WO2013111045A1 - Use of waste products for detecting amyloidogenic proteinaceous material in living mammals - Google Patents

Use of waste products for detecting amyloidogenic proteinaceous material in living mammals Download PDF

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WO2013111045A1
WO2013111045A1 PCT/IB2013/050530 IB2013050530W WO2013111045A1 WO 2013111045 A1 WO2013111045 A1 WO 2013111045A1 IB 2013050530 W IB2013050530 W IB 2013050530W WO 2013111045 A1 WO2013111045 A1 WO 2013111045A1
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amyloidogenic
proteinaceous material
method according
proteins
amyloid
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PCT/IB2013/050530
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French (fr)
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Tristan BOLMONT
Patrick FRAERING
Theo Lasser
Mitko DIMTROV
Taoufiq HARACH
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Ecole Polytechnique Federale De Lausanne (Epfl)
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896Neurological disorders, e.g. Alzheimer's disease
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701Details
    • G01N2333/4709Amyloid plaque core protein
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/28Neurological disorders
    • G01N2800/2814Dementia; Cognitive disorders
    • G01N2800/2821Alzheimer

Abstract

Method for detecting amyloidogenic proteinaceous material induced by a proteopathy, for instance Alzheimer's disease, characterized by the fact that said amyloidogenic proteinaceous material is detected in a mammal waste product, such as feces.

Description

USE OF WASTE PRODUCTS FOR DETECTING AMYLOIDOGENIC PROTEINACEOUS MATERIAL IN LIVING MAMMALS

FIELD OF THE INVENTION

The present invention relates to the detection of amyloidogenic proteinaceous material, in particular, but not exclusively, for diagnosis purpose.

STATE OF THE ART

Proteins are involved in almost every biological process in a living organism. They are synthesized on ribosomes as linear chains of amino acids from information encoded within the cellular DNA. In order to perform their biological function these chains of amino acids must fold into the native three- dimensional structures that are characteristic of the individual proteins. How and whether a protein folds is influenced primarily by its amino acid sequence and the cellular environment surrounding the amino acid chain. Mutations, abnormal physiological concentrations, coupled with prolonged time and certain biochemical conditions are thought to destabilize the native three- dimensional state, or divert soluble proteins from their normal folding pathway, often leading to their aggregation into stable insoluble amyloid deposits. Numerous degenerative diseases arise due to the buildup of insoluble misfolded protein deposits. These proteopathies include neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and also bovine spongiform encephalopathy and its human equivalent Creutzfeld-Jakob disease, in addition to diverse systemic amyloidosis (Table 1 ).

No sequence or structural similarities are apparent between any of the proteins that display the ability to form amyloids. Despite these differences, the fibrils formed by different polypeptides share a number of structural characteristics. For example, X-ray fiber diffraction studies indicate that the peptide backbone of the fibers adopts a cross β- structure. In this structure, the individual β-strands are oriented perpendicular to the long axis of the fiber, while the hydrogen bonds are oriented parallel with the long axis of the fiber. Amyloid fibers are also resistant to proteolysis, and display characteristic apple-green birefringence when stained with the histological dye Congo red and seen under polarized light. There are also striking similarities in the aggregation behavior of many misfolded proteins, even if their propensity to aggregate can vary markedly between different sequences. Amyloid fibrils are thought to form through self-assembly of protein monomers via a nucleation- dependent pathway initiated in partially denatured states of amyloidogenic proteins.

Figure imgf000004_0001

Table 1. List of diseases resulting from amyloid formation Alzheimer's disease (AD) is one of these protein conformational diseases and the leading cause of dementia in the Western world. Postmortem, it is characterized by two major neuropathological features: extracellular deposition of Αβ peptides and intracellular aggregates of neurofibrillary lesions made of hyperphosphorylated tau proteins. The observation that rare, early-onset familial forms of AD are caused by mutations in the amyloid precursor protein (APP), presenilin-1 (PS1 ), or presenilin-2 (PS2) gene, all of which increase the production of Αβ, led to the so-called Αβ amyloid cascade hypothesis. This hypothesis proposes that the aggregation of polymerized forms of Αβ in soluble multimeric and/or insoluble senile plaque deposits in the brain is an early and critical event that triggers a cascade of pathological events leading to hyperphosphorylation and somatodendritic segregation of tau, formation of neurofibrillary lesions, neuroinflammation, degeneration of brain cells and, finally, dementia.

