Method for the in vitro diagnosis of a neurodegenerative disease

Abstract

The invention relates to a method for the early in vitro diagnosis of a neurodegenerative disease in a human or an animal subject, the method comprising the step of detecting the presence of at least one marker chosen from among forms derived from amyloid beta peptides (Aβ) chosen from among the oligomers of these peptides and the prefibrillar and fibrillar aggregate forms of these peptides, and forms derived from phosphorylated tau proteins chosen from among the hyperphosphorylated forms of these proteins, the aggregate forms of these proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, the presence of the one or more markers being detected in a stool sample from this subject.

Description

[0001] DESCRIPTION

[0002] TITLE: METHOD FOR THE IN VITRO DIAGNOSIS OF A NEURODEGENERATIVE DISEASE

[0003] The present invention relates to the detection of neurodegenerative diseases (NDDs), and more specifically to the detection of these diseases at an early stage.

[0004] Neurodegenerative disease is a chronic, progressive disease that affects the nervous system and leads to irreversible cognitive, behavioral, sensory, and motor disorders. Alzheimer's disease is the most common form of dementia in humans, accounting for 60 to 70% of all cases, followed by Parkinson's disease, followed by diseases such as Lou Gehrig's disease and Huntington's disease. They are all characterized by a progressive loss of neuron function and a major risk factor: aging.

[0005] Several neurodegenerative diseases, and in particular the aforementioned diseases, are classified as proteinopathies, in that they are associated with the presence of proteins in an abnormally aggregated and / or misfolded form, the accumulation of which in the brain leads to toxicity. Belonging to this group of pathologies are tauopathies which have in common an abnormal accumulation of tau proteins in the form of clusters and which share clinical and pathological similarities. The invention relates to the diagnosis of such diseases and particularly those associated with at least one of the amyloid beta and tau proteins.

[0006] The present invention is described with reference to tauopathies, in particular Alzheimer's disease, but is not limited thereto and extends to all MNDs meeting the above definition.

[0007] Alzheimer's disease (AD) is an incurable neurodegenerative disease that begins with a long asymptomatic phase and is characterized by a progressive decline in cognitive abilities accompanied by neuronal and synaptic loss.

[0008] There is currently no curative treatment for Alzheimer's disease and the failure of recent therapeutic strategies is largely the result of late diagnosis of the disease when the first characteristic signs are already visible.

[0009] The diagnosis of AD has, however, evolved considerably in recent years with the identification of the concomitant presence of two types of cerebral lesions, characteristic of the disease: extracellular deposits of amyloid peptides (Ap), in particular Ap42, designated as amyloid plaques or senile plaques, and intraneuronal deposits of hyperphosphorylated tau proteins (P-tau), associated with neurofibrillary degeneration (NFD). Thus, to date, the detection of AD relies on neuropsychological tests, the measurement of amyloid peptides and hyperphosphorylated tau proteins in the cerebrospinal fluid (CSF), morphological imaging (MRI) and positron emission tomography (PET) technology to detect functional tracers (radioactive fluorodeoxyglucose PET or 18 FDG) or molecular (PET-amyloid and PET-tau).

[0010] Detection of AD markers, Ap and tau, is performed in CSF because it contains proteins from the brain, but CSF collection can be difficult depending on the patient's age and health status because it requires a lumbar puncture, an invasive procedure.

[0011] The interest of these markers lies in their synergistic use to refine the positive diagnosis and begin to provide prognostic information to the patient. However, even if they are very sensitive and specific for the positive diagnosis and for detecting pathophysiological lesions from the stage of memory complaints or minor cognitive disorders, well before cognitive decline and loss of autonomy, the diagnosis occurs in the clinical phase of AD, whereas it is preceded by a preclinical phase of around 20 years or more, during which no or only subtle symptoms of the disease are observed. The identification of markers in this phase of the disease would allow:

[0012] 1) to delay the onset of the disease or even prevent AD dementia; indeed, current treatments can only help improve clinical symptoms, but cannot delay or reverse the progression of AD;

[0013] 2) more upstream intervention of therapeutic strategies that could have greater effectiveness; and

[0014] 3) systematic monitoring of patients and the effectiveness of treatments.

[0015] Studies have been conducted on detecting a molecular signature of AD in a bodily fluid such as blood or saliva, which can be collected minimally invasively.

[0016] Regarding the Ap marker, it is produced in the brain and is indeed present in blood plasma. However, it is also produced in platelets and peripheral tissues, making it difficult to distinguish between brain-derived and produced Ap in plasma. In addition, plasma Ap42 concentrations are at least 100 times lower than in CSF (typically, in the order of a few pg / ml) and are diluted by the high concentration of other nonspecific protein components. Finally, plasma Ap peptides, due to their hydrophobic nature, tend to bind to erythrocytes and other plasma proteins, which may lead to epitope masking and thus prevent peptide detection.

