WO2017010931A1

WO2017010931A1 - Biomarkers for atypical parkinsonism

Info

Publication number: WO2017010931A1
Application number: PCT/SE2016/050719
Authority: WO
Inventors: Per Svenningsson; Andrea BOMAN; Katarina KÅGEDAL; Adam BOXER; Samuel SVENSSON
Original assignee: Karin & Sten Mortstedt Cbd Solutions Ab
Priority date: 2015-07-16
Filing date: 2016-07-14
Publication date: 2017-01-19

Abstract

The invention relates to biomarkers which are useful for the diagnosis and prediction of atypical parkinsonism in mammalian animals such as humans. In particular, the invention relates to the use of lysosomal network proteins chosen from Early Endosomal Antigen 1 (EEA1); Lysosomal-associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP-2); Microtubule-associated protein light chain 3 (LC3);and Lysozymeas biomarkers in methods and kits for diagnosing, predicting or treating atypical parkinsonism in mammals. The invention further relates to methods for identifying compounds which are useful for the diagnosis, prediction or treatment of atypical parkinsonism.

Description

BIOMARKERS FOR ATYPICAL PARKINSONISM

TECHNICAL FIELD The invention relates to biomarkers which are useful for the diagnosis and prediction of atypical parkinsonism, in mammalian animals such as humans. In particular, the invention relates to the use of lysosomal network proteins as biomarkers in methods and kits for diagnosing, predicting or treating atypical parkinsonism in mammals. The invention further relates to methods for identifying compounds which are useful for the diagnosis, prediction or treatment of atypical parkinsonism.

BACKGROUND ART

Parkinson's disease (PD) makes up about 80% of parkinsonism cases. About 10% of parkinsonism cases can be symptomatic; e.g. drug-induced PD etc. Another 10% is caused by other neurodegenerative diseases, so called "atypical parkinsonism" or "parkinson-plus syndromes" (APS). These diseases rarely respond to dopaminergic medication, have a more rapid course, generally have a worse prognosis, and may be difficult to differential diagnose in the initial stages of the diseases. Examples of APS are e.g. corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA) and Pick's disease (Ali, K. & Morris, H.R. (2015) Practical neurology 15: 14-25; Mark, M.H. (2001) Neurologic clinics 19:607-627, vi).

Clinical diagnosis is based on physical manifestations of the disease and is hampered by the fact that the symptoms for APS overlap with many other common neurodegenerative diseases like PD, Alzheimer's disease (AD), frontotemporal dementia (FTD), dementia with Lewy bodies (DLB), and diseases with mixed pathologies (see e.g. Joutsa, J. et al. (2014) Parkinsonism & related disorders 20:840-844). Currently there is no bio-marker for diagnosis of either PD or APS, and definitive diagnosis is only possible post-mortem at autopsy by the presence of specific neuropathological findings. PD, which is a synucleinopathy (McCann, H. et al. (2014) Parkinsonism & related disorders 20 Suppl l :S62-67), can be distinguished from PSP and CBD, which are 4R tauopathies (Kouri, N. et al. (2011) Nature Reviews Neurology 7:263-272; Dickson, D.W. et al. (2010) Current opinion in neurology 23:394-400), by identification of Lewy bodies for PD, and by a Gallyas-Braak staining method to identify astroglial inclusions and tauopathy found in CBD and PSP. One of the noted differences is that tauopathy in PSP results in tuft-shaped astrocytes and doughnut-shaped astrocytic plaques found in CBD (Hattori, M. et al. (2003) Acta neuropathology 106: 143-149; Scaravilli, T, et al. (2005) Movement disorders 20 Suppl 12:S21-28).

Current biomarker studies for atypical parkinsonism have focused on markers for general neurodegeneration in cerebrospinal fluid (CSF): Total tau (T-Tau) and neurofilament light chain (NFL); markers for inflammation: monocyte chemoattractant protein- 1 and YKL-40; amyloid-β related markers: soluble amyloid precursor protein a and β (sAPPa and β),

(Αβι-42); and on phosphorylated tau (P-Tau) and a- synuclein. Several studies have reported increased NFL in APS, however this marker is increased in many other neurodegenerative diseases and cannot be used to distinguish between different forms of APS (Sako, W. et al. (2015) Journal of the neurological sciences 352:84-87). Results of T and P-Tau levels in CBD and PSP have so far been inconclusive (Magdalinou, N. et al. (2014) Journal of neurology, neurosurgery, and psychiatry 85: 1065-1075).