To date there is no definitive diagnosis for AD in living patients. Confirmation of the presence of AD requires a neuropathological examination at autopsy, demonstrating the presence of cerebral Αβ amyloid plaques and neurofibrillary tau tangles. To make the diagnosis of AD and to exclude other causes of dementia, clinicians rely on histories from patients and informants; physical, neurological, and psychiatric examinations; neuropsychological testing; laboratory assessments; and a variety of other diagnostic tests including neuroimaging (e.g., fMRI, PET-PIB scan). Genetic testing of diseased mutated proteins (APP, PS1 , PS2) in rare, early-onset familial forms of AD cases are also used, as are testing of genetic risk factors (ApoE4) in sporadic AD cases. To serve as diagnostic biomarkers of AD, biochemical changes that reflect the presence of disease-related proteins Αβ and tau also have been extensively studied in the cerebrospinal fluid (CSF) and blood. Both amyloidogenic proteins are particularly relevant to the pathology of AD and thus may provide diagnostically useful information. However, while some clinical testing using CSF and blood biomarkers have shown encouraging results, others have not. Conventional diagnosis techniques for AD have some important limitations, leading to the absence of a definitive premortem diagnosis of AD. To date, there is no definitive diagnosis of AD in living patients. Confirmation of the presence of AD requires a postmortem neuropathological demonstration of the presence of Αβ amyloid plaques and neurofibrillary tau tangles. Nevertheless, several studies have reported that a combination of the currently available diagnosis techniques (including neuropsychological, CSF/blood biomarker and neuroimaging assessments) may lead, at best, to up to 70-80 % confidence in diagnosis of middle to advanced AD cases. There is no diagnosis test that allows for sensitive assessment of the early stages of AD pathology and development.

Low confidence in the premortem diagnosis of many central and peripheral proteopahies using conventional diagnosis techniques. For instance, there is no reliable biochemical diagnosis for tau-containing progressive supranuclear palsy (or for the frontotemporal dementia syndrome) in living patients. Definitive diagnosis occurs at autopsy, following a neuropathological confirmation of the presence of neurofibrillary tangles in frontal and temporal cortices as well as a biochemical assessment of the pathological tau species. Parkinson's disease is a neurodegenerative movement disorder characterized by the progressive degeneration of the dopaminergic neurons from the substantia nigra and the appearance of intraneuronal inclusions named Lewy bodies in the surviving neurons. The major component of these eosinophilic Lewy bodies is aggregated forms of the protein cc-syn. Diagnosis of Parkinson's disease is difficult to obtain before late stages of pathology where > 90 % loss of dopaminergic neurons in the substantia nigra is observed.

SUMMARY OF THE INVENTION

One objective of the present invention is to improve the diagnostic of proteopathies. Another objective is to offer an early stage diagnostic of such diseases, when the subject is still living.

Other objectives are to facilitate and to reduce the duration of the diagnostic.

The invention is based on the surprising finding that amyloidogenic proteinaceous material induced by a proteopathy are present in the subject waste products, in particular the feces.

The invention therefore concerns a method for detecting amyloidogenic proteinaceous material induced by a proteopathy, which is characterized by the fact that said amyloidogenic proteinaceous material is detected in a mammals waste products.

Preferred embodiments of the invention are defined in the dependent claims.

The following terms and related definitions are used in the present document.

Amyloid: Amyloids are insoluble fibrillar protein aggregates that share specific structural traits. Amyloids arise from at least 20 misfolded proteins and polypeptides present naturally in the body. These inappropriately folded structures alter their proper configuration such that they erroneously interact with one another or other cell components forming insoluble fibrils. Amyloids have been associated with the pathology of more than 20 serious human diseases in that, abnormal accumulation of amyloid fibrils in organs may lead to amyloidosis, and may play a role in various neurodegenerative disorders

Birefringence: Birefringence, or double refraction, is the decomposition of a ray of light into two rays when it passes through certain anisotropic materials, such as boron nitride or crystals of calcite.

Proteinaceous: Of, relating to, consisting of, resembling, or pertaining to protein, or pertaining to any material having a protein base.

