WO2014128329A1 - Method for the diagnosis and/or prognosis of alzheimer disease - Google Patents

Abstract

The invention relates to the use of the heterodimeric complex of presenilin comprising the N-y C-terminal fragments of same as a biomarker of Alzheimer disease. The invention also relates to a method and to a kit for the diagnosis and/or the prognosis of said disease by means of the use of said biomarker.

Description

 METHOD OF DIAGNOSIS AND / OR FORECAST OF DISEASE OF

 ALZHEIMER

DESCRIPTION

The present invention falls within the field of Alzheimer's disease, specifically within the methods of diagnosis and / or prognosis of this type of disease, which is based on the detection of presenilin 1 complexes in isolated biological samples.

STATE OF THE PREVIOUS TECHNIQUE

Alzheimer's disease (AD) is characterized at the brain level by two abnormal structures or proteinaceous deposits, amyloid plaques and neurofibrillary tangles. The plaques are composed of the β-amyloid peptide (Αβ), while the tangles consist of abnormally hyperphosphorylated tau protein (P-tau) clusters. Αβ and tau / P-tau are recognized biomarkers for AD and have been extensively researched. Analysis in cerebrospinal fluid (CSF) of the levels of Αβ1-42 (Αβ42) and tau species (total levels, T-tau, and P-tau forms) has demonstrated high sensitivity and specificity for the diagnosis of EA (Blennow et al., 2006, Lancet, 368: 387-403). However, there is a continuous search for new biomarkers in the CSF for use in support of clinical diagnosis, especially for the initial stages of AD, as well as for use in clinical trials.

Having an early and accurate diagnosis of AD is essential in advancing the treatment of this dementia. Numerous laboratories have reported increases in the levels of T-tau and P-tau in the CSF, but these markers also increase in other processes that occur with dementia, then there is a lack of specificity.

Regarding the other classic marker of AD, Αβ42, its generation is increased in the brain EA, however, and due to the increasing deposition of the peptide, there is a paradox that its CSF levels are reduced (Blennow et al. , 2010, Nat Rev Neurol., 6: 131-144).

 These classic biomarkers have shown value in the diagnosis of AD at an early level, but the need to identify other specific biochemical markers

i that allow retinal diagnosis persists. In addition, in the field of biomarkers of AD, one of the biggest deficits is the follow-up of therapies.

To date, most of the proteins involved in the pathological processing of the amyloid precursor protein (APP) are known, processing generated by the β-amyloid peptide. Αβ is generated through the successive action of two enzymes that act on APP, the enzymes β-secretase and γ-secretase (Thinakaran and Koo, 2008, J Biol Chem., 283: 29615-29619).

The β-secretase activity is mainly due to a protein called BACE1 (from "beta-site amyloid precursor protein cleaving enzyme 1"; Vassar et al., 2009, J Neurosa., 29: 12787-12794). BACE1 is present in the CSF as a soluble truncated form (Holsinger et al., 2004; Ann. Neurol., 55: 898-899). As BACE1 contains a single transmembrane domain, its presence in the CSF was not surprising and was characterized almost a decade ago. In this context, it has been proposed that an increase in BACE1 levels in the CSF of AD would reflect the elevation of BACE1 protein levels and activity in the pathological brain; although there is no consensus on whether this enzyme would be equally affected at different stages of the disease (Rosen et al., 2012, Neuromolecular Med., 14: 65-73).

In contrast, γ-secretase activity is due to a complex consisting of four subunits: presenilin-1 (PS1) (or presenilin-2), nicastrin, APH-1 ("previous pharynx-defective 1") and PEN -2 ("presenilin enhancer 2"). Presenilin-1 (PS1) is the catalytic subunit of the complex, it is an aspartyl protease with nine transmembrane domains.

DESCRIPTION OF THE INVENTION

The present invention proposes the use of the heterodimeric complex comprising the C-terminal (CTF) and N-terminal (NTF) fragments of presenilin 1 for the diagnosis and / or prognosis of Alzheimer's disease (AD), and therefore provides a alternative method of diagnosis and / or prognosis of AD based on the use of said heterodimeric complex.

Presenilin-1 (PS1) is the active component of the γ-secretase complex responsible for proteolytic processing of the β-amyloid precursor protein or APP, generating the Αβ peptide whose anomalous expression is one of the triggers of Alzheimer's disease. PS1 is a transmembrane protein that contains multiple hydrophobic regions, so its presence in cerebrospinal fluid (CSF) has not been measured to date.

Maturation of PS1 to its active form leads to endoproteolytic cleavage that cleaves the protein into two parts or N- and C-terminal fragments. Thus, by extracting the brain enzyme or cells in culture in the presence of detergents, only a small proportion of PS1 is extracted as its entire form.

The inventors of the present invention have detected that PS1 is present in the human CSF as hetero-com bundles of approximately 100-150 kDa of molecular mass composed of the C-terminal and N-terminal fragments of said protein. They have also shown that the level of stability of these complexes is significantly higher in the CSF of patients suffering from AD compared to cognitively normal subjects.