[0017] As for the P-tau marker, secreted in the CSF, it is also present in blood plasma. A semi-sensitive test for this marker phosphorylated on threonine 181 (similar to the most widely used CSF test) with electrochemiluminescence detection has been developed. This test has shown that the plasma concentration of P-tau was higher in patients with Alzheimer's dementia than in controls (2 to 4 times). This increase appears in the preclinical phase of the disease, but can also occur episodically in other circumstances such as acute hypoxic brain injury in the case of concussion and during anesthesia, which has led to the rejection of this test.

[0018] Other candidate proteins in the blood have been found by the proteomic approach (Kitamura Y. et al., Plasma protein profiling for potential biomarkers in the early diagnosis of Alzheimer's disease, Neurological Research. Taylor & Francis, 39(3), pp231-238, (2017) but they did not provide satisfactory results.

[0019] Document WO2019 / 192969A1 describes a method for diagnosing or prognosing, at a so-called early stage, a disease associated with an amyloid peptide or protein in a biological sample, consisting of specifically detecting aggregates of this peptide or protein therein, or determining their number, concentration or size. A biological sample may be a sample of blood, urine, synovial fluid, stool or saliva. This method may be supplemented by a step consisting of detecting the tau protein, possibly phosphorylated, in said sample, or determining its number, concentration or size.

[0020] Document WO2021 / 239700A2 discloses a method for detecting oligomers and aggregated forms of the Ap peptide as pathological markers of a neurodegenerative disease, in particular Alzheimer's disease, in a stool sample, the method making it possible to determine the number, concentration and size of the aggregates. The choice of stool (or feces) considered as a reflection of the presence of these markers in the brain, cerebrospinal fluid and blood, spares the individual invasive sampling and the need for healthcare professionals to carry out the sampling. This method is applicable to the diagnosis, in particular early diagnosis, of Alzheimer's disease.

[0021] The accumulation of oligomeric and aggregated forms of amyloid peptides and phosphorylated forms of tau proteins are indeed markers of a neurodegenerative disease and the more specific their detection is compared to monomeric forms and the more sensitive it is, the earlier the diagnosis is made. But, as previously indicated, the preclinical phase is very long and this diagnosis occurs late, these aggregated forms being already ubiquitous and the clinical phase of the disease already initiated.

[0022] To date, there is still a crucial need to identify viable markers for AD that track disease progression over time and whose concentrations can change in response to disease treatment.

[0023] Surprisingly, a viable and reliable molecular signature of AD has been found to be present in the stool (or feces) of a human or animal at an early stage. These markers are beta-amyloid peptides (Ap) and phosphorylated tau proteins (P-tau) and their respective derivative forms, variations of which can characterize an early stage of proteinopathy-type neurodegenerative disease such as tauopathy, including Alzheimer's disease.

[0024] Thus, the invention relates to a method for the in vitro diagnosis of a neurodegenerative disease, in a human or animal individual, comprising the step which consists in detecting the presence of at least one marker chosen from the forms derived from beta-amyloid peptides (Ap) selected from the oligomers of said peptides and the prefibrillar and fibrillar aggregated forms of said peptides; and the forms derived from phosphorylated tau proteins (P-tau) selected from the hyperphosphorylated forms of said proteins, the aggregated forms of said proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, the presence of the marker(s) being detected in a stool (or feces) sample of said individual.

[0025] According to one implementation of the method of the invention, the neurodegenerative disease is a tauopathy, and the presence of at least one marker chosen from said forms derived from phosphorylated tau proteins is detected in a stool sample of said individual. According to another implementation of the method of the invention, the presence of at least one marker chosen from said forms derived from beta-amyloid peptides and at least one marker chosen from said forms derived from phosphorylated tau proteins is determined.

[0026] In all the objects of the invention described in the present text, the expression "at least one marker chosen from the forms derived from beta-amyloid peptides (Ap) and the forms derived from phosphorylated tau proteins (P-tau)", it should be understood that said object involves one or more markers chosen indifferently from the forms derived from beta-amyloid peptides (Ap) and the forms derived from phosphorylated tau proteins (P-tau). When it involves several markers, any combination thereof can be envisaged.

[0027] The detection of these markers in stool, which has the advantage of being less invasive and less expensive than their detection in CSF, is also reproducible and sensitive. Unexpectedly, it has proven to be specific for the early diagnosis of AD, and even able to reveal subjects at risk of developing AD. In addition, the biomarkers analyzed after sampling have been shown to be stable over time, and their concentration does not vary up to 7 days post-sampling, indicating that there is little, if any, degradation of the sample during this period. This is particularly the case for the tau protein and its derived forms, which degrade rapidly in a liquid biological sample, particularly aqueous, such as blood or serum (Schôll et al., Biomarkers for tau pathology, Molecular and Cellular Neuroscience, 97, pp. 18-33, 2019) and (Randall et al., Tau proteins in serum predict neurological outcome after hypoxic brain injury from cardiac arrest: results of a pilot study. Resuscitation 84, pp351-356, 2013). The authors observed that, in stools, these markers can be detected several days after sampling, even a week, without any change over time, either in their structure or in their concentration. Given the practicality of a stool sample that can be taken by the individual himself and then deposited or sent to a laboratory for analysis, this discovery is a real step forward in the diagnosis of tauopathies.

