ES2889914B2 - GLYCOSYLATION PATTERN OF sAPPalpha AND/OR sAPPbeta AS DIAGNOSTIC BIOMARKER OF ALZHEIMER'S DISEASE, METHOD AND KIT BASED ON THE SAME - Google Patents

Description

DESCRIPTION

GLYCOSYLATION PATTERN OF sAPPa AND/OR sAPPp AS DIAGNOSTIC BIOMARKER OF ALZHEIMER'S DISEASE, BASED METHOD AND KIT

IN THE SAME

TECHNICAL SECTOR

The invention relates to in vitro methods for diagnosing Alzheimer's disease in which the protein glycosylation pattern is determined. In particular, the invention relates to an in vitro method in which the glycosylation pattern of sAPPa and/or sAPPp, fragments generated in the proteolytic processing of the amyloid protein precursor (APP), is determined.

BACKGROUND OF THE INVENTION

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, as amyloid plaques, and, at the intracellular level, as neurofibrillary tangles. AD is a tauopathy in which neurofibrillary tangles of abnormally hyperphosphorylated cytoskeletal protein tau (P-tau) lead to neuronal collapse and death. AD is the only tauopathy that presents with amyloid fibrillar deposits, consisting mainly of p-amyloid peptide (Ap).

Ap is a 40-42 amino acid peptide product of the proteolytic processing of the transmembrane protein known as amyloid precursor protein (APP). APP processing can be carried out by different pathways that coexist under normal physiological conditions, the non-amyloidogenic pathway and the amyloidogenic pathway. Both pathways produce long N-terminal fragments (NTFs ).

In the non-amyloidogenic pathway, with the sequential action of a-secretase (ADAM10) and ^y -secretase, Ap is not produced. With the action of a-secretase, the NTF sAPPa fragment and the C-terminal fragment (CTF , from the English C-terminal fragment) APP-CTF83. With the action of Y-secretase, the APP-CTF83 fragment produces the AICD and P3 fragments.

The amyloidogenic pathway, directed by the sequential action of the enzymes p-secretase (BACE1) and Y-secretase, generates Ap. With the action of p-secretase, the NTF sAPPp fragment and the CTF APP-CTF99 fragment are produced. With the action of Y-secretase, the APP-CTF99 fragment produces the AICD and Ap fragments.

Today it is accepted that the true pathological "effectors" of AD are the Ap oligomers and P-tau and that an excess of oligomeric forms of Ap (especially the 42-amino acid form: Ap42) is the primary determinant of AD. neurotoxicity in AD. The determination of Ap42 in cerebrospinal fluid (CSF) has been proposed as a diagnostic marker (along with tau and P-tau, good markers but lacking specificity against other tauopathies and neurological disorders).

Regarding Ap 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 Ap in the brain, which in turn fibrillates, forms plaques and is sequestered in said amyloid plaques, for which reason it reaches the CSF in lower amounts than those determined in subjects without plaques, without AD . This circumstance makes it difficult for CSF Ap to be considered an early or progression marker.

The levels of the NTF fragments of APP, sAPPp and sAPPa, mentioned above, have been proposed as alternative markers for Ap. 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 reported increased sAPPp, but others reported no change or even decreased sAPPp. Something equivalent has occurred with the levels of sAPPa; some studies reported decreased sAPPa, while other studies reported either no change or increased sAPPa. Studies that examined both sAPPa and sAPPp reported that both APP NTFs showed the same trend, either increased or decreased CSF levels, when expected to show opposite trends due to imbalance of processing pathways (Perneczky et al ., 2014).

In addition, it has been determined that the complete APP protein (without proteolytic processing) is also present in the CSF coexisting with sAPPa and sAPPp and that all species form heteromers, so ELISA analyzes for determining levels of sAPPa and sAPPp, even with specific antibodies, do not reliably quantify the species separately (Cuchillo-Ibañez et al., 2015).

Subsequently, a Western blot study was carried out, preventing the heteromers from distorting the interpretation of the results, in which no altered levels of sAPPa or sAPPp were found (Lopez-Font et al., 2017).