Thus, the present invention is a solution to the need to provide an alternative method of diagnosis and / or prognosis of Alzheimer's disease, detectable in a biological sample.

In addition, the method described in the present invention provides the advantage that it allows to carry out a diagnosis and / or prognosis of AD useful in the follow-up of therapies for said disease. The potential for secretases to be targets of new EA treatments makes it particularly interesting that the detection of the stability of PS1 heterodimeric complexes, as proposed in the present invention, represents a biomarker in clinical trials, in the monitoring of treatment and disease progression, where the use of current biomarkers is more discussed.

 Therefore, in a first aspect, the present invention relates to the use of the heterodimeric complex comprising the C-terminal and N-terminal fragments of PS1 for the diagnosis and / or prognosis of AD.

"Heterodimeric complex" means a complex that is formed by two different protein subunits. In the present invention the heterodimeric complex is composed of the monomers of the N-terminal fragment and the C-terminal fragment of the PS1 protein. "Presenilin 1" or "PS1", as explained above, is the active component or catalytic subunit of the γ-secretase complex responsible for proteolytic processing of the precursor protein of β-amyloid or APP, generating the Αβ peptide whose anomalous expression It is one of the triggers of AD. PS1 is an aspartyl protease with nine transmembrane domains that contains multiple hydrophobic regions. Preferably, the PS1 referred to in the present invention is the protein of SEQ ID NO: 1 or reference P49768.1 in the UniProtKB / Swiss-Prot database. The N-terminal or NTF fragment of PS1 is the fragment comprised between amino acid residues 1 to 298 of SEQ ID NO: 1. The C-terminal fragment or CTF of PS1 is the fragment comprised between amino acid residues 299 to 467 of the SEQ ID NO: 1.

The term "Alzheimer's disease" or "EA", as used in the present invention, refers to a neurodegenerative disease that manifests as cognitive impairment and behavioral disorders. It is characterized in its typical form by a progressive loss of memory and other mental abilities, as nerve cells degenerate and / or die and different areas of the brain atrophy.

"Diagnosis" means the procedure by which the presence or absence of AD in a subject is identified. The term "prognosis" refers to the procedure by which a prediction of the events that will occur in the development or course of AD is established, including an increase in the severity of the disease and the ability to respond to a given treatment in a subject. sick person suffering from AD

In a second aspect, the present invention relates to a method for in vitro diagnosis and / or prognosis of AD, hereinafter "method of the invention", which comprises the following steps: a) detecting heterodimeric complexes of Presenilin 1 comprising the C-terminal and N-terminal fragments thereof in a biological sample isolated from a subject,

 b) determine the stability of the complexes detected in step (a), c) compare the value obtained in step (b) with a control value, and

d) assign the subject of step (a) to the group of patients suffering from AD or to the group of patients with a poor prognosis of AD when the value obtained in step (b) is significantly greater than the control value.

The expression "detect the heterodimeric complexes of presenilin 1" in an isolated biological sample of a subject, as used in the present invention, refers to the measure of the presence of the C-terminal and N-terminal fragments of the complex Presenilin 1 heterodimeric in a biological sample. These complexes have a molecular mass of 100-150 kDa.

The detection of step (a) can be performed by means of protein or fragment detection techniques known to those skilled in the art, such as, but not limited to, by incubation with a specific antibody that recognizes the heterodimeric complex of PS1 comprising the N-terminal and C-terminal fragments thereof, or by incubation with an antibody that recognizes the N-terminal fragment, or by incubation with an antibody that recognizes the C-terminal fragment, or by incubation simultaneous with a specific antibody against the C-terminal fragment of PS1 and a specific antibody against the N-terminal fragment of PS1, in assays such as Western blot, electrophoresis gels, immunoprecipitation, protein arrays, immunofluorescence, immunohistochemistry, ELISA or any other protein detection method; by incubation with a specific ligand; by NMR or any other imaging technique; or, for example, by chromatographic techniques combined with mass spectrometry.

Electrophoresis can be, but not limited to, capillary electrophoresis, paper electrophoresis, agarose gel electrophoresis, polyacrylamide gel electrophoresis, isoelectric focusing or two-dimensional electrophoresis. Electrophoresis can be performed under denaturing or native conditions.

Chromatographic techniques can be based on the separation of molecules by their charge, size, molecular mass, by hydrophobicity, polarity or redox potential. The chromatography technique can be, but not limited to, liquid chromatography (partition chromatography, adsorption chromatography, exclusion chromatography or ion exchange chromatography), gas chromatography or supercritical fluid chromatography. Protein arrays technology is based, for example, on the fixation on a solid support of a molecule that recognizes the heterodimeric complexes of PS1 referred to in the present invention. The antibody-based microarray is the most common protein microarray. In this case, the antibodies are fixed on the solid support (the term chip can also be used to refer to microarray). These antibodies are used to capture molecules that allow the detection of proteins from biological samples. The term "solid support" as used in the present invention refers to a wide variety of materials, for example, but not limited to, ion exchange or adsorption resin, glass, plastic, latex, nylon, gel, esters of cellulose, paramagnetic spheres or the combination of some of them.