[0028] The present invention makes a real contribution to the diagnosis of neurodegenerative diseases, such as Alzheimer's disease.

[0029] As mentioned above, beta-amyloid peptides (Ap) and phosphorylated tau proteins and their respective derivative forms are a group of known and well-identified molecules. Their definitions are recalled below.

[0030] Beta-amyloid peptide (Ap) is a 38- to 42-amino acid structure resulting from the enzymatic cleavage of the amyloid precursor protein (APP) involving beta- or gamma-secretase enzymes. The gene encoding this protein is located on chromosome 21. The soluble beta-amyloid peptide has the ability to oligomerize into soluble oligomers of various sizes and to aggregate to form prefibrillar structures and fibrillar amyloid plaques, which are insoluble. Thus, by derived form of the beta-amyloid peptide, we mean oligomers of the beta-amyloid peptide regardless of their size, and the prefibrillar and fibrillar aggregated forms (Chen, Gf., et al. Amyloid beta: structure, biology and structure-based therapeutic development. Acta Pharmacol Sin 38, 1205-1235 (2017).It should be noted that, according to a particular implementation of the invention, namely detection by DotBIot, these different conformations can be distinguished. This is an additional advantage for the invention, because the oligomeric forms, which are not yet aggregated, are the most toxic. In the definition of derived form of the Ap peptide, also included are modified peptides resulting from one or more post-translational modifications such as a truncated Ap peptide.

[0031] Tau is a protein encoded by the microtubule-associated protein tau (MAPT) gene on human chromosome 17 and is described in Xia et al. Molecular Neurodegeneration 16, 37 (2021). It is a water-soluble protein and has approximately 80 potential phosphorylation sites, on the amino acids serine and threonine. In a healthy individual, some of these sites are phosphorylated under physiological conditions, but in certain pathological conditions, and particularly in MND, the protein is highly phosphorylated (referred to as P-tau, phosphorylated tau, or hyperphosphorylated tau). According to the invention, phosphorylated tau refers to an abnormal phosphorylation of certain previously identified key sites of the tau protein.Phosphorylated tau protein derivatives include hyperphosphorylated forms found at disease sites, aggregated tau proteins, and modified proteins resulting from one or more post-translational modifications such as truncated tau protein.

[0032] The derived forms of the Ap peptide or the tau protein resulting from post-translational modification(s) are a concept known to those skilled in the art. As examples, reference may be made to the following articles:

[0033] Carroll T, Guha S, Nehrke K and Johnson GVW; Tau Post-Translational Modifications: Potentiators of Selective Vulnerability in Sporadic Alzheimer's Disease. Biology 2021, 10, 1047;

[0034] Alquezar C, Arya S and Kao AW; Tau Post-translational Modifications: Dynamic Transformers of Tau Function, Degradation, and Aggregation. Forehead. Neurol. 2021, 11:595532; And

[0035] Barykin EP, Mitkevich VA, Kozin SA and Makarov AA; Amyloid p Modification: A Key to the Sporadic Alzheimer's Disease? Forehead. Broom. 2017, 8:58.

[0036] The Ap peptide and the tau protein are water-soluble entities; as previously indicated, they tend to form oligomers which, as they increase in size, become less and less water-soluble, evolving into aggregates which are insoluble in aqueous media.

[0037] As a marker selected from beta-amyloid peptides (Ap) and phosphorylated tau proteins (P-tau), as well as their respective derived forms, it is understood that they are in the free state, or in a state combined with one or more other entities such as other proteins / peptides, not preventing their detection.

[0038] In a variant of the invention, beta-amyloid peptides (Ap) and phosphorylated tau proteins (P-tau), as non-specific monomers for an early diagnosis of a neurodegenerative disease, may be used as marker(s) in calculating a ratio of derived forms to all forms of said peptide or protein; all forms including peptides (Ap) and derived forms thereof as described above or phosphorylated tau proteins (P-tau) and derived forms thereof as described above, respectively.

[0039] According to a preferred implementation of each of the methods, kits and uses of the invention, at least one marker chosen from said forms derived from beta-amyloid peptides, and at least one marker chosen from said forms derived from phosphorylated tau proteins, is detected.

