WO2017198554A1

WO2017198554A1 - Method for the diagnosis of neurodegenerative diseases

Info

Publication number: WO2017198554A1
Application number: PCT/EP2017/061407
Authority: WO
Inventors: Claudia Martini; Maria Letizia Trincavelli; Simona Daniele
Original assignee: Universita' Di Pisa
Priority date: 2016-05-16
Filing date: 2017-05-11
Publication date: 2017-11-23
Also published as: ITUA20163473A1

Abstract

Disclosed is an in vitro method for the determination of neurodegenerative diseases, in particular Parkinson's disease and Alzheimer's disease, based on measuring, in a peripheral blood sample, the heteromeric complexes of a-synuclein with β-amyloid 1-42 and a-syn with tau protein. Further subjects of the invention are an immunochemical assay for the quantitation of said heteromeric complexes and a diagnostic kit for conducting the assay.

Description

METHOD FOR THE DIAGNOSIS OF NEURODEGENERATIVE DISEASES

The present invention relates to an in vitro method for the determination of neurodegenerative diseases, in particular Parkinson's disease and Alzheimer's disease. The method according to the invention is based on measuring, in a peripheral blood sample, the heteromeric complexes of a-synuclein (hereinafter called "a-syn") with β-amyloid 1-42 (hereinafter called "Αβ") and of a-syn with tau protein (hereinafter called "tau"). Further objects of the invention are an enzyme immunoassay for the quantitation of said heteromeric complexes and a diagnostic kit for conducting the assay.

INTRODUCTION AND PRIOR ART

Neurodegenerative diseases (NDs), most of which are still incurable, affect over 7 million people in Europe. The current cost of treatment is about€ 130 billions a year, representing one of the main medical and social challenges facing European society. NDs are difficult to diagnose, because many of them progress for years before the symptoms appear. The availability of a sensitive diagnostic kit is therefore crucial to ensure early diagnosis of the disease and optimisation of the treatment, leading to a significant reduction in patient management costs.

Protein misfolding and aggregation play a fundamental role in many NDs.

Various research groups have demonstrated that the Αβ, tau and a-syn proteins mutually promote their accumulation or aggregation {Science 2003, 300:636-640; Trends Neurosci 2004, 27:129-134), and that these protein-protein interactions are involved in the physiopathology of diseases like Alzheimer's disease (AD), Parkinson's disease (PD) and Lewy body dementia (Alzheimer & Research & Therapy 2012, 4:1).

For example, AD is associated with the presence of plaques of Αβ and neurofibrils consisting of tau protein (Acta Neuropathol.2013;125:699-709), whereas PD is characterised by cellular inclusions consisting of a-syn and tau protein (J Neuropathol Exp Neurol. 1996;55(3):259-72).

The levels of a-syn, Αβ and tau (total, oligomeric or phosphorylated) in cerebrospinal fluid are currently used as diagnostic markers for AD and other NDs (Clin Chem Lab Med.2006;44: 1472-80; Ann Neurol 1995, 389:649-652; Arch Neurol 2001, 58:373-379), PD (Front Aging Neurosci 2014; 6:1-8).

The use of blood as a source of biomarkers for NDs is still under study. Although some of the data are controversial, a variation in the levels of tau (especially phosphorylated tau) or Αβ in various blood fractions of patients suffering from AD or PD is reported in the literature (Neurosci Lett.2000;285(l):49-52; Alzh Res and Ther 2013; 5:8-10; IntJAlz dis 2012; 2012:1-9; Jn of Ah Dis 2011; 25:103-109; Sci Rep 2013; 3: 2540).

a-syn, Αβ and tau proteins do not normally exist in the same subcellular compartment of healthy cells, thus limiting their potential for direct interaction (Proc Natl Acad Sci USA 2001, 98: 12.245-12.250). In pathological states, however, the location of said proteins changes, allowing direct interactions to take place in damaged or diseased cells. For example, some studies demonstrate that a-syn forms heteromeric complexes with Αβ in transgenic models of NDs and in the brains of patients suffering from Alzheimer's disease (PLoS One 2008, 3: e3135). In the same way, a-syn can interact with tau, promoting its polymerisation and forming heteromeric protein aggregates in neuronal cells isolated from patients suffering from Alzheimer's disease (Science 2003, 300:636-640; Trends Neurosci 2004, 27:129-134; PLoS One 2011, 6:e26609).