AD and parkinsonism are examples of protein misfolding diseases that affects protein clearance in the brain. The pathological changes of PD may appear as early as three decades before the appearance of clinical signs. Chaperone mediated autophagy plays a significant role in a-synuclein degradation in PD (Alvarez-Erviti, L. et al. (2010) Archives of neurology 67: 1464-1472), and some of the earliest changes in AD are connected to the lysosomal network, which consists of an interconnected vesicular network of endosomes, lysosomes and autophagosomes (Ihara, Y. et al. Cold Spring Harb PerspectMed 2012;2:a006361).

It has previously been shown that certain lysosomal network proteins are increased in CSF from Alzheimer's disease (AD) patients: early endosomal antigen 1 (EEA1); lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP -2); microtubule- associated protein 1 light chain 3 (LC3); and Rab3 (Armstrong, A. et al. (2014) Neuromolecular medicine 16: 150-160).

There is a need for novel tools for investigating the disease mechanisms for atypical parkinsonism diseases. For instance, there is a need for biomarkers that may be useful in the diagnosis of the various forms of atypical parkinsonism (APS).

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1: The lysosomal network proteins LAMP-1 and LAMP-2 are downregulated in the CSF of PD patients. A) Representative western blots of CSF from controls (C) and Parkinson disease (PD) patients analyzed for the lysosomal network proteins EEAl, LAMP-1, LAMP-2, LC3, Rab3 and lysozyme. B) Densitometric quantification of the scanned western blots from C (n=18) and PD (n=18) patients. The protein levels are normalized to a standard CSF sample loaded on each gel. The bars represent the mean ±SD: *p < 0.05.

Fig. 2: The lysosomal network proteins LAMP-1, LAMP-2, LC3 and lysozyme are upregulated in the CSF of pathologically confirmed CBD patients. A) Representative western blots of CSF from controls (C) and pathologically confirmed corticobasal degeneration (CBD) patients analyzed for the lysosomal network proteins EEAl, LAMP-1, LAMP-2, LC3, Rab3 and lysozyme. B) Densitometric quantification of the scanned western blots from C (n=10) and pathologically CBD (n=7) patients. The protein levels are normalized to a standard CSF sample loaded on each gel. The bars represent the mean ±SD: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 3: The lysosomal network proteins EEAl and Lysozyme are upregulated in the CSF of pathologically confirmed PSP patients. A) Representative western blots of CSF from controls (C) and pathologically confirmed progressive supranuclear palsy (PSP) analyzed for the lysosomal network proteins EEAl, LAMP-1, LAMP-2, LC3, Rab3 and lysozyme. B) Densitometric quantification of the scanned western blots from C (n=10) and pathologically confirmed PSP (n=8). The protein levels are normalized to a standard CSF sample loaded on each gel. The bars represent the mean ±SD: **p < 0.01; ***p < 0.001. DISCLOSURE OF THE INVENTION

It has surprisingly been found that a panel of five lysosomal network proteins (EEA1, LAMP-1, LAMP-2, LC3, and lysozyme) are useful as biomarkers for atypical parkinsonism. The lysosomal proteins LAMP-1 and LAMP-2 were significantly decreased in Parkinson's disease (PD) patients, LAMP-1, LAMP-2, LC3 and lysozyme levels were significantly increased in pathologically diagnosed CBD patients. EEA1 levels were significantly decreased, and lysozyme levels were significantly increased, in pathologically diagnosed PSP patients.

Consequently, in a first aspect this invention provides a method for diagnosing or predicting atypical parkinsonism in a mammal, preferably a human, said method comprising:

(a) obtaining a biological sample, such as a sample of cerebrospinal fluid (CSF), of a mammal;

(b) detecting and/or quantifying the amount of at least one biomarker in the sample, wherein the biomarker is selected from the group consisting of

Early Endosomal Antigen 1 (EEA1);

Lysosomal-associated membrane protein 1 (LAMP-1);

Lysosomal-associated membrane protein 2 (LAMP-2);

Microtubule-associated protein 1 light chain 3 (LC3); and

Lysozyme; and

(c) comparing the amount of the biomarker in the sample with a reference amount of the biomarker; wherein a significant difference between the amount of the biomarker in the sample and the reference amount is indicative of atypical parkinsonism, or a predisposition thereto.