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 finished its synthesis ). Carbohydrates can be attached to proteins by N-glycosylation, in which they bind to the nitrogen of the amino acid side chains asparagine or arginine, or by O-glycosylation, in which they bind to the oxygen of the hydroxyl group of the side chains of amino acids. the amino acids serine, threonine or tyrosine.

Glycosylation is a specific process of the cell type and the moment of cell development, and that shows alterations associated with some pathologies. Glycosylation also determines the interaction of proteins, their functionality and subsequent processing. The pattern of glycosylation 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 exposed terminal sugars on glycoproteins with very high specificity.

WO2012056008A1 describes the O-glycosylation of an amino acid of Ap as a biomarker for the diagnosis of AD and describes, among others, the following techniques to detect the O-glycosylated amino acid in Ap: ELISA, mass spectrometry, emission tomography positron (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 sAPPp glycosylation patterns as biomarkers for the diagnosis of AD.

On the other hand, the O-glycosylation of specific Ap residues in CSF and the differences in the levels of glycosylation of said residues between patients with AD and control subjects have been characterized (Halim et al., 2011). This document, focused on Ap glycosylation, however, does not describe anything about the glycosylation patterns of sAPPa or sAPPp, with highly N-glycosylated residues, apart from O-glycosylation; nor of their usefulness as biomarkers for the diagnosis of AD.

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.

DESCRIPTION OF THE INVENTION

Throughout the description and claims, the term "comprises", "comprising" and its variants are not of a limiting nature and, therefore, are not intended to exclude other technical characteristics. The term "comprise", "comprising" and its variants, throughout the description and claims, also includes, specifically, the term "consists of", "consisting of" and its variants.

As used in this description and 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.

Unless otherwise defined, all technical and scientific terms used throughout the description and claims have the same meaning as is commonly understood by a person skilled in the field of the invention.

For 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 at risk of having, said disease. Subjects who are affected by said disease can be identified by the symptoms that accompany 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 clinical examination of routine, or may be a subject at risk of developing the disease mentioned.

For purposes of this invention, the term "sample" refers to a body fluid sample, cell sample, tissue sample, or lavage/rinse fluid sample obtained from an external body surface or Preferably, the samples are samples of cerebrospinal fluid, urine, blood, whole blood, plasma, serum, lymphatic fluid, saliva, cells and tissues.

For the purposes of the present invention, "glycosylation pattern of sAPPa and/or sAPPp" refers, in general, to the set of carbohydrates linked to sAPPa and/or sAPPp. In the present invention, any recognition method can be used. patterns known in the state of the art, so that differences between the glycosylation pattern of sAPPa and/or sAPPp of the subject with respect to a reference glycosylation pattern are detected Advantageously, a numerical value or a range of values can be obtained binding assays that depend on the glycosylation pattern, allowing comparison between the glycosylation pattern of sAPPa and/or sAPPp of the subject with respect to the glycosylation pattern of reference sAPPa and/or sAPPp In the present invention, binding assays can be used to lectins to determine the glycosylation pattern of sAPPa and/or sAPPp In said lectin binding assays, a percentage or fraction of sAPPa and/or sAPPp bound to a lectin can be obtained, said percentage The percentage or fraction of sAPPa and/or sAPPp depends on the glycosylation pattern of sAPPa and/or sAPPp and allows the comparison between the glycosylation pattern of sAPPa and/or sAPPp in the subject with respect to the glycosylation pattern of sAPPa and/or sAPPp of reference.

For the purposes of the present invention, the term "compare" refers to contrasting the glycosylation pattern of sAPPa and/or sAPPp 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 sAPPp bound to lectins is compared with a reference percentage or fraction of sAPPa and/or sAPPp bound to lectins; an intensity signal obtained from the glycosylation pattern of sAPPa and/or sAPPp bound to lectins in a sample is compared with the same type of intensity signal of said glycosylation pattern of sAPPa and/or or lectin-bound sAPPp in a reference sample. Referred comparison can be carried out manually or computer assisted. 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 result of the identification referred to in this document may be provided automatically in a suitable output format.

In the present patent, the term "reference sAPPa and/or sAPPp glycosylation pattern", is derived from samples from healthy subjects, for whom it is known that they do not have Alzheimer's disease. A glycosylation pattern of sAPPa and/or o Suitable reference sAPPp may be determined, by the methods of the present invention, from a reference sample to be analyzed together, i.e. simultaneously, or subsequently, with the test sample Preferably, a cut-off value may be used as a reference value associated with the reference sAPPa and/or sAPPp glycosylation pattern.