[0040] By early stage according to the invention, we mean a stage at which mild symptoms of dementia have not yet appeared or appear, without a direct link having been established with a neurodegenerative disease. For example, it may correspond to the stage at which the first memory disorders are detected or at which other disorders are observed, in particular during screening campaigns such as those implemented from the age of 50 for cancers. The authors also discovered that at this early stage, said derived forms of beta-amyloid peptides and said derived forms of phosphorylated tau proteins are not accumulated in the brain. This means that at this early stage, the brain is still capable of eliminating these derived forms; the latter may therefore be present there but not in a pathological manner since they do not accumulate there.It appears that at this early stage according to the invention, biological tissues and liquids do not contain the same forms of molecules, monomeric and soluble, or aggregated and insoluble. Compared to the state of the art and in particular compared to the aforementioned documents, WO2019 / 192969A1 and WO2021 / 239700A2, the invention is based on the demonstration of a process of elimination of the derived forms of Ap peptides and tau proteins produced in the brain, in the stools where they can be detected before their toxic accumulation in the brain.

[0041] The presence of these derived forms in the brain can be determined by medical imaging such as positron emission tomography (PET); those skilled in the art can refer to the following documents:

[0042] Brain imaging using PET and radiotracers will reflect reliably on fibrillar deposits of amyloid in the brain that are > 1 mm in size (20-22) 20. Saint-Aubert, L., Lemoine, L., Chiotis, K., Leuzy, A., Rodriguez-Vieitez, E., and Nordberg, A. (2017) Molecular neurodegeneration 12, 19 Garibotto, V., Herholz, K., Boccardi, M., Picco, A., Varrone, A., Nordberg, A., Nobili, F., Ratib, O., and Geneva Task Force for the Roadmap of Alzheimer's, B. (2017) Neurobiology of aging 52, 183-195

[0043] Chiotis, K., Saint-Aubert, L., Boccardi, M., Gietl, A., Picco, A., Varrone, A., Garibotto, V., Herholz, K., Nobili, F., Nordberg, A., and Geneva Task Force for the Roadmap of Alzheimer's, B. (2017) Neurobiology of aging 52, 214-227.

[0044] It was further found that the said derived forms of Ap peptides and phosphorylated tau proteins detected in the stool are different from those present in the intestine, suggesting that they reach the stool by a route other than the digestive tract. This is corroborated by the study described in A. Lionnet et al, Acta Neuropathologica Communications (2018) 6:65.

[0045] In a variant, the method of the invention comprises the following steps: said stool sample of said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one of said markers, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, and the presence of said complex is detected, where appropriate, the presence of said complex determining whether the individual is affected by a neurodegenerative disease. According to a variant of the invention, this method allows the in vitro diagnosis of Alzheimer's disease in an individual.

[0046] As mentioned above, one of the advantages of the invention is to obtain an early diagnosis. In this respect, the tested individual may be asymptomatic or present some minor cognitive disorders that may not be directly associated with MND.

[0047] The invention also relates to a method for monitoring the condition of a human or animal individual suspected of suffering from a neurodegenerative disease, comprising the following steps: a first stool sample from said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one marker chosen from beta-amyloid peptides, forms derived from said peptides, phosphorylated tau proteins and forms derived from said proteins, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, the content of said complex in the first sample is measured, then a second stool sample from said individual is brought into contact, said second sample being taken after the first sample, in the presence of said antibody or part of an antibody,under conditions allowing the formation of a complex between said antibody or part of antibody and said marker, the content of said complex is measured in the second sample, a variation between the contents of said complex in the first sample and in the second sample, indicating progression of the disease or regression of the disease.,

[0048] In a variant of this method, the antibody or antibody part specifically recognizes at least one beta-amyloid peptide or a derived form of a beta-amyloid peptide and forms a complex therewith, and a content of said complex in the second sample higher than the content of this same complex in the first sample indicates progression of the disease, at an early stage, or a content of said complex in the second sample lower than the content of this same complex in the first sample indicates regression of the disease.

[0049] According to another variant of a method for monitoring the condition of an individual, which can be combined with the previous one or one, the antibody or part of an antibody specifically recognizes at least one soluble phosphorylated tau protein or a form derived from a phosphorylated tau protein and forms a complex with it, and a content of said complex in the second sample higher than the content of this same complex in the first sample indicates progression of the disease, at an early stage, or a content of said complex in the second sample lower than the content of this same complex in the first sample indicates regression of the disease.

[0050] The method for monitoring the condition of a human or animal individual suspected of suffering from a neurodegenerative disease as presented above is preferably included at an early stage of the disease up to a stage where the individual presents disorders which have just been associated with MND, as opposed to a late stage of the disease. It has indeed been observed that at a late stage of the disease, an individual may experience alterations in the elimination of plaques, which results in a decrease in the content of markers of the invention in the stool, which, at this stage, should not be interpreted as a regression of the disease.

[0051] Any of the variants of a monitoring method above may be implemented on a stool sample from an individual being treated for said disease, in particular allowing the effectiveness of the treatment to be assessed.