Various methods are known in the art for detecting and quantifying a biomarker of choice. In a preferred aspect, detection/quantification is performed using an immunological method involving an antibody, or a fragment thereof, capable of specific binding to the peptide biomarker. Antibodies with high specificity for each of the disclosed biomarkers are commercially available (see Table II). Suitable immunological methods include; radioimmunoassays (RIA), sandwich, direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), Fluorescence immunoassays (FIA), western blotting,

immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots). Other suitable methods include the use of biosensor systems, e.g. systems based on surface plasmon resonance (SPR) techniques, such as Biacore™ systems and automated immunoassays such as the Gyros™ or Luminex™ technology platforms. Alternatively, detecting and/or quantifying biomarkers can be performed by one or more antibody-independent methods such as SELDI (-TOF), MALDI (-TOF), polyacrylamide gel electrophoresis (PAGE), Mass spectrometry (MS), reverse phase liquid chromatography (RPLC), size permeation (gel filtration), ion exchange chromatography, affinity chromatography, HPLC, UPLC or other LC or LC-MS-based methods.

The term "diagnosis" as used herein encompasses identification, confirmation, and/or characterization of atypical parkinsonism, or predisposition thereto. By "predisposition" it is meant that a subject does not currently have the disorder, but is liable to be affected by the disorder in time. The term "predicting" or "prediction" refers to a statement that it is expected that a patient is liable to be affected by atypical parkinsonism, but also encompasses the prediction that a patient is not liable to be affected by the said disorder.

In the present context, the term "biomarker" refers to a measurable indicator of a biological state or condition. Peptide biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and uses thereof are valuable for identification of new drug treatments and for discovery of new targets for drug treatment. Detection and/or quantification of peptide biomarkers may be performed by detection of the peptide biomarker or of a fragment thereof, e.g. a fragment with C- terminal truncation, or with N-terminal truncation. The term "reference amount" of a biomarker refers to the amounts present in a normal control biological sample from a normal subject. Consequently, the reference amount is an amount determined from at least one sample obtained from at least one healthy subject without atypical parkinsonism. Preferably, the reference level is obtained by analysis of several samples from different healthy subjects. In addition, reference amounts can be determined from historical data from a patient or a group of patients. Suitably, the samples from a subject undergoing diagnosis or monitoring will be taken over a time course. Samples may be taken prior to and/or during and/or following therapy for atypical parkinsonism.

In the present context, the term "significant difference" refers to a statistically significant difference. It will be understood that statistical significance is attained when a p-value is less than the significance level. In statistics, the p-value is the probability of obtaining at least as extreme results given that the null hypothesis (the hypothesis that there is no relationship between two measured phenomena) is true, whereas the significance level is the probability of rejecting the null hypothesis given that it is true. According to the present invention, the significance level is set to 0.05 (5%). More preferably, the significance level is 0.01 or 0.001. The term "biological sample" includes samples from whole blood, blood serum, plasma, cerebrospinal fluid (CSF), urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof. Biological samples also include tissue homogenates, tissue sections and biopsy specimens from a live subject, or taken post-mortem. Preferably, the biological sample is from cerebrospinal fluid (CSF).

In one aspect of the invention, the APS is corticobasal degeneration (CBD). For diagnosis of CBD, a biomarker chosen from lysozyme, LC3, LAMP-1 and LAMP -2, including combinations thereof, can be used. A significant increase in the amount of any one of the said biomarkers or combinations of biomarkers is indicative of CBD, or a predisposition thereto. Consequently, a combination of biomarkers can be chosen from:

Lysozyme and LC3;

Lysozyme and LAMP-1 ;

Lysozyme and LAMP-2; Lysozyme, LC3 and LAMP-1;

Lysozyme, LC3 and LAMP-2;

Lysozyme, LAMP-1 and LAMP-2; and

Lysozyme, LC3, LAMP-1 and LAMP-2.

In another aspect, APS is progressive supranuclear palsy (PSP). For diagnosis of PSP, a biomarker chosen from lysozyme and EEA1 can be used. A significant increase in the amount of Lysozyme in the sample or a significant decrease in the amount of EEA1 in the sample is indicative of PSP, or a predisposition thereto. Preferably, a significant increase in the amount of Lysozyme in the sample together with a significant decrease in the amount of EEA1 in the sample is indicative of PSP, or a predisposition thereto.