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 depending on the glycosylation pattern of sAPPa and/or sAPPp in the sample to be analyzed. , with respect to a numerical value or range of values dependent on the glycosylation pattern of sAPPa and/or sAPPp, 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 p-value, for example, through binomial tests Preferred confidence intervals are at least 90%, when 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.

In the present patent, the term "indicative" refers to a difference between the glycosylation pattern of sAPPa and/or sAPPp in the sample to be analyzed, with respect to to the glycosylation pattern of sAPPa and/or sAPPp in a suitable reference sample, makes it possible to diagnose whether a subject has Alzheimer's disease.

For the purposes of the present invention, "Western blot", also called "immunoblot" or "electroblotting", refers to an analytical technique used in cell and molecular biology to identify specific proteins in a complex mixture of proteins, such as the one shown presented in cell or tissue extracts.The technique uses the following three steps: separation by size, transfer to a solid support and, finally, visualization by protein binding to appropriate primary or secondary antibodies.

For 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 linked to an enzyme (peroxidase, alkaline phosphatase, etc.) capable of of generating 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 that, in turn, Once it is recognized by a secondary antibody bound to said enzyme, the antigen can be detected indirectly in the sample by means of color changes measured by spectrophotometry.

For the purposes of the present invention, "alternative splicing " refers to a procedure in which different isoforms of mRNA and proteins, which may have different functions, are obtained from a primary mRNA or pre-mRNA transcript. process occurs primarily in eukaryotes.

For purposes of the present invention, the term "pan-specific antibodies" refers to antibodies with specificity against a specific domain uniquely present in a target, eg, the unique 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:

(a) determining, in a biological sample of said subject, the glycosylation pattern of sAPPa and/or sAPPp,

(b) comparing said sAPPa and/or sAPPp glycosylation pattern to a reference sAPPa and/or sAPPp glycosylation pattern, wherein a difference in said comparison is indicative of a positive diagnosis of Alzheimer's disease in said subject.

In an embodiment of the method of the invention, said biomarker is sAPPa.

In an 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.

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 lectin binding, immunohistochemistry, Western blot and flow cytometry.

In a preferred embodiment of the method of the invention, said glycosylation pattern is determined by lectin binding assays.

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.

In one embodiment of the method of the invention, in said lectin binding assays the amount or concentration of sAPPa and/or sAPPp is detected, 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 sAPPp is carried out by ELISA or Western blot.

In one embodiment of the method of the invention, sAPPa and/or sAPPp are derived from any of the variants of the amyloid precursor protein (APP). Preferably, said APP variants are selected from the group consisting of: APP695, APP751 and APP770.

In one embodiment of the method of the invention, a decrease in a subject, with respect 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 of healthy subjects, in the level of sAPPp derived from APP695, not bound to Con A or PHA lectins, is indicative of a positive diagnosis of Alzheimer's disease; and wherein an increase in a subject, relative to a reference value derived from samples from healthy subjects, in the level of sAPPp derived from the combination of the APP-KPI variants, APP751 and APP770, not bound to Con A lectins or PHA, is indicative of a positive diagnosis of Alzheimer's disease in said subject.

Information on the sequence of human APP and on APP proteolytic processing variants, along with information on amino acids with potential glycosylation, is accessible in the UniProtKB protein database (Accession No. P05067). According to this registry of human APP in the UniProtKB protein database, the following amino acids of APP may have potential glycosylation (numbering for the major variant APP695):

N-glycosylation: amino acids 542, 571

O-glycosylation: amino acids 633, 651, 652, 659, 663, 667, 681.

Amino acids 1-17 of APP correspond to the signal peptide. The sAPPp 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 UniProtKB protein database (Accession No. P05067).

The present invention also provides the sAPPa and/or sAPPp biomarkers for use in an in vitro method for diagnosing Alzheimer's disease in which the glycosylation pattern of sAPPa and/or sAPPp in a biological sample is determined.

In an embodiment of the biomarkers for use of the invention, said biomarker is sAPPa.

