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WO2012175674A1 - Diagnosis and/or prognosis of a neurodegenerative disease - Google Patents

Diagnosis and/or prognosis of a neurodegenerative disease Download PDF

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Publication number
WO2012175674A1
WO2012175674A1 PCT/EP2012/062089 EP2012062089W WO2012175674A1 WO 2012175674 A1 WO2012175674 A1 WO 2012175674A1 EP 2012062089 W EP2012062089 W EP 2012062089W WO 2012175674 A1 WO2012175674 A1 WO 2012175674A1
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Prior art keywords
disease
parkinson
biomarker
subject
expression level
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PCT/EP2012/062089
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French (fr)
Inventor
Markus Otto
Stefan Lehnert
Sarah JESSE
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Baden Wuerttemberg Stiftung gGmbH
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Baden Wuerttemberg Stiftung gGmbH
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical 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
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease, particularly Parkinson's disease (PD) or Parkinson's disease dementia (FDD), which comprises the determination of the expression profile of a biomarker indicative for said disease.
  • the present invention further relates to a molecule for detecting said biomarker for diagnosing and/or prognosing a neurodegenerative disease. It also relates to means for diagnosing and/or prognosing a neurodegenerative disease and a kit for diagnosing and/or prognosing a neurodegenerative disease.
  • PD Parkinson's disease
  • PD patients have a 6-fold higher risk for the development of a dementia than healthy persons of the same age [2].
  • Up to half of all PD patients show a mild cognitive impairment in the early disease-stages [3], About one third of them later develop a dementia which is sometimes also accompanied by changes in personality [4].
  • This dementive syndrome normally develops within 8-10 years and has severe consequences on the course of the disease.
  • the dementia Apart from the obvious strain on the person's social environment the dementia also goes along with a worse prognosis as far as disease- progression and life-expectancy are concerned [5]. Therefore, early treatment of dementia is critical since early therapy of the cognitive deficits is considered to be crucial to its success [6] ⁇
  • Parkinson's disease dementia is neuropathologically characterized by the presence of cortical Lewy bodies which also occur in patients with Lewy-body-dementia [7- 10], These Lewy bodies contain alpha-synuclein, a presynaptic filament protein which is expressed i high amounts in the terminal ends of neurons. The fact that these inclusions are detected mostly in living cells rather than in apoptotic cells suggests that the inclusions play a protective role by sequestering toxic molecules [11]. A possible link between aggregation, neurotoxicity and disease-propagation might be that neurotoxic oligomers of alpha-synuclein can be transformed to non-toxic oligomers which have a higher aggregation- tendency [12, 13].
  • Several studies to improve the early diagnosis of FDD in cerebrospinal fluid and serum have been undertaken [14]. However the results are of limited use for the prognosis of disease progression.
  • diagnosis and/or prognosis of a neurodegenerative disease such as PD or PDD and the differential diagnosis and/or prognosis between the neurodegenerative diseases PD and PDD is still performed by physicians on the basis of the patient's medical history and neurological examination.
  • iTRAQ cerebrospinal fluid optimised for cerebrospinal fluid
  • NDC's cerebrospinal fluid
  • the present inventors found differentially regulated proteins. They verified them in a larger group of patients using a m ass-spec trometry-based technique, the so-called multiple-reaction- monitoring (MRM), which uses specific, synthetic peptides to quantify proteins in a complex mixture.
  • MRM multiple-reaction- monitoring
  • MRM-approach itself has been used for some time in the field of pharmacokinetics, but has only very recently been applied as a quantitative proteomic tool [15-21] due to the challenging development of the method.
  • MRM or the smglc-analyte analogue selected-reaction-monitoring (SUM) are highly specific methods as exclusive peptides of the protein(s) of interest are first selected via their intact mass and then further fragmented and characterized via their specific fragments. Therefore, at least two correct ion- masses are required for a single measurement, thus, enhancing sensitivity by reducing cross- reactivity and background (for a detailed review see: [22]).
  • SUM smglc-analyte analogue selected-reaction-monitoring
  • patients having a neurodegenerative disease such as PD or PDD can be identified on the basis of the expression level of at least one biomarker selected from the group consisting of Netrin G l .
  • CSF cerebrospinal fluid
  • the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of:
  • the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160 for diagnosing and/or prognosing a neurodegenerative disease.
  • a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160 for diagnosing and/or prognosing a neurodegenerative disease.
  • the present invention relates to ' means for diagnosing and/or prognosing a neurodegenerative disease comprising at least one molecule according to the second aspect.
  • the present invention relates to a kit for diagnosing and/or prognosis a neurodegenerative disease comprising
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
  • the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of:
  • biomarker e.g. 1, 2, 3, or 4 biomarker(s) selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B- I QO and Golgin-160 in a biological sample from a subject, and
  • the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of;
  • biomarker Netrin Gl (ia) the biomarker Netrin Gl , (ib) the biomarker Tyrosin-protein phosphatase non- receptor-type 13, (ic) the biomarker Apolipoprotein B- 100, (id) the biomarker Golgin- 160, (ie) the biomarkers Netrin Gl and Tyrosin-protein phosphatase non-receptor-type 13, (if) the biomarkers Netrin Gl and Apolipoprotein B-100, (ig) the biomarkers Netrin Gl and Golgin-160, (ih) the biomarkers Tyrosin-protein phosphatase non- receptor-type 13 and Apolipoprotein B-100, (ij) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160, (ik) the biomarkers Apolipoprotein B-100 and Golgin-160, (il) the biomarkers Netr
  • neurodegenerative disease refers to a range of conditions which primarily affect the neurons in the brain. Neurons are the building blocks of the nervous system which includes the brain and. spinal cord. They normally don't reproduce or replace themselves, so when they become damaged or die they cannot be replaced by the body. Thus, particularly, the term “neurodegenerative disease” refers to a range of conditions which leads to a progressive loss of structure or function of neurons, including death of neurons. This causes problems with, movement (called ataxias), or mental functioning (called dementias). Examples of neurodegenerative diseases include, but are not limited to, Parkinson's, Alzheimer's, and Huntington's disease.
  • diagnosis a neurodegenerative disease means determining whether a subject shows signs of or suffers from a neurodegenerative disease.
  • prognosing a neurodegenerative disease means predicting whether a subject will show signs of or suffer from a neurodegenerative disease in the future, but preferably also means predicting the course of a neurodegenerative disease of a subject already showing signs of or suffering from a neurodegenerative disease.
  • Netrin Gl belongs to a family of proteins which are involved in the process of axonal guiding through which growing axons are guided towards their correct targets.
  • Netrin Gl was first described in the year 2000 as a glycosyl phosphatidylinositol (GPi) anchored member of that family. Its expression in the brain reaches a maximum at perinatal stages in most regions. However in the cerebellum, Netrin Gl is only expressed in certain regions which are implicated in controlling motor activity.
  • the term "Netrin Gl” encompasses Netrin Gl variants, e.g. all non-naturally or naturally occurring variants such as Netrin Gl homologues. particularly orthologues or paralogues.
  • the Netrin Gl variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1 (human Netrin Gl , Accession number: AAH30220.1).
  • the Netrin Gl variants have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66. 67.
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 420, or more amino acids, preferably over the whole length of the Netrin Gl amino acid sequence.
  • the biomarker Tyrosin-protein phosphatase non-receptor-type 13 belongs to the protein tyrosine phosphatase (PTP) family.
  • PTPs are known to be signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation.
  • PTPN13 is a large protein that possesses a PTP domain at C- terminus, and multiple non-catalytic domains, which include a domain with similarity to band 4.1 superfamily of cytoskeletal-associated proteins, a region consisting of five PDZ domains, and a leucine zipper motif.
  • PTPN13 was found to interact with, and dephosphorylate Fas receptor, as well as IkappaBalpha through the PDZ domains, which suggested its role in Fas mediated programmed cell death, PTPN13 was also shown to interact with GTPase- activating protein, and, thus, may function as a regulator of Rho signaling pathway.