The detection of step (a) of the method of the invention can be carried out by any of the aforementioned techniques or by any combination thereof. Complexes can be detected by assessing their presence or absence. The detection can be carried out by means of the specific recognition of the complexes by means of any probe and / or any antibody.

In a preferred embodiment, step (a) of the method of the invention is carried out by Western blot. As will be shown later in the examples, under certain conditions of manipulation of the biological sample and / or the test conditions the stability and detection of the heterodimeric complexes of PS1 can be influenced. Therefore, in a more preferred embodiment, the isolated biological sample is processed at a temperature not exceeding 50 ° C for further analysis by Western blot in step (a) of the method of the invention. These conditions prevent the destabilization of the complexes, even in the presence of a denaturing agent for electrophoresis such as SDS, so that the stability results thereof that will be obtained later in step (b) will not be altered by the test conditions. and sample manipulation. In an even more preferred embodiment, the isolated biological sample is processed in the presence of SDS and at a temperature not exceeding 50 ° C for further analysis by Western blot in step (a) of the method of the invention.

In the present description, "Western blot" is understood as an analytical technique used to detect the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1 in a sample by its antigen-antibody binding, by chemiluminescence, fluorescence, or enzymatic activity among other methods. The PS1 protein is expressed in many peripheral organs, in addition to the brain. Therefore, the term "isolated biological sample", as used in the present invention, refers to an isolated sample of an organism that can come from a physiological fluid and / or any cell or tissue of an organism. In a preferred embodiment, the biological sample isolated from step (a) of the method of the invention is cerebrospinal fluid (CSF). In a more preferred embodiment, the biological sample is lumbar cerebrospinal fluid. In another preferred embodiment, the biological sample is ventricular cerebrospinal fluid. In another preferred embodiment, the isolated biological sample is not plasma.

The term "cerebrospinal fluid", as used herein, refers to a transparent colored liquid, which bathes the brain and spinal cord. Numerous diseases alter its composition and its study is important, and often determining, in diseases of the central or peripheral nervous system. Those diseases include neurodegenerative diseases such as Alzheimer's. There are different ways to obtain a sample of cerebrospinal fluid. Cisternal or suboccipital puncture involves placing a needle under the occipital bone (back of the skull). To obtain lumbar CSF, a lumbar puncture, commonly called a spinal tap, is the most common method. To obtain ventricular CSF, the ventricular puncture can be used in which a hole is drilled in the skull and a needle is inserted directly into one of the ventricles of the brain, or the ventricular CSF can be collected from a probe, such as a shunt or ventricular drain.

The stability of the heterodimeric complexes of PS1 in step (b) of the method of the present invention can be determined, this data being used as a reference to compare them with the data obtained in a control sample and look for some significant deviation. This significant deviation can be assigned to the diagnosis and / or prognosis of AD in the individual from whom the problem sample comes.

The term "determine stability" as used in the present invention refers to the definition of how stable the heterodimeric complex formed by the C-terminal and N-terminal fragments of presenilin 1 is. The stability of said complex It is determined by techniques that involve the exposure of the biological sample to denaturing conditions and subsequent evaluation of how these conditions have contributed to changes in the stability of the complexes. The Stability determination can be carried out by, for example, but not limited to, sucrose gradient ultracentrifugation, native electrophoresis or molecular exclusion chromatography. The use of, for example, but not limited to, changes in ionic strength, viscosity, temperature, pH, or the presence of destabilizing agents such as detergents, enzymes and enzyme inhibitors, or reducing agents, can directly affect the stability and detectability of PS1 complexes.

In a preferred embodiment of the method, the determination of the stability of the complexes in step (b) is carried out by a first step of sucrose gradient ultracentrifugation in the presence of detergents and a second Western blot step. The 100-150 kDa complexes identified by ultracentrifugation in sucrose gradients containing, preferably, Brij97 detergent, are more stable during subsequent electrophoretic analysis under denaturing conditions. While heavier complexes, such as those of about 200 and 250 kDa, turn out to be more unstable and are resolved in the electrophoretic analysis as 50 kDa components.

The term "sucrose gradient ultracentrifugation" as used in the present invention refers to the separation of the particles based on their flotation density, which is directly related to the size and mass of the particles. The sample is arranged above (or below) a linear or continuous density gradient, and prepared from a very high concentration of sucrose. When centrifuging each particle will move down (or up, if the sample has been deposited to the bottom) until it reaches a position where its density is equal to that of its surroundings (neutral buoyancy situation). Once the gradient is fractionated, it is examined in which fractions the particle in question is located and its position is determined by its sedimentation coefficient (S) whose determination, according to the method of Martin and Ames (1961, J. Biol. Chem ., 236: 1372-1379) is performed by comparing the distance traveled by the particle under analysis with the migration of a standard protein of known sedimentation coefficient.