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.

In one embodiment of the biomarkers for use of the invention, the biological sample has been obtained from a subject.

The present invention also provides a diagnostic kit for Alzheimer's disease, comprising reagents for determining the glycosylation pattern of sAPPa and/or sAPPp in a biological sample.

In one embodiment of the kit of the invention, said reagents comprise the Con A lectin and/or PHA.

In one embodiment, the kit of the invention also comprises specific antibodies against sAPPa and/or sAPPp.

In one embodiment, the kit of the invention further comprises at least one buffer solution.

Examples of buffers are: phosphate buffer, phosphate buffer saline, acetate buffer, borate-chloride buffer, carbonate buffer, glycine buffer and Tris buffer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 . Increased APP expression in AD brain tissue. Relative expression of APP mRNA analyzed by qRT-PCR in frontal cortex tissue of control subjects (C) (n = 7) and subjects with AD (stage V-VI on the Braak and Braak scale, n = 7). The values were calculated from relative standard curves, normalized to 18Sa ribosomal subunit mRNA from the same cDNA. A p value <0.001 was obtained with respect to C.

Figure 2 . Schematic representation and biochemical characterization of NTF and CTF fragments of APP. ( A ) Schematic representation of the proteolytic fragments sAPPa, sAPPp, CTFa and CTFp generated by α-secretase (non-amyloidogenic pathway) and p-secretase (amyloidogenic pathway) (not drawn to scale). The location of the KPI domain present in variants APP751 and APP770, but not in APP695, is indicated. Epitopes for the antibodies used in this study are also shown. ( B ) To assess the identity of higher molecular mass sAPPa and sAPPp species derived from KPI variants, two brain extracts were processed in parallel and tested separately by electrophoresis under denaturing conditions with the indicated antibody to identify immunoreactive bands. anti-sAPPa antibody (generated in mouse), anti-sAPPp antibody (generated in rabbit) and anti-KPI (generated in rabbit). The band that coincides with all the antibodies is indicated by an arrow and the non-coincident band by an asterisk. 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 anti-sAPPp antibody, after deglycosylation treatment by incubation with specific N- and O-glycosidases, combined with Sialidase A, are represented under number 2. under denaturing conditions. The results of the control (buffer without the glycosidases) are represented under the number 1. The Western blot was resolved by simultaneously incubating with sAPPa (mouse) and sApPPp (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 "Binding" the bands that co-localize are shown). Positions corresponding to a molecular mass of 100 and 130 kDa are indicated.( D ) To characterize CTFa and CTFp, CHO-PS70 cells treated with the Y-secretase inhibitor, DAPT, or vehicle alone (DMSO) were analyzed. ; control: Ctrl) in parallel to control brain extracts (human frontal cortex: Cx).Immunoblots of CHO-PS70 extracts were detected simultaneously with two different antibodies: the C-terminal antibody generated in rabbit and recognizing a common domain to CTFa and CTFp; and the antibody generated in rat 2D8 and that recognizes the N-terminal domain of Ap which, therefore, only detects CTFp (the band that accumulates after treatment with DAPT is indicated by an arrow). Positions corresponding to a molecular mass of 6.5, 10 and 14 kDa are indicated.

Figure 3 . The sAPPa and sAPPp variants remain unchanged in AD brain tissue. ( A ) Western blot of human frontal cortex samples from control subjects (n=7) and AD subjects (n=7) detected with antibodies against sAPPa. ( B ) Densitometric quantification of Western blot bands from control subjects (n=7) and AD subjects (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 vinculin as loading control. Calculations were performed in duplicate. ( C ) Western blot of human frontal cortex samples from control subjects (n=7) and AD subjects (n=7) detected with antibodies against sAPPp. ( D ) Densitometric quantifications of Western blot bands from control subjects (n=7) and AD subjects (n=7) detected with antibodies against sAPPp, of the APP-695 and APP-KPI variants, with equivalent amounts of protein loaded in each lane and using vinculin as loading control. Calculations were performed in duplicate. ( E ) Plot of the ratio obtained by dividing the values for the sAPP species (sAPPa or sAPPp) derived from the APP-KPI variant by those derived from APP695 for each sample. None of the comparisons resulted in statistically significant differences between the EA and C samples.

