La determinación de Ab42 en el líquido cefalorraquídeo (LCR) se ha propuesto como marcador diagnóstico (junto a tau y P-tau, buenos marcadores pero que carecen de especificidad frente a otras tauopatías y desórdenes neurológicos). Today it is accepted that the true 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. The determination of Ab42 in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker (together with tau and P-tau, good markers but lacking specificity against other tauopathies and neurological disorders).
Respecto al Ab como biomarcador, se da la paradoja de que lo que existe es una disminución en sus valores en el LCR de EA. Ello sería el resultado del aumento de la producción de Ab en el cerebro, que a su vez fibrila, forma placas y queda secuestrado en dichas placas amiloides, por lo que llega al LCR en cantidades menores de las determinadas en sujetos sin placas, sin EA. Esta circunstancia hace difícil que el Ab en LCR pueda ser considerado un marcador temprano o de progresión. Regarding Ab as a biomarker, there is the paradox that what exists is a decrease in its values in the CSF of AD. This would be the result of the increased production of Ab in the brain, which in turn fibrillates, forms plaques and is sequestered in said amyloid plaques, thus reaching the CSF in smaller amounts than those determined in subjects without plaques, without AD. . This circumstance makes it difficult for Ab in CSF to be considered an early or progression marker.
Se han propuesto los niveles de los fragmentos NTF de APP, eARRb y el sAPPa, antes mencionados, como marcadores alternativos al Ab. Pese a las altas expectativas, los resultados no han sido alentadores, por la poca consistencia en los resultados de diversos estudios. Algunos de dichos estudios describieron aumento de eARRb, pero otros describieron que no había ningún cambio o incluso disminuciones de eARRb. Algo equivalente ha ocurrido con los niveles de sAPPa; algunos estudios describieron disminución de sAPPa, mientras que otros estudios describieron o bien que no había cambios o aumentos de sAPPa. Los estudios que examinaron ambos, sAPPa y eARRb, describieron que ambos NTF de APP mostraban la misma tendencia, fuese aumento o disminución en sus niveles en LCR, cuando lo esperable es que muestren tendencias opuestas por desequilibrio de las vías de procesamiento (Perneczky et al., 2014). The levels of the NTF fragments of APP, eARRb and the sAPPa, mentioned above, have been proposed as alternative markers to Ab. Despite high expectations, the results have not been encouraging, due to the lack of consistency in the results of various studies. Some of these studies described an increase in eARRb, but others described that there was no change or even a decrease in eARRb. Something equivalent has happened with the levels of sAPPa; some studies described decrease in sAPPa, while other studies described either no change or increase in sAPPa. The studies that examined both sAPPa and eARRb, described that both APP NTFs showed the same trend, be it an increase or decrease in their CSF levels, when it is expected that they show opposite trends due to an imbalance of the processing pathways (Perneczky et al. ., 2014).
Además, se ha determinado que la proteína completa de APP (sin procesar proteolíticamente) está también presente en el LCR coexistiendo con sAPPa y eARRb y que todas las especies forman heterómeros, por lo que los análisis de ELISAs para determinar niveles de sAPPa y eARRb, aun contando con anticuerpos específicos, no cuantifican las especies separadamente de forma fiable (Cuchillo-lbañez et al., 2015). In addition, it has been determined that the complete APP protein (proteolytically unprocessed) is also present in the CSF coexisting with sAPPa and eARRb and that all species form heteromers, therefore the ELISA analyzes to Determining levels of sAPPa and eARRb, even with specific antibodies, do not reliably quantify the species separately (Cuchillo-lbañez et al., 2015).
Posteriormente, se realizó un estudio por Western blot, evitando que los heterómeros falsearan la interpretación de los resultados, en el que no se evidenciaron niveles alterados de sAPPa o eARRb (Lopez-Font et al., 2017). Subsequently, a Western blot study was carried out, preventing the heteromers from falsifying the interpretation of the results, in which no altered levels of sAPPa or eARRb were evidenced (Lopez-Font et al., 2017).
La glicosilación es un proceso de adición de glúcidos a proteínas, que puede darse como una modificación cotraduccional (ocurre paralela a la síntesis de la proteína cuando el ribosoma se encuentra asociado al retículo endoplásmico) o postraduccional (ocurre cuando la proteína ya ha terminado su síntesis). Los glúcidos pueden unirse a las proteínas mediante N-glicosilación, en la que se unen al nitrógeno de las cadenas laterales de los aminoácidos asparagina o arginina o mediante O-glicosilación, en la que se unen al oxígeno del grupo hidroxilo de las cadenas laterales de los aminoácidos serina, treonina o tirosina. 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.
La glicosilación es un proceso específico del tipo celular y del momento del desarrollo celular, y que muestra alteraciones asociadas a algunas patologías. La glicosilación también determina la interacción de las proteínas, su funcionalidad y posterior procesamiento. El patrón de glicosilación de proteínas y/o péptidos como biomarcadores ha sido descrito para el diagnóstico de la EA. Así, US2002022242A1 describe el patrón de glicosilación de la enzima butirilcolinesterasa como un biomarcador para el diagnóstico de la EA. Dicho documento describe la detección del patrón de glicosilación de la butirilcolinesterasa mediante la unión a lectinas. Las lectinas son proteínas que reconocen con muy alta especificidad azúcares terminales expuestos en glicoproteínas. 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. Thus, 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 describe la O-glicosilación de un aminoácido del Ab como un biomarcador para el diagnóstico de la EA y describe, entre otras, las siguientes técnicas para detectar el aminoácido O-glicosilado en el Ab: ELISA, espectrometría de masas, tomografía por emisión de positrones (PET), resonancia magnética, radioinmunoensayos, ensayos de unión a lectinas, inmunohistoquímica, Western blot y citometría de flujo. Sin embargo, no se describe en este documento nada acerca de la utilidad de los patrones de glicosilación de sAPPa o de eARRb como biomarcadores para el diagnóstico de la EA. Por otro lado, se han caracterizado la O-glicosilación de residuos específicos de Ab en LCR y las diferencias en los niveles de glicosilación de dichos residuos entre pacientes con EA y sujetos control (Halim et al., 2011). Este documento, centrado en la glicosilación de Ab, no describe, sin embargo, nada acerca de los patrones de glicosilación de sAPPa o de eARRb, con residuos altamente N-glicosilados, además de la O-glicosilación; ni de su utilidad como biomarcadores para el diagnóstico de la EA. 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. However, nothing is described in this document about the utility of sAPPa or eARRb glycosylation patterns as biomarkers for the diagnosis of AD. On the other hand, the O-glycosylation of specific Ab residues in CSF and the differences in the levels of glycosylation of these residues between AD patients and control subjects have been characterized (Halim et al., 2011). This document, focused on the glycosylation of Ab, does not, however, describe anything about the glycosylation patterns of sAPPa or eARRb, with highly N-glycosylated residues, in addition to O-glycosylation; nor of their usefulness as biomarkers for the diagnosis of AD.
A pesar de los avances recientes en el desarrollo de biomarcadores para el diagnóstico de la EA, existe actualmente una necesidad de desarrollar nuevos biomarcadores para el diagnóstico de esta enfermedad. Despite recent advances in the development of biomarkers for the diagnosis of AD, there is currently a need to develop new biomarkers for the diagnosis of this disease.
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
A lo largo de la descripción y las reivindicaciones, el término "comprende", "que comprende" y sus variantes no son de naturaleza limitativa y, por lo tanto, no pretenden excluir otras características técnicas. El término "comprende", "que comprende" y sus variantes, a lo largo de la descripción y las reivindicaciones, incluye también, específicamente, el término "consiste en", "que consiste en" y sus variantes. Throughout the description and claims, the term "comprises", "comprising" and their variants are not limiting in nature and, therefore, are not intended to exclude other technical characteristics. The term "comprises", "comprising" and their variants, throughout the description and claims, also specifically includes the term "consists of", "consisting of" and their variants.
Como se usa en esta descripción y en las reivindicaciones, las formas singulares “el”, “la” incluyen referencias a las formas plurales a menos que el contenido indique claramente lo contrario. Así, por ejemplo, la referencia a "una célula" incluye una combinación de dos o más células, y similares. As used in this description and in the claims, the singular forms "the", "the" include references to the plural forms unless the content clearly indicates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.
A menos que se defina lo contrario, todos los términos técnicos y científicos utilizados a lo largo de la descripción y reivindicaciones, tienen el mismo significado que el comúnmente entendido por un experto en el campo de la invención. Unless otherwise defined, all technical and scientific terms used throughout the description and claims have the same meaning as commonly understood by one of ordinary skill in the field of the invention.
A efectos de la presente invención, el término “sujeto” se refiere a un humano. Más preferentemente, dicho sujeto tiene la enfermedad de Alzheimer o se sospecha que tiene, o está en riesgo de tener, dicha enfermedad. Los sujetos que están afectados por dicha enfermedad pueden ser identificados por los síntomas que acompañan a la enfermedad, que son conocidos en el estado de la técnica. Sin embargo, un sujeto que se sospecha que está afectado por la enfermedad mencionada puede ser también un sujeto aparentemente sano, por ejemplo, investigado mediante un examen clínico de rutina, o puede ser un sujeto que corre el riesgo de desarrollar la enfermedad mencionada. For the purposes of the present invention, 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.