In yet a further aspect the invention provides a method for diagnosing or predicting Parkinson's disease (PD) in a mammal, wherein at least one biomarker is LAMP-1 or LAMP-2. A significant decrease in the amount of LAMP-1 or LAMP-2 in the sample is indicative of PD, or a predisposition thereto. Preferably, a significant decrease in the amounts of both LAMP-1 and LAMP-2 in the sample is indicative of PD, or a predisposition thereto. The invention further provides a method of monitoring efficacy of a therapy in a mammal having, suspected of having, or suspected of being predisposed to atypical parkinsonism, said method comprising detecting and/or quantifying, in a biological sample, preferably of cerebrospinal fluid (CSF) of said mammal, a biomarker is selected from the group consisting of: Early Endosomal Antigen 1 (EEA1); Lysosomal- associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP-2); Microtubule-associated protein 1 light chain 3 (LC3); and Lysozyme. The said method can preferably be used as a method of monitoring efficacy of a clinical trial. As described herein, the term "atypical parkinsonism" preferably refers to CBD and/or PSP. The said biomarkers, in various combinations as described herein, can be used for specifically distinguishing the said medical conditions.

It will be understood that the methods according to the invention may comprise the detection and quantification of additional biomarkers. Such additional biomarkers could be chosen from known biomarkers such as markers for general neurodegeneration in cerebrospinal fluid (CSF), e.g. total tau (T-Tau) or neurofilament light chain (NFL); markers for inflammation, e.g. monocyte chemoattractant protein-1 (MCP-1) or YKL- 40; amyloid-β related markers, e.g. soluble amyloid precursor protein a and β (sAPPa and β) or amyloid -β _1-42 (Αβ_1-42); as well as phosphorylated tau (P-Tau) and a-synuclein. Additional biomarkers could also include other lysosomal network proteins, such as one or more of the proteins listed in Table 1 in Armstrong, A. et al. (2014) Neuromolecular medicine 16: 150-160.

A further aspect of the invention is the use of Early Endosomal Antigen 1 (EEA1); Lysosomal-associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP-2); Microtubule-associated protein 1 light chain 3 (LC3); and Lysozyme as a specific panel of analyte biomarkers for the diagnosis of atypical parkinsonism, or a predisposition thereto. As described herein, the term "atypical parkinsonism" preferably refers to CBD and/or PSP. The said biomarkers, in various combinations as described herein, can be used for specifically distinguishing the said medical conditions.

The invention further provides a kit for the diagnosis or prediction of atypical parkinsonism, said kit comprising (a) at least one reagent capable of detecting at least one biomarker; and (b) instructions for using the at least one reagent in an assay for detecting the presence of the at least one biomarker, wherein the biomarker is selected from the group consisting of: Early Endosomal Antigen 1 (EEA1); Lysosomal- associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP-2); Microtubule-associated protein 1 light chain 3 (LC3); and Lysozyme. As described herein, the term "atypical parkinsonism" preferably refers to CBD and/or PSP. The said biomarkers, in various combinations as described herein, can be used for specifically distinguishing the said medical conditions.

The said reagent preferably comprises an antibody or a biosensor. The term "antibody" as used herein includes polyclonal, monoclonal, bispecific, humanized or chimeric antibodies, single chain antibodies, Fab fragments and F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies and epitope- binding fragments of any of the above. The term "antibody" as used herein also refers to immunoglobulin molecules and immunologically-active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. The immunoglobulin molecules of the invention can be of any class (e.g., IgG, IgE, IgM, IgD and IgA) or subclass of immunoglobulin molecule. The invention further provides the use of the biomarkers, as described above, as targets for the identification of active compounds that may be useful in the treatment of atypical parkinsonism. Consequently, the invention provides a method for identifying a compound useful for the diagnosis, prediction or treatment of atypical parkinsonism, said method comprising:

(a) screening compounds for specific interaction with a biomarker mRNA or biomarker protein; and

(b) observing an altered activity or expression of the biomarker mRNA or biomarker protein, wherein the biomarker is selected from the group consisting of: Early

Endosomal Antigen 1 (EEA1); Lysosomal-associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP -2); Microtubule-associated protein 1 light chain 3 (LC3); and Lysozyme. As described herein, the term "atypical parkinsonism" preferably refers to CBD and/or PSP. The said biomarkers, in various combinations as described herein, can be used for specifically distinguishing the said medical conditions.