The destabilizing effect of the detergents used in the sucrose gradient ultracentrifugation step will subsequently determine the stability of the heterodimeric complexes, so that those more stable complexes will better resist detergent-mediated destabilization. Examples of Detergents useful in this step of the method of the invention are, but are not limited to, Tween 20, CHAPS, Triton X-100 or Brij 97. In a preferred embodiment, the detergent is Brij 97.

The subsequent passage of Western blot will allow the identification of the different PS1 complexes based on their molecular mass. Performing electrophoresis under denaturing and reducing conditions [in the presence of SDS and β-mercaptoethanol, or other agents such as urea or DTT (dithiothreitol)] will also allow analyzing the stability of the components or subunits of these complexes.

In another preferred embodiment the control value of the method of the invention is the value of the stability of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1 in a biological sample isolated from a healthy subject.

"Control value" means any value or range of values derived from the determination of the stability of the heterodimeric complexes comprising the C-terminal and N-terminal fragments of presenilin 1 in a biological control sample from a healthy individual or in a mixture of biological samples derived from a control group.

In the present description, "control group" means a group of healthy individuals, of the same or similar age as the subject studied, from which values or ranges of stability values of heterodimeric complexes comprising the C fragments have been obtained. -terminal and N-terminal PS1 derived from the determination of stability of said complexes in a collection of biological samples from said healthy individuals, and which are representative of the population in which the method of the invention is to be applied. The determination of the stability of the heterodimeric complexes comprising the C-terminal and N-terminal fragments of PS1 must be carried out in the same way as that performed on the subject to be studied, and will be obtained from the same type of isolated biological sample that from the subject to be studied in step (a) of the method of the invention.

By "healthy", "healthy individual" or "healthy subject" is understood in the present invention that subject or individual who does not suffer from AD.

"Healthy population" in the present invention is understood as a set of individuals or subjects who do not have AD. "Healthy individuals representative of the population in which the method of the invention is to be applied" means those subjects who do not suffer from AD, at the time of extraction of the isolated biological sample to be analyzed and who as a group have a pattern similar in terms of, for example, but not limited to, race, age or gender distribution, than the population of patients or subjects to whom the method of the invention is to be applied.

The term "comparison", as used in the present invention, refers to the comparison of the stability of the complexes determined in step (b) with a control value. The comparison described in section (c) of the method of the invention can be performed manually or assisted by a computer.

An amount "significantly greater" than a control value can be established by a person skilled in the art by using different statistical tools, for example, but not limited, by determining confidence intervals, determining the p-value, Student's test. two-tailed, Mann-Whitney U test, Fisher discriminant functions, using Kruskal-Wallis analysis with Dunn post-hoc multiple comparisons test, one-way ANOVA with Bonferroni post-hoc multiple comparisons test , Kursal-Wallis U tests, or ROC (acronym for Receiver Operating Characteristic).

The isolated biological sample can be taken from a human, but also from non-human mammals, such as, but not limited to, rodents, ruminants, felines or canids. In another preferred embodiment of the method of the invention, the subject is a human.

The method of the invention can also be useful for determining the efficacy of a certain treatment that is being administered to a subject suffering from AD, and thus establishing a prognosis that helps clinical decision making. Therefore, in a more preferred embodiment of the method, the human is being subjected to a therapeutic treatment against AD.

The term "therapeutic treatment" refers to the set of means of any kind whose purpose is the cure or relief of the disease or the symptoms of AD, which may be, but not limited to, treatment with acetylcholinesterase inhibitors (such such as tacrine, rivastigmine, galantamine or donepezil), and / or with NMDA receptor antagonists (such as memantine); or from developing therapies (for example, but not limited to, active or passive immunization against Αβ, β-secretase inhibitors, γ-secretase inhibitors, or Θ8Κ-3β kinase inhibitors).

In a third aspect, the present invention relates to a method of obtaining useful data for the diagnosis and / or prognosis of AD which comprises the following steps: a) detecting the presenilin 1 heterodimeric complexes comprising the C-terminal fragments and N-terminal thereof in an isolated biological sample of a subject,

 b) determine the stability of the complexes detected in step (a), and c) compare the value obtained in step (b) with a control value.

The methods included in the present invention may additionally include a step in which, if it is determined that the subject has EA or a poor prognosis of AD, the treatment for the subject is determined.

In a fourth aspect, the present invention relates to a kit for the diagnosis and / or prognosis of AD comprising antibodies for the detection of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1, hereinafter hereinafter "kit of the invention".