  • the term "Tyro sin-protein phosphatase non-receptor-type 13" encompasses Tyrosin-protei phosphatase non-receptor-type 13 variants, e.g. all non-naturally or naturally occurring variants such as Tyrosin-protein phosphatase non-receptor-type 13 homologues, particularly orthologues or paralogues.
  • the Tyrosin-protein phosphatase non-receptor-type 13 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 2 (human PTPN 13, Accession number: AAH39610.1).
  • the Tyrosin-protein phosphatase non-receptor-type 13 variants have an amino acid sequence which is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 2.
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more amino acids, preferably over the whole length of the Tyrosin-protein phosphatase non- receptor-type 13 amino acid sequence.
  • the biomarker Apolipoprotein B-100 is the primary Apolipoprotein of low-density lipoproteins (LDL or "bad cholesterol"), which is responsible for carrying cholesterol to tissues. In the brain, Apolipoprotein B-100 can lead to neurod egen erati on through increasing the levels of serum lipids thus leading to cerebrovascular lesions.
  • the term "Apolipoprotein B-100” encompasses Apolipoprotein B-100 variants, e.g. all non-naturally or naturally occurring variants such as Apolipoprotein B-100 homologues, particularly orthologues or paralogues.
  • the Apolipoprotein B-100 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 3 (human Apolipoprotein B-100 precursor, Accession number: P_000375.2) or SEQ ID NO: 4 (human Apolipoprotein B- 100, Accession number: A A51758.1).
  • the Apolipoprotein B-100 variants have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or more amino acids, preferably over the whole length of the above Apolipoprotein B-100 amino acid sequences.
  • Golgins are a large family of proteins which have different functions in connection with the Golgi-apparatus.
  • the biomarker Golgin A3 protein also known as Golgin-160, because of its molecular weight, is involved in intra-Golgi vesicle mediated transport.
  • Golgin-160 encompasses Golgin-160 variants, e.g. all non-naturally or naturally occurring variants such as Golgin-160 homologues, particularly orthologues or paralogues.
  • the Golgin-160 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 5 (human Golgin subfamily A member 3 isoform 1, Accession number: NP 005886.2).
  • the Golgin-160 variants have an amino acid sequence which is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 5.
  • sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more amino acids, preferably over the whole length of the Golgin-160 amino acid sequence.
  • the above-mentioned terms encompass all types of isoforms of said biomarkers.
  • Alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.iik/emboss/align index.htrril) using standard settings, preferably for Align EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • ClustalW www.ebi.ac.uk/clustalw
  • Align http://www.ebi.ac.iik/emboss/align index.htrril
  • Align EMBOSS ::needle
  • Matrix Blosum62
  • Gap Open 10.0 Gap Extend 0.5.
  • Gene expression is the process by which information from a gene is used for the synthesis of a functional gene product. Gene expression occurs in two major stages. The first is transcription. In this process, the gene is copied to produce a R A molecule (a primary transcript) with essentially the same sequence as the gene. Most eukaryotic genes are divided into exons and introns, and only the exons carry information required for protein synthesis. Most primary transcripts are, therefore, processed by splicing to remove intron sequences and. generate a mature transcript or messenger RNA (mRNA) that only contains exons. In the second step, the final protein encoded by said mRNA is produced. This stage is also known as translation.
  • mRNA messenger RNA
  • the term "expression level" of Netrin Gl, Tyro sin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160 refers to the expression level of the gene of Netrin Gl , Tyro sin-protein phosphatase non- receptor-type 13, Apolipoprotein B- 100 or Golgin-160.
  • the expression level of the gene of Netrin G l , Tyrosin-protein phosphatase non-rcceptor-type 13, Apolipoprotein B-100 or Golgin-160 may be determined on the mRNA level (transcriptional level) or protein level (translationai level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via immunohistochemistry) or mRNA (e.g. via in situ hybridization).
  • the amount of the Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160 mRNA or protein is determined.
  • the subject is identified as experiencing a neurodegenerative disease or being prone thereto, if the expression level of the above-mentioned biomarker is altered compared to a control.
  • an altered expression level is an expression level changed to a higher (increased) level or lower (decreased) level in a subject experiencing a neurodegenerative disease or being prone thereto compared to a control.
  • an altered, more preferably an increased or a decreased, expression level is considered to be present for the purpose of the present invention when the expression profile of the tested subject differs by at least 1%, 5% or 10%, preferably by at least 20% or 30%, more preferably by at least 50% or 70%, most preferably by at least 80%, 90%, 100%, or more than 100%, e.g.
  • subject may mean a subject suspected to experience a neurodegenerative disease such as PD or PDD.
  • the subject may be diagnosed to experience a neurodegenerative disease such as PD or PDD, i.e. be diseased.
  • subject as mentioned in the method above, may also mean a subject which already experiences a neurodegenerative disease such as PD or PDD.
  • the subject may be retested for experiencing a neurodegenerative disease such as PD or PDD and may be diagnosed to still experience a neurodegenerative disease such as PD or PDD, e.g. a more severe or pronounced form, level or stage of a neurodegenerative disease such as PD or PDD.
  • the term "subject”, as mentioned in the method above, may further mean a subject suspected to develop a neurodegenerative disease such as PD or PDD.
  • the subject may be prognosed to develop a neurodegenerative disease such as PD or PDD in the future.
  • the subject may be a subject which already suffers from PD but which is suspected to have developed a dementia in the course of the disease.
  • the "subject”, as mentioned in the method above, may further be a human or another mammal, e.g. a rodent (e.g. rat, hamster, or mouse) or monkey, or may be another animal than a mammal, e.g. an avian.
  • the subject is a human or another mammal.
  • the subject to be diagnosed and/or prognosed with the method of the present invention may also be designated as "test subject” herein.
  • control may refer to a value/data of a (control) subject known to experience a neurodegenerative disease such as PD or PDD (positive control), i.e. be diseased.
  • control as mentioned in the method above, may also refer to a value/data of a (control) subject known to not experience a neurodegenerative disease such as PD and/or PDD (negative control), i.e. be healthy.
  • Said (control) subject may be a human or another mammal, e.g. a rodent (e.g. rat, hamster, or mouse) or monkey, or may be another animal than a mammal, e.g. an avian.
  • said (control) subject is a human or another mammal.
  • the subject known to experience a neurodegenerative disease or known to not experience a neurodegenerative disease may also be designated as "control subject” herein.
  • control subject a "(control) subject” that is known to be healthy, i.e. not suffering from a neurodegenerative disease such as PD and/or PDD, may possibly suffer from another disease not known/tested, e.g. migraine.
  • control (value) is
  • biomarker e.g. 1, 2, 3, or 4 biomarkcr(s) selected from the group consisting of Netrin Gl , Tyrosin- protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Goigin-160 of a healthy subject, and/or
  • biomarker e.g. 1, 2, 3, or 4 biomarker(s) selected from the group consisting of Netrin Gl , Tyro sin -protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience a neurodegenerative disease (e.g. a specific form, level or stage of a neurodegenerative disease).
  • control (value) is an average control (value). It is particularly preferred that the control (value) is an. average control (value) of at least 2 to 40 (control) subjects, more preferably of at least 10 to 40 (control) subjects, and most preferably of at least 15 to 40 (control) subjects, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 (control) subjects.
  • control (value) is an average control (value). It is particularly preferred that the control (value) is an. average control (value) of at least 2 to 40 (control) subjects, more preferably of at least 10 to 40 (control) subjects, and most preferably of at least 15 to 40 (control) subjects, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39
  • both results (values) are preferably achieved with the same methods, more preferably carried out under the same method/process conditions.
  • results (values) achieved with the same methods and more preferably under comparable, most preferably identical method/process conditions are compared to identify a subject experiencing a neurodegenerative disease.
  • the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). It is also preferred that the method of the present invention relates to the differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • PD Parkinson's disease
  • PDD Parkinson's disease dementia
  • the present invention relates to a method for diagnosing and/or prognosing Parkinson's disease (PD). Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD) comprising the steps of:
  • biomarker e.g. 1, 2, 3, or 4 biomarker(s) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject, and
  • differentiated diagnosing and “differential prognosing” between PD and PDD relate to the discrimination between both disease states based on observations made with respect to the expression level of at least one of the biomarkers selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160.