Figure 4 . CTFa and CTFp 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 (to full-length APP) antibody. ( B ) Densitometric quantification of CTFp ( B ) and CTFa ( C ) species, using GAPDH as a loading control to ensure equivalent amounts of protein were loaded in each lane. Calculations were performed in duplicate. ( D ) Graph of the ratio obtained for each sample by dividing the immunoreactivity of CTFp 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 sAPPa fragment derived from APP695 or APP-KPI variants from brain tissues of 7 control subjects (C) and 7 AD subjects that did not bind to immobilized PHA lectins ( A ) or With A ( B ). Graphical representation of the percentage of sAPPp fragment derived from APP695 or APP-KPI variants from brain tissues of 7 control (C) and 7 AD subjects that did not bind to immobilized PHA ( C ) or ConA ( D ) lectins. ). The p values are indicated in the figure (ns, not significant).

Figure 6 . Comparison of sAPPa and sAPPp glycosylation in brain tissue from control and AD subjects. Graphical representation of the percentage of sAPPa fragment derived from APP695 or APP-KPI variants from brain tissues of 7 control (C) and 7 AD subjects that did not bind to immobilized PHA ( A ) or ConA ( B ) lectins. ). Graphical representation of the percentage of sAPPp fragment derived from APP695 or APP-KPI variants from brain tissues of 7 control (C) and 7 AD subjects that did not bind to immobilized PHA ( C ) or ConA ( D ) lectins. ). The p-values are indicated in the figure.

Figure 7 . Comparison of brain glycosylation of sAPP between control subjects and subjects with AD. Graphical representation of the percentage of sAPPa fragment derived from APP695 ( A ) and APP-KPI ( B ) in the brain tissues of 7 control subjects ( C ) and 7 AD subjects that did not bind to immobilized PHA and Con A lectins. Graphical representation of the percentage of sAPPp fragment derived from APP695 ( C ) and APP-KPI ( D ) in the brain tissues of 7 control subjects (C) and 7 AD subjects that did not bind to immobilized PHA and Con A lectins. p-values are indicated in the figure (ns, not significant).

DESCRIPTION OF METHODS OF IMPLEMENTATION

Materials and methods

Biological samples

The study was approved by the ethics committee of the Miguel Hernández University of Elche (Alicante, Spain), and was carried out in accordance with the Declaration of Helsinki. The samples were received anonymously and identified only based on the code of the tissue bank.

Frozen samples of cerebral cortex from 7 AD patients (3 men and 4 women, age range 81 ± 12 years) and 7 healthy controls (3 men and 4 women, age range 65 ± 12 years). 15 years), were obtained from the neurological tissue bank of the CIEN Foundation-Project Research Unit Alzheimer (UIPA; Madrid), and the tissue bank of the Region of Murcia, both coordinated by the neuropathologist Dr. Alberto Rábano (CIEN-UIPA Foundation).

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 tissue interval was between 1.5 and 6 hours, with no significant differences in both groups. Control subjects were negative on histopathology, and had no history of neurological or psychiatric symptoms or impaired memory.

Processing of biological samples

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; pH7.4; 500mM NaCl; 5mM EDTA; 1% (w/v) Nonidet P-40; 0.5% (w/v) Triton X-100, supplemented with a cocktail of protease inhibitors (Sigma P834). The homogenate was sonicated using a Misonix Microson ultrasonic cell disruptor sonicator in 3 sets 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 bovine serum albumin (BSA) as a standard according to the bicinchoninic acid method (BCA; Pierce).

Cells that overexpress APP

CHO-PS70 cells stably over-expressing wild-type human APP (variant 751) and the Y-secretase catalytic subunit, presenilin-1, were cultured for 48 hours in six-well plates (350,000 cells/well). ) in Dulbecco's modified Eagle's medium (DMEM), supplemented with GlutaMAX™ (Gibco® Life Technologies, Paisley, UK), 5% fetal bovine serum (FBS; Gibco) and 100 µg/ml penicillin/streptomycin (Gibco ). Cells were treated with the ^y -secretase inhibitor DAPT (5 jM, 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 dimethyl sulfoxide (DMSO). After an 18-h exposure of cells to inhibitor, cells were washed twice with cold phosphate buffered saline (PBS) and 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 extracts were frozen at -80°C for further analysis.