[0052] Thus, the invention also relates to a method for screening a medicament for treating a neurodegenerative disease, in an individual suspected of suffering from such a disease, comprising the following steps: a first stool sample from said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one marker chosen from beta-amyloid peptides, the forms derived from said peptides, phosphorylated tau proteins and the forms derived from said proteins, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, the content of said complex in the first sample is measured, then a second stool sample from said individual is brought into contact, said second sample being taken after the first sample, in the presence of said antibody or part of an antibody,under conditions allowing the formation of a complex between said antibody or part of antibody and said marker, the content of said complex in the second sample is measured, a variation between the contents of said complex in the first sample and of said complex in the second sample, indicating the effectiveness or ineffectiveness of said drug.,

[0053] In a variant of this method, the antibody or antibody part specifically recognizes at least one beta-amyloid peptide or a derived form of a beta-amyloid peptide and forms a complex therewith, and the content of said complex in the second sample being lower than the content of this complex in the first sample indicates efficacy of the drug.

[0054] In another variant of the method, which may be combined with the above variant(s), the antibody or antibody portion specifically recognizes at least one phosphorylated tau protein or a derived form of a phosphorylated tau protein and forms a complex therewith, and a content of said complex in the second sample lower than the content of this complex in the first sample indicates efficacy of the drug.

[0055] The time between the collection of the first and second samples will vary depending on the process used. Thus, in a process for monitoring the condition of an individual undergoing therapeutic treatment, this time may be short and the process repeated more frequently than in a diagnostic process. However, since the process is non-invasive, it is possible to take multiple samples for individualized and close monitoring.

[0056] According to any of the above variants, the non-specific monomeric forms of Ap peptides and / or P-tau proteins, as non-specific monomers for an early diagnosis of a neurodegenerative disease, may be used as marker(s) in the calculation of the ratio of derived forms of said AP peptides or said P-tau proteins / all forms of said Ap peptides or said P-tau proteins.

[0057] The invention further relates to a kit for the in vitro detection of a neurodegenerative disease, at the early stage, of a human or animal individual, comprising at least one antibody or part of an antibody specifically recognizing at least one marker chosen from the forms derived from beta-amyloid peptides (Ap) selected from the oligomers of said peptides and the prefibrillar and fibrillar aggregated forms of said peptides;and among the derived forms of phosphorylated tau proteins selected from hyperphosphorylated forms of said proteins, aggregated forms of said proteins and modified phosphorylated tau proteins resulting from one or more post-translational modifications, and the means allowing the extraction, from a stool sample of said individual, of at least one marker chosen from beta-amyloid peptides (Ap), the derived forms, phosphorylated tau proteins and the derived forms of said proteins, and the formation of a complex between said antibody or part of antibody and said marker;

[0058] The kit of the invention may also comprise antibodies recognizing the monomeric forms of Ap peptides and / or P-tau proteins, non-specific for an early diagnosis of a neurodegenerative disease, these being able to be used as marker(s) in the calculation of the ratio of derived forms of said Ap peptides / total quantities of said Ap peptides and their derived forms, or of the ratio of derived forms of said P-tau proteins / total quantities of said P-tau proteins and their derived forms.

[0059] The invention also relates to the use of a marker chosen from the forms derived from beta-amyloid (Ap) peptides selected from the oligomers of said peptides and the prefibrillar and fibrillar aggregated forms of said peptides; and from the forms derived from phosphorylated tau proteins selected from the hyperphosphorylated forms of said proteins, the aggregated forms of said proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, for the in vitro diagnosis of a neurodegenerative disease, in an individual, said marker being detected in the stools of said individual. According to one use of the invention, the diagnosis is carried out at an early stage of the disease. In particular, it is intended for the in vitro diagnosis of Alzheimer's disease.

[0060] The detection of the derived forms of Ap peptides and phosphorylated tau proteins can be carried out by techniques within the reach of those skilled in the art. Thus, the AT8 antibody makes it possible to specifically detect hyperphosphorylated tau protein. There are antibodies against toxic oligomeric amyloid peptide (such as Apo, E22P), anti-fibrillar amyloid peptide (such as Ap-F, OC); the use of these antibodies is illustrated in the examples which follow.

[0061] The present invention is illustrated in the following example and with the support of the figures according to which:

[0062] [Fig. 1] and [Fig. 2] represent the quantity, determined by densitometric analysis and expressed as relative intensity, of toxic oligomeric amyloid peptides (E22P), prefibrillar amyloid peptides (OC) and P-tau proteins (Tau-P Thr181 , Tau-P Thr205) of a fraction of soluble peptides and proteins [Fig. 1] and a fraction of insoluble peptides and proteins [Fig. 2], extracted from feces of wild-type mice (WT) and AD transgenic mice (APP / PS1-21), as a function of time expressed in months: at 1 month, Fig. 1A and Fig. 2A; at 4 months, Fig. 1 B and Fig. 2B; at 6 months, Fig. 1C and Fig. 2C; at 9 months, Fig. 1 D and Fig. 2D.

[0063] Example

[0064] In a transgenic (TG) mouse model of Alzheimer's disease (AD) (APP / PS1-21 mice), the temporal progression (from 1 to 9 months) of AD was monitored in feces, brain, and intestines, respectively.