A efectos de la presente invención, el término “muestra” se refiere a una muestra de un fluido corporal, a una muestra de células, a una muestra de un tejido o a una muestra de fluido de lavado/enjuague obtenida de una superficie corporal externa o interna. Preferentemente, las muestras son muestras de líquido cefalorraquídeo, orina, sangre, sangre completa, plasma, suero, líquido linfático, saliva, células y tejidos. For the purposes of the present invention, the term "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. Preferably, the samples are samples of cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
A efectos de la presente invención, “patrón de glicosilación de sAPPa y/o eARRb” se refiere, de forma general, al conjunto de glúcidos unidos a sAPPa y/o eARRb. En la presente invención, se puede utilizar cualquier método de reconocimiento de patrones conocido en el estado de la técnica, de forma que se detecten diferencias entre el patrón de glicosilación de sAPPa y/o eARRb del sujeto respecto a un patrón de glicosilación de referencia. Ventajosamente, se puede obtener un valor numérico o un rango de valores numéricos que dependan del patrón de glicosilación, permitiendo la comparación entre el patrón de glicosilación de sAPPa y/o eARRb del sujeto respecto al patrón de glicosilación de de sAPPa y/o eARRb de referencia. En la presente invención, se pueden utilizar ensayos de unión a lectinas para determinar el patrón de glicosilación de sAPPa y/o eARRb. En dichos ensayos de unión a lectinas, se puede obtener un porcentaje o fracción de sAPPa y/o eARRb ligada a una lectina, dicho porcentaje o fracción de sAPPa y/o eARRb depende del patrón de glicosilación de sAPPa y/o eARRb y permite la comparación entre el patrón de glicosilación de sAPPa y/o eARRb en el sujeto respecto al patrón de glicosilación de sAPPa y/o eARRb de referencia. For the purposes of the present invention, "glycosylation pattern of sAPPa and / or eARRb" refers, generally, to the set of carbohydrates linked to sAPPa and / or eARRb. In the present invention, 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. Advantageously, 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. In the present invention, lectin binding assays can be used to determine the glycosylation pattern of sAPPa and / or eARRb. In said lectin binding assays, 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.
A efectos de la presente invención, el término “comparar” se refiere a contrastar el patrón de glicosilación de sAPPa y/o eARRb en la muestra que se va a analizar con el patrón de glicosilación en una muestra de referencia adecuada, tal como se especifica más adelante en la presente descripción. La comparación se refiere a la de los parámetros o valores correspondientes a dichos patrones de glicosilación, por ejemplo, un porcentaje o fracción de sAPPa y/o eARRb ligada a lectinas se compara con un porcentaje o fracción de referencia de sAPPa y/o eARRb ligada a lectinas; una señal de intensidad obtenida del patrón de glicosilación de sAPPa y/o eARRb ligada a lectinas en una muestra se compara con el mismo tipo de señal de intensidad de dicho patrón de glicosilación de sAPPa y/o eARRb ligada a lectinas en una muestra de referencia. La comparación referida puede ser llevada a cabo manualmente o asistida por ordenador. En el caso de una comparación asistida por ordenador, el valor de la cantidad determinada puede compararse con los valores correspondientes a las referencias adecuadas que se almacenan en una base de datos mediante un programa informático. En consecuencia, el resultado de la identificación a que se hace referencia en el presente documento podrá facilitarse automáticamente en un formato de salida adecuado. For the purposes of the present invention, 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. In the case of a computer-assisted comparison, 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.
En la presente patente, el término “patrón de glicosilación de sAPPa y/o eARRb de referencia”, se deriva de muestras de sujetos sanos, para los cuales se sabe que no padecen la enfermedad de Alzheimer. Un patrón de glicosilación de sAPPa y/o eARRb de referencia adecuado puede determinarse, mediante los métodos de la presente invención, a partir de una muestra de referencia para analizarse juntos, es decir, simultáneamente, o posteriormente, con la muestra de prueba. Preferiblemente, se puede usar un valor de corte como un valor de referencia asociado al patrón de glicosilación de sAPPa y/o eARRb de referencia. In the present patent, 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. Preferably, a cut-off value can be used as a reference value associated with the reference sAPPa and / or eARRb glycosylation pattern.
A efectos de la presente invención, el término “diferencia en la comparación” puede corresponder a una disminución o a un aumento de un valor numérico o rango de valores dependiente del patrón de glicosilación de sAPPa y/o eARRb en la muestra que se va a analizar, respecto a un valor numérico o rango de valores dependiente del patrón de glicosilación de sAPPa y/o eARRb, en una muestra de referencia adecuada, tal como se especificó anteriormente. Preferiblemente, aunque no necesariamente, dicha diferencia, es estadísticamente significativa, es decir, una disminución estadísticamente significativa, o un aumento estadísticamente significativo. Se puede determinar si una diferencia es estadísticamente significativa, utilizando diversas herramientas de evaluación estadística, bien conocidas en la técnica, por ejemplo, determinación de intervalos de confianza y determinación del valor p, por ejemplo, a través de pruebas binomiales. Los intervalos de confianza preferidos son al menos 90%, al menos 95%, al menos 97%, al menos 98% o al menos 99%. Los niveles de significación de las pruebas estadísticas son, preferiblemente, 0,1 , 0,05, 0,01 , 0,005 o 0,0001. For the purposes of the present invention, 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. Preferably, although not necessarily, 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.
En la presente patente, el término “indicativa” se refiere a que, una diferencia entre el patrón de glicosilación de sAPPa y/o eARRb en la muestra que se va a analizar, respecto al patrón de glicosilación de sAPPa y/o eARRb en una muestra de referencia adecuada, permite diagnosticar si un sujeto tiene la enfermedad de Alzheimer. In the present patent, 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.
A efectos de la presente invención, “Western blot”, también denominado “inmunoblot” o “electrotransferencia”, se refiere a una técnica analítica usada en biología celular y molecular para identificar proteínas específicas en una mezcla compleja de proteínas, tal como la que se presenta en extractos celulares o de tejidos. La técnica utiliza las siguientes tres etapas: separación por tamaño, transferencia a un soporte sólido y, finalmente, visualización mediante unión de proteínas a anticuerpos primarios o secundarios apropiados. For the purposes of the present invention, "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.
A efectos de la presente invención, “ensayo por inmunoabsorción ligado a enzimas (ELISA)” se refiere a una técnica de inmunoensayo en la cual un antígeno inmovilizado se detecta mediante un anticuerpo unido a una enzima (peroxidasa, fosfatasa alcalina, etc.) capaz de generar un producto detectable a partir de un sustrato, por medio de un cambio de color o algún otro tipo de cambio, provocado por la acción enzimática sobre dicho sustrato. En dicha técnica puede existir un anticuerpo primario que reconoce al antígeno y que a su vez es reconocido por un anticuerpo secundario unido a dicha enzima. El antígeno se puede detectar indirectamente en la muestra mediante los cambios de color medidos por espectrofotometría. For the purposes of the present invention, "enzyme-linked immunosorbent assay (ELISA)" 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. In this technique there may be 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.
A efectos de la presente invención, “splicing alternativo” se refiere a un procedimiento en el que se obtienen, a partir de un transcrito primario de ARNm o pre-ARNm, distintas isoformas de ARNm y proteínas, las cuales pueden tener funciones diferentes. Este proceso ocurre principalmente en eucariotas. For the purposes of the present invention, "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.
A efectos de la presente invención, la expresión “anticuerpos pan-específicos” se refiere a anticuerpos con especificidad frente a un dominio específico presente únicamente en una diana, por ejemplo, el dominio exclusivo que diferencia varias formas o variantes de una proteína. For the purposes of the present invention, the term "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.
El problema técnico a resolver consiste en el desarrollo de un método de diagnóstico in vitro para el diagnóstico de la EA basado en nuevos biomarcadores. La presente invención, tal y como se define en las reivindicaciones, proporciona a una solución a dicho problema técnico. 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.
La presente invención proporciona un método in vitro de diagnóstico de la enfermedad de Alzheimer en un sujeto, que comprende: The present invention provides an in vitro method of diagnosing Alzheimer's disease in a subject, comprising:
(a) determinar, en una muestra biológica de dicho sujeto, el patrón de glicosilación de sAPPa y/o eARRb, (a) determining, in a biological sample from said subject, the glycosylation pattern of sAPPa and / or eARRb,
(b) comparar dicho patrón de glicosilación de sAPPa y/o eARRb con un patrón de glicosilación de sAPPa y/o eARRb de referencia, en el que una diferencia en dicha comparación es indicativa de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto. (b) comparing said sAPPa and / or eARRb glycosylation pattern with a reference sAPPa and / or eARRb glycosylation pattern, wherein a difference in said comparison is indicative of a positive diagnosis of Alzheimer's disease in said subject.
En una realización del método de la invención, dicho biomarcador es sAPPa. In an embodiment of the method of the invention, said biomarker is sAPPa.