Proteinopathy: Proteopathy refers in medicine to a class of diseases in which certain proteins become structurally abnormal, and thereby disrupt the function of cells, tissues and organs of the body. Frequently the proteins fail to fold into their normal configuration; in this misfolded state, the proteins can become toxic in some way (a gain of toxic function) or they can lose their normal function. The proteopathies (also known as proteinopathies, protein conformational disorders, or protein misfolding diseases) include diseases such as prion diseases, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and a wide range of other central and peripheral disorders. The concept of proteopathy can trace its origins to the mid-19th century, when, in 1854, Rudolf Virchow coined the term amyloid ("starch-like") to describe a substance in cerebral corpora amylacea that exhibited a chemical reaction resembling that of cellulose. In 1859, Friedreich and Kebule demonstrated that, rather than consisting of cellulose, "amyloid" actually is rich in protein. Subsequent research has shown that many different proteins can form amyloid, and that all amyloids have in common birefringence in cross- polarized light after staining with the dye Congo Red, as well as a fibrillar ultrastructure when viewed with an electron microscope.

Proteolysis: Proteolysis is the directed degradation or digestion, of proteins by cellular enzymes called proteases or by intramolecular digestion.

Seeded proteinopathies: Some proteins can be induced to form abnormal assemblies by exposure to the same or similar protein assembly that has folded into a disease-causing conformation, a process called 'seeding' or 'permissive templating'. In this way, the disease state can be brought about in a susceptible host by the introduction of diseased tissue extract from an afflicted donor. The most known form of such inducible proteopathy is prion disease (e.g., creutzfeldt jakob), which can be transmitted by exposure of a host organism to purified prion protein in a disease-causing conformation. There is now evidence that other proteopathies can be induced by a similar mechanism, including but not restriceted to Αβ amyloidosis, tauopathy and synucleinopathy. In all of these instances, an aberrant form of the protein itself appears to be the pathogenic agent. In some cases, the deposition of one type of protein can be experimentally induced by aggregated assemblies of other proteins that are rich in β-sheet structure, possibly because of structural complementarity of the protein molecules. There is also experimental evidence for cross-seeding between prion protein and Αβ or between tau and Αβ.

Waste product: An unusable or unwanted substance or material produced during or as a result of a process, such as metabolism. Material discarded as useless in the process of producing something. Feces, urine, and other material excreted in the life process. In this document, waste product also refers to saliva, hairs, tears and perspiration.

It should be underlined that to date, (i) the presence in the human feces of cerebrally-derived aggregates or amyloid in general has never been demonstrated, (ii) the amount of amyloid in the feces has never been correlated with the severity of disease progression in organs (e.g., in the brain), (iii) there is no diagnosis test evaluating the presence of amyloid in the fecal matters as a diagnosis tool for the disease.

Waste products such as fecal matters are easily accessible from the human's digestive tract. Indeed, feces can easily be collected after they are expelled from a human's digestive tract through the anus during defecation. Furthermore, defecation occurs naturally in humans from once every two or three days to several times a day (depending on the individual and the circumstances).

Unlike brain biopsies or the collection of CSF and blood samples, collection of fecal matters is a non-invasive process. For CSF collection, lumbar puncture is the most commonly used method. Alternative methods of CSF collection include a cisternal puncture using a needle placed below the occipital bone (back of the skull). It can be dangerous because it is so close to the brain stem and is always done with fluoroscopy. Ventricular puncture, although rarer, may be recommended and is usually done in the operating room. A hole is drilled in the skull, and a needle is inserted directly into one of brain's ventricles. Risks of lumbar puncture include: bleeding into the spinal canal, headache after the test, hypersensitivity (allergic) reaction to the anesthetic, infection introduced by the needle going through the skin. Brain herniation may occur if this test is done on a person with a mass in the brain (such as a tumor or abscess). This can result in brain damage or death. Damage to the nerves in the spinal cord may occur, particularly if the person moves during the test.

Human fecal matters may be collected as a stool test for detection of amyloidogenic material for AD and other proteopathies. A stool test is one where fecal matter is collected for analysis to diagnose the presence or absence of a given medical condition. Stool tests are already used to diagnose conditions such as colorectal cancer or stomach cancer. Stool tests are commonly used in microbiology tests: parasitic diseases such as ascariasis, Hookworm, Strongyloidiasis and Whipworm can be diagnosed by examining stools under a microscope for the presence of worm larvae or eggs. Some bacterial diseases can be detected with a stool culture. Stool tests are used as well as chemical tests: A fecal pH test may be used determine lactose intolerance or the presence of an infection. Steatorrhea can be diagnosed using a fecal fat test that checks for the malabsorption of fat. Faecal Elastase levels are becoming the mainstay of pancreatitis diagnosis.