[0065] 1) Follow-up in the stool

[0066] Feces from wild-type (WT) and APP / PS1 mice were collected and proteins from these samples were extracted.

[0067] Procedure 1.1) Stool collection

[0068] Mice are housed in a clean cage for two hours, after which the droppings are collected and counted before being frozen at -80°C. Then the samples are thawed, 5-6 droppings (100 mg) are transferred to a tube and placed overnight in a 56°C incubator to be dehydrated. The dry material is weighed again to determine the % water content, which partly reflects intestinal function.

[0069] 1.2) Protein extraction

[0070] Dried stools are taken up in 1 ml of RIPA buffer (50 mM Tris-HCl pH8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing a cocktail of protease and phosphatase inhibitors. The homogenate is incubated for 10 minutes on ice before being subjected to 5 sonication cycles under the following conditions: 10 seconds of sonication (Amplitude 45) + 10 seconds in ice. The homogenate is then centrifuged for 10 minutes at 4°C at 14,000 rpm. The supernatant is collected in a clean tube. It contains soluble proteins.

[0071] The pellet is resuspended in 1 mL of 5M guanidine HCl / 50 mM Tris-HCl pH 8 buffer containing a cocktail of protease and phosphatase inhibitors. The homogenate is again subjected to 5 sonication cycles under the following conditions: 10 seconds of sonication (Amplitude 45) + 10 seconds in ice. The homogenate is then centrifuged for 10 minutes at 4°C at 14,000 rpm. The supernatant is collected in a clean tube. It contains the insoluble proteins.

[0072] The protein content of each fraction is determined using the BCA Protein Assay Kit (Pierce™).

[0073] 1.3) Analysis of fractions by DotBIot

[0074] Ten µg of protein were absorbed by capillary action onto a nitrocellulose membrane (Biorad apparatus) and processed for DotBIot analysis with antibodies against toxic oligomeric amyloid peptide (Apo, E22P), fibrillar amyloid peptide (Ap-F, OC) and anti-Tau phosphorylated on Threonine 181 (pTau Thr181 ) or Threonine 205 (pTau Thr205 ). Normalization of the results is carried out using an anti-GAPDH antibody or amido-black staining for the soluble fraction or after staining with Ponceau Red for the insoluble fraction.

[0075] 2) Monitoring in the brain and in the intestines

[0076] Proteins and peptides are recovered from different tissues and organs

[0077] Operating mode

[0078] 2.1) Dissection

[0079] At the different ages studied, mice were sacrificed by cervical dislocation. Brains and intestines from wild-type (WT) and APP / PS1 mice were collected. The brain was extracted from the skull and dissected to separate the cortex and hippocampus. The intestines were recovered and compartmentalized as follows: duodenum, jejunum, ileum, and colon. These samples were separated into 2 batches before being frozen (in liquid nitrogen and then stored at -80°C, in the OCT):

[0080] - one of the batches will be used for microscopic analysis after preparation of a 30 µm section)

[0081] - the other batch will be used for biochemical experiments (Western Blot) or molecular biology (QPCR).

[0082] 2.2) Protein extraction

[0083] Intestinal and brain samples are taken up in 1 ml of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS) containing a cocktail of protease and phosphatase inhibitors. The tissues are ground using an automated program on a Milteny grinder. The homogenate is then centrifuged for 10 minutes at 4°C at 2,500 rpm. The supernatant is collected in a clean tube. It contains the proteins. The protein content of each fraction is determined using the BCA protein assay kit (Pierce™).

[0084] 2.3) Analysis of lysates by Western Blot

[0085] Fifteen μg of protein are run on a SDS-PAGE polyacrylamide gel (BioRad) at 100 volts for one hour and 30 minutes. The gel is then electrotransferred onto a polyvinylidene fluoride membrane (BioRad) and processed for analysis with anti-oligomeric amyloid peptide (Apo, A11), anti-fibrillary amyloid peptide (Ap-F, OC) and anti-Tau phosphorylated on Threonine 205 (pTau) antibodies. Thr205 ) or Threonine 181 (pTau Thr181 ).

[0086] 2.4 Confocal microscopy

[0087] Paraformaldehyde (PFA)-fixed tissues from WT and APP / PS1 mice were immersed in 20% sucrose in PBS overnight, then frozen in chilled PFA and stored at -30°C. Sections (20-30 μm thick) were cut with a cryostat microtome (HM 500M, Microm). Sections were blocked by incubation with phosphate-buffered saline (PBS) containing 0.1% (v / v) Triton X100 and 3% BSA for 30 min at room temperature. The tissue sections were then incubated overnight at 4°C with primary antibodies directed against the different proteins of interest, washed 3 times with PBS containing 0.2% Tween20 (PBS-T) and incubated for 2 h at room temperature with the secondary antibodies conjugated to different fluorochromes.After three washes in PBS-T, nuclei were stained by incubation in Hoescht's solution (1:200) for 5 minutes and slides were mounted in fluorescent mounting medium (Dako).