  • Parkinson's disease refers to a chronic (persistent) disorder of part of the brain. It is named after the person who first described it. It mainly affects the way the brain co-ordinates the movements of the muscles in various parts of the body.
  • the main symptoms of Parkinson's disease are, for example, stiffness, shaking (tremor), and slowness of movement. Symptoms typically become gradually worse over time.
  • Parkinson's disease dementia denotes the impairment of one or more cognitive processes, particularly related to memory, in subjects showing signs of or suffering from Parkinson's disease, also known as Parkinson disease, Parkinson's, idiopathic parkinsonism, primary parkinsonism, PD or paralysis agitans.
  • the control (value) is particularly preferred that in a method for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD), the control (value) is a method for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD), the control (value) is
  • the method of the present invention is for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD) comprising the steps of:
  • the expression level of the biomarker Netrin Gl and/or Tyrosin- protein phosphatase non-receptor-type 13 in a biological sample from a subject determined with the above-mentioned method, is increased compared to a control which is the expression level of the biomarker Netrin Gl and/or Tyrosin-protem phosphatase non-receptor-type 13 known to be present in a healthy subject, the subject is identified as experiencing FDD or being prone thereto.
  • the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-rcceptor-type 13 in a biological sample from a subject determined with the above-mentioned method, is increased compared to a control which is the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a subject experiencing Parkinson's disease (PD), the subject is identified as experiencing FDD or being prone thereto.
  • PD Parkinson's disease
  • the method of the present invention is for diagnosing and or prognosing Parkinson's disease dementia (FDD) and that the control is
  • the subject is identified as experiencing Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), if the expression level of the biomarker Netrin Gl is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g.
  • the expression level of the biomarkcr Netr is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above- mentioned respective control.
  • the subject is identified as experiencing Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), if the expression level of the biomarkcr Tyrosin-protein phosphatase non-receptor-type 13 is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g.
  • the expression level of the biomarkcr Tyrosin-protein is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 105, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned respective control.
  • Netrin Gl As well as Tyrosin-protein phosphatase non-receptor-type 13 as biomarkers.
  • the subject is identified as experiencing
  • Parkinson's disease dementia or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), (i) if the expression level of the biomarker Netrin Gl is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g.
  • the expression level of the biomarker Tyrosin- protein phosphatase non-receptor-type 13 is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g. Is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13. 14, 15, 16. 17, 18, 19, 20. 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35,
  • the (average) expression level of the biomarker Netrin Gl and or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a subject experiencing Parkinson's disease dementia (PDD) is preferably used as a control.
  • the method of the present invention is for diagnosing and/or prognosing Parkinson's disease (PD) comprising the steps of:
  • the expression level of the biomarker Apolipoprotein B-100 in a biological sample from a subject determined with the above-mentioned method, is decreased compared to a control which is the expression level of the biomarker Apolipoprotein B-100 known to be present in a healthy subject, the subject is identified as experiencing PD or being prone thereto.
  • the expression level of the biomarker Apolipoprotein B-100 in a biological sample from a subject determined with the above-mentioned method, is decreased compared to a control which is the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD), the subject is identified as experiencing PD or being prone thereto.
  • a control which is the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD)
  • the method of the present invention is for diagnosing and/or prognosing Parkinson's disease (PD) and that the control is (iia) the expression level of the biomarker Apolipoprotein B-100 known to be present in a healthy subject, and
  • the subject is identified as experiencing Parkinson's disease (PD) or being prone thereto, particularly as experiencing Parkinson's disease (PD), if the expression level of the above-mentioned biomarker is decreased by at least 1%, at least 5%, or at least 10 %, more preferably at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably at least 60 % , at least 70%, at least 80%, and most preferably at least 90 %, 95 %, 99%, 100% or more, e.g.
  • the expression level of the biomarker Apolipoprotein B-100 is decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned control
  • the (average) expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease (PD) is preferably used as a control.
  • the method of the present invention is for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) comprising the steps of:
  • a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) comprising the steps of:
  • the expression level of the biomarker Golgin-160 in a biological sample from a subject is decreased compared to a control which is the expression level of the hiomarker Golgin- 160 known to be present in a healthy subject, the subject is identified as experiencing Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto.
  • PD Parkinson's disease
  • PDD Parkinson's disease dementia
  • the subject is identified as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD), if the expression level of the above-mentioned hiomarker is decreased by at least 1%, at least 5%, or at least 10 %, more preferably at least 20 %, at least 30%. at least 40% or at least 50 %, still more preferably at least 60 % , at least 70%, at least 80%, and most preferably at least 90 %, 95 %, 99%, 100% or more, e.g.
  • the expression level of the hiomarker Golgin-160 is decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned control.
  • a comprehensive diagnosis and/or prognosis of a neurodegenerative disease preferably involves the analysis, whether the test subject suffers from PD or PDD. Therefore, the expression profile of at least one hiomarker indicative for PD (e.g. overlookipoprotein B- 100) and at least one biomarker indicative for PDD (e.g. Netrin Gl or Tyrosin-protein phosphatase non-receptor-type 13) is preferably determined.
  • the expression profile of (i) Netrin Gl and AINipoprotein 13- 100, (ii) Tyrosin-protein phosphatase non-receptor-type 13 and ANAipoprotein B-100, or (iii) Netrin Gl , Tyrosin- protein phosphatase non-receptor-type 13 and ANAipoprotein B-100 is determined.
  • the term "increased compared to a control” may also mean increased compared to an expression level of zero. This may be the case where a specific biomarker is not present/detectable in the biological sample from a control subject, e.g. subject known to be healthy or known to suffer from a neurodegenerative disease such as PD or PDD, but present/detectable in the biological sample from the test subject, e.g. subject which is suspected to experience or develop a neurodegenerative disease such as PD or PDD.
  • the term "biological sample”, as used herein, refers to any biological sample comprising the above-mentioned biomarkers.
  • the biological sample may be any sample comprising cells or the products of cells derived from a subject. It may be a body fluid sample, a tissue sample (e.g. explant or section), or a cell sample (e.g. ccll(s) or cell colonies).
  • said biological sample may be an. explant sample, a section sample, a single cell sample, a cell colony sample, a ceil culture sample, a blood sample, a urine sample, or a sample from another peripheral source.
  • the biological samples may be mixed or pooled, e.g. a biological sample may be a mixture of a blood sample and a urine sample.
  • the biological sample may be provided by removing cell colonies, an explant, or a section from a subject, but may also be provided by using a previously isolated sample.
  • a tissue sample may be removed from a subject by conventional biopsy techniques or a blood sample may be taken from a subject by conventional blood collection techniques.
  • the biological sample is a body fluid sample, a tissue sample, a cell colony sample, a single cell sample or a cell culture sample. More preferably, the tissue sample is a section or an explant sample.
  • the tissue sample from a subject has a weight of between 0.1 and 500 mg, more preferably of between 0.5 and 250 mg, and most preferably of between 1 and 50 mg, i.e. 0.1, 0.2, 03, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg.
  • the cell sample (e.g. cell colony sample or cell culture sample) from a subject consists of between 10 2 and 10 10 cells, more preferably of between 10 3 and 10 7 cells, and most preferably of between 10 4 and 10 6 cells, i.e. 10 2 , 10 3 , 1 4 , 10 ⁇ 10 6 , 10 7 , 10*, 10 9 , or 10 10 cells.
  • body fluid sample refers to a liquid sample derived from the body of a subject, e.g. human or animal.
  • Said body fluid sample may be a blood, urine, sputum, breast milk, cerebrospinal fluid (CSF), cerumen (earwax), endolymph, perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid, saliva, sebum (skin oil), or a sweat sample including components or fractions thereof.
  • CSF cerebrospinal fluid
  • a blood sample more preferably a whole blood sample or serum sample, an urine sample, or a saliva sample including components or fractions thereof.
  • a “body fluid sample” may be provided by removing a body liquid from a subject, but may also be provided by using previously isolated body fluid sample material.