Enzyme-linked immunosorbent assay (ELISA) tests for quantitation of the total, phosphorylated or mono-oligomeric levels of a-syn, Αβ or tau protein are to be found on the market and in the literature. However, enzyme immunoassays for the quantitation of tau-a-syn or Αβ-α-syn heteromeric complexes are not known.

The presence of these protein complexes has been demonstrated in biological fluids and tissues using co-immunoprecipitation/western blot techniques or NMR analysis (Neurochem Res 2006, 31:. 1153-1162; Clin Chem Lab Med.2006;44: 1472-80; Arch Neurol. 1999;56:673-80; Neurosci Lett.2000;285(l):49-52). These techniques are rather laborious, expensive, and do not generate fully quantitative results. They are therefore unsuitable for use in diagnostic kits. DESCRIPTION OF THE INVENTION

It has now surprisingly been discovered that neurodegenerative disease can be determined at an early stage by quantitation of the a-syn-Αβ and/or a-syn-tau heteromeric complexes from a blood sample. The data obtained in patients suffering from neurodegenerative diseases, especially Parkinson's disease, demonstrate the efficacy of said complexes as peripheral markers of disease. Moreover, the results collected from a sample of sporty individuals, who are known to suffer from neurodegenerative diseases to a lesser extent than sedentary individuals, indicate that the same heteromeric complexes constitute prognostic biomarkers for NDs.

In particular, by analysing blood samples taken from patients suffering from neurodegenerative diseases, a significant increase in the levels of α-syn-tau complex, and a reduction in a-syn-Αβ complex in the red blood cells, compared with healthy volunteers, were observed. The levels of α-syn-tau complex observed in the red blood cells and platelets of sporty individuals were also lower than in sedentary individuals. These findings, taken as a whole, confirm the diagnostic value of the a-syn-Αβ and α-syn-tau complexes and the prognostic value of the α-syn-tau complex.

A first aspect of the invention therefore provides an in vitro method for the diagnosis or prognosis of neurodegenerative disease in an individual, wherein said method comprises:

(1) determining the amount of α-syn-tau complex in a sample of red blood cells or platelets isolated from the individual tested

(2) comparing said amount with a reference value corresponding to the quantity of the same complex present in the red blood cells or platelets of healthy volunteers,

wherein a higher amount than the reference value found in the red blood cells of the individual tested indicates the presence of neurodegenerative disease or an increased risk of developing neurodegenerative disease, while a higher amount found in the platelets of the individual tested indicates an increased risk of developing neurodegenerative disease.

Another aspect of the invention provides an in vitro method for the diagnosis of neurodegenerative disease in an individual, which comprises:

(1) determining the amount of a-syn-Αβ complex in a sample of red blood cells or platelets isolated from the individual tested;

(2) comparing said amount with a reference value corresponding to the amount of the same complex present in the red blood cells or platelets of healthy volunteers;

where the finding of a lower amount than the reference value in the red blood cells of the individual tested indicates the presence of neurodegenerative disease.

As used herein, the term "neurodegenerative diseases" includes Parkinson's disease, Alzheimer's disease, dementia, in particular frontotemporal dementia, and mild cognitive impairment.

The amount of a-syn-Αβ and a-syn-tau complexes in the samples tested can be determined by known methods. The interaction between a-syn and Αβ has been demonstrated by multidimensional NMR spectroscopy (Neurochem Res 2006; 31:1153- 1162) and molecular dynamics studies {Plos One 2014; 9: el06883). The direct bond of - syn to tau has been determined by affinity chromatography techniques and binding assays (J Biol Chem 1999; 274, : 25481-25489). Moreover, in biological fluids and tissues, the presence of said heteromeric complexes of a-syn has been demonstrated by co- immunoprecipitation/western blot or immunofluorescence techniques (PLoS One 2008, 3: e3135; Science 2003, 300:636-64).