[0088] 2.5) Monitoring in the brain The morphological study of spines and synaptic density is carried out by confocal microscopy using the Thy1-eYFP APP / PS1-21 mouse model. The distribution of Ap and P-tau proteins is also analyzed by confocal microscopy with antibodies specific for the different proteins. The presence of amyloid plaques is detected by confocal microscopy using thioflavin-T. The expression levels of these different proteins are determined by Western Blot, DotBIot and / or Elisa.

[0089] 2.6) Follow-up in the intestines

[0090] As in the brain, the distribution of Ap and P-tau proteins is analyzed by confocal microscopy with antibodies specific for the different proteins. This technique also allows us to understand the structure and morphology of the villi and intestinal crypts. The expression levels of these different proteins are determined by Western Blot, DotBIot. The inflammatory context will be validated by QPCR and Elisa assays of different cytokines.

[0091] 3) Results

[0092] Analysis of fecal Ap peptides (toxic oligomeric forms E22P and pre-fibrillar OC by specific conformational antibodies) and Tau-P proteins Thr181 and Tau-P Thr205revealed a significant difference in soluble fractions extracted from feces between WT and TG animals, as early as 1 month, before any detectable accumulation in the brain [Fig. 1A]. Indeed, transgenic mice eliminate significantly more toxic Apo (E22P), pTau Thr181 and pTau Thr205 than WT animals, at 1 month. These differences are maintained at 2 months (results not shown). From 4 months [Fig. 1 B] and [Fig. 1C], the differences between WT and TG animals are less significant. At 9 months [Fig. 1 D], the difference in the elimination of toxic Apo forms (E22P) and pTau Thr181 between WT and TG animals persist and increase. Similarly, at this age, a significant elimination of pre-fibrillar forms of Ap (OC) is observed in TG animals.

[0093] In the insoluble fractions, an increase in the detection of Tau-P proteins is observed Thr181 , Tau-P Thr205and Ap-F (OC) in TG animals compared to WT animals at 4 months of age [Fig. 2B]. From 6 months of age [Fig. 2C] and [Fig. 2D], WT animals eliminate pTau identically Thr181 , or even more than pTau Thr205 or Ap-F (OC) than transgenic animals.

[0094] In the brain, amyloid plaques are detected in the cortex and hippocampus of mice from 3 months of age. These observations are accompanied by morphological changes and a decrease in synapse density.

[0095] At the intestinal level, from 6 weeks, an accumulation of the toxic oligomeric form of Ap (Apo) and the pre-fibrillar form (A pF) is observed. However, there are no amyloid plaques at the intestinal level. This accumulation is associated with a disorganization of the epithelial barrier. These observations suggest a modification of the barrier properties leading to an alteration of the nutrient absorption capacities and / or the transit motility of TG mice and the development of an inflammatory context.

[0096] These results demonstrate that, unexpectedly, the gastrointestinal tract mirrors what is happening in the brain at the level of Ap and tau. According to the invention, robust molecular and histological changes are observed, heralding AD in the intestine, even before the appearance of the first symptoms, i.e. before any brain damage and specifically to this disease.

[0097] The use of feces compared to blood or saliva markers has the advantage of being much earlier and more sensitive (the eliminated Ap peptides and tau proteins are stable and in significant quantities).

[0098] The measurement of these markers in stools therefore constitutes a powerful tool for the early diagnosis of AD and monitoring of disease progression.

[0099] Taken together, these observations indicate that intestinal changes parallel the progression of AD brain disease. The intestine also reveals an accumulation of Ap peptides and phosphorylated tau proteins in the feces that was detected before any accumulation in the brain. Indeed, an accumulation and then elimination of soluble proteins associated with intestinal morphological changes, followed by a clearance of insoluble proteins in the feces were observed.

Claims

CLAIMS 1. Method for the in vitro diagnosis of a neurodegenerative disease, in a human or animal individual, at an early stage, comprising the step of detecting the presence of at least one marker chosen from the forms derived from beta-amyloid (Ap) peptides selected from the oligomers of said peptides and the prefibrillar and fibrillar aggregated forms of said peptides, and the forms derived from phosphorylated tau proteins selected from the hyperphosphorylated forms of said proteins, the aggregated forms of said proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, said method being characterized in that the presence of the marker(s) is detected in a stool sample of said individual.

2. Method according to claim 1 for the in vitro diagnosis of a tauopathy, in a human or animal individual, at an early stage, comprising the step which consists in detecting the presence of at least one marker chosen from the forms derived from the phosphorylated tau proteins selected from the hyperphosphorylated forms of said proteins, the aggregated forms of said proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, in a stool sample of said individual.