En una realización del método de la invención, la muestra biológica está seleccionada del grupo que consiste en: líquido cefalorraquídeo, orina, sangre, sangre completa, plasma, suero, líquido linfático, saliva, células y tejidos. In one embodiment of the method of the invention, the biological sample is selected from the group consisting of: cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
En una realización del método de la invención, se determina dicho patrón de glicosilación por una técnica seleccionada del grupo que consiste en: ELISA, espectrometría de masas, tomografía por emisión de positrones (PET), resonancia magnética nuclear (RMN), radioinmunoensayos, ensayos de unión a lectinas, inmunohistoquímica, Western blot y citometría de flujo. In one embodiment of the method of the invention, 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.
En una realización preferente del método de la invención, se determina dicho patrón de glicosilación por ensayos de unión a lectinas. In a preferred embodiment of the method of the invention, said glycosylation pattern is determined by lectin binding assays.
En una realización más preferente del método de la invención, dichas lectinas son Concanavalina A de Canavalia ensiformis (Con A) y/o PHA de Phaseolus vulgaris. In a more preferred embodiment of the method of the invention, said lectins are Concanavalin A from Canavalia ensiformis (Con A) and / or PHA from Phaseolus vulgaris.
En una realización del método de la invención, en dichos ensayos de unión a lectinas se detecta la cantidad o concentración de sAPPa y/o eARRb discriminando entre ligados o no ligados a lectinas. En una realización del método de la invención, la detección de la cantidad o concentración de sAPPa y/o eARRb se lleva a cabo por ELISA o Western blot. In one embodiment of the method of the invention, in said lectin binding assays the amount or concentration of sAPPa and / or eARRb is detected by discriminating between bound or not bound to lectins. In one embodiment of the method of the invention, the detection of the amount or concentration of sAPPa and / or eARRb is carried out by ELISA or Western blot.
En una realización del método de la invención, sAPPa y/o eARRb derivan de cualquiera de las variantes del precursor de la proteína amiloide (APP). Preferentemente, dichas variantes del APP se seleccionan del grupo que consiste en: APP695, APP751 y APP770. In one embodiment of the method of the invention, sAPPa and / or eARRb are derived from any of the variants of the amyloid protein precursor (APP). Preferably, said APP variants are selected from the group consisting of: APP695, APP751 and APP770.
En una realización del método de la invención, una disminución en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, de los niveles de sAPPa derivado de APP695 y derivado de la combinación de las variantes APP-KPI, APP751 y APP770, no ligado a las lectinas Con A o PHA, es indicativa de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto; una disminución en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, del nivel de eARRb derivado de APP695, no ligado a las lectinas Con A o PHA, es indicativa de un diagnóstico positivo de enfermedad de Alzheimer; y en el que un aumento en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, del nivel de eARRb derivado de la combinación de las variantes APP-KPI, APP751 y APP770, no ligado a las lectinas Con A o PHA, es indicativo de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto. In one embodiment of the method of the invention, 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.
Información sobre la secuencia del APP humano y sobre las variantes de procesamiento proteolítico de APP, junto con información sobre aminoácidos con glicosilación potencial, es accesible en la base de datos de proteínas UniProtKB (N° de acceso P05067). Según dicho registro del APP humano en la base de datos de proteínas UniProtKB, los siguientes aminoácidos de APP pueden tener glicosilación potencial (numeración para la variante mayoritaria APP695): Information on the sequence of human APP and on proteolytic processing variants of APP, along with information on amino acids with potential glycosylation, is accessible in the protein database UniProtKB (Accession No. P05067). According to said registry of human APP in the protein database UniProtKB, the following amino acids of APP may have potential glycosylation (numbering for the majority variant APP695):
N-glicosilación: aminoácidos 542, 571 N-glycosylation: amino acids 542, 571
O-glicosilación: aminoácidos 633, 651, 652, 659, 663, 667, 681. O-glycosylation: amino acids 633, 651, 652, 659, 663, 667, 681.
Los aminoácidos 1-17 del APP corresponden al péptido señal. El fragmento eARRb corresponde a los aminoácidos 18-671 del APP. El fragmento sAPPa corresponde a los aminoácidos 18-687. Esta información está también accesible en la base de datos de proteínas UniProtKB (N° de acceso P05067). La presente invención también proporciona los biomarcadores sAPPa y/o eARRb para uso en un método in vitro de diagnóstico de la enfermedad de Alzheimer en el que se determina el patrón de glicosilación de sAPPa y/o eARRb en una muestra biológica. 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.
La presente invención también proporciona el uso de los biomarcadores sAPPa y/o eARRb en el diagnóstico in vitro de la enfermedad de Alzheimer, en el que se determina el patrón de glicosilación de sAPPa y/o eARRb en una muestra biológica. 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.
En una realización de los biomarcadores para uso de la invención, dicho biomarcador es sAPPa. In one embodiment of the biomarkers for use of the invention, said biomarker is sAPPa.
En una realización de los biomarcadores para uso de la invención, la muestra biológica está seleccionada del grupo que consiste en: líquido cefalorraquídeo, orina, sangre, sangre completa, plasma, suero, líquido linfático, saliva, células y tejidos. In one embodiment of the biomarkers for use of the invention, the biological sample is selected from the group consisting of: cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells, and tissues.
En una realización de los biomarcadores para uso de la invención, la muestra biológica ha sido obtenida de un sujeto. In one embodiment of the biomarkers for use of the invention, the biological sample has been obtained from a subject.
La presente invención también proporciona un kit de diagnóstico de la enfermedad de Alzheimer, que comprende reactivos para determinar el patrón de glicosilación de sAPPa y/o eARRb en una muestra biológica, en el que dichos reactivos comprenden la lectina Con A y/o PHA y anticuerpos específicos frente a sAPPa y/o eARRb. 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.
En una realización del kit de la invención, dichos anticuerpos específicos son el anticuerpo policlonal IBL-a, específico frente a sAPPa y el anticuerpo monoclonal IBL- b, específico frente a eARRb. In one embodiment of the kit of the invention, said specific antibodies are the polyclonal antibody IBL-a, specific against sAPPa and the monoclonal antibody IBL-b, specific against eARRb.
En una realización, el kit de la invención además comprende al menos una disolución tampón. In one embodiment, the kit of the invention further comprises at least one buffer solution.
Ejemplos de tampones son: tampón fosfato, tampón fosfato salino, tampón acetato, tampón borato-cloruro, tampón carbonato, tampón glicina y tampón Tris. BREVE DESCRIPCIÓN DE LOS DIBUJOS Examples of buffers are: phosphate buffer, phosphate saline buffer, acetate buffer, borate-chloride buffer, carbonate buffer, glycine buffer, and Tris buffer. BRIEF DESCRIPTION OF THE DRAWINGS
Figura 1. Aumento de la expresión de APP en tejido cerebral de EA. Expresión relativa de ARNm de APP analizada por qRT-PCR en tejido de corteza frontal de sujetos control (C) (n = 7) y sujetos con EA (etapa V-VI en la escala Braak y Braak, n = 7). Los valores se calcularon a partir de curvas estándar relativas, se normalizaron al ARNm de la subunidad ribosomal 18S a partir del mismo ADNc. Se obtuvo un valor p <0,001 respecto a C. Figure 1. Increased expression of APP in AD brain tissue. Relative expression of APP mRNA analyzed by qRT-PCR in frontal cortex tissue from control subjects (C) (n = 7) and subjects with AD (stage V-VI on the Braak and Braak scale, n = 7). Values were calculated from relative standard curves, normalized to 18S ribosomal subunit mRNA from the same cDNA. A p value <0.001 was obtained with respect to C.