In a particularly preferred embodiment of the present invention, the a-syn-Αβ and a-syn-tau complexes are determined by solid-phase enzyme immunoassay (ELISA), comprising the following steps:

(1) preparing a sample of biological fluid;

(2) placing the sample in contact with an antibody specific for a-syn;

(3) placing the sample of step (2) in contact with an antibody specific for tau or Αβ, wherein said antibody is bonded to an enzyme able to develop a colorimetric reaction when exposed to a suitable substrate; (4) placing the sample of step (3) in contact with said substrate of the enzyme and measuring the colour thus produced.

Alternatively, steps (2) and (3) can be reversed, in which case antibodies specific for tau or Αβ are used in step (2), and an antibody specific for a-syn bonded to an enzyme able to develop a colorimetric reaction when exposed to a suitable substrate is used in step (3)·

The biological fluid sample can be a sample of blood or separated fractions of blood, such as red blood cells or platelets, suitably treated, e.g. suspended in phosphate buffer containing sodium dodecyl sulphate (SDS).

The anti-a-syn, anti- Αβ and anti-tau antibodies usable in the enzyme immunoassay according to the invention are commercially available (suppliers: Sigma Aldrich and DBA Italia), and are described in the literature in protocols for identification of the individual proteins or complexes thereof (Neurobiol ofdis, 2015; 79: 81-99; BBA 2013 1832: 1249- 59; Biophys J. 2009; 96: 4200-4211).

Peroxidase or alkaline-phosphatase can be used as enzyme markers bound to the secondary antibody, preferably peroxidase (Horse Radish Peroxidase, HRP) which, by catalysing the oxidation of a substrate by hydrogen peroxide, generates a coloured product that can be measured spectrophotometrically.

In a procedure for the conduct of the enzyme immunoassay, the blood samples (red blood cells and platelets), after suitable preparation, can be added to plastic platelet wells coated with anti-a-syn antibody. The samples are then incubated with an antibody specific for tau protein (to detect tau-a-syn complexes) or for Αβ (to detect Αβ-α-syn complexes). After suitable washes, the antigen-antibody complex is detected by a secondary antibody labelled with peroxidase. After the addition of a suitable substrate, the samples develop a colour which is spectrophotometrically quantified to measure the quantity of the protein complexes present in the sample.

An alternative strategy involves coating the wells with an antibody specific for tau (to detect tau-a-syn complexes) or for Αβ (to detect Αβ-α-syn complexes). In this case, after incubation with the test samples, a primary antibody specific for a-syn is used. The antigen-antibody complex is then detected as described above.

To determine the reference values to be used in the diagnostic/prognostic method according to the present invention, the quantities of the a-syn- Αβ and a-syn-tau complexes are measured as described, from a statistically significant pool of biological samples obtained from healthy volunteers (not suffering from a neurodegenerative disease). By operating in this way it is possible to establish the reference value for α-syn-tau complex in the red blood cells and platelets, amounting to 2.35 ± 0.17/mg of protein and 0.170 ± 0.030^g of protein respectively. The reference value for the a-syn-Αβ complex, amounting to 4.54 ± 0.20/mg of protein and 0.336 ± 0.048^g of protein in red blood cells and platelets respectively, is determined in the same way.

A further aspect of the invention relates to a diagnostic kit containing the reagents and instructions for conducting the enzyme immunoassay described herein. In particular the kit can contain, in separate containers, anti-a-syn and anti-Αβ or anti-tau antibodies, optionally labelled with an enzyme able to produce a colorimetric reaction, the enzyme substrate and buffer solutions. The kit can also contain samples with a known (standard) concentration of tau-a-syn or Αβ-α-syn, so that a calibration line can be plotted in parallel to the analysis of the blood samples.

Compared with other known techniques, the sandwich ELISA to which the invention relates determines the α-syn-tau and a-syn- Αβ levels quantitatively, cheaply and quickly.