RNA isolation and qRT-PCR analysis

Total RNA was isolated from brain tissue samples using the 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 (mRNA) molecules by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). First-strand complementary DNA (cDNA) was obtained by reverse transcription of total RNA (1.5 ^g), using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Life Technologies Paisley, UK), from according to the kit manufacturer's instructions. Polymerase chain reaction (PCR) was performed on the StepOne™ Real-Time PCR System (Applied Biosystems), using TaqMan Gene Expression Assays (Hs00169098-m1 for APP and Hs03003631 for APP). -g1 for 18S; Thermo Fisher) and TaqMan PCR Master Mix. Transcription levels were calculated by the 2"ACt comparative method with respect to the 18S ribosomal subunit cDNA.

western blot

Brain tissue samples (30 µg per well) were boiled at 95°C for 5 minutes, separated by electrophoresis on 7.5% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) in Tris-Tricine and subsequently transferred to 0.45 μm nitrocellulose membranes (Schleicher & Schuell Bioscience, GmbH, Dassel, Germany). The immunoreactive signal of APP bands in Western blots was quantified. APP species in the samples were detected using: a rabbit C-terminal anti-APP polyclonal antiserum (1:1000; Sigma Aldrich, St. Louis, MO, USA; designated here as Sigma-Ct); a rat monoclonal antibody called 2D8 raised against the N-terminal domain of Ap, thus that detects CTFp but not CTFa (1:50) (Willem et al., 2015), a polyclonal rabbit anti-sAPPp antiserum specific for the C-terminus of sAPPp (1:100; IBL, Hamburg, Germany; referred to here as IBL -p); a mouse anti-sAPPa monoclonal antibody specific for the C-terminus of sApPPa (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, anti-vinculin mouse monoclonal antibody, sc-73614 Santa Cruz; 1:1000 rabbit anti-vinculin antiserum, Sigma V4139) and GAPDH (1:10000 anti-GAPDH mouse monoclonal antibody; Proteintech 60004- 1) were used as loading controls. Band intensities were analyzed using Image Studio Lite (LI-COR) software. The antibodies used had a cross-reactivity between the sAPPa and sAPPp fragments of less than 1.5%, and none of the antibodies cross-reacted 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).

Enzymatic de-glycosylation

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 single O-linked glycans (including polysialylated) from the glycoproteins.

lectin binding

Cerebral cortex samples were incubated at 4°C overnight with terminal sugar-specific lectins: the Canavalia ensiformis lectin Concanavalin A (Con A) (Sigma; Catalog Number C9017), with high specificity for terminal mannose residues. , or the Phaseolus vulgaris lectin 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 by 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 maintained under the same conditions, but not incubated with a lectin. All analyzes were performed in duplicate.

Statistic analysis

All data were analyzed in SigmaStat (Version 2.0; SPSS Inc.), applying a Student's t-test (two-tailed) or Mann-Whitney U-test for individual comparisons, and determining exact p-values (p-values < 0.05 were considered significant). Results are presented as means ± standard error of the mean (SEM) and the correlation between variables was evaluated by linear regression analysis.

Example 1. Increased expression of APP in the brain of AD subjects qRT-PCR assays were performed to determine the expression of APP messenger RNA (mRNA) in AD, using primers corresponding to sequences in exons 10-11 of APP and that are common to the main cerebral 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 the brain tissue of AD subjects than in the brain tissue of 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 blots of brain tissue from AD subjects, a method that allowed discriminating different APP species, especially those with different molecular masses. However, sAPPa and sAPPp differ from each other by only 16 amino acids (representing 1-2 kDa in molecular mass), and sAPPa or sAPPp have been predicted to be only ~5-10 kDa smaller than full-length APP. , and therefore are not 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 be able to distinguish sAPPa or sAPPp, the discrimination of these proteins by SDS-PAGE electrophoresis, using pan-specific antibodies raised against the unique C-terminal domain of sAPPa and sAPPp.