The term "antibody for the detection of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, that is, molecules that contain a binding site of antigen that specifically binds (immunoreacts) with the heterodimeric complexes of PS1, or with the C-terminal and / or N-terminal fragments of presenilin 1 components of said complexes. The antibodies against the N-terminal or NTF fragment of PS1 and the antibodies against the C-terminal fragment or CTF of PS1 are capable of recognizing, by their individual or joint use, the heterodimeric complex of PS1 referred to in the present invention. Therefore, a preferred embodiment, the kit of the invention comprises specific antibodies against the N-terminal or NTF fragment of PS1 and / or specific antibodies against the C-terminal fragment or CTF of PS1. Antibodies against the N-terminal or NTF fragment of PS1 may be antibodies against the fragment comprised between amino acid residues 1 to 298 of SEQ ID NO: 1 or against fragments comprised within this fragment. In a preferred embodiment, the antibody against the N-terminal fragment or NTF of PS1 is an antibody against the fragment comprised between amino acid residues 1 to 65 of SEQ ID NO: 1. In another preferred embodiment, the antibody against fragment N - terminal or NTF of PS1 is an antibody against the fragment comprised between amino acid residues 21 to 34 of SEQ ID NO: 1. In another preferred embodiment, the antibody against the N-terminal or NTF fragment of PS1 is an antibody against fragment between amino acid residues 1 and 20 of SEQ ID NO: 1.

Antibodies against the C-terminal or CTF fragment of PS1 may be antibodies against the fragment comprised between amino acid residues 299 to 467 of SEQ ID NO: 1 or against fragments comprised within this fragment. In a preferred embodiment, the antibody against the C-terminal fragment or CTF fragment of PS1 is an antibody against the fragment comprised between amino acid residues 301 to 317 of SEQ ID NO: 1. In another preferred embodiment, the antibody against fragment C -terminal or CTF of PS1 is an antibody against the fragment comprised between amino acid residues 303 to 316 of SEQ ID NO: 1.

In another preferred embodiment, the kit of the invention comprises a specific antibody against the C-terminal fragment of presenilin 1 and a specific antibody against the N-terminal fragment of presenilin 1. In a more preferred embodiment, the kit of the invention further comprises detergents, reducing agents, denaturing agents and / or pH regulating compounds.

Examples of detergents that the kit of the invention may comprise are, but not limited to, Tween 20, CHAPS, Triton X-100 or Brij 97. In a preferred embodiment, the detergent is Brij 97.

"PH regulating compounds" means substances that regulate and adjust pH, essentially acids and bases. By "reducing agents" we generally mean that which gives electrons to an oxidizing agent and therefore participates in oxidation-reduction reactions, and more specifically the agent that breaks di-sulfide bonds, separating the protein in its sub-units Examples of reducing agents that the kit of the invention may comprise are, although without limit ourselves, β-mercaptoethanol or DTT (dithiothreitol). These agents are usually used in combination with denaturing agents such as urea or SDS detergent, among others; agents that destabilize the formation of hydrogen bridge bonds between amino acids, displaying the native structure of the proteins.

The kit of the invention may comprise, without any limitation, labeled or unlabeled primers, labeled or unlabeled probes, buffers, agents to prevent contamination, marker compounds such as, but not limited to, fluorochromes, etc. On the other hand, the kit of the invention can include all the supports and containers necessary for its implementation and optimization. The kit of the invention may contain positive and negative controls. Preferably, this kit further comprises the instructions for carrying out the detection and determination of the stability of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1 as described in the invention.

In a fifth aspect, the present invention relates to the use of the kit of the invention to carry out the method of the invention.

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Fig. 1. Represents the detection of PS1 complexes in human CSF by alternative antibodies. (A) Schematic representation of holoprotein PS1 with the detail of epitopes for anti-PS1 antibodies used in this study. (B) Sample of post-mortem ventricular CSF resolved with the indicated anti-PS1 antibodies.

Fig. 2. It shows that PS1 is present in human CSF and that high temperatures during sample preparation for electrophoretic analysis affect the stability of PS1 complexes. (A) Samples of human ventricular CSF obtained post-mortem from individuals without dementia (non-demented controls, ND) and with EA were analyzed by Western blotting with an anti-NTF-PS1 antibody (Calbiochem) and an anti-CTF-PS1 antibody (00/2). (B) Samples of human CSF were immunoprecipitated with anti-NTF antibody 98/1 and precipitated proteins (IP) were analyzed by Western blotting with anti NTF and CTF antibodies of PS1. Samples incubated with A-Sepharose protein with the omission of the 98/1 antibody were analyzed in parallel as negative controls. (C) The effect on heating PS1 complexes was determined for different periods of time, at 98 ° C and 50 ° C. Western blots were resolved with Calbiochem's anti-NTF antibody PS1.

Fig. 3. It shows that stable PS1 complexes are increased in ventricular and post-mortem CSF of patients with AD. Total immunoreactivity of the 100 and 150 kDa PS1 complexes determined by the Calbiochem anti-NTF antibody (A) or the 00/2 anti-CTF antibody (B) showed higher values in ventricular CSF CS samples (10 cases represented in white circles) than in samples of ND controls (5 cases, in black circles). (C) Eight of the 10 cases available for the EA group and 4 of the 5 cases for the ND group had sufficient volume for ultracentrifugation fractionation in 5-20% sucrose density gradients (w / v), in the presence of Brij 97 detergent. The aliquots obtained were analyzed by Western blotting under denaturing conditions to examine the PS1 complexes (Calbiochem anti NTF antibody). The internal markers were β-galactosidase (G), catalase (C) and alkaline phosphatase (P). The panel below (EA) shows the individual values for the ratio between stable complexes (resolved as aggregates of 100 and 150 kDa in fractions 2-7, close to catalase) and unstable complexes (heavier aggregates that settle in fractions 8-12, but they are resolved as aggregates of only 50 kDa). * Significantly different (p <0.05) compared to the ND group, according to the evaluation of the Mann-Whitney U test.