DESCRIPTION OF FIGURES

Figure 1. tau-a-syn heteromeric complexes. The tau-a-syn complex was prepared by incubating 0.5 μg of each protein, a-syn was then immunoprecipitated using a specific monoclonal antibody; the resulting sample was suspended in running buffer, and the tau- a-syn complex was detected by immunoblot using a specific anti-tau antibody, tau protein alone was used as positive control of the experiment. The image shows three independent experiments. Figure 2. Direct correlation between absorbance and concentration of a-syn-tau complex. Different quantities of recombinant a-syn-tau complex (incubation at 37°C for 60 minutes in 2 mM SDS) were incubated in wells pre-absorbed with anti-a-syn antibody. The data are expressed as specific absorbance, and represent the mean ± SD of 5 different experiments.

Figure 3. "Theoretical" and "real" calibration lines of a-syn-tau complex.

Different quantities of recombinant α-syn-tau complex (incubation at 37°C for 60 minutes in 2 mM SDS) were incubated in wells pre-absorbed with anti-a-syn antibody. After incubation with the samples, the supernatants were collected, and the remaining quantity of tau was assayed on them with a commercial ELISA. The correlation between the real quantity of α-syn-tau complex and the absorbance was determined in this way. The data are expressed as specific absorbance, and represent the mean ± SD of 5 different experiments.

Figure 4. Λβ-α-syn heteromeric complexes. The Αβ-α-syn complex was prepared by incubating 0.5 μg of each protein, a-syn was then immunoprecipitated using a specific monoclonal antibody; the resulting sample was suspended in running buffer, and the Αβ- α-syn complex was detected by immunoblot using a specific anti-Αβ antibody. Αβ protein alone was used as positive control of the experiment.

Figure 5. Direct correlation between absorbance at 450 nm and concentration of a-syn-Αβ complex. Different quantities of a-syn- Αβ complex (incubation at 37°C for 20 hours in 2 mM SDS) were incubated in wells pre-absorbed with anti-Αβ antibody. The data are expressed as specific absorbance, and represent the mean ± SD of 5 different experiments.

Figure 6. "Theoretical" and "real" calibration lines of a-syn-Λβ complex.

Different quantities of a-syn-Αβ complex (incubation at 37°C for 20 hours in 2 mM SDS) were incubated in wells pre-absorbed with anti-Αβ antibody. After incubation with the samples, the supernatants were collected, and the remaining quantity of a-syn was assayed on them with a commercial ELISA. The correlation between the real quantity of a-syn-Αβ complex and the absorbance was determined in this way. The data are expressed as specific absorbance, and represent the mean ± SD of 5 different experiments.

Figure 7. Physiological range of a-syn-tau complex. The red blood cell (panel A) or platelet (panel B) fractions of healthy volunteers, after being suitably solubilised, were incubated in wells pre-absorbed with anti-a-syn antibody. The quantity of a-syn-tau complex was determined as described above. The data are expressed as ng of heteromeric complex/mg of total proteins for red blood cells, or ng of heteromeric complex^g of total proteins for platelets.

Figure 8. Physiological range of a-syn-Αβ complex. The fractions of red blood cells or platelets of healthy volunteers, after being suitably solubilised, were incubated in wells pre-absorbed with anti-Αβ antibody. The quantity of a-syn-Αβ complex was determined as described above. The data are expressed as ng of heteromeric complex/mg of total proteins for red blood cells, or ng of heteromeric complex^g of total proteins for platelets.

Figure 9. Levels of heteromeric complexes of syn in patients suffering from PD. The red blood cell fractions of healthy volunteers or PD patients, after being suitably solubilised, were incubated in wells pre-absorbed with the relevant antibody. The quantities of a-syn-Αβ or a-syn-tau complex were determined as described above. The data are expressed as ng of heteromeric complex/mg of total proteins.

Figure 10. Levels of a-syn-tau complex in sedentary and sporty individuals. The red blood cell fractions of healthy volunteers (sporty or sedentary), after being suitably solubilised, were incubated in wells pre-absorbed with anti-a-syn antibody. The quantity of α-syn-tau complex was determined as described above. The data are expressed as ng of heteromeric complex/mg of total proteins.

DETAILED DESCRIPTION OF THE INVENTION

Determination of tau-a-syn complexes

The tau-a-syn complexes were determined by incubating known, increasing concentrations of human, tau and a-syn recombinant proteins.