  • said "body fluid sample” may allow for a non-invasive diagnosis/and or prognosis of a subject. It is preferred that the body fluid sample from a subject has a volume of between 0.1 and 20 ml, more preferably of between 0.2 and 10 ml, more preferably between 0.4 and 8 ml and most preferably between 1 and 5 ml, I.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml.
  • the expression level is determined with an immunoassay, gel electrophoresis, spectrometry, chromatography, in situ hybridization, or a combination thereof.
  • the immunoassay is an enzyme immunoassay, more preferably an ELISA, or a Western Blot (also designated as immunoblot).
  • the gel electrophoresis preferably is ID (One-dimensional) or 2D (Two- dimensional) gel electrophoresis.
  • the spectrometry preferably is mass spectrometry (MS), more preferably tandem mass spectrometry (MS/MS).
  • the chromatography preferably is liquid chromatography (LC, or alternative HPLC) or affinity chromatography, e.g.
  • the in situ hybridization preferably is a silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), or fluorescence in situ hybridization (FISH),
  • the chromatography is preferably combined with spectrometry, more preferably mass spectrometry (MS), and is even more preferably liquid chromatography-mass spectrometry (LC-MS, or alternative HPLC-MS) and most preferably liquid chrornatography- tandem mass spectrometry (LC-MS/MS, or alternative HPLC -MS/MS).
  • LC-MS liquid chromatography-mass spectrometry
  • LC-MS liquid chromatography-mass spectrometry
  • LC-MS/MS liquid chrornatography- tandem mass spectrometry
  • MS mass analysis capabilities of mass spectrometry
  • LC-MS is a powerful technique as it has very high sensitivity and selectivity. Generally its application is oriented towards the specific detection and potential identification of molecules in the presence of other molecules, e.g. in a complex mixture.
  • the gel electrophoresis is preferably combined with an immunoassay and is more preferably a 2D immunoblot.
  • ID gel electrophoresis the biological sample is preferably beforehand purified, e.g. with affinity chromatography.
  • ID gel electrophoresis includes protein separation techniques such as Sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE), native gel electrophoresis and isoelectric focusing.
  • SDS-PAGE Sodium dodecyl sulfate polyacryl amide gel electrophoresis
  • the SDS-PAGE is a technique for separating proteins based on their ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. The addition of the SDS detergent to these samples gives the proteins the same electrical charge.
  • SDS-PAGE allows for separation of proteins from a wide range of samples including cells, tissues and whole blood.
  • Native Gel Electrophoresis for example, is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio.
  • the main types of native gels used in protein electrophoresis are polyacrylamide gels and agarose gels. It should be noted that unlike SDS-PAGE type electrophoreses, native gel electrophoresis does not use a charged denaturing agent. The proteins being separated, therefore, differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two.
  • isoelectric focusing (also known as electrofocusing), for example, is a technique to separate the proteins by isoelectric point. Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If the proteins are positively charged, they will be pulled towards the more negative end of the gel and if the proteins are negatively charged they will be pulled to the more positive end of the gel.
  • the proteins applied in the IEF will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).
  • IEF is preferably carried out using immobilized pH gradient (lPG) gels, or iPG strips, more preferably dry and rehydratabie iPG strips.
  • Microfluidic chip based isoelectric focusing may also be used (Sommer and Hatch, Electrophoresis. 2009 Mar;30(5):742-5.).
  • the ID gel electrophoresis is isoelectric focusing (IEF) or SDS-PAGE as first dimension to separate the proteins according to their isoelectric point (pi).
  • 2D gel electrophoresis refers to a form of gel electrophoresis commonly used to analyze proteins in two dimensions.
  • 2D gel electrophoresis begins with ID electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first.
  • ID electrophoresis proteins are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a 2D gel.
  • the two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass.
  • the first dimension is isoelectric focusing (IEF) and the second dimension is SDS-PAGE.
  • the 2D gel electrophoresis is isoelectric focusing (IEF) as first dimension and SDS-PAGE as second dimension to separate the proteins according to their isoelectric point (pi) and according to their protein mass.
  • the proteins separated with gel electrophoresis can then be detected by a variety of means known to the person skilled in the art.
  • silver and Coomassie Blue staining are used.
  • a silver colloid is applied to the gel.
  • the silver binds to cysteine groups within the protein.
  • the silver is darkened by exposure to ultra-violet light. The darkness of the silver can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes.
  • Western blotting is a technique which allows the detection of specific proteins (native or denatured) from extracts made from cells or tissues or body liquid samples, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane (typically nitrocellulose or PVDF) via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to imrnunohistochemistry, but without a need for fixation. Detection is typically performed using reporter enzyme linked antibodies, e.g. peroxidase linked antibodies to catalyze a ehemiluminescent reaction or alkaline phosphatase linked antibodies to catalyze a colori metric reaction.
  • reporter enzyme linked antibodies e.g. peroxidase linked antibodies to catalyze a ehemiluminescent reaction or alkaline phosphatase linked antibodies to catalyze a colori metric reaction.
  • Western blotting is a routine molecular biology method that can be used to semiquantitatively or quantitatively compare protein levels between extracts. The size separation prior to blotting allows the protein molecular weight to be gauged as compared with known molecular weight markers.
  • Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions).
  • the col ri metric detection method may depend on incubation of the Western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody.
  • Development of the blot may be then stopped by washing away the soluble dye.
  • Protein levels may be evaluated through densitometry (how intense the stain is) or spectrophotometry. Further, ehemiluminescent detection methods may depend on incubation of the Western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which capture a digital image of the Western blot. The image may be analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density.
  • the enzyme-linked immunosorbent assay or ELISA is a method for quantitatively or semi-quantitatively determining protein concentrations from, blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 6-wclls per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.
  • mass spectrometry refers to the use of an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer.
  • laser desorption mass spectrometry refers to the use of a laser as an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer.
  • the mass spectrometry may be a matrix-assisted laser desorption ionization mass spectrometry or MALDI.
  • the analyte is typically mixed with a matrix material that, upon drying, co-crystallizes with the analyte.
  • the matrix material absorbs energy from the energy source which otherwise would fragment the labile biomolecuies or analytes.
  • the mass spectrometry may also be a surface- enhanced laser desorption/ionization mass spectrometry or SELDI.
  • SELDI the surface on which the analyte is applied plays an active role in the analyte capture and/or desorption.
  • the biological sample used in the method of the first aspect of present invention may have undergone chromatographic or other chemical processing.
  • the "apparent molecular mass” refers to the molecular mass (in Daltons)-to- charge value, m/z, of the detected ions. How the apparent molecular mass is derived is dependent upon the type of mass spectrometer used. With a time of-ttight mass spectrometer, the apparent molecular mass is a function of the time from ionization to detection.
  • the term "signal” refers to any response generated by a biomolecule such as protein under investigation. For example, the term signal refers to the response generated by a biomolecule hitting the detector of a mass spectrometer. The signal intensity correlates with the amount or concentration of the biomolecule.
  • the signal is defined by two values: an apparent molecular mass value and an intensity value generated as described.
  • the mass value is an elemental characteristic of the biomolecule, whereas the intensity value accords to a certain amount or concentration of the biomolecule with the corresponding apparent molecular mass value.
  • the “signal” always refers to the properties of the biomolecule.
  • tandem mass spectrometry refers to multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation.
  • one mass analyzer can isolate one peptide from many entering a mass spectrometer.
  • a second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID).
  • CID collision-induced dissociation
  • a third mass analyzer sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap.
  • CID collision-induced dissociation
  • ECD electron capture dissociation
  • ETD electron transfer dissociation
  • IRMPD infrared multiphoton dissociation
  • BIRD blackbody infrared radiative dissociation
  • ED electron-detachment dissociation
  • SID surface-induced, dissociation
  • Tandem mass spectrometry enables a variety of experimental sequences.
  • Many commercial mass spectrometers are designed to expedite the execution of such routine- sequences as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), and precursor ion scan.
  • SRM selected reaction monitoring
  • MRM multiple reaction monitoring
  • precursor ion scan the first analyzer allows only a single mass through and the second analyzer monitors for a single user defined fragment ion.