Figura 2. Representación esquemática y caracterización bioquímica de fragmentos NTF y CTF de APP. (A) Representación esquemática de los fragmentos proteolíticos sAPPa, eARRb, CTFa y OTRb generados por la a-secretasa (vía no amiloidogénica) y la b- secretasa (vía amiloidogénica) (no dibujado a escala). Se indica la localización del dominio KPI presente en las variantes APP751 y APP770, pero no en APP695. También se muestran los epítopos para los anticuerpos utilizados en este estudio. (B) Para evaluar la identidad de las especies sAPPa y eARRb de mayor masa molecular derivadas de las variantes de KPI, dos extractos cerebrales se procesaron en paralelo y se probaron por separado mediante electroforesis en condiciones desnaturalizantes con el anticuerpo indicado para identificar las bandas inmunoreactivas al anticuerpo anti- sAPPa (generado en ratón), al anticuerpo qhΐί-eARRb (generado en conejo) y al anti- KPI (generado en conejo). Se señala mediante una flecha la banda coincidente con todos los anticuerpos y mediante asterisco la banda no coincidente. Se indican las posiciones correspondientes a una masa molecular de 100 y 130 kDa. (C) Se representan bajo el número 2 los resultados con extractos de corteza frontal, analizados con el anticuerpo anti-sAPPa y el anticuerpo qhΐί-eARRb, tras tratamiento de de- glicosilación mediante incubación con N- y O-glicosidasas específicas, combinadas con Sialidasa A en condiciones desnaturalizantes. Se representan bajo el número 1 los resultados del control (tampón sin las glicosidasas). El Western blot se resolvió incubando simultáneamente con los anticuerpos sAPPa (ratón) y eAbRRb (conejo) y anticuerpos secundarios fluorescentes. La fluorescencia de los anticuerpos secundarios (IRDye 800CW cabra anti-conejo y IRDye 680RD cabra anti-ratón) se detectó con un aparato Odyssey CLx para fluorescencia simultánea (en el panel marcado con el término “Unión” se muestran las bandas que co-localizan). Se indican las posiciones correspondientes a una masa molecular de 100 y 130 kDa. (D) Para caracterizar CTFa y OTRb, se analizaron células CHO-PS70 tratadas con el inhibidor de la y-secretasa, DAPT, o el vehículo solo (DMSO; control: Ctrl) en paralelo para controlar los extractos cerebrales (corteza frontal humana: Cx). Las inmunotransferencias de extractos de CHO-PS70 se detectaron simultáneamente con dos anticuerpos diferentes: el anticuerpo C-terminal generado en conejo y que reconoce un dominio común a CTFa y (IPTb; y el anticuerpo generado en rata 2D8 y que reconoce el dominio N-terminal de Ab que, por lo tanto, solo detecta (IPTb (se indica mediante flecha la banda que se acumula tras tratamiento con DAPT). Se indican las posiciones correspondientes a una masa molecular de 6,5, 10 y 14 kDa. Figure 2. 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. (B) To assess the identity of the higher molecular mass sAPPa and eARRb species derived from the KPI variants, two brain extracts were processed in parallel and tested separately by electrophoresis under denaturing conditions with the indicated antibody to identify the immunoreactive bands. to anti-sAPPa antibody (generated in mouse), to qhΐί-eARRb antibody (generated in rabbit) and to anti-KPI (generated in rabbit). The band coinciding with all antibodies is indicated by an arrow and the non-coincident band by an asterisk. The positions corresponding to a molecular mass of 100 and 130 kDa are indicated. (C) The results with frontal cortex extracts, analyzed with the anti-sAPPa antibody and the qhΐί-eARRb antibody, after deglycosylation treatment by incubation with specific N- and O-glycosidases, combined with Sialidase A under denaturing conditions. Control results (buffer without glycosidases) are represented under number 1. The Western blot was resolved by incubating simultaneously with the sAPPa (mouse) and eAbRRb (rabbit) antibodies and fluorescent secondary antibodies. The fluorescence of the secondary antibodies (IRDye 800CW goat anti-rabbit and IRDye 680RD goat anti-mouse) was detected with an Odyssey CLx apparatus for simultaneous fluorescence (in the panel marked with the term “Union” the bands that co-localize ). The positions corresponding to a molecular mass of 100 and 130 kDa are indicated. (D) To characterize CTFa and OTRb, CHO-PS70 cells treated with the y-secretase inhibitor, DAPT, or vehicle alone (DMSO; control: Ctrl) were analyzed in parallel to control the extracts. cerebral (human frontal cortex: Cx). 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.
Figura 3. Las variantes sAPPa y eARRb permanecen inalteradas en tejido cerebral de EA. (A) Western blot de muestras de corteza frontal humana de sujetos control (n=7) y sujetos con EA (n=7) detectados con anticuerpos contra sAPPa. (B) Cuantificación densitométrica de las bandas de Western blot de sujetos control (n=7) y sujetos con EA (n = 7) detectados con anticuerpos contra sAPPa, de las variantes APP-695 y APP-KPI, con cantidades equivalentes de proteína cargadas en cada carril y utilizando vinculina como control de carga. Los cálculos se realizaron por duplicado. (C) Western blot de muestras de corteza frontal humana de sujetos control (n=7) y sujetos con EA (n=7) detectados con anticuerpos contra eARRb. (D) Cuantificaciones densitométricas de las bandas de Western blot de sujetos control (n=7) y sujetos con EA (n = 7) detectados con anticuerpos contra eARRb, de las variantes APP-695 y APP-KPI, con cantidades equivalentes de proteína cargadas en cada carril y utilizando vinculina como control de carga. Los cálculos se realizaron por duplicado. (E) Gráfico de la relación obtenida dividiendo los valores para las especies sAPP (sAPPa o eARRb) derivadas de la variante APP-KPI por los derivados de APP695 para cada muestra. Ninguna de las comparaciones resultó en diferencias estadísticamente significativas entre las muestras EA y C. Figure 3. The sAPPa and eARRb variants remain unchanged in AD brain tissue. (A) Western blot of human frontal cortex samples from control subjects (n = 7) and subjects with AD (n = 7) detected with antibodies against sAPPa. (B) Densitometric quantification of the Western blot bands of control subjects (n = 7) and subjects with AD (n = 7) detected with antibodies against sAPPa, of the APP-695 and APP-KPI variants, with equivalent amounts of protein loaded in each lane and using vinculine as load control. The calculations were carried out in duplicate. (C) Western blot of human frontal cortex samples from control subjects (n = 7) and subjects with AD (n = 7) detected with antibodies against eARRb. (D) Densitometric quantifications of the Western blot bands of control subjects (n = 7) and subjects with AD (n = 7) detected with antibodies against eARRb, of the APP-695 and APP-KPI variants, with equivalent amounts of protein loaded in each lane and using vinculine as load control. The calculations were carried out in duplicate. (E) Graph of the relationship obtained by dividing the values for the sAPP species (sAPPa or eARRb) derived from the APP-KPI variant by the APP695 derivatives for each sample. None of the comparisons resulted in statistically significant differences between samples EA and C.
Figura 4. CTFa y OTRb permanecen inalterados en tejido cerebral de EA. (A) Western blot de tejido de la corteza frontal humana de sujetos control (n = 7) y EA (n = 7), detectado con un anticuerpo C-terminal (de la APP de longitud completa). (B) Cuantificación densitométrica de las especies ΰTRb (B) y CTFa (C), utilizando GAPDH como control de carga para asegurar que se cargaron cantidades equivalentes de proteína en cada carril. Los cálculos se realizaron por duplicado. (D) Gráfico de la relación obtenida para cada muestra dividiendo la inmunorreactividad de ΰTRb por la de CTFa. No hubo diferencias estadísticamente significativas evidentes. Figura 5. Comparación de la glicosilación APP695 y APP-KPI en tejido cerebral de sujetos control y con EA. Representación gráfica del porcentaje del fragmento sAPPa derivado de las variantes APP695 o APP-KPI de los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA (A) o Con A (B). Representación gráfica del porcentaje del fragmento eARRb derivado de las variantes APP695 o APP-KPI de los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA (C) o Con A (D). Los valores de p están indicados en la figura (ns, no significativo). Figure 4. CTFa and OTRb remain unchanged in AD brain tissue. (A) Western blot of human frontal cortex tissue from control (n = 7) and EA (n = 7) subjects, detected with a C-terminal antibody (from full-length APP). (B) Densitometric quantification of the ΔTRb (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 ΰTRb 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. Graphical representation of the percentage of the sAPPa fragment derived from the APP695 or APP-KPI variants of the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA (A) or Con A (B ). Graphical representation of the percentage of the eARRb fragment derived from the APP695 or APP-KPI variants of the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA (C) or Con A (D ). The p values are indicated in the figure (ns, not significant).
Figura 6. Comparación de la glicosilación de sAPPa y eARRb en tejido cerebral de sujetos control y sujetos con EA. Representación gráfica del porcentaje del fragmento sAPPa derivado de las variantes APP695 o APP-KPI de los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA (A) o Con A (B). Representación gráfica del porcentaje del fragmento eARRb derivado de las variantes APP695 o APP-KPI de los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA (C) o Con A (D). Los valores p están indicados en la figura. Figure 6. Comparison of glycosylation of sAPPa and eARRb in brain tissue of control subjects and subjects with AD. Graphical representation of the percentage of the sAPPa fragment derived from the APP695 or APP-KPI variants of the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA (A) or Con A (B ). Graphical representation of the percentage of the eARRb fragment derived from the APP695 or APP-KPI variants of the brain tissues of 7 control subjects (C) and 7 subjects with AD that did not bind to the immobilized lectins of PHA (C) or Con A (D ). The p-values are indicated in the figure.
Figura 7. Comparación de la glicosilación cerebral de sAPP entre sujetos control y sujetos con EA. Representación gráfica del porcentaje del fragmento sAPPa derivado de APP695 (A) y APP-KPI (B) en los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA y Con A. Representación gráfica del porcentaje del fragmento eARRb derivado de APP695 (C) y APP-KPI (D) en los tejidos cerebrales de 7 sujetos control (C) y 7 sujetos con EA que no se unieron a las lectinas inmovilizadas de PHA y Con A. Los valores p están indicados en la figura (n.s., no significativo). 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).