The formation of the heteromers was confirmed by co-immunoprecipitation/western blot assays. Figure 1 shows a representative example of the results obtained. The samples obtained with known, increasing concentrations of the two monomers were assayed by the ELIS A "sandwich" technique. A direct correlation between absorbance and concentration of a-syn-tau heteromers, in a range of equal concentrations between 10 ng/100 μΐ and 1000 ng/100 μΐ of each protein, was obtained from these experiments (Figure 2). The absorbance of the blanks, obtained in the absence of anti-tau antibody, was less than 20% of the total value.

Different concentrations of one protein compared with the other gave rise to similar results to those obtained with equivalent quantities (1 :1) of each protein, suggesting that the heteromer contains stoichiometric quantities of a-syn and tau.

To determine the free ("unbound") portion of tau after formation of the complex with a-syn, a standardised commercial ELISA was used. The results demonstrate that the percentage of tau not bound to a-syn falls within the 16-29% range.

These data allowed the correction of the correlation line between absorbance and concentration of heteromers, called the "a-syn-tau calibration line", corrected for the free proportion of tau (Figure 3).

Determination of β-amyloid-a-syn complexes

The Αβ-α-syn complex was prepared by incubating known, increasing concentrations of recombinant human proteins, Αβ and a-syn, for different times.

The formation of the heteromers was confirmed by co- immunoprecipitation/western blot assays (Figure 4).

When the optimum incubation time had been identified, the procedure used for tau- a-syn was reversed, i.e. the sample was placed in wells coated with anti-Αβ antibody, and the a-syn antibody labelled with peroxidase was then added. This approach solved the problem of the aspecific bond of Αβ by exploiting the lipophilicity of the protein to ensure optimum anchorage to the plate.

The absorbance under the experimental conditions used increased on a straight-line basis with the concentration of Αβ-α-syn complex (Figure 5).

To determine the free ("unbound") portion of a-syn after formation of the complex with beta, a standardised commercial ELISA was used.

The experiment conducted with a standardised commercial ELISA demonstrated that the percentage of Αβ not bound to a-syn fell within the 5-20% range.

These data allowed the absorbance obtained to be correlated with the real concentrations of the heteromeric complexes (Figure 6).

Assay of a-syn-Αβ and a-syn-tau complexes in blood samples obtained from healthy volunteers

The heteromeric complexes of a-syn with tau and Αβ were assayed in 80 blood samples from healthy volunteers, taken according to the applicable legislation. The platelets and red blood cells were separated by centrifugation, according to the protocols reported in the literature.

Experiments were conducted for the quantitation of the total proteins in the two different blood fractions and the heteromeric complexes of a-syn with the ELISAs described above, using increasing, known quantities of Αβ-α-syn or tau-a-syn in parallel.

The Αβ-α-syn and tau-a-syn complexes proved detectable in both blood fractions, confirming the correctness of our approach. As shown in Figures 7A and 7B, the values (expressed as mean value ± standard error of the mean of tau-a-syn in the healthy volunteers amounted to 2.35 ± 0.17/mg of protein and 0.170 ± 0.030^g of protein in red blood cells and platelets respectively.

The levels of Αβ-α-syn complexes in healthy volunteers amounted to 4.54 ±

0.20/mg of protein and 0.336 ± 0.048^g of protein in red blood cells and platelets respectively (Figures 8 A and 8B).

These experiments enabled us to determine the physiological range of the heteromeric complexes studied (see Table).

Assay of a-syn-Αβ and a-syn-tau complexes in blood samples obtained from patients suffering from neurodegenerative diseases

As reported for the healthy volunteers, the fraction of red blood cells was isolated from 15 patients suffering from Parkinson's disease (PD). The results obtained (Figure 9 and Table) demonstrated significantly higher tau-a- syn complex levels in the PD patients than the controls. Conversely, the -syn-Αβ levels were lower in the PD patients than the healthy volunteers (Figure 9 and Table).

In view of the difference in the levels of the heteromeric complexes between the PD patients and the controls, these complexes can be identified as new peripheral biomarkers for the disease.