  • MRM allows for multiple user defined fragment ions.
  • SRM and MRM are most often used with scanning instruments where the second mass analysis event is duty cycle limited.
  • Precursor ion scan refers to monitoring for a specific loss from, the precursor ion.
  • the first and second mass analyzers scan across the spectrum as partitioned by a user defined m/z value. This experiment is used to detect specific motifs within unknown molecules.
  • the mass spectrometry is an electrospray ionization mass spectrometry (ESI-MS), a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), or an electron capture dissociation mass spectrometry (ECD-MS).
  • ESI-MS electrospray ionization mass spectrometry
  • MALDI-MS matrix-assisted laser desorption/ionization mass spectrometry
  • ECD-MS electron capture dissociation mass spectrometry
  • the mass spectrometry employs tandem mass tags
  • TMT isobaric tags for relative and absolute quantitation
  • ICATs isotope-eoded affinity tags
  • ITRAQ is a non-gel-based technique used to quantify proteins from different sources in a single experiment. It uses isotope-coded covalent tags. The method is based on the covalent labeling of the N-terminus and side-chain amines of peptides from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments.
  • the diagnosis comprises (i) determining the presence or occurrence of a neurodegenerative disease such as PD or PDD, (ii) monitoring the course of a neurodegenerative disease such as PD or PDD, (iii) staging of a neurodegenerative disease such as PD or PDD, (iv) measuring the response of a subject with a neurodegenerative disease such as PD or PDD to therapeutic intervention, and/or (v) classification of a subject with a neurodegenerative disease such as PD or PDD.
  • a neurodegenerative disease such as PD or PDD
  • the prognosis comprises (i) predicting or estimating the occurrence, preferably the severity of occurrence, of a neurodegenerative disease such as PD or PDD, and/or (ii) predicting or estimating the response of a subject with a neurodegenerative disease such as PD or PDD to therapeutic intervention.
  • the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl , ⁇ yrosin-protem phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing a neurodegenerative disease, preferably in a biological sample from a subject.
  • a biomarker selected from the group consisting of Netrin Gl , ⁇ yrosin-protem phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing a neurodegenerative disease, preferably in a biological sample from a subject.
  • the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). It is also preferred that the molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160 is for the differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • PD Parkinson's disease
  • PDD Parkinson's disease dementia
  • the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • the present invention relates to a molecule for detecting the biomarker Netrin Gl or Tyrosin-protein phosphatase non-receptor-type 13 for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (FDD),
  • PDD Parkinson's disease dementia
  • the present invention relates to a molecule for detecting the biomarker Apolipoprotein B-100 for diagnosing and/or prognosing Parkinson's disease (PD).
  • PD Parkinson's disease
  • the present invention relates to a molecule for detecting the biomarker Golgin-160 for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
  • a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
  • the molecule is able to detect or detects the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determinant(s)) of said biomarker.
  • Said molecule may be a protein such as an antibody, a polypeptide such as an antibody fragment, a peptide such as a mass spectrometry probe, a polynucleotide, or a small molecule.
  • the molecule is able to bind or binds the biomarker Netrin Gl,
  • Tyrosin-protein phosphatase non-receptor-type 13 Apolipoprotein B-100 or Golgin-160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determmant(s)) of said biomarker.
  • Said molecule may be a protein such as an antibody, a polypeptide such as an antibody fragment, a polynucleotide, or a small molecule.
  • peptide refers to a short polymer of amino acids linked by peptide bonds. It has the same peptide bonds as those in proteins, but is commonly shorter in length.
  • the shortest peptide is a dipeptide, consisting of two amino acids joined by a single peptide bond. There can also be a tripeptide, tetrapeptide, pentapeptide, etc.
  • a peptide has an amino end and a carboxyl end, unless it is a cyclic peptide.
  • a peptide has a length of between 2 to 20 ammo acids, more preferably of between 5 to 20 amino acids and most preferably of between 7 to 15 amino acids, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
  • polypeptide refers to a part of a protein which is composed of a single linear chain of amino acids bonded together by peptide bonds.
  • the polypeptide has a length of more than 20 amino acids, more than 30 amino acids, or more than 40 amino acids. More preferably, the polypeptide has a length of between 21 and 200 amino acids, most preferably of between 50 and 100 amino acids, e.g.
  • protein may refer to a protein which comprises one or more polypeptides that resume a secondary and tertiary structure and additionally refers to a protein that is made up of several amino acid chains, i.e. several subimits, forming quaternary structures.
  • the protein has sometimes non-peptide groups attached, which can be called prosthetic groups or co factors.
  • polynucleotide may refer to a molecule which comprises at least 10 nucleotides and not more than 80 nucleotides, wherein the nucleotides are covalentiy linked together.
  • said polynucleotide is a molecule of 10 to 70 nucleotides or 15 to 60 nucleotides in length, more preferably of 20 to 40 nucleotides or 25 to 35 nucleotides in length, i.e.
  • small molecule refers to a low molecular weight organic compound which is by definition not a polymer.
  • a small molecule may bind with high affinity to a biopolymer such as a protein and, thus, may allow the detection of said biopolymer.
  • the upper molecular weight limit for a small molecule is usually about 800 Daltons.
  • the molecule e.g. protein or polypeptide, which is able to bind or binds the biomarker Netrin Gl , Apolipoprotein B 100, TyTosin-protein phosphatase non-receptor-type 13 or Go!gm- 160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determinant(s)) of said biomarker, is an antibody or a fragment thereof, a synthetic polypeptide, a recombinant pol peptide, preferably a darpin or an anticalin, or a polynucleotide. Said molecule is specific for said biomarker.
  • synthetic peptide or polypeptide refers to a synthetically produced peptide or polypeptide.
  • a peptide or polypeptide is synthetically produced by adding the amino acid from, the carboxylate groups forward, as opposed to ribosomal production, wherein, synthesizing starts with the amino group.
  • a synthetic polypeptide or peptide may be produced using liquid-phase synthesis or solid-phase peptide synthesis (SPPS), preferably Fmoc or Boc.
  • SPPS liquid-phase synthesis or solid-phase peptide synthesis
  • recombinant peptide or polypeptide refers to a genetically engineered polypeptide or peptide, i.e.
  • a polypeptide or peptide with a sequence manipulated by man usually a recombinant polypeptide or peptide is produced from recombinant DNA (e.g. DNA coding for said polypeptide or peptide comprised in a vector such as an expression vector), for example, in a host organism such as a bacterial cell or yeast cell.
  • recombinant DNA e.g. DNA coding for said polypeptide or peptide comprised in a vector such as an expression vector
  • darpin refers to a genetically engineered antibody mimetic protein typically exhibiting highly specific and high-affinity target protein binding. It is derived from natural ankyrin proteins and consists of at least three, usually four or five repeat motifs of these proteins. Its molecular mass is about 14 or 18 kDa for four- or five-repeat DARPins, respectively.
  • antigens e.g. to proteins or to small molecules
  • antigens e.g. to proteins or to small molecules
  • antigens e.g. to proteins or to small molecules
  • antigens e.g. to proteins or to small molecules
  • antigens e.g. to proteins or to small molecules
  • the size is about 180 amino acids and the mass is about 20 kDa.
  • said polynucleotide may be a molecule of at least 10 nucleotides and of not more than 80 nucleotides covalently linked together.
  • said polynucleotide is a molecule of 10 to 70 nucleotides or 15 to 60 nucleotides in length, more preferably of 20 to 40 nucleotides or 25 to 35 nucleotides in length, i.e.
  • Said polynucleotide may be single or double stranded, or may contain portions of both double and single stranded sequences.
  • Said polynucleotide may be a polymer of deoxyribormcleotide or ribonucleotide bases, including DNA (e.g. cDNA and genomic DNA) and R A (e.g. mRNA or cRNA) molecules, both sense and anti-sense strands.
  • Said polynucleotide may be obtained by chemical synthesis methods.