DESCRIPCIÓN DE MODOS DE REALIZACIÓN DESCRIPTION OF IMPLEMENTATION MODES
Materiales y métodos Materials and methods
Muestras biológicas Biological samples
El estudio fue aprobado por el comité de ética de la Universidad Miguel Hernández de Elche (Alicante, España), y se llevó a cabo en conformidad con la declaración de Helsinki. Las muestras se recibieron anonimizadas e identificadas sólo en base al código del banco de tejidos. Las muestras congeladas de corteza cerebral de 7 pacientes con la EA (3 hombres y 4 mujeres, con un rango de edad 81 ± 12 años) y 7 pacientes sanos como controles (3 hombres y 4 mujeres, con un rango de edad de 65 ± 15 años), fueron obtenidas del banco de tejidos neurológicos de la Fundación CIEN-Unidad de Investigación Proyecto Alzheimer (UIPA; Madrid), y del banco de tejidos de la Región de Murcia, ambos coordinados por el neuropatólogo Dr. Alberto Rábano (Fundación CIEN-UIPA). The study was approved by the ethics committee of the Miguel Hernández de Elche University (Alicante, Spain), and was carried out in accordance with the Declaration of Helsinki. The samples were received anonymized and identified only on the basis of the tissue bank code. The frozen samples of the cerebral cortex from 7 patients with AD (3 men and 4 women, with an age range of 81 ± 12 years) and 7 healthy patients as controls (3 men and 4 women, with an age range of 65 ± 12 years). 15 years), were obtained from the neurological tissue bank of the CIEN Foundation-Alzheimer Project Research Unit (UIPA; Madrid), and from the Murcia Region tissue bank, both coordinated by neuropathologist Dr. Alberto Rábano (CIEN Foundation -UIPA).
Todos los pacientes con la EA cumplían criterios clínicos para probable EA (NINCDS- ADRDA). Los pacientes con la EA se correspondían a casos esporádicos que fueron seleccionados en base a su historial clínico y diagnóstico neuropatológico basado en los criterios de diagnóstico CERAD, del inglés Consortium to Establish a Registry for Alzheimer’s Disease. Los casos fueron categorizados como estadios V-VI durante el análisis neuropatológico siguiendo la escala de Braak y Braak (Braak y Braak, 1991). El intervalo postmortem medio del tejido fue entre 1 ,5 y 6 horas, sin diferencias significativas en ambos grupos. Los sujetos control resultaron negativos en su análisis histopatológico, y no tenían antecedentes de síntomas neurológicos o psiquiátricos ni de alteración en la memoria. All patients with AD met clinical criteria for probable AD (NINCDS-ADRDA). 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.
Procesamiento de las muestras biológicas Processing of biological samples
Las muestras de tejido cerebral recolectadas en los bancos de tejidos se mantuvieron en todo momento a -80°C. Posteriormente, se descongelaron lentamente en hielo y se homogeneizaron en un tampón de extracción (10% p/v) Tris-HCI 50 mM; pH 7,4; NaCI 500 mM; EDTA 5 mM; 1% (p/v) Nonidet P-40; 0,5% (p/v) Tritón X-100, complementado con un cóctel de inhibidores de proteasas (Sigma P834). El homogeneizado se sonicó utilizando un sonicador Microson ultrasonic cell disruptor Misonix en 3 series de 10 pulsos. Tras sonicar, las muestras se centrifugaron a 70.000*g a 4°C durante 1 hora. Por último, se recolectó el sobrenadante, se alicuotó y se almacenó a -80°C hasta el momento de su utilización. La concentración total de proteína de los distintos extractos se determinó utilizando como estándar albúmina de suero bovino (BSA) de acuerdo con el método del ácido bicinconínico (BCA; Pierce). 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. After sonication, the samples were centrifuged at 70,000 * g at 4 ° C for 1 hour. Finally, the supernatant was collected, aliquoted, and stored at -80 ° C until use. The total protein concentration of the different extracts was determined using as standard bovine serum albumin (BSA) according to the bicinchoninic acid method (BCA; Pierce).
Células que sobre-expresan APP Cells that overexpress APP
Las células CHO-PS70 que sobre-expresan de manera estable la APP humana (variante 751) de tipo salvaje y la subunidad catalítica de g-secretasa, presenilina-1, se cultivaron durante 48 horas en placas de seis pocilios (350000 células/pocillo) en medio Eagle modificado por Dulbecco (DMEM), suplementado con GlutaMAX ™ (Gibco® Life Technologies, Paisley, Reino Unido), 5% de suero fetal bovino (FBS; Gibco) y 100 pg/ml de penicilina/estreptomicina (Gibco). Las células se trataron con el inhibidor de y- secretasa DAPT (5 mM, LY-374973 t-butil éster de (N-[N-(3,5-difluorofenacetilo)-l-alanil]- S-fenilglicina; Calbiochem®, Merck KGaA). Las células de control se trataron solo con el mismo volumen de dimetilsulfóxido (DMSO). Después de una exposición de 18 h de las células al inhibidor, las células se lavaron dos veces con tampón fosfato solución salino (PBS) frío y se volvieron a suspender en 100 pl de tampón de extracción (descrito más arriba) enfriado con hielo complementado con un cóctel de inhibidores de proteasa (descrito más arriba). Los lisados celulares se sonicaron y centrifugaron durante 1 hora a 70000 x g y 4 °C, y los extractos se congelaron a -80 °C para futuros análisis. CHO-PS70 cells stably overexpressing wild-type human APP (variant 751) and the catalytic g-secretase subunit, presenilin-1, were cultured for 48 hours in six-well plates (350,000 cells / well ) in the middle Eagle Modified by Dulbecco (DMEM), supplemented with GlutaMAX ™ (Gibco® Life Technologies, Paisley, UK), 5% fetal calf serum (FBS; Gibco) and 100 pg / ml penicillin / streptomycin (Gibco). 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). After 18 hr exposure of the cells to the inhibitor, the cells were washed twice with cold phosphate buffered saline (PBS) and They were resuspended in 100 µl 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.
Aislamiento de ARN y análisis qRT-PCR RNA isolation and qRT-PCR analysis
El ARN total se aisló de las muestras de tejido cerebral usando el reactivo TRIzol y el sistema de purificación de ARN PureLink™ Micro-to-Midi Total RNA Purification System (Invitrogen), de acuerdo con las instrucciones del fabricante de dicho kit y se realizó un análisis de moléculas de ARN mensajero (ARNm) por reacción en cadena de la polimerasa cuantitativa con transcriptasa inversa (qRT-PCR). El ADN complementario (ADNc) de primera cadena se obtuvieron por transcripción inversa del ARN total (1 ,5 pg), utilizando el kit de transcripción inversa High Capacity cDNA Reverse Transcríption Kit (Applied Biosystems; Life Technologies Paisley, Reino Unido), de acuerdo con las instrucciones del fabricante de dicho kit. La reacción en cadena de la polimerasa (PCR) se realizó en el sistema de PCR en tiempo real StepOne ™ Real-Time PCR System (Applied Biosystems), utilizando los ensayos de expresión génica TaqMan Gene Expression Assays (Hs00169098-m1 para APP y Hs03003631-g1 para 18S; Thermo Fisher) y la mezcla maestra de la PCR TaqMan PCR Master Mix. Los niveles de transcripción se calcularon mediante el método comparativo 2 ACt con respecto al ADNc de la subunidad ribosomal 18S. Total RNA was isolated from brain tissue samples using TRIzol reagent and the PureLink ™ 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). First strand complementary DNA (cDNA) 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. The polymerase chain reaction (PCR) was performed in the StepOne ™ Real-Time PCR System (Applied Biosystems), using the TaqMan Gene Expression Assays (Hs00169098-m1 for APP and Hs03003631). -g1 for 18S; Thermo Fisher) and the TaqMan PCR Master Mix. The levels of transcription were calculated by the comparative method 2 ACt with respect to the cDNA of the ribosomal subunit 18S.