Influence of sporting activity on levels of a-syn-Αβ and a-syn-tau complexes

The findings reported in the literature suggest that in animals, regular physical exercise prevents and slows the neurodegeneration rate, probably by promoting neurogenesis or activating anti-oxidative signal pathways {Frontiers in aging Neuroscience, 2010; 25:1-8).

Our data collected from the population of healthy volunteers demonstrated that the levels of tau-a-syn complexes were significantly lower in the sporty individuals than the sedentary ones (Figure 10 and Table). These data confirm that the heteromeric syn complex levels represent valid predictive biomarkers for NDs.

Table. Physiological and pathological ranges of α-syn-tau and a-syn-Αβ levels.

The levels were determined in platelets or red blood cells, as indicated. The data represent the mean ± standard error of the mean; the values are expressed as ng of heteromeric complex/mg of total proteins for red blood cells, or ng of heteromeric complex^g of total proteins for platelets.

Claims

1. An in vitro method for the diagnosis or prognosis of neurodegenerative diseases in a subject, which comprises:

(1) determining the amount of a-syn-tau complex in a sample of red blood cells or platelets from said subject,

(2) comparing said amount with a reference value corresponding to the amount of the same complex contained in red blood cells or platelets from healthy individuals,

wherein an increase in the amount of complex in the red blood cells of the subject compared to the reference value is indicative of the presence of a neurodegenerative disease or of a higher risk of developing a neurodegenerative disease in said subject, while an increase in the amount of complex in the platelets of the subject compared to the reference value is indicative of a higher risk of developing a neurodegenerative disease in said subject.

2. An in vitro method for the diagnosis of neurodegenerative diseases in a subject, which comprises:

(1) determining the amount of a-syn-Αβ complex in a sample of red blood cells isolated from said subject,

(2) comparing said amount with a reference value corresponding to the amount of the same complex contained in red blood cells from healthy individuals, wherein a decrease in the amount of complex in the red blood cells of the subject compared to the reference value is indicative of the presence of a neurodegenerative disease in said subject.

3. Method according to claim 1 or 2, wherein said neurodegenerative disease is selected from Parkinson's disease, Alzheimer's disease, dementia, particularly frontotemporal dementia, and mild cognitive deficit.

4. Method according to claims 1-3, wherein the amount of a-syn-Αβ or a-syn-tau complex is determined by the following steps:

(1) providing a biological fluid sample;

(2) contacting said sample with an antibody specific for a-syn;

(3) contacting the sample from step (2) with an antibody specific for tau or Αβ, wherein said antibody is bound to an enzyme which is able to produce a colorimetric reaction when exposed to a suitable enzyme substrate;

(4) contacting the sample from step (3) with said enzyme substrate and measuring the colour produced by the enzyme-substrate reaction.

5. Method according to claims 1-3, wherein the amount of a-syn-Αβ and a-syn-tau complexes is determined by the following steps:

(1) providing a biological fluid sample;

(2) contacting said sample with an antibody specific for Αβ or tau;

(3) contacting the sample from step (2) with an antibody specific for a-syn, wherein said antibody is bound to an enzyme which is able to produce a colorimetric reaction when exposed to a suitable enzyme substrate;

6. Method according to claims 4 and 5, further comprising the interpolation of the amount of complex present in the sample onto a calibration curve obtained with known amounts of the same complex.

7. Method according to claims 4 and 5, wherein said biological fluid sample consists of a preparation of red blood cells or platelets.

8. Method according to claims 4 and 5, wherein the enzyme able to produce a colorimetric reaction is a peroxidase enzyme.

9. Method according to claims 1-8, wherein the reference value corresponding to the amount of a-syn-Αβ or a-syn-tau complex contained in red blood cells or platelets from healthy individuals is selected from:

(a) α-syn-tau in red blood cells: 2.35 ± 0.17/mg of protein, (b) α-syn-tau in platelets: 0.170 ± 0.030/μ of protein,

(c) a-syn-Αβ in red blood cells: 4.54 ± 0.20/mg of protein,

(d) a-syn-Αβ in platelets: 0.336 ± 0.048^g of protein.