Fig. 4. Shows the immunoprecipitation of PS1 in lumbar CSF samples.

Non-pathological CSF samples were immunoprecipitated with anti-NTF-PS1 antibody called 98/1, and precipitated proteins (IP) were analyzed by Western blotting with Calbiochem anti-NTF-PS1 antibody. Negative controls of CSF samples incubated with A-Sepharose protein, with omission of the antibody are also shown.

Fig. 5. It shows that stable PS1 complexes are increased in the lumbar and ante-mortem CSF of patients with probable AD. (A) Immunoreactivity Total of the 100 and 150 kDa PS1 complexes determined by the anti-NTF Calbiochem antibody showed similar values in lumbar CSF samples of EA (12 cases represented in white circles) and ND controls (12 cases, in black circles) . (B) Eight of the 12 cases available for both groups had sufficient volume for fractionation by ultracentrifugation in sucrose density gradients and were analyzed as described in Fig. 3. * Significantly different (p <0.05) regarding the ND group, according to the evaluation of the Mann-Whitney U test.

EXAMPLES

The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the use of the heterodimeric complex of presenilin 1 for the diagnosis and / or prognosis of AD. These specific examples provided serve to illustrate the nature of the present invention and are included for illustrative purposes only, and therefore should not be construed as limitations on the invention claimed herein. Therefore, the examples described below show the invention without limiting its scope.

Example 1. Samples and methods.

The origin of the samples studied in the present invention and all the methods used to carry out the examples described below are described below.

Samples:

Post-mortem CSF: these ventricular CSFs were obtained post-mortem at the Tissue Bank, CIEN Foundation (Madrid, Spain), Alcorcón Hospital Foundation (Madrid, Spain) and Alzheimur Foundation (Murcia, Spain). Samples contaminated with blood were excluded from the analysis. The CSF was centrifuged at 1,000 * g for 15 min to remove the cells and insoluble material, before the biochemical analyzes. The cases of AD [n = 10 (5 women and 5 men), with an age of 77 ± 2 years (mean ± standard error of the mean; SEM)] were selected based on the clinical history of dementia and neuropathological diagnosis based on the CERAD criterion (Mirra et al., 1994, J Neuropath Exp Neuroi, 53: 303-315). The samples of the control cases without dementia (ND) and similar age did not show clinical or pathological characteristics of dementia [n = 5 (1 woman and 4 men), 74 ± 3 years]. They were used only samples of a post-mortem interval of less than 14 hours, without differences between the groups.

Ante-mortem CSF: lumbar CSF samples were obtained from 12 patients in mild or moderate phase of probable AD (5 men and 7 women, 71 ± 5 years) and 12 healthy volunteers (5 men and 7 women, 66 ± 9 years ) from Huddinge University Hospital (Stockholm, Sweden). All patients with AD met the NINCDS-ADRDA criteria for "probable" AD (McKhann et al., 1984, Neurology, 34: 939-944). The controls had no history or symptoms of neurological or psychiatric disorders, or subjective memory complaints, and had an MMSE score of 28 or higher. The CSF samples were aliquotted and frozen at -80 ° C until use.

Western blotting and immunoprecipitation assays:

 CSF samples (30 μΙ) were denatured at 50 ° C for 15 min for PS1 analysis. The samples were resolved by electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE).

Electrophoresis-separated proteins were transferred to nitrocellulose membranes (Schleicher & Schuell Bioscience GmbH) and analyzed with PS1 antibodies against N-terminal amino acids (aa): aa 1-65 (Calbiochem antibody), aa 21- 34 (from Thermo Scientific), or aa 1-20 (antibody 98/1, Evin et al., 2001, Biochemistry, 40: 8359-8368); or with antibodies against the C-terminal loop region: aa 303-316 (Sigma), or aa 301-317 (antibody 00/2; Evin et ai, 2001, Biochemistry, 40: 8359-8368) of SEQ ID NO: 1. All Western blots tested with the different antibodies were performed separately, avoiding striping of the membranes. Transfers were incubated with the corresponding secondary antibody conjugated to horseradish peroxidase and the signal was detected using an ECL Plus detection reagent, according to the manufacturer's instructions (GE Healthcare) on a Luminescent Image Analyzer LAS-1000 Plus (FUJIFILM) ). The signal detected in a control CSF sample was used to normalize the immunoreactive signal of each antibody. To estimate semi-quantitatively the intensity of immunoreactive bands was measured by densitometry using the Science Lab Image Gauge v 4.0 software provided by FUJIFILM.