EXPERIMENTAL METHODS

Study population

CSF samples (Set 1) from PD patients (n=18) who met the criteria for PD as defined by The United Kingdom PD Society Brain Bank (Hughes, A.J. et al. (1992) Journal of neurology, neurosurgery, and psychiatry 55: 181-184) were sampled at the Karolinska Hospital, Stockholm, Sweden. The patients were matched in age and gender with controls (C) suffering from benign neurological conditions, such as tension headache and unclassified sensory disturbances (n=18). The characteristics of the patients included in CSF set 1 are shown in Table I.

CSF samples (Set 2) from clinically diagnosed and pathologically confirmed PSP (n=8), CBD (n=7), were sampled at the University of California, San Francisco Memory and Aging Center. The patients were matched in age and gender with controls (n=10). Patients with PSP were clinically diagnosed with possible or probable PSP according to the National Institute of Neurological Disorders and Stroke-Society for Progressive Supranuclear Palsy (NINDS-SPSP) criteria (Litvan, I. et al. (1996) Neurology 47: 1-9). Patients with CBD were diagnosed according to the Armstrong criteria (Armstrong, M.J. et al. (2013) Neurology 80:496-503). The patients were matched in age and gender, with controls (n=l 1) suffering from benign neurological conditions, such as tension headache and unclassified sensory disturbances. Pathological confirmation of the disease was performed on postmortem brains according to the previously described method (Gallyas F. (1971) Acta Morphologica Academiae Scientiarum Hungaricae 19: 1 -8). The characteristics of the patients included in CSF set 2 are presented in Table I.

CSF sampling

At the Karolinska Hospital, a standardized lumbar puncture (LP) procedure was performed at the L4-5 vertebrae level with the patient in a supine position. Between 12 and 15 mL CSF from the first portion was collected in order to minimize the gradient influence on the proteins, peptides and neurotransmitters. All CSF samples were aliquoted within an hour after LP and stored at -80°C until assayed. At the University of California, San Francisco, LPs were performed fasting and sitting up, in accordance with the Alzheimer's Disease Neuroimaging Initiative (ADNI) recommended procedure

(http://www. adni-info. org/ADNIStudyProcedures/LumbarPunctures. aspx), and occurred within a maximum of 5 months of the clinical assessment. Samples were collected in the morning after a 12 h fast, into sterile 13 mL polypropylene tubes. The first 2 mL were discarded. CSF was then transferred into 1 mL aliquots and put on dry ice for an hour before storing at -80°C.

Determination ofbiomarkers by immunoblotting (Western blotting)

20 CSF was mixed with loading buffer (0.1 M Tris, pH 6.8, 6% glycerol, 4% SDS, 0.2% bromophenol blue, 1.6% β-mercaptoethanol, 50 mM DTT), and the samples were heated to +95°C. Proteins were separated by SDS-PAGE (200 V, 90 mA/gel, 1 h) and blotted onto a 0.45 um nitrocellulose membrane in transfer buffer (Tris-Base 25 mM, Glycine 192 mM, Methanol 20%) for 1 h at 100 V and +4°C. The membrane was blocked in 5% dry milk or 5% BSA in TBS-Tween and probed with a primary antibody (diluted in 5% dry milk or 5% BSA in TBS-Tween) overnight at 4°C. The primary antibodies used are shown in Table II.

The membranes were washed and probed with horseradish peroxidase-conjugated antibodies (P0449, P0448 and P0447, Dako, Glostrup, Denmark) for 1 h at room temperature. After washing, immunodetection of the bound antibodies was performed using Amersham™ ECL™ detection systems (GE Healthcare, Pittsburgh, PA, USA) and exposure to photographic film. The films were scanned and the immunoblots were quantified using the Image J program (available at http://rsbweb.nih.gov/ij/). The relative amount of protein corresponding to an immunoreactive band was calculated as a product of average optical density of the area of the band. All antibodies confirmed to recognize their epitope via western blot analysis of cell lysates. Equal sample loading was verified by Ponceau S (Sigma Aldrich, St. Louis, MO, USA) staining of total protein in each lane on the membranes. 20 uL of a standard CSF sample was loaded on each gel for normalization between the gels. Each gel was loaded with both control and patient samples. Any difference in blot intensity, due to development differences between the paired gels, was removed by subtracting the blot background for each sample separately.