Western blot Western blot
Las muestras de tejido cerebral (30 pg por pocilio) se hirvieron a 95°C durante 5 minutos, se separaron por electroforesis en geles de poliacrilamida con dodecilsulfato sódico (en inglés, SDS-PAGE) al 7,5% en Tris-Tricina y posteriormente se transfirieron a membranas de nitrocelulosa de 0,45 pm (Schleicher & Schuell Bioscience, GmbH, Dassel, Germany). Se cuantificó la señal inmunorreactiva de las bandas APP en las transferencias Western ( Western blots). Las especies de APP en las muestras se detectaron usando: un antisuero policlonal anti-APP C-terminal de conejo (1:1000; Sigma Aldrich, St. Louis, MO, EE.UU.; denominado aquí Sigma-Ct); un anticuerpo monoclonal de rata llamado 2D8 producido contra el dominio N-terminal de Ab, por lo que detecta (IPTb pero no CTFa (1:50) (Willem et al., 2015), un antisuero policlonal de conejo qhΐί-eARRb específico para el C-terminal de eARRb (1:100; IBL, Hamburgo, Alemania; denominado aquí IBL-b); un anticuerpo monoclonal de ratón anti-sAPPa específico para el C-terminal de eAbRRa (1:100; IBL; denominado aquí IBL-a); y un antisuero policlonal de conejo anti-KPI específico para el dominio KPI de APP (1:500; Millipore; denominado aquí como KPI). Vinculina (1:2000, anticuerpo monoclonal de ratón anti-vinculina, sc-73614 Santa Cruz; antisuero de conejo anti-vinculina 1:1000, Sigma V4139) y GAPDH (1:10000 anticuerpo monoclonal de ratón anti-GAPDH; Proteintech 60004-1) se utilizaron como controles de carga. Las intensidades de las bandas se analizaron utilizando el programa informático Image Studio Lite (LI-COR). Los anticuerpos utilizados tenían una reactividad cruzada entre los fragmentos sAPPa y eARRb inferior al 1,5%, y ninguno de los anticuerpos reacciona de forma cruzada con la APP de longitud completa. Las transferencias se detectaron utilizando anticuerpos secundarios conjugados apropiados (IRDye 680RD cabra anti-ratón (Número de catálogo 925-68180); IRDye 800CW cabra anti-conejo (Número de catálogo 925- 32211); IRDye 680RD cabra anti-conejo (Número de catálogo 925-68071) o IRDye 800CW cabra anti-rata (Número de catálogo 926-32210); todos de LI-COR Biosciences GmbH, Bad Homburg, Alemania) y se analizaron en un sistema de imagen infrarrojo Odyssey Clx (LI-COR). 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. Louis, MO, USA; referred to herein as Sigma-Ct); a rat monoclonal antibody called 2D8 raised against the N-terminal domain of Ab, thus detecting (IPTb but not CTFa (1:50) (Willem et al., 2015), a polyclonal rabbit qhΐί-eARRb antiserum specific for the C-terminus of eARRb (1: 100; IBL, Hamburg, Germany; referred to herein as IBL-b); a mouse anti-sAPPa monoclonal antibody specific for the C-terminus of eAbRRa (1: 100; IBL; referred to herein as IBL -a); and a polyclonal rabbit anti-KPI antiserum specific for the KPI domain of APP (1: 500; Millipore; referred to herein as KPI). 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. Blots were detected using appropriate conjugated secondary antibodies (IRDye 680RD goat anti-mouse (Catalog number 925-68180); IRDye 800CW goat anti-rabbit (Catalog number 925-32211); IRDye 680RD goat anti-rabbit (Catalog number 925-68071) or IRDye 800CW goat anti-rat (Catalog Number 926-32210); all from LI-COR Biosciences GmbH, Bad Homburg, Germany) and analyzed on an Odyssey Clx Infrared Imaging System (LI-COR).
De-glicosilación enzimática Enzymatic de-glycosylation
Las glicoproteínas solubilizadas de cerebro se trataron con un kit de de-glicosilación enzimática de ProZyme (GK80110), de acuerdo con las instrucciones del fabricante de dicho kit, y luego se sometieron a SDS-PAGE y análisis por Western blot. Los extractos de la corteza frontal se desnaturalizaron y se de-glicosilaron mediante incubación con N-Glicanasa, O-Glicanasa y Sialidasa A. Este tratamiento elimina todos los glicanos unidos a N y los glicanos simples unidos a O (incluidos los polisialilados) de las glicoproteínas. Unión a lectinas 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
Las muestras de corteza cerebral se incubaron a 4°C durante toda la noche con lectinas específicas de azúcares terminales: la lectina de Canavalia ensiformis Concanavalina A (Con A) (Sigma; Número de catálogo C9017), con alta especificidad por residuos de mañosa terminales, o la lectina de Phaseolus vulgaris PHA (Vector; Número de catálogo AL113), con alta especificidad por residuos de galactosa terminales, inmovilizadas en sefarosa (Con A) o agarosa (PHA). La fracción de glicoproteína no ligada a las lectinas se separó por centrifugación y se analizó en Western blot utilizando anticuerpos contra sAPPa y sAPPp. La proporción de APP no unida a lectina se calculó como la relación entre la inmunoreactividad de APP no unida a lectina y la inmunoreactividad total, obtenida de una alícuota mantenida en las mismas condiciones, pero no incubada con una lectina. Todos los análisis se realizaron por duplicado. 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). 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.
Análisis estadístico Statistic analysis
Todos los datos se analizaron en SigmaStat (Versión 2.0; SPSS Inc.), aplicando una prueba t de Student (de dos colas) o una prueba U de Mann-Whitney para comparaciones individuales, y determinando los valores p exactos (los valores p <0.05 fueron considerados significativos). Los resultados se presentan como las medias ± error estándar de la media (SEM) y la correlación entre las variables se evaluó mediante análisis de regresión lineal. All data were analyzed in SigmaStat (Version 2.0; SPSS Inc.), applying a Student's t-test (two-tailed) or a Mann-Whitney U test for individual comparisons, and determining the exact p-values (p-values < 0.05 were considered significant). The results are presented as the means ± standard error of the mean (SEM) and the correlation between the variables was evaluated by linear regression analysis.
Ejemplo 1. Aumento de la expresión de APP en el cerebro de sujetos con EAExample 1. Increased expression of APP in the brain of subjects with AD
Se realizaron ensayos de qRT-PCR para determinar la expresión del ARN mensajero (ARNm) de APP en EA, usando cebadores que correspondían a secuencias en los exones 10-11 de APP y que son comunes a las principales variantes cerebrales. Por lo tanto, se determinaron los niveles totales de ARNm de las variantes APP695, APP751 y APP770. En consecuencia, los niveles de transcritos de APP en su conjunto fueron significativamente mayores en el tejido cerebral de sujetos con EA que en el tejido cerebral de sujetos control (C) (p<0,001; Figura 1). QRT-PCR assays were performed to determine the expression of APP messenger RNA (mRNA) in EA, using 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).
Ejemplo 2. Caracterización de sAPPa, sAPPp, CTFa and CTFp en el tejido cerebral de sujetos con EA Example 2. Characterization of sAPPa, sAPPp, CTFa and CTFp in the brain tissue of subjects with AD
Se caracterizaron el sAPPa o sAPPp en Western blot del tejido cerebral de sujetos con EA, un método que permitió discriminar diferentes especies de APP, especialmente aquellas con diferentes masas moleculares. Sin embargo, sAPPa y sAPPp se distinguen entre sí sólo en 16 aminoácidos (lo que representa 1-2 kDa en masa molecular), y se ha predicho que sAPPa o eARRb son sólo -5-10 kDa más pequeños que la APP de longitud completa, y por lo tanto, no se distinguen por separación electroforética. Además, pequeñas diferencias en la migración electroforética también pueden atribuirse a diferencias en la glicosilación, o incluso reflejar formas inmaduras de la proteína. En consecuencia, para poder distinguir sAPPa o eARRb, se abordó la discriminación de estas proteínas por electroforesis SDS-PAGE, utilizando anticuerpos pan-específicos generados contra el dominio exclusivo C-terminal de sAPPa y eARRb. The 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. However, 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. Furthermore, small differences in electrophoretic migration can also be attributed to differences in glycosylation, or even reflect immature forms of the protein. Consequently, in order to distinguish sAPPa or eARRb, the discrimination of these proteins was addressed by SDS-PAGE electrophoresis, using pan-specific antibodies generated against the exclusive C-terminal domain of sAPPa and eARRb.
Tanto para sAPPa y el eARRb, en el cerebro existen 3 variantes de splicing alternativo, la de 695 aminoácidos, APP695, mayoritariamente expresada en neuronas, y las variantes APP751 y APP770, expresadas en células gliales, que presentan el dominio inhibidor de serín proteasa tipo Kunitz KPI (del inglés Kunitz-type serine protease ίh ί ϋoή. Los NTF de APP sAPPa y el eARRb se distinguen electroforéticamente y por Western blot con anticuerpos pan-específicos contra el extremo C-terminal exclusivo de sAPPa y eARRb. En las muestras de tejido cerebral, las diferentes especies de APP y sus fragmentos N-terminales largos se detectaron en varias bandas que migraron en el rango 100-130 kDa. Las diferencias en la masa molecular reflejan variantes alternativas de la especie predominante neuronal, la especie denominada APP695, y las isoformas APP751 y APP770, ambas portadoras del dominio KPI y prácticamente indistinguibles entre ellas por tamaño. Todas las variantes sufren el mismo procesamiento por secretasas y generan los mismos tipos de fragmentos, incluidos el sAPPa y el eARRb. La Figura 2A muestra una representación esquemática de la longitud completa de APP y de los fragmentos N- y C-terminales determinados en este ejemplo, así como los epítopos reconocidos por los diferentes anticuerpos utilizados. Las especies de APP- NTF de cerebro se caracterizaron en Western blot, utilizando anticuerpos pan- específicos contra los dominios C-terminales específicos de sAPPa o eARRb (Figura 2B). Cuando se utilizó un anticuerpo anti-KPI, solo las especies de mayor masa molecular mostraron tamaños compatibles con las bandas inmunorreactivas de KPI. For both 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 ίh ί ϋoή. 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. Differences in molecular mass reflect alternative variants of the predominant neuronal species, the species named APP695. and the APP751 and APP770 isoforms, both carriers of the KPI domain and practically indistinguishable from each other by size. All variants undergo the same po processing r secretases and generate the same types of fragments, including sAPPa and eARRb. Figure 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.