Immunoprecipitations were performed at 4 ° C by incubation overnight of 150 μΙ of CSF with the primary anti-N-terminal antibody of PS1 98/1, previously coupled to protein A-Sepharose by pimelimidate dihydrochloride. dimethyl (Sigma-Aldrich Co). The precipitated proteins were washed with phosphate-saline buffer (PBS) and eiuido with 0.1 M glycine buffer at pH 2.5. After pH neutralization, the supernatants were denatured for electrophoretic analysis in Laemmli buffer at 50 ° C for 15 min and subjected to SDS-PAGE; The separated proteins were transferred to nitrocellulose membranes. The membranes were incubated with anti-N-terminal (Calbiochem) and anti-C-terminal (00/2) antibodies of PS1.

Ultracentrifugation in sucrose gradients:

PS1 complexes were analyzed by ultracentrifugation at 250,000 * g in a continuous sucrose gradient (5-20% w / v) centrifuged 4 hours at 4 ° C in a Beckman TLS 55 rotor. CSF aliquots (50 μΙ) were loaded carefully on the top of the gradient containing 2 ml of 50 mM Tris-HCI buffer (pH 7.4), containing 0.15 M NaCI, 50 mM MgCI ₂ and 0.5% (w / v) Brij detergent 97. After centrifugation, -14 fractions were collected manually from the top of the gradient tubes. As internal markers, enzymes of easy determination and known sedimentation coefficient, β-galactosidase, catalase and alkaline phosphatase were used.

Determination of T-tau, P-tau and A342 by ELISA:

 The levels of total tau (T-tau), phosphorylated tau (P-tau) and Αβ42 in CSF were determined using commercial ELISA kits (Innogenetics, Ghent, Belgium), following the manufacturers' protocols (Blennow et al., 1995, Mol. Chem. Neuropathology, 26: 231-245; Andreasen et al., 1999, Arch. Neurol., 56: 673-680; Vanmechelen et ai, 2000, Neurosa Lett., 285: 49-52).

Statistic analysis:

 All data were analyzed with the SigmaStat program package (Version 2.0, SPSS Inc.) using the Student's t-test or the Mann-Whitney U test, to determine the exact values of the p-value that reflects the statistical significance. (p values <0.05 were considered significant to mark differences between groups). The correlation between variables was evaluated by linear regression analysis. All results are presented as mean ± SEM (standard error of the mean).

Example 2. NTF and CTF PS1 are present as high molecular weight complexes in the CSF. Maturation of PS1 to its active form leads to endoproteolytic cleavage that cleaves the protein into two parts or Ny C-terminal fragments (NTF and CTF). Thus, by extracting the enzyme from the brain or cells in culture in the presence of detergents, only a small proportion of PS1 is extracted as its entire form. Thus, when the presence of PS1 in human CSF was tested by Western blot (first in post-mortem ventricular CSF), different anti-PS1 NTF and CTF antibodies were used. In Fig. 1A, a schematic representation of the structure of PS1 and the epitopes for antibodies used in this study can be observed. Western blot results using an anti-PS1 NTF antibody (Calbiochem) revealed the presence of a weak band of 29 kDa corresponding to the expected size for PS1-NTF, but also, surprisingly, the majority of immunoreactivity corresponded with bands of approximately 100 and 150 kDa (Fig. 2). Development with the PS1-CTF antibody called 00/2 also detects complexes of 100 to 150 kDa, and a weak band of approximately 20 kDa, corresponding to PS1-CTF (Fig. 2A).

To further confirm the identity of PS1 complexes in human CSF, immunoprecipitation experiments were performed followed by Western blotting (Fig. 2B). Samples of post-mortem CSF were immunoprecipitated using antibody 98/1, an antibody against PS1-NTF that has been proven effective in immunoprecipitation of human PS1. Western blot analysis of the bound fraction, both with anti NTF (Calbiochem) and anti CTF (00/2) antibody, confirmed the identity of the 100-150 kDa complexes of PS1, also revealing 50 kDa species, probably added of an NTF and a CTF of PS1, and not the complete holoprotein of PS1 in its native form. The complexes were not observed in the negative immunoprecipitation controls, where the anti-PS1 antibody was omitted (Fig. 2B). The use of alternative antibodies against NTF and CTF confirmed the specificity of the PS1 signal in CSF samples (Fig. 1B).

Different experimental conditions and the test protocol, especially factors such as the use of detergents, can affect the molecular interactions necessary for the formation of PS1 complexes and their subsequent detection. Since the denaturation temperature before electrophoresis is not standardized, the effect of different temperatures and heating time on the preparation of samples for electrophoresis was determined, and how this affects the stability / detectability of PS1 complexes. The high temperature used during sample preparation for electrophoresis (98 ° C compared to 50 ° C), is reflected in a total loss of PS1 immunoreactivity, particularly that of high molecular weight complexes (Fig. 2C). These experiments demonstrate that the manipulation of samples and variables in the test condition can influence the stability and detection of PS1 complexes. Thus, a large part of previous studies conducted with denatured samples at 98 ° C may underestimate and fail to detect PS1 complexes. In this study, all analyzes were performed on previously frozen samples at -80 ° C; avoiding freezing-defrosting cycles and controlling during the preparative denaturation of the electrophoresis the temperature at 50 ° C.