Statistical Analyses

The mean value and standard deviation (SD) were calculated for all data. The parametric Mann-Whitney U test was used to test for significant differences between groups. Correlation analysis to measure dependence between two quantities was performed using the Spearman's rank correlation coefficient. Statistical significance was defined for p-values of less than 0.05 (*), 0.01 (**) and 0.001 (***). All statistical analyses were performed using GraphPad Prism version 5.00 for Windows (GraphPad Software, San Diego, CA USA). EXAMPLES OF THE INVENTION

EXAMPLE 1 : Decreased levels of the lysosomal network proteins LAMP-1 and LAMP-2 in PD CSF

Levels of the lysosomal network proteins EEAl, LAMP-1, LAMP-2, LC3, Rab3 and lysozyme were determined in controls and in PD CSF (Table I, CSF set 1) using immunoblotting. LAMP-1 and LAMP-2 levels were significantly decreased in the PD samples as compared with the control CSF samples (Fig. 1A and IB). There was a 23% decrease in LAMP-1 levels, and a 30% decrease in LAMP-2 levels in PD CSF. Equal sample loading was confirmed via Poncaeu staining of total protein in each lane. EEAl, LC3, Rab3 and lysozyme were present at detectable levels in the CSF, but no significant differences were observed between controls and PD patients. EXAMPLE 2 : Changed levels of lysosomal network proteins EEA- 1 , LAMP- 1 , LAMP- 2, LC3 and lysozyme discriminates between pathologically confirmed CBD and PSP CSF

Levels of the lysosomal network proteins in CSF from clinically diagnosed CBD and PSP patients, where the diagnosis had been pathologically confirmed post-mortem, were investigated. LAMP-1, LAMP-2, LC3 and lysozyme levels were significantly increased in pathologically confirmed CBD patient CSF, compared with control samples (Fig. 2A and 2B): LAMP-1 by 66%, LAMP-2 and LC3 by 280%, and lysozyme by 456%.

A different pattern emerged in the pathologically confirmed PSP patients, where the EEAl level was significantly decreased by 29% and the lysozyme level was significantly increased by 211% in CSF compared with control samples (Fig. 3A and 3B). ^A 3

TABLE I

Study demographics. Data are presented as mean and (range).

Age Gender Disease Levodopa CSF CSF CSF CSF P- (years) (M:F) duration equivalent NFL Αβ42 T-tau taul81P

(years) dose (ng/L) (ng/L) (ng L)

(ng/L)

(LED)

CSF set 1

PL) (n=18) 61 15:3 4 1 14 973 182 41

(39-80) (1-14) (0-2012) (438- (22- (16-90)

1600) 422)

C (R=18) 62 14:4 n a n/a

(46-83)

CSF set 2

Pathologically 64 4:3 6 0 7324 288 91 26 confirmed (61-70) (5-8) (4935- (203- (55- (17-36)

9440)

CBD (n ~ ) 389) 140)

Pathologically 69 2:6 10 150 3783 344 84 28 confirmed (55 - (4-17) (0-600) (1669- (169- (63- (19-36)

PSP (n=8) 88) 6104) 409) 127)

C (n=10) 59 4:6 ti/a n/a 417 72 16

(52-68) (310- (43- (15-42)

425) 108)

TABLE II

Biomarkers quantified by immunoblotting.

Human protein Antibody

(Catalog No.)

EEA1 E4156

Sigma-Aldrich, St. Louis, MO, USA

LAMP-1 9835-01

Southern Biotech, Birmingham, AL, USA

LAMP-2 9840-01

Southern Biotech, Birmingham, AL, USA

LC3 NB600-1384

(MAP1LC3) Novus Biologicals, Littleton, CO, USA

Lysozyme A0099

(Lysozyme C) Dako, Glostrup, Denmark

Rab3 107011

Synaptic Systems GmbH, Goettingen, Germany

Claims

A method for diagnosing or predicting atypical parkinsonism (APS) in a mammal, said method comprising:

(a) obtaining a biological sample from a mammal;

Early Endosomal Antigen 1 (EEAl);

Lysosomal-associated membrane protein 1 (LAMP-1);

Lysosomal-associated membrane protein 2 (LAMP-2);

Microtubule-associated protein 1 light chain 3 (LC3); and

Lysozyme; and

(c) comparing the amount of the biomarker in the sample with a reference amount of the biomarker; wherein a significant difference between the amount of the biomarker in the sample and the reference amount is indicative of parkinsonism, or a predisposition thereto.