3. Method according to claim 1, characterized in that it comprises the step which consists in detecting the presence of at least one marker chosen from said forms derived from beta-amyloid peptides (Ap) and a marker chosen from said forms derived from phosphorylated tau proteins.

4. Method according to any one of claims 1 to 3, characterized in that it comprises the following steps: said stool sample of said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one of said markers, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, the presence of a said complex is detected, where appropriate, the presence of a said complex determining whether the individual is affected by a neurodegenerative disease.

5. Method according to any one of the preceding claims, for the in vitro diagnosis of Alzheimer's disease, in an individual.

6. Method according to any one of the preceding claims, characterized in that the individual is asymptomatic.

7. Method for monitoring the condition of a human or animal individual suspected of suffering from a neurodegenerative disease, comprising the following steps: a first stool sample from said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one marker chosen from beta-amyloid peptides (Ap), the derived forms of said peptides, phosphorylated tau proteins and the derived forms of said proteins, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, the content of a said complex in the first sample is measured, then a second stool sample from said individual is brought into contact, said second sample being taken after the first sample, in the presence of said antibody or part of an antibody, under conditions allowing the formation of a complex between said antibody or part of an antibody and said marker, the content of a said complex in the second sample is measured,a variation between the contents of said complex in the first sample and said complex in the second sample, indicating disease progression or disease regression., 8. Method according to claim 7, characterized in that the antibody or part of antibody specifically recognizes at least one beta-amyloid peptide or a form derived from a soluble beta-amyloid peptide and forms a complex with it, and a content of said complex in the second sample higher than the content of this same complex in the first sample indicates progression of the disease, at an early stage, or a content of said complex in the second sample lower than the content of this same complex in the first sample indicates regression of the disease.

9. Method according to claim 7 or 8, characterized in that the antibody or part of antibody specifically recognizes at least one soluble phosphorylated tau protein or a form derived from a phosphorylated tau protein and forms a complex with it, and a content of said complex in the second sample higher than the content of this same complex in the first sample indicates progression of the disease, at an early stage, or a content of said complex in the second sample lower than the content of this same complex in the first sample indicates regression of the disease.

10. Method according to any one of claims 7 to 9, characterized in that said individual is treated for said disease.

11. Method for screening a drug for treating a neurodegenerative disease, in an individual suspected of suffering from such a disease, comprising the following steps: a first stool sample from said individual is brought into contact with at least one antibody or part of an antibody specifically recognizing at least one chosen marker among beta-amyloid peptides (Ap), the derived forms of said peptides, phosphorylated tau proteins and the derived forms of said proteins, under conditions allowing the formation of a complex between said antibody or part of antibody and said marker, the content of a said complex in the first sample is measured, then a second stool sample of said individual is brought into contact, said second sample being taken after the first sample, in the presence of said antibody or part of antibody, under conditions allowing the formation of a complex between said antibody or part of antibody and said marker, the content of a said complex in the second sample is measured, a variation between the contents of said complex in the first sample and of said complex in the second sample, indicating the effectiveness or ineffectiveness of said drug.

12. Method according to claim 11, characterized in that the antibody or part of antibody specifically recognizes at least one beta-amyloid peptide or a form derived from a soluble beta-amyloid peptide and forms a complex with it, and the content of said complex in the second sample lower than the content of this complex in the first sample indicates an effectiveness of the drug.

13. Method according to claim 11 or 12, characterized in that the antibody or part of antibody specifically recognizes at least one phosphorylated tau protein or a form derived from a soluble phosphorylated tau protein and forms a complex therewith, and a content of said complex in the second sample lower than the content of this complex in the first sample indicates an efficacy of the drug.

14. Kit for the in vitro detection of a neurodegenerative disease, at the early stage, of a human or animal individual, comprising at least one antibody or part of an antibody specifically recognizing at least one marker chosen from the forms derived from beta-amyloid (Ap) peptides selected from the oligomers of said peptides and the prefibrillar and fibrillar aggregated forms of said peptides; and from the forms derived from phosphorylated tau proteins selected from the hyperphosphorylated forms of said proteins, the aggregated forms of said proteins and the modified phosphorylated tau proteins resulting from one or more post-translational modifications, and the means allowing the extraction, from a stool sample of said individual, of at least one marker as defined above, and the formation of a complex between said antibody or part of an antibody and said marker, 15. Use of a marker selected from forms derived from beta-amyloid (Ap) peptides selected from oligomers of said peptides and aggregated prefibrillar and fibrillar forms of said peptides; and from forms derived from phosphorylated tau proteins selected from hyperphosphorylated forms of said proteins, aggregated forms of said proteins and modified phosphorylated tau proteins resulting from one or more post-translational modifications, for the in vitro diagnosis of a neurodegenerative disease, in an individual, said marker being detected in the stools of said individual.

16. Use according to claim 15, characterized in that the diagnosis is carried out at an early stage of the disease.

17. Use according to claim 15 or 16, characterized in that the neurodegenerative disease is Alzheimer's disease.

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