DETAILED DESCRIPTION OF THE INVENTION

The invention will be better understood below, in a detailed description which includes some examples.

The unique figure summarizes the different steps of the method according to the invention wherein feces are first collected and then prepared according to the selected detection technique. Amyloidogenic proteinaceous material (if any) is detected and a diagnosis is established.

The presence of Αβ amyloid proteins is univoquely detected in human fecal samples from diseased patients compared to control patients. The detection in the fecal matters is achieved using a combination of biochemical/biophysical techniques after preparation of the human samples. The three main techniques that allow for the specific detection of Αβ amyloid in the fecal samples are immunoblot, ELISA and mass spectrometry analysis. These are complemented by Thioflavin T binding fluorescence assay.

The presence of Αβ amyloid proteins is univoquely detected by immunoblot of prepared human fecal samples from AD patients as compared to control non-demented patients. Fecal samples (1 g) are dissolved in TBS (1 :1 w/v) (pH 7) with Hiedolph homohenizer at 900rpm for 5min and centrifuged at 13000 x g (30 min). The TBS-soluble fraction is collected, then the pellet is dissolved in TBS-Triton-X100 (1 %) and centrifuged again at 13000 x g. The TBS-Triton-soluble fraction is collected. The prepared fecal fractions are analyzed by immmunoblotting. Prepared fecal samples are diluted in 4X sample buffer containing 0.5 M Tris-HCI; 4 % (w/v) SDS; 20% (w/v) glycerol; 3.33% (v/v) DTT; 0.0053% (w/v) bromophenol blue, then boiled for 20min at 95C°, sonicated for 10min in a sonicator bath and subsequently loaded on a 4-12% Bis-Tricine gels. To separate Αβ40 from Αβ42, the prepared fecal samples are diluted 1 :4 in 0.48 M Bis-Tris; 0.21 M Bicine; 1 .32% (w/v) SDS; 20% (w/v) sucrose; 3.33% (v/v) DTT; 0.005% (w/v) bromophenol blue, and then boiled for 20 minutes and subjected to 10% Bis- Tricine 8M Urea SDS-PAGE. Human AD and aged non-demented brain extracts as well as synthetic Αβ1 -40 and Αβ1 -42 are used as controls. After migration, proteins are transferred onto a PVDF membrane, which is heated for 3min in a microwave oven, then blocked for 1 hour at room temperature and finally probed with monoclonal antibody specific to human Αβ1 -17. The secondary antibody is goat anti-mouse IgG conjugated with a fluorophore with detection wavelength at 680nm, and developed with a Licor system (Li-cor Nebraska, US). Quantification of the signal (optical density) is done using the public domain software ImageJ. The presence of Αβ amyloid proteins is unequivocally detected by Enzyme-Linked Immunosorbent Assay (ELISA) in prepared human fecal samples from AD patients as compared to control non-demented patients. Fecal samples (1 g) are dissolved in TBS (1 :1 w/v) (pH 7) with Hiedolph homohenizer at 900rpm for 5min and centrifuged at 13000 x g (30 min). The TBS-soluble fraction is collected, then the pellet is dissolved in TBS-Triton-X100 (1 %) and centrifuged again at 13000 x g. The TBS-Triton- soluble fraction is collected. For a quantitative assessment of Αβ peptides, Enzyme-Linked Immunosorbent Assay is performed. This biochemical technique has been commonly used in clinics as a standard test for detection or titration of antigens or antibodies. In certain diseases such as HIV this is considered first screening test because of its high sensitivity and ease of manipulation. The aforementioned fractionation samples are probed with ELISA immunoassay kit for detection of human Αβ1 -40 and Αβ1 -42 (Invitrogen), following manufacturer's instructions. A monoclonal antibody specific for the N-terminus of human Αβ is coated onto the 96 wells plate. During the first incubation, standards of known human Αβ40 content, controls, and fecal samples are pipetted into the wells and co-incubated with a rabbit antibody specific for the C-terminus of the 1 -40/42 Αβ sequence. Bound rabbit antibody is detected by the use of a horseradish peroxidase-labeled anti- rabbit antibody. Samples are quantified by standard curve generated from the recombinant Αβ40 Fluoresence signal was read on Tecan Sapphire II 96plate reader system (Tecan Mannedorf, Switzerland).