Dado que las variantes sAPPa y eARRb derivadas del APP695 (no KPI) tampoco mostraron co-localización, se exploró si esas diferencias en masa molecular eran atribuibles a glicosilación (si la extensión de la glicosilación entre los distintos NTF variaba). Para ello, se de-glicosiló APP de manera completa, observando que, tras de- glicosilación, todas las bandas identificadas con anticuerpos sAPPa y eARRb sí co localizaban (Fig. 2C). Since the sAPPa and eARRb variants derived from APP695 (not KPI) also did not show co-localization, it was explored whether these differences in molecular mass were attributable to glycosylation (if the extent of glycosylation between the different NTFs varied). To do this, APP was completely de-glycosylated, observing that, after de- glycosylation, all bands identified with sAPPa and eARRb antibodies did co-localize (Fig. 2C).
El CTFa y el (IPTb no difieren entre las variantes de APP y se caracterizaron usando extractos de células CHO-PS70 que sobre-expresan de manera estable la APP humana de tipo salvaje y la subunidad catalítica de g-secretasa, presenilina-1. 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.
Los extractos de estas células tratadas con el inhibidor de la g-secretasa, DAPT, se ensayaron con el anticuerpo C-terminal, proporcionando evidencia de la acumulación de CTFa (también llamado C83 por su longitud de aminoácidos) y OTRb (también llamado C99 por su longitud de aminoácidos) en los extractos celulares, y de bandas coincidentes en homogeneizados cerebrales cargados en paralelo (Figura 2D). Cuando se ensayó con el anticuerpo 2D8 de rata generado contra el dominio N-terminal de Ab, CTFa y ΰTRb podían discriminarse ya que el epítopo reconocido por este anticuerpo está ausente en CTFa (ver esquema en la Figura 2A). 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). When tested with the rat 2D8 antibody raised against the N-terminal domain of Ab, CTFa and ΔTRb could be discriminated since the epitope recognized by this antibody is absent in CTFa (see scheme in Figure 2A).
Después de esta caracterización bioquímica, se evaluaron las diferentes isoformas de sAPPa y eARRb en extractos de tejido cerebral. No se detectaron diferencias entre las muestras C y EA en los niveles relativos de sAPPa (Figura 3A y 3B) o eARRb (Figura 3C y 3D) derivados de APP695 o APP-KPI. A pesar de las grandes diferencias en las proporciones APP695/APP-KPI para las especies sAPPa y eARRb, no había diferencias evidentes en estas proporciones entre las muestras de C y EA (Figura 3E). La acumulación de bandas inmunorreactivas sAPPa-695 y sAPPa-KPI se correlacionó tanto en extractos de tejido C (r=0.76; p=0,048) como EA (r=0,96; p<0.001), aunque esta correlación no fue significativa para las especies eARRb (C: r=0, 19; p=0,69; EA: r=0,57; p=0, 17). No hubo correlaciones significativas entre sAPPa y eARRb en las isoformas APP695 o APP-KPI en sujetos con C o EA. After this biochemical characterization, the different isoforms of sAPPa and eARRb were evaluated in extracts of brain tissue. No differences were detected between samples C and EA in the relative levels of sAPPa (Figure 3A and 3B) or eARRb (Figure 3C and 3D) derived from APP695 or APP-KPI. Despite the large differences in the APP695 / APP-KPI ratios for the sAPPa and eARRb species, there were no obvious differences in these ratios between the C and EA samples (Figure 3E). The accumulation of immunoreactive bands sAPPa-695 and sAPPa-KPI was correlated both in extracts of tissue C (r = 0.76; p = 0.048) and EA (r = 0.96; p <0.001), although this correlation was not significant for eARRb species (C: r = 0.19; p = 0.69; EA: r = 0.57; p = 0.17). There were no significant correlations between sAPPa and eARRb in the APP695 or APP-KPI isoforms in subjects with C or AD.
Para evaluar si los niveles de APP-CTF se alteraron en extractos cerebrales de pacientes con EA, se utilizó un anticuerpo generado contra el dominio C-terminal original de la APP de longitud completa. La inmunoreactividad para CTFa y ΰTRb en el tejido cerebral (Figura 4A, Figura 4B y Figura 4C) fue similar entre los sujetos con C y EA. Además, la relación ΰTRbLPTa no indicó diferencias entre los grupos EA y C (Figura 4D). Ejemplo 3. Unión de sAPPa and eARRb a lectinas To assess whether APP-CTF levels were altered in brain extracts from AD patients, an antibody raised against the original full-length APP C-terminal domain was used. Immunoreactivity for CTFa and ΔTRb in brain tissue (Figure 4A, Figure 4B and Figure 4C) was similar between subjects with C and AD. Furthermore, the ΰTRbLPTa relationship did not indicate differences between groups EA and C (Figure 4D). Example 3. Binding of sAPPa and eARRb to lectins
Para comparar el patrón de glicosilación de sAPPa y eARRb, se incubaron muestras de extractos de corteza frontal de sujetos C (n= 7) y sujetos con la EA (n= 7) con la lectina Con A, que tiene alta especificidad por residuos de mañosa terminales, y la lectina PHA, que tiene alta especificidad por residuos de galactosa terminales, inmovilizadas en sefarosa (Con A) o agarosa (PHA). Se utilizaron anticuerpos pan-específicos para determinar qué porcentaje de sAPPa y eARRb no era reconocido por cada una de las lectinas (Tabla 1). To compare the glycosylation pattern of sAPPa and eARRb, samples of frontal cortex extracts from subjects C (n = 7) and subjects with AD (n = 7) were incubated with lectin Con A, which has high specificity for residues of terminal mannose, and PHA lectin, which has high specificity for terminal galactose residues, immobilized on sepharose (Con A) or agarose (PHA). Pan-specific antibodies were used to determine what percentage of sAPPa and eARRb was not recognized by each of the lectins (Table 1).
Tabla 1
Figure imgf000022_0001
Table 1
Figure imgf000022_0001
Los fragmentos eARRb derivados tanto de APP695 como de APP-KPI mostraron diferencias en su interacción con ambas lectinas, tanto en el grupo control como en el de pacientes con la EA (Figuras 5A-5D). Esto puede deberse al origen celular diferente para las isoformas derivadas de la especie APP695 (neuronal) y las APP-KPI (mayormente glial), y a que cada tipo celular tiene una maquinaria de glicosilación particular. Sin embargo, los distintos fragmentos sAPPa exhibieron un patrón similar de unión a Con A o PHA en ambos grupos. Lo que resultó más interesante fue constatar que el patrón de unión a lectinas variaba al comparar sAPPa y eARRb, tanto para las especies derivadas de APP695, como para las derivadas de APP-KPI, y en ambos grupos, control y con la EA (Figuras 6A-6D). Este resultado, junto con las evidencias de una extensión diferente en glicosilación entre sAPPa y eARRb derivada de la misma variante (Figura 2C), sugiere que diferentes glicoformas de APP se procesan preferencialmente por la vía no amiloidogénica (a-secretasa) o por la amiloidogénica (b- secretasa). Además, esta determinación se da tanto en neuronas (APP695) como en células gliales (APP-KPI). Para estudiar si la glicosilación de APP difiere en extractos cerebrales de sujetos con la EA en comparación con los sujetos control, se confrontó el patrón de glicosilación de sAPPa y eARRb (Figuras 7A-7D). Se determinaron diferencias significativas entre el grupo control y el grupo con la EA en cuanto a la interacción del sAPPa derivado de la APP695 con Con A y PHA; y también con Con A para el sAPPa derivado de las especies APP-KPI. Existían diferencias en la proporción de eARRb que no se unió ni a Con A ni a PHA en muestras de pacientes con la EA en comparación con controles en la especie APP695 y en las especies APP-KPI, aunque dichas diferencias no eran estadísticamente significativas con número de sujetos del presente ejemplo y con la precisión alcanzada en el presente ejemplo. No obstante, no es descartable que se puedan detectar diferencias con mayor significación estadística utilizando técnicas más precisas o un mayor número de pacientes. Los resultados de este ejemplo indicaron que la glicosilación de APP en el cerebro con la EA está alterada y afecta principalmente a las glicoformas procesadas por la vía no amiloidogénica. 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. What was more interesting was to verify that the lectin binding pattern varied when comparing sAPPa and eARRb, both for species derived from APP695 and for those derived from APP-KPI, and in both groups, control and with AD (Figures 6A-6D). This result, together with the evidence of a different extension in glycosylation between sAPPa and eARRb derived from the same variant (Figure 2C), suggests that different APP glycoforms are preferentially processed by the non-amyloidogenic (α-secretase) or by the amyloidogenic pathway. (b-secretase). Furthermore, this determination occurs both in neurons (APP695) and in glial cells (APP-KPI). To study whether glycosylation of APP differs in brain extracts of subjects with AD compared to control subjects, the glycosylation pattern of sAPPa and eARRb was confronted (Figures 7A-7D). Significant differences were determined between the control group and the group with AD regarding the interaction of sAPPa derived from APP695 with Con A and PHA; and also with Con A for sAPPa derived from APP-KPI species. There were differences in the proportion of eARRb that bound neither Con A nor PHA in samples of patients with AD compared to controls in the APP695 species and in the APP-KPI species, although these differences were not statistically significant with number of subjects of the present example and with the precision achieved in the present example. However, it cannot be ruled out that differences with greater statistical significance can be detected using more precise techniques or a greater number of patients. The results of this example indicated that glycosylation of APP in the brain with AD is impaired and mainly affects glycoforms processed by the non-amyloidogenic pathway.