  • Said polynucleotide as a single polynucleotide strand may provide a probe that is capable of binding to or hybridizing with and, thus, detecting a target of complementary sequence, such as the nucleotide sequence of the mRNA of the above-mentioned biomarkers, e.g. through one or more types of chemical bonds, usually through complementary base pairing and hydrogen bond formation.
  • Said polynucleotide as a probe may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind.
  • Said label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • suitable labels include 32P, fluorescent dyes, electron- dense reagents, enzymes (e.g., as commonly used in an ELBA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
  • a label may be incorporated into nucleic acids at any position, e.g. at the 3' end, at the 5' end or internally.
  • the polynucleotide is selected from the group consisting of a polynucleotide probe, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), a microR A (miR A), and a small interfering RNA (si RNA).
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • miR A microR A
  • si RNA small interfering RNA
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • GNAs glycol nucleic acids
  • TAAs threose nucleic acids
  • the one or more polynucleotide(s) is (are) selected from the group consisting of a primer(s) (e.g. a primer pair), preferably a primer(s) for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing. More preferably, the primers (e.g. a primer pair) are for real time polymerase chain reaction (RT-PCR), and most preferably for quantitative real time polymerase chain reaction (qRT-PCR).
  • Polynucleotides as primers have preferably a length of between 15 and 35 nucleotides and more preferably of between 18 and 30 nucleotides.
  • the synthetic polypeptides or peptides, recombinant polypeptides or peptides. preferably darpins or anticalins, lectins or small molecules according to the invention may be selected by routine screening of existing libraries, e.g. small molecule libraries. Suitable standard screening methods, e.g. phage display for polypeptides or peptides, are well known to the person skilled in the art. That said molecules are able to detect/determine the above- mentioned biomarkers, e.g. by binding said biomarkers, can easily be tested by the person skilled in the art with methods known to the person skilled in the art, e.g. by fluorescence resonance energy transfer (FRET), co-immunoprecipitarion or an Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA).
  • FRET fluorescence resonance energy transfer
  • ELISA Enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • the binding of the above-mentioned molecule to the biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin- 160 may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment. Therefore, the biomarker selected from the group consisting of Netrin G l , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160 may e immobilized on the surface of an ELISA plate and contacted with the above-mentioned molecule.
  • ELISA enzyme-linked immunosorbent assay
  • Binding of the molecule may be verified, for example, for proteins, polypeptides, peptides, and epitope-tagged compounds, by antibodies specific for said molecule or the epitope-tag. These antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to said enzyme that - in combination with the appropriate substrates - carries out chemiiuminescent reactions (e.g. horseradish peroxidase) or colorimetric reactions (e.g. alkaline phosphatase). In another embodiment, binding of molecules that cannot be detected by antibodies might be verified by labels directly coupled to the molecules. Such labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiiuminescent compounds and bioluminescent compounds.
  • the above-mentioned molecule might be immobilized on the ELISA plate and contacted with the the biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160. Binding of said biomarker may be verified by an antibody specific for said biomarker and chem i I urn i n escence or colorimetric reactions as described above.
  • antibody or fragment thereof 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. Also comprised are ii nunoglobu!in-!ike proteins that are selected through techniques including, for example, phage display to specifically bind to a target molecule or target protein.
  • the immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • antibodies and fragments thereof include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g. scFv), Fab fragments, F(ab3 ⁇ 4 fragments, fragments produced by a Fab expression library, diabodies or tetrabodies (Holliger P. et al., 1993), nanobodies, anti-idiotypic (anti-Id) antibodies, and epitopc-binding fragments of any of the above.
  • the antibody fragments are mammalian, preferably human antigen-binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab3 ⁇ 4, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH 1 , CH2, and CH3 domains.
  • the antigen-binding fragments may also comprise any combination of variable domain(s) with a hinge region, CL, CHI, CH2, and CH3 domains.
  • Antibodies usable in the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, simian (e.g. chimpanzee, bonobo, macaque), rodent (e.g. mouse and rat), donkey, sheep rabbit, goat, guinea pig, camel, horse, or chicken. It is particularly preferred that the antibodies are of human or murine origin.
  • "human antibodies” include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
  • Antibodies according to the second aspect of the invention may be produced by methods well known, in the art or may simply be ordered to be made commercially. Means of preparing and characterizing antibodies or antibody fragments are also well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
  • the antibody that recognizes the target antigen here the biomarker selected from the group consisting of etrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-! OO and Golgin- i 60, is generally called the "primary antibody”.
  • Said antibody may be labeled with a detectable taglabel in order to allow direct detection of the target antigen.
  • Said detectable tag/label may be an enzymatic, fluorescent or radioisotope tag/label.
  • the primary antibody is not labeled for direct detection. Instead a "secondary antibody” that has been labeled with a detectable tag/label (e.g.
  • the enzymatic, fluorescent or radioisotope tag/label is applied in a second step to probe for the primary antibody, which is bound to the target antigen.
  • the primary antibody or the secondary antibody may be labeled with an affinity tag such as biotin.
  • Tt is preferred that the biomarker specific molecule is able to bind or binds the amino acid sequence according to SEQ ID NO: 1 to 5 or a variant thereof (see above).
  • the molecule of the second aspect may be directly or indirectly labeled, e.g. with biotin to which a streptavidin complex may bind.
  • label means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
  • useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
  • the molecule, e.g. peptide, for detecting the biomarker selected, from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-1 0 is a mass spectrometry peptide (probe).
  • the terms mass spectrometry probe or mass spectrometry peptide are interchangeable used herein.
  • Said probe is a synthetic peptide analog to a native peptide of the biomarker Netrin Gl , Tyrosin- protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 which is cleavable with a protease (e.g. trypsin protease).
  • Said probe enables protein identification and absolute protein quantitation of said biomarker with mass spectrometry, preferably with HPLC-MS or HPCL-MS/MS, particularly supported by multiple reaction monitoring (MRM). It preferably incorporates one stable isotope labeled amino acid, creating a slight increase (e.g. 6-10 daltons) in molecular weight.
  • the native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 and the synthetic peptide co-elute chromatographically, co-migrate el ectrophoret i cally , and ionize with the same intensity. Nevertheless, by mass spectrometry, the native peptide of the biomarker Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 and the synthetic peptide can easily be distinguished.
  • a known amount of the synthetic peptide is added to a sample of a subject.
  • the sample is then digested (e.g. by a protease such as trypsin protease) and analyzed by mass spectrometry, preferably by HPLC-MS or H PLC -MS/MS.
  • Extracted ion chromatograms are generated for the native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160 and the synthetic peptide internal standard.
  • the quantity of native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 is calculated. It also allows quantitation of the amount of the biomarker Netrin Gl, Tyrosin- protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in the sample of the subject.
  • the techniques for selecting and preparing the synthetic peptide are well known in the art. For example, the skilled person knows how to select precursor- fragment -ion- transitions under the aspect of optimal selectivity and sensitivity.
  • the mass spectrometry peptide (probe) has an amino acid sequence according to SEQ ID NO: 6 to SEQ ID NO: 9.
  • the present invention relates to a composition or set comprising a molecule for detecting the bioraarker Netrin Gl, a molecule for detecting the biomarker Tyrosin-protein phosphatase non-receptor-type 13, a molecule for detecting the biomarker Apolipoprotein B-100 and/or a molecule for detecting the biomarker Golgin-160 for diagnosing and'or prognosing a neurodegenerative disease, preferably in a biological sample from a subject.
  • the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). All preferred and particularly preferred embodiments previously described in the context of the molecule of the second aspect of the invention are similarly preferred in the context of this aspect of the invention.
  • the composition or set comprises a molecule(s) for detecting (i) the biomarker Netrin Gl, (ii) the biomarker Tyrosin-protein phosphatase non-receptor-type 13, (iii) the biomarker Apolipoprotein B-100, (iv) the biomarker Golgin-160, (v) the biomarkers Netrin Gl and Tyrosin-protein phosphatase non-receptor-type 13, (vi) the biomarkers Netrin Gl and Apolipoprotem B-100, (vii) the biomarkers Netrin Gl and Golgin-160, (viii) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Apolipoprotein B-100, (ix) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160, (x) the biomarkers Apolipoprotein B-100 and Golgin-160, (x
  • Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160 (xiii) the biomarkers Tyrosin-protein phosphatase non-reee tor-type 13, Apolipoprotein B-100 and Golgin-160, (xiv) the biomarkers Apolipoprotein B-100, Netrin Gl and Golgin-160, or (xv) the biomarkers Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, preferably in a biological sample from a subject.