The presence of Αβ amyloid proteins is unequivocally detected by mass spectrometry analysis of human fecal samples from AD patients. A defined volume (1 .5mL) of each soluble fraction from crude fecal extractions was subjected to immunoprecipitation coupled with mass spectrometric analysis (IP/MS). An antigen-specific antibody was used as a capture antibody in immunoprecipitation procedure. Antigen/antibody complex was pulled down using protein G agarose beads. After overnight incubation at 4C° on a rotating wheel, the beads were washed 3 times with washing buffer (0.1 % N-octyl-beta-pyranoside, 150mM NaCI, 50mM Tris, pH 7) and further eluted with TFA/acetonitrle/water (1 :20:20 v/v). 2μΙ_ of the eluate was mixed with a-cyanic acid at 1 :1 ratio and deposited on a reading plate for MS analysis. MALDI-TOF apparatus was used in a mode reflectron for accurate detection of amyloidogenic peptides. Both internal and external calibrations were made using synthetic peptides. Nano-HPLC coupled to an electrospray ionisation (ABI LTQ ion trap) source was used as a complementary procedure to identify proteins of interest. 10μΙ_ of each immunoprecipitaded fraction was injected in a reverse phase column and progressively eluted with a gradient of acetonitril/methanol/water. Protein mass was searched using Expasy proteomics server.

The presence of Αβ amyloid proteins is unequivocally detected by ThT binding fluorescence assay of human fecal samples from AD patients.

ThT binding assay is performed by mixing aliquots of prepared fecal samples with 10-20 uM ThT dye and 50 mM glycine-NaOH, pH 8.5, in Nunc 384-well fluorescence plates. ThT fluorescence is measured in an AnalystADfluorometer (Molecular Devices) at excitation and emission wavelengths of 450 and 485 nm, respectively. The samples are analyzed in triplicates.

Claims

1 . Method for detecting amyloidogenic proteinaceous material induced by a proteopathy characterized by the fact that said amyloidogenic proteinaceous material is detected in a mammal waste product.
2. Method according to claim 1 wherein said product are human feces.
3. Method according to claim 1 or 2 wherein the proteopathy is Alzheimer's disease.
Method according to anyone of the previous claims wherein said amyloidogenic proteinaceous material comprises Αβ amyloid proteins.
Method according to anyone of the previous claims wherein said amyloidogenic proteinaceous material comprises tau amyloid proteins.
Method according to anyone of the previous claims wherein said amyloidogenic proteinaceous material comprises cc-syn amyloid proteins.
7. Method according to anyone of the previous claims wherein said amyloidogenic proteinaceous material comprises any mammal proteins that form amyloid.
8. Method according to anyone of the previous claims wherein said amyloidogenic proteinaceous material is detected by a biochemical and/or a biophysical technique.
9. Method according to claim 7 wherein said technique is selected in the following group : Immunoblot; Enzyme-linked immunosorbent assay (ELISA) analysi; Thioflavin T binding fluorescence assay; mass spectrometry.
PCT/IB2013/050530 2012-01-26 2013-01-21 Use of waste products for detecting amyloidogenic proteinaceous material in living mammals WO2013111045A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011009967A1 (en) * 2009-07-23 2011-01-27 Universidad Complutense De Madrid Kit and method for the pre-mortem in vitro detection of alzheimer’s disease
WO2011057029A1 (en) * 2009-11-04 2011-05-12 Novartis Ag Positively charged species as binding reagents in the separation of protein aggregates from monomers
WO2012006540A2 (en) * 2010-07-08 2012-01-12 University Of Maryland, Baltimore Improving the efficiency of prion conversion in vitro and sensitivity of prion detection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011009967A1 (en) * 2009-07-23 2011-01-27 Universidad Complutense De Madrid Kit and method for the pre-mortem in vitro detection of alzheimer’s disease
EP2458385A1 (en) * 2009-07-23 2012-05-30 Universidad Complutense De Madrid Kit and method for the pre-mortem in vitro detection of alzheimer disease
WO2011057029A1 (en) * 2009-11-04 2011-05-12 Novartis Ag Positively charged species as binding reagents in the separation of protein aggregates from monomers
WO2012006540A2 (en) * 2010-07-08 2012-01-12 University Of Maryland, Baltimore Improving the efficiency of prion conversion in vitro and sensitivity of prion detection

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
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