In the post-mortem ventricular CSF samples, PS1 levels were analyzed by antibodies to the NTF and CTF. PS1 immunoreactivity for 100 + 150 kDa complexes (summed immunoreactivity of both bands and detected with either antibody, Fig. 3A-B) is significantly higher in the EA group compared to NDC subjects. In addition, fractionation by ultracentrifugation in sucrose gradients showed that in EA cases highly stable PS1 complexes are more abundant compared to the situation in healthy subjects (analyzed in 8 of the 10 EA cases and in 4 of the 5 ND of those with sufficient volume; Fig. 3C).

Example 3. The levels of highly stable PS1 complexes are increased in the ante-mortem lumbar CSF of EA.

Also abundant bands of PS1 of approximately 100 and 150 kDa were detected in the lumbar CSF. The 50 kDa PS1 species were detected only in immunoprecipitates of NTF-PS1 and probably correspond to NTF and CTF PS1 heterodimers (probably aggregates of an NTF and a PSF CTF) originating from the 100 and 150 complexes after elution at acidic pH of the bound fraction: see immunoprecipitation protocol and result in Fig. 4. In the CSF no appreciable levels of NTF monomers of 29 kDa were detected (corresponding to the expected size for PS1-NTF). Results in CSF of post-mortem samples of ventricular CSF, showed that the levels of PS1 complexes are increased in cases with AD, particularly those of the most stable complexes. Therefore, a similar study was carried out in lumbar CSF samples of probable EA cases and control subjects. In the lumbar CSF, no significant changes were observed in the total PS1 levels between cases of probable AD and ND subjects (Fig. 5A). However, fractionation by ultracentrifugation in sucrose gradients yes it offered significant differences between the probable EA cases and control regarding the levels of the highly stable PS1 complexes (analyzed in 8 of the 12 EA and NDC cases of which there was sufficient volume; Fig. 5B).

Lumbar CSFs were characterized by determining classic Alzheimer's biomarkers, showing the group of probable AD levels of T-tau (692 ± 1 15 pg / mL with respect to ND: 311 ± 27 pg / mL) and P-tau (126 ± 21 pg / mL with respect to the ND: 57 ± 4 pg / mL) and low levels of Αβ42 (335 ± 18 pg / ml_ with respect to the ND: 911 ± 76 pg / mL). The ratio of highly stable PS1 complexes showed a positive correlation with the more specific markers of AD, Αβ42 (r = 0.51, p = 0.04) and P-tau (r = 0.57, p = 0.02).

Therefore, the results in lumbar CSF suggest that an early and significant phenomenon is the change in the formation dynamics of the heterodimeric complexes of PS1, which would be revealed in its stability, and this change seems to be a better marker to discriminate pathological samples that the mere use of total levels of heterodimeric complexes of PS1.

Claims

CLAIMS Use of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1 for the diagnosis and / or prognosis of Alzheimer's disease. Method for in vitro diagnosis and / or prognosis of Alzheimer's disease comprising the following stages:  a) detecting the presenilin 1 heterodimeric complexes comprising the C-terminal and N-terminal fragments thereof in an isolated biological sample of a subject,  b) determine the stability of the complexes detected in step (a), c) compare the value obtained in step (b) with a control value, and d) assign the subject of step (a) to the group of patients suffering from Alzheimer's disease or the group of patients with a poor prognosis of Alzheimer's disease when the value obtained in stage (b) is significantly greater than the control value. The method according to claim 2, wherein the control value is the stability value of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1 in a biological sample isolated from a healthy subject. The method according to any of claims 2 or 3, wherein the isolated biological sample is cerebrospinal fluid. The method according to any of claims 2 to 4, wherein the isolated biological sample is lumbar cerebrospinal fluid. The method according to any of claims 2 to 4, wherein the isolated biological sample is ventricular cerebrospinal fluid. The method according to any of claims 2 to 6, wherein the detection of step (a) is carried out by Western blot. The method according to claim 7, wherein the isolated biological sample is processed in the presence of SDS, and at a temperature not exceeding 50 ° C for analysis by Western blot. The method according to any of claims 2 to 8, wherein the determination of the stability of the complexes in step (b) is carried out by a first step of sucrose gradient ultracentrifugation in the presence of detergents and a second Western step blot The method according to any of claims 2 to 9, wherein the subject is a human. The method according to claim 10, wherein the human is being subjected to a therapeutic treatment against Alzheimer's disease. A kit for the diagnosis and / or prognosis of Alzheimer's disease comprising antibodies for the detection of the heterodimeric complex comprising the C-terminal and N-terminal fragments of presenilin 1. The kit according to claim 12 comprising a specific antibody against to the C-terminal fragment of presenilin 1 and a specific antibody against the N-terminal fragment of presenilin 1. The kit according to any of claims 12 or 13 further comprising detergents, reducing agents, denaturing agents and / or pH regulating compounds. Use of the kit according to any of claims 12 to 14, to carry out the method according to any of claims 2 to 1 1.