  • the present invention relates to means for diagnosing and/or prognosing a neurodegenerative disease comprising at least one molecule (e.g. 1 , 2, 3, or 4 molecule(s)) according to the second aspect.
  • a neurodegenerative disease comprising at least one molecule (e.g. 1 , 2, 3, or 4 molecule(s)) according to the second aspect.
  • said means allows the detection/determination of the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of etrin Gl, Tyrosin-protein phospliatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160, preferabl in a biological sample from a subject, and, thus, the diagnosis and/or prognosis of a neurodegenerative disease.
  • biomarker e.g. 1, 2, 3, or 4 biomarker(s)
  • biomarker selected from the group consisting of etrin Gl, Tyrosin-protein phospliatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160
  • the expression level of the gene of Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13, Apolipoprotein B-100 or Golgin-160 may be determined on the mRNA level (transcriptional level) or protein level (translational level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via tmmunohistochemistry) or mRNA (e.g. via in situ hybridization).
  • the amount of the Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13, Apolipoprotein B-100 or Golgin- 160 mRNA or protein is determined.
  • the above-mentioned neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (FDD).
  • said means are for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • said means comprise a solid support. It is preferred that said means further comprises means for immobilising the at least one molecule according to the second aspect of the present invention on said solid support or for attaching the at least one molecule according to the second aspect of the present invention to said solid support.
  • the solid support may be made of the following materials: glass (including modified or function ah zed glass), plastics (including acrylics, polystyrene, polypropylene, polyethylene, polybutylcne, polyurethanes, teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals or mixtures/combinations thereof.
  • the solid support may be planar, e.g. a slide, chip, matrix, or array, although also other configurations of the solid support may be possible as well, e.g. tubes, beads, or microspheres.
  • the at least one molecule according to the second aspect of the present invention is attached to or immobilized on the solid support.
  • said means consists of a solid support to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized. More preferably, said means comprise a biochip. ' m i croarray or a set of beads. It is preferred that said means further comprises means for immobilising the at least one molecule according to the second aspect of the present invention on the biocMp or beads of said set of beads or for attaching the at least one molecule according to the second aspect of the present invention to the biochip or beads of said set of beads.
  • the at least one molecule according to the second aspect of the invention is attached to or immobilized on the biochip/microarray or the at least one molecule according to the second aspect of the invention is attached to or immobilized on the beads of said set of beads.
  • said means consists of (i) a biochip to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized, or (ii) beads of a set of beads to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized.
  • said beads or microspheres have a mean diameter of between 2 to 20 microns, preferably 4 to 10 microns, most preferably 5 to 7 microns, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns.
  • biochip ' and “microarray” are interchangeable used herein.
  • attachment or "'immobilized”., as used herein, refer to the binding between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, and may mean that the binding between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, is sufficient to be stable under conditions of binding, washing, analysis and removal.
  • the binding may be covalent or non-covalent. Covalent bonds may be formed directly between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, or may be formed by a cross linker or by inclusion of specific reactive groups on either the solid support, e.g.
  • Non-covalent binding may be electrostatic, hydrophilic arid hydrophobic interactions or combinations thereof.
  • Immobilization or attachment may also involve a combination of covalent and non-covalent interactions.
  • the above-mentioned means comprises or consists of the molecule composition or set as described above.
  • at least one molecule e.g. 1, 2, 3, or 4 molecule(s)
  • the second aspect of the invention is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
  • the above-mentioned molecule composition or set (e.g. composition or set comprising 2, 3, or 4 biomarker(s)) is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least two biornarkers (e.g. 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
  • biornarkers e.g. 2, 3, or 4 biomarker(s)
  • the means according to the third aspect of the invention is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least one biomarker (e.g. 1. 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
  • biomarker e.g. 1. 2, 3, or 4 biomarker(s)
  • the present invention relates to a kit for (use in) the diagnosis and/or prognosis of a neurodegenerative disease comprising
  • biomarker e.g. 1, 2, 3, or 4 biomarker(s) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, and optionally
  • kit of parts in short a kit is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.
  • the expression level of at least one biomarker e.g. 1 , 2, 3, or 4 biomarker(s) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 is determined in a biological sample from a subject. This may then allow the diagnosis and/or prognosis of a neurodegenerative disease.
  • biomarker e.g. 1 , 2, 3, or 4 biomarker(s)
  • the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 is determined in a biological sample from a subject. This may then allow the diagnosis and/or prognosis of a neurodegenerative disease.
  • the expression level of the gene of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoproteiii B-100 or Golgin- 1 60 may be determined on the mR A level (transcriptional level) or protein level, (translational level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via immunohistochemistry) or mRNA (e.g. via in situ hybridization).
  • the amount of the Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotcin B-100 or Golgin- 160 mRNA or protein is determined.
  • the above-mentioned neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
  • said means are for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • said means comprises or consists of
  • At least one molecule e.g. 1 , 2, 3, or 4 rnolecule(s) according to the second aspect of the present invention, and/or
  • the above-mentioned means (alternatively or additionally) comprises or consists of a molecule composition or set as defined above.
  • kits comprise materials desirable from a commercial and user standpoint such as a bnffer(s), a reagent(s) and/or a dilnent(s). Said materials may be ⁇ useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein. phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160. It is further particularly preferred that the kits comprise reporter- means such as an affinity tag binding protein, for example, a biotin binding molecule (e.g. avidin or streptavidin) bound to a detectable label, e.g.
  • a biotin binding molecule e.g. avidin or streptavidin
  • reporter-means may also be useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin G l , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Goigin- 160.
  • the above-mentioned data carrier may be a graphically data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronically data carrier such as a floppy disk, a compact disk (CD), or a digital versatile disk (DVD),
  • the access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database.
  • said data carrier comprises a control (value), particularly to allow the interpretation of information obtained when performing the above-mentioned method for diagnosing and/or prognosing a neurodegenerative disease, preferably for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • a control value
  • said control (value) may allow for the diagnosis and/or prognosis of Parkinson's disease (PD), Parkinson's disease dementia (PDD) or for differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD),
  • PD Parkinson's disease
  • PPD Parkinson's disease dementia
  • said control (value) it is referred to the first aspect of the invention.
  • the data carrier comprises instructions for the method according to the first aspect of the present invention, the molecule according to the second aspect of the present invention and/or the means according to the third aspect of the present invention in order to diagnose and/or prognose a neurodegenerative disease, preferably to diagnose and/or prognose Parkinson's disease (PD), Parkinson's disease dementia (PDD) or to differential diagnose and/or prognose between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
  • Said data carrier may further comprise the following:
  • quality information material such as information about the lot/batch number of the means for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, and/or of the kit, (iv) information concerning the detailed composition of the buffer(s), dilucnt(s) and reagent(s) -useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160,
  • the present invention relates to the use of the molecule according to the second aspect, the means according the third aspect, or the kit according to the fourth aspect in the method of the first aspect.
  • the present invention relates to the use of a gel- free mass- spectrometry approach with isotope-labelled samples (iTRAQ) or multiple-reaction- monitoring (MRM) for the determination of a biomarker for diagnosing and/or prognosing a neurodegenerative disease, preferably Parkinson's disease (PD) or Parkinson's disease dementia (FDD), in a cerebrospinal fluid (CSF) sample.
  • iTRAQ isotope-labelled samples
  • MRM multiple-reaction- monitoring
  • Fig. 1 HPLC of CSF-samples. Elution diagrams for the two-dimensional HPLC-pre- fractionation of the iTRAQ-samples. a: Strong-cation-exchange column - individual runs, b-d: reversed-phase chromatography of the fractions collected after SCX-separation. Fig. 2: M M-Measurements. Quantitative analysis of MRM-data in PD, FDD and
  • a Proteins with a significant difference between NDC and FDD.