LISTA DE REFERENCIAS BIBLIOGRÁFICAS LIST OF BIBLIOGRAPHIC REFERENCES
Braak y Braak. (1991). Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol, 82(4), 239-259. doi:10.1007/BF00308809 Braak and Braak. (1991). Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol, 82 (4), 239-259. doi: 10.1007 / BF00308809
Cuchillo-lbañez et al. (2015). Heteromers of amyloid precursor protein in cerebrospinal fluid. Mol Neurodegener, 10(2). doi: 10.1186/1750-1326-10-2 Cuchillo-lbañez et al. (2015). Heteromers of amyloid precursor protein in cerebrospinal fluid. Mol Neurodegener, 10 (2). doi: 10.1186 / 1750-1326-10-2
Halim et al. (2011). Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid beta-peptides in human cerebrospinal fluid. Proc Nati Acad Sci U S A, 108(29), 11848-11853. doi: 10.1073/pnas.1102664108 Halim et al. (2011). Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein / amyloid beta-peptides in human cerebrospinal fluid. Proc Nati Acad Sci U S A, 108 (29), 11848-11853. doi: 10.1073 / pnas.1102664108
Lopez-Font et al. (2017). Alterations in the Balance of Amyloid-b Protein Precursor Species in the Cerebrospinal Fluid of Alzheimer's Disease Patients. J Alzheimers Dis, 57(4), 1281-1291. doi:10.3233/JAD-161275 Lopez-Font et al. (2017). Alterations in the Balance of Amyloid-b Protein Precursor Species in the Cerebrospinal Fluid of Alzheimer's Disease Patients. J Alzheimers Dis, 57 (4), 1281-1291. doi: 10.3233 / JAD-161275
Perneczky et al. (2014). Soluble amyloid precursor proteins and secretases as Alzheimer's disease biomarkers. Trends Mol Med, 20(1), 8-15. doi:10.1016/j.molmed.2013.10.001 Perneczky et al. (2014). Soluble amyloid precursor proteins and secretases as Alzheimer's disease biomarkers. Trends Mol Med, 20 (1), 8-15. doi: 10.1016 / j.molmed.2013.10.001
Willem et al. (2015). h-Secretase Processing of APP inhibits neuronal activity in the hippocampus. Nature, 526(7573), 443-447. doi:10.1038/nature14864 Willem et al. (2015). h-Secretase Processing of APP inhibits neuronal activity in the hippocampus. Nature, 526 (7573), 443-447. doi: 10.1038 / nature14864

Claims

REIVINDICACIONES
1. Un método in vitro de diagnóstico de la enfermedad de Alzheimer en un sujeto, que comprende: 1. An in vitro method of diagnosing Alzheimer's disease in a subject, comprising:
(a) determinar, en una muestra biológica de dicho sujeto, el patrón de glicosilación de los biomarcadores sAPPa y/o eARRb, (a) determining, in a biological sample from said subject, the glycosylation pattern of the sAPPa and / or eARRb biomarkers,
(b) comparar dicho patrón de glicosilación de los biomarcadores sAPPa y/o eARRb con un patrón de glicosilación de sAPPa y/o eARRb de referencia, en el que una diferencia en dicha comparación es indicativa de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto. (b) comparing said glycosylation pattern of the sAPPa and / or eARRb biomarkers with a reference sAPPa and / or eARRb glycosylation pattern, in which a difference in said comparison is indicative of a positive diagnosis of Alzheimer's disease in said subject.
2. El método según la reivindicación 1 , en el que dicho biomarcador es sAPPa. 2. The method according to claim 1, wherein said biomarker is sAPPa.
3. El método según la reivindicación 1 ó 2, en el que la muestra biológica está seleccionada del grupo que consiste en: líquido cefalorraquídeo, orina, sangre, sangre completa, plasma, suero, líquido linfático, saliva, células y tejidos. The method according to claim 1 or 2, wherein the biological sample is selected from the group consisting of: cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells and tissues.
4. El método según cualquiera de las reivindicaciones 1 a 3, en el que se determina dicho patrón de glicosilación por una técnica seleccionada del grupo que consiste en: ELISA, espectrometría de masas, tomografía por emisión de positrones (PET), resonancia magnética nuclear (RMN), radioinmunoensayos, ensayos de unión a lectinas, inmunohistoquímica, Western blot y citometría de flujo. The method according to any of claims 1 to 3, wherein 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, lectin-binding assays, immunohistochemistry, Western blot, and flow cytometry.
5. El método según la reivindicación 4, en el que se determina dicho patrón de glicosilación por ensayos de unión a lectinas. The method according to claim 4, wherein said glycosylation pattern is determined by lectin binding assays.
6. El método según la reivindicación 5, en el que dichas lectinas son Concanavalina A de Canavalia ensiformis (Con A) y/o PHA de Phaseolus vulgaris. The method according to claim 5, wherein said lectins are Concanavalin A from Canavalia ensiformis (Con A) and / or PHA from Phaseolus vulgaris.
7. El método según la reivindicación 5 ó 6, en el que, en dichos ensayos de unión a lectinas, se detecta la cantidad o concentración de sAPPa y/o eARRb discriminando entre ligados o no ligados a lectinas. The method according to claim 5 or 6, wherein, in said lectin binding assays, the amount or concentration of sAPPa and / or eARRb is detected by discriminating between lectin-bound or non-lectin-bound.
8. El método según la reivindicación 7, en el que la detección de la cantidad o concentración de sAPPa y/o eARRb se lleva a cabo por ELISA o Western blot.8. The method according to claim 7, wherein the detection of the amount or concentration of sAPPa and / or eARRb is carried out by ELISA or Western blot.
9. El método según cualquiera de las reivindicaciones 1 a 8, en el que sAPPa y/o eARRb derivan de cualquiera de las variantes del precursor de la proteína amiloide (APP). The method according to any of claims 1 to 8, wherein sAPPa and / or eARRb are derived from any of the amyloid protein precursor (APP) variants.
10. El método según cualquiera de las reivindicaciones 1 a 9, en el que dichas variantes del APP se seleccionan del grupo que consiste en: APP695, APP751 y APP770.The method according to any one of claims 1 to 9, wherein said APP variants are selected from the group consisting of: APP695, APP751 and APP770.
11. El método según cualquiera de las reivindicaciones 1 a 10, en el que una disminución en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, de los niveles de sAPPa derivado de APP695 y derivado de la combinación de las variantes APP-KPI, APP751 y APP770, no ligado a las lectinas Con A o PHA, es indicativa de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto; en el que una disminución en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, del nivel de eARRb derivado de APP695, no ligado a las lectinas Con A o PHA, es indicativa de un diagnóstico positivo de enfermedad de Alzheimer; y en el que un aumento en un sujeto, respecto a un valor de referencia derivado de muestras de sujetos sanos, del nivel de eARRb derivado de la combinación de las variantes APP-KPI, APP751 y APP770, no ligado a las lectinas Con A o PHA, es indicativo de un diagnóstico positivo de enfermedad de Alzheimer en dicho sujeto. The method according to any of claims 1 to 10, wherein 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 APP-KPI variants, APP751 and APP770, not bound to Con A or PHA lectins, is indicative of a positive diagnosis of Alzheimer's disease in said subject; in which 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.
12. Los biomarcadores sAPPa y/o eARRb para uso en un método in vitro de diagnóstico de la enfermedad de Alzheimer en el que se determina el patrón de glicosilación de sAPPa y/o eARRb en una muestra biológica. 12. 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.
13. Los biomarcadores para uso según la reivindicación 12, en el que en el que dicho biomarcador es sAPPa. The biomarkers for use according to claim 12, wherein said biomarker is sAPPa.
14. Un kit de diagnóstico de la enfermedad de Alzheimer, que comprende reactivos para determinar el patrón de glicosilación de los biomarcadores sAPPa y/o eARRb en una muestra biológica, en el que dichos reactivos comprenden la lectina Con A y/o PHA y anticuerpos específicos frente a sAPPa y/o eARRb. 14. An Alzheimer's disease diagnostic kit, comprising reagents for determining the glycosylation pattern of sAPPa and / or eARRb biomarkers in a biological sample, wherein said reagents comprise Con A and / or PHA lectin and antibodies specific against sAPPa and / or eARRb.
15. El kit según la reivindicación 14, en el que dicho biomarcador es sAPPa. The kit according to claim 14, wherein said biomarker is sAPPa.
16. El kit según la reivindicación 14 ó 15, en el que dichos anticuerpos específicos son el anticuerpo policlonal IBL-a, específico frente a sAPPa y el anticuerpo monoclonal IBL-b, específico frente a eARRb. 16. The kit according to claim 14 or 15, wherein said specific antibodies are polyclonal antibody IBL-a, specific against sAPPa and monoclonal antibody IBL-b, specific against eARRb.
17. El kit según cualquiera de las reivindicaciones 14 a 16, que además comprende al menos una disolución tampón. 17. The kit according to any of claims 14 to 16, further comprising at least one buffer solution.
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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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