For both sAPPa and sAPPp, there are 3 alternative splicing variants in the brain: the 695 amino acid variant, APP695, mainly expressed in neurons, and the variants APP751 and APP770, expressed in glial cells, which have the serine protease inhibitor domain. Kunitz KPI ( Kunitz-type serine protease inhibitor). The APP NTFs sAPPa and sAPPp are distinguished electrophoretically and by Western blot with pan-specific antibodies against the exclusive C-terminus of sAPPa and sAPPp. In the brain tissue samples, the different APP species and their long N-terminal fragments were detected in several bands that migrated in the 100-130 kDa range. The differences in molecular mass reflect alternative variants of the predominant neuronal species, the species designated APP695, and the APP751 and APP770 isoforms, both of which carry the KPI domain and are virtually indistinguishable from each other by size. All variants undergo the same processing by secretases and generate the same types of fragments, including sAPPa and sAPPp. 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 on Western blot, using panspecific antibodies against the specific C-terminal domains of sAPPa or sAPPp (Figure 2B). When an anti-KPI antibody was used, only the higher molecular mass species showed sizes compatible with the KPI immunoreactive bands.

Since the sAPPa and sAPPp variants derived from APP695 (non-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). For this, APP was completely de-glycosylated, observing that, after deglycosylation, all the bands identified with sAPPa and sAPPp antibodies did co-localize (Fig. 2C).

CTFa and CTFp do not differ between APP variants and were characterized using extracts from CHO-PS70 cells that stably overexpress wild-type human APP and the Y-secretase catalytic subunit, presenilin-1.

Extracts from these cells treated with the Y-secretase inhibitor, DAPT, were assayed with the C-terminal antibody, providing evidence for the accumulation of CTFa (also called C83 after its amino acid length) and CTFp (also called C99 after amino acid length). their amino acid length) in cell extracts, and from matching bands in parallel-loaded brain homogenates (Figure 2D). When tested with the rat 2D8 antibody raised against the N-terminal domain of Ap, CTFa and CTFp could be discriminated as the epitope recognized by this antibody is absent in CTFa (see scheme in Figure 2A).

After this biochemical characterization, the different isoforms of sAPPa and sAPPp in brain tissue extracts were evaluated. No differences were detected between samples C and EA in the relative levels of sAPPa (Figure 3A and 3B) or sAPPp (Figure 3C and 3D) derived from APP695 or APP-KPI. Despite the large differences in the APP695/APP-KPI ratios for the sAPPa and sAPPp species, there were no apparent differences in these ratios between the C and EA samples (Figure 3E). The accumulation of sAPPa-695 and sAPPa-KPI immunoreactive bands was correlated in both C (r=0.76; p=0.048) and EA (r=0.96; p<0.001) tissue extracts, although this correlation was not significant for the sAPPp species (C: r=0.19; p=0.69; EA: r=0.57; p=0.17). There were no significant correlations between sAPPa and sAPPp in the APP695 or APP-KPI isoforms in subjects with C or AD.

To assess whether APP-CTF levels were altered in brain extracts from AD patients, an antibody raised against the original C-terminal domain of full-length APP was used. Immunoreactivity for CTFa and CTFp in brain tissue (Figure 4A, Figure 4B, and Figure 4C) was similar between C and AD subjects. Furthermore, the CTFp/CTFa ratio did not indicate differences between the EA and C groups (Figure 4D).

Example 3. Binding of sAPPa and sAPPp to lectins

To compare the glycosylation pattern of sAPPa and sAPPp, samples of frontal cortex extracts from C subjects (n= 7) and AD subjects (n= 7) were incubated with the Con A lectin, which has high specificity for A nucleotide residues. terminal mannose, and the lectin PHA, 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 sAPPp was not recognized by each of the lectins (Table 1).

Table 1

The sAPPp 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-KPI species (mostly glial), and to the fact that each cell type has a particular glycosylation machinery. However, the different sAPPa fragments exhibited a similar pattern of binding to Con A or PHA in both groups. What was most interesting was to verify that the lectin binding pattern varied when comparing sAPPa and sAPPp, both for the 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 extent of glycosylation between sAPPa and sAPPp derived from the same variant (Figure 2C), suggests that different APP glycoforms are preferentially processed by the non-amyloidogenic pathway (α-secretase) or by the amyloidogenic pathway. (psecretase). In addition, this determination occurs both in neurons (APP695) and in glial cells (APP-KPI).