  • b proteins with a significant regulation between PD and N ' DC.
  • the clinical characteristics such as the minimental status test (MMST), the Hoehn&Yahr stages for PD as well as CSF tan protein concentrations are given in Table 1 (see below).
  • the diagnosis of all patients was done according to the consensus criteria for PD/PDD [35] as well as the DSM-IV criteria and was established in cooperation by neurologists and neuropsychologists, both blinded with respect to the neurochemical outcomes.
  • Table 1 Listing of detailed patient parameters in all groups investigated. Data are
  • CSF samples were depleted of albumine and immunoglobuline G with the Albumin and Ig G removal kit (GE-Healthcare, Little Chalfont, UK) according to the manufacturer's instructions.
  • Total protein concentration was measured before and after depletion using a BCA-assay.
  • samples for each group (PD, PDD and NDC) were analysed, as well as a pool consisting of equal amounts of all samples.
  • the sample compositions of each iTRAQ run were as follows: The pool was labelled with 114, PDD with 115, PD with 116 and NDC with 117.
  • Proteins were pelleted with 6 volumes of ice-cold-actone for Ih at — 20°C, centrifuged for 30 minutes at 13,000 rpm (4°C) and air-dried. Pellets were stored at 80°C until further analysis. Protein Digestion and iTRAQ Labelling
  • the pellet was resuspended in 30 ⁇ xL dissolution buffer containing 2 ,uL denaturant (iTRAQ reagent kit, Applied Biosystems). Equal protein amounts of each disease state were reduced with 3 mM TCEP for 30 mm at 60°C and alkylated with 6 mM iodoacetamide at room temperature for 20 min in the dark. Proteins were digested with 1 ⁇ g LysC (Wako Chemicals) at 37°C for 3 h and incubated over night after adding 10 ⁇ . water with 1 ⁇ g trypsin (Promega) at 37°C. Digested samples were labelled with the iTRAQ reagents according to the manufacturer's instructions (Applied Biosystems). The four iTRAQ samples (114: Pool; 115: FDD; 116: PD; 117: NDC) were combined and dried by vacuum oentrif ligation. Two-dimensional Peptide Separation
  • MS/MS data were analyzed using ProteinPilot 2.0.1 (Applied Biosystems) which uses the Paragon algorithm to perform database matching for protein identification, protein grouping to remove redundant hits and comparative quantitation.
  • the Swiss-Pro t Homo sapiens protein database was used for ail searches. Data were normalized for loading error by bias corrections calculated using ProteinPilot. All reported data were based on 95% confidence for protein identification as determined by ProteinPilot.
  • Peptide selection and measurement protocol For each of the above-mentioned proteins, a peptide which was present in as many iT AQ runs as possible with high intensities was chosen and synthezised (Thermo Scientific, Ulm, Germany) from the iTRAQ measurements since those peptides promised the highest success rate.
  • the peptides are given in Table 2.
  • Peptides were reconstituted to a concentration of 0.5 pg iil with double-distilled water and stored at -80°C until further use.
  • Table 2 Regulated proteins in the iTRAQ-measurement with, selected peptides.
  • the samples were prefractionated using an HPLC-system (Agilent 1200 Binary Pump, Agilent 1200 Micro Vacuum Degasser and Agilent 1200 Thermostatted Column Compartment (Agilent Technologies, Morges, Switzerland) with a CI S-column (Symmetry CI 8, 5 ⁇ ⁇ ⁇ , NanoEase Trap Column (Waters Corporation, Massachusetts, USA)) coupled online to the mass- spectrometer.
  • HPLC-system Agilent 1200 Binary Pump, Agilent 1200 Micro Vacuum Degasser and Agilent 1200 Thermostatted Column Compartment (Agilent Technologies, Morges, Switzerland) with a CI S-column (Symmetry CI 8, 5 ⁇ ⁇ ⁇ , NanoEase Trap Column (Waters Corporation, Massachusetts, USA) coupled online to the mass- spectrometer.
  • Peptides were eluted using an Acetonitrile gradient (3 minutes 5%, gradient to 50% within 1 minute, 11 minutes 50%, gradient to 100% within 1 minute and 100% for 3 minutes) containing 0.1 % formic acid with a flow rate of 20 ⁇ per minute. Peptide-counts were calculated with AnalystTM as areas under the curve. For standard curves, the peptides were diluted 1 :1000 with 50% Acetonitrile in 0.1% formic acid and further dilutions were made down to approximately 1 n.M per peptide. All measurements were carried out in duplicates and linear regressions were used to calculate the standard-curves. Sample concentrations are given in Table 3.
  • Peptides for each protein were chosen according to iTR AQ results. An overview of the peptides is shown in Table 2. For each peptide an optimised measurement protocol was established and specificity of the protocols was checked using different mixtures of the synthetic peptides to exclude false positive measurements (data not shown). A ten point standard curve was calculated for each peptide using 2 independent measurements. The coefficient of variation was also calculated separately for each peptide using 4 independent measurements of an identical CSF-sample. These coefficients were well below 20% (data not shown).
  • Protein concentrations were calculated based on their unmodified molecular weight.
  • Parkinson's dementia is so far diagnosed only according to clinical criteria [37].
  • cognitive decline and even dementia is often neglected since usually in the early stages of the disease the classical Parkinson symptoms dominate the clinical picture [38],
  • the proteins identified and verified in this study are interesting candidates. Said candidates are Tyrosinc-protein phosphatase non-receptor-type 13 (PTPN 13), Netrin Gl , Apolipoprotein B- 100 and Golgin- 160.
  • PTPN 13 Tyrosinc-protein phosphatase non-receptor-type 13
  • Netrin Gl Netrin Gl
  • Apolipoprotein B- 100 Golgin- 160.
  • Netrin Gl belongs to a family of proteins which are involved in the process of axonal guiding through which growing axons are guided towards their correct targets. Netrin Gl was first described in the year 2000 as a glycosyl phosphatidyiinositol (GPI) anchored member of that family. Its expression in the brain reaches a maximum at perinatal stages in most regions. However in the cerebellum, Netrin Gl is only expressed in certain regions which are implicated in controlling motor activity.
  • GPI glycosyl phosphatidyiinositol
  • Golgins are a large family of proteins which have different functions in connection with the Golgi-apparatus.
  • the Golgin A3 protein also known as Golgin-160 because of its molecular weight is thought to be involved in intra-Golgi vesicle mediated transport.
  • Parkinson dementia syndromes a review, CNS Neurosci Ther 15 (2), 157 (2009).

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Abstract

The present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease, particularly Parkinson's disease (PD) or Parkinson's disease dementia (PDD), which comprises the determination of the expression profile of a biomarker indicative for said disease. The present invention further relates to a molecule for detecting said biomarker for diagnosing and/or prognosing a neurodegenerative disease. It also relates to means for diagnosing and/or prognosing a neurodegenerative disease and a kit for diagnosing and/or prognosing a neurodegenerative disease.

Description

DIAGNOSIS AND/OR PROGNOSIS OF A NEURODEGENERATIVE DISEASE
The present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease, particularly Parkinson's disease (PD) or Parkinson's disease dementia (FDD), which comprises the determination of the expression profile of a biomarker indicative for said disease. The present invention further relates to a molecule for detecting said biomarker for diagnosing and/or prognosing a neurodegenerative disease. It also relates to means for diagnosing and/or prognosing a neurodegenerative disease and a kit for diagnosing and/or prognosing a neurodegenerative disease.
BACKGROUND OF THE INVENTION
Neurodegenerative diseases such as Parkinson's disease (PD) have a prevalence which increases with age [1]. It is noteworthy that PD patients have a 6-fold higher risk for the development of a dementia than healthy persons of the same age [2]. Up to half of all PD patients show a mild cognitive impairment in the early disease-stages [3], About one third of them later develop a dementia which is sometimes also accompanied by changes in personality [4]. This dementive syndrome normally develops within 8-10 years and has severe consequences on the course of the disease. Apart from the obvious strain on the person's social environment the dementia also goes along with a