The method according to claim 1 wherein at least one biomarker is Lysozyme, and wherein a significant increase in the amount of Lysozyme in the sample is indicative of APS, or a predisposition thereto.

The method according to claim 1 or 2 wherein the APS is corticobasal degeneration (CBD).

The method according to claim 3 wherein at least one additional biomarker is chosen from the group consisting of LC3, LAMP-1 and LAMP-2, and wherein a significant increase in the amount of an additional biomarker in the sample is indicative of CBD, or a predisposition thereto.

The method according to claim 1 or 2 wherein the APS is progressive

supranuclear palsy (PSP).

The method according to claim 5 wherein an additional biomarker is EEAl, and wherein (i) a significant increase in the amount of Lysozyme in the sample; and (ii) a significant decrease in the amount of EEA1 in the sample is indicative of PSP, or a predisposition thereto.

A method of monitoring efficacy of a therapy in a mammal having, suspected of having, or suspected of being predisposed to atypical parkinsonism (APS), said method comprising detecting and/or quantifying, in a biological sample of said mammal, a biomarker selected from the group consisting of:

Early Endosomal Antigen 1 (EEA1);

Lysosomal-associated membrane protein 1 (LAMP-1);

Lysosomal-associated membrane protein 2 (LAMP-2);

Microtubule-associated protein 1 light chain 3 (LC3); and

Lysozyme.

The method according to any one of claims 1 to 7, comprising detecting and/or quantifying, in a biological sample of said mammal, at least one additional lysomal network protein.

The method according to any one of claims 1 to 8, comprising detecting and/or quantifying, in a biological sample of said mammal, at least one additional biomarker chosen from the group consisting of total tau (T-Tau), neurofilament light chain (NFL), monocyte chemoattractant protein-1 (MCP-1), YKL-40; soluble amyloid precursor protein a and β (sAPP a and β), amyloid-Pi-42 (Αβι-42), phosphorylated tau (P-Tau), and a-synuclein.

The method according to any one of claims 1 to 9, wherein the said biological sample is a sample of cerebrospinal fluid (CSF).

The method according to any one of claims 1 to 10 wherein the mammal is a human.

Use of Early Endosomal Antigen 1 (EEA1); Lysosomal-associated membrane protein 1 (LAMP-1); Lysosomal-associated membrane protein 2 (LAMP-2); Microtubule-associated protein 1 light chain 3 (LC3); and Lysozyme as a specific panel of analyte biomarkers for the diagnosis of atypical parkinsonism (APS), or a predisposition thereto.

A kit for the diagnosis or prediction of atypical parkinsonism (APS), said kit comprising

(a) at least one reagent capable of detecting at least one biomarker; and

(b) instructions for using the at least one reagent in an assay for detecting the presence of the at least one biomarker;

wherein the biomarker is selected from the group consisting of:

Early Endosomal Antigen 1 (EEA1);

Lysosomal-associated membrane protein 1 (LAMP-1);

Lysosomal-associated membrane protein 2 (LAMP-2);

Microtubule-associated protein 1 light chain 3 (LC3); and

Lysozyme.

The kit according to claim 13 wherein at least one reagent is a monoclonal or polyclonal antibody.

The kit according to claim 13 wherein at least one reagent is a biosensor.

A method for identifying a compound useful for the diagnosis, prediction or treatment of atypical parkinsonism (APS), said method comprising:

(a) screening compounds for specific interaction with a biomarker mRNA or biomarker protein;

(b) observing an altered activity or expression of the biomarker mRNA or biomarker protein, wherein the biomarker is selected from the group consisting of:

Early Endosomal Antigen 1 (EEA1);

Lysosomal-associated membrane protein 1 (LAMP-1);

Lysosomal-associated membrane protein 2 (LAMP-2);

Microtubule-associated protein 1 light chain 3 (LC3); and

Lysozyme.