To study whether APP glycosylation differs in brain extracts from AD subjects compared to control subjects, the glycosylation pattern of sAPPa and sAPPp was compared (Figures 7A-7D). Significant differences were determined between the control group and the AD group in terms of 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 sAPPp that did not bind to Con A or to PHA in samples from patients with AD compared to controls in the APP695 species and in the APP-KPI species, although these differences were not statistically significant with the number of subjects in the present example and with the precision achieved in the present example. However, it is not ruled out that differences with greater statistical significance can be detected using more precise techniques or a larger number of patients. The results of this example indicated that APP glycosylation in the AD brain is impaired and primarily affects glycoforms processed by the non-amyloidogenic pathway.

LIST OF BIBLIOGRAPHICAL REFERENCES

Braak and Braak. (1991). Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol, 82(4), 239-259. doi:10.1007/BF00308809

Cuchillo-Ibañez 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 Natl Acad Sci USA, 108(29), 11848-11853. doi:10.1073/pnas.1102664108

Lopez-Font et al. (2017). Alterations in the Balance of Amyloid-p 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

Willem et al. (2015). n-Secretase processing of APP inhibits neuronal activity in the hippocampus. Nature, 526(7573), 443-447. doi:10.1038/nature14864

Claims (17)

1. An in vitro method of diagnosing Alzheimer's disease in a subject, comprising: (a) determining, in a biological sample of said subject, the glycosylation pattern of the sAPPa and/or sAPPp biomarkers, (b) comparing said glycosylation pattern of the sAPPa and/or sAPPp biomarkers with a reference sAPPa and/or sAPPp glycosylation pattern, wherein a difference in said comparison is indicative of a positive diagnosis of Alzheimer's disease in said subject. 2. The method according to claim 1, wherein said biomarker is sAPPa. 3. 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. 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. The method according to claim 4, wherein said glycosylation pattern is determined by lectin binding assays. The method according to claim 5, wherein said lectins are Concanavalin A from Canavalia ensiformis (Con A) and/or PHA from Phaseolus vulgaris. The method according to claim 5 or 6, wherein, in said lectin binding assays, the amount or concentration of sAPPa and/or sAPPp is detected by discriminating between bound or unbound lectins. 8. The method according to claim 7, wherein the detection of the amount or concentration of sAPPa and/or sAPPp is carried out by ELISA or Western blot. 9. The method according to any of claims 1 to 8, wherein sAPPa and/or sAPPp are derived from any of the variants of the amyloid precursor protein (APP). 10. The method according to any of claims 1 to 9, wherein said APP variants are selected from the group consisting of: APP695, APP751 and APP770. 11. The method according to any of claims 1 to 10, wherein a decrease in a subject, with respect 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; wherein a decrease in a subject, relative to a reference value derived from healthy subject samples, in the level of APP695-derived sAPPp, not bound to Con A or PHA lectins, is indicative of a positive diagnosis of Alzheimer's disease ; and wherein an increase in a subject, relative to a reference value derived from samples from healthy subjects, in the level of sAPPp derived from the combination of the APP-KPI variants, APP751 and APP770, not bound to Con A lectins or PHA, is indicative of a positive diagnosis of Alzheimer's disease in said subject. 12. The sAPPa and/or sAPPp biomarkers for use in an in vitro method for diagnosing Alzheimer's disease in which the glycosylation pattern of sAPPa and/or sAPPp in a biological sample is determined. 13. The biomarkers for use according to claim 12, wherein said biomarker is sAPPa. 14. A diagnostic kit for Alzheimer's disease, comprising reagents to determine the glycosylation pattern of the sAPPa and/or sAPPp biomarkers in a biological sample, wherein said reagents comprise the Con A lectin and/or PHA and antibodies specific against sAPPa and/or sAPPp. 15. The kit according to claim 14, wherein said biomarker is sAPPa. 16. The kit according to claim 14 or 15, wherein said specific antibodies are the polyclonal antibody IBL-a, specific against sAPPa and the monoclonal antibody I BL-p, specific against sAPPp. 17. The kit according to any of claims 14 to 16, further comprising at least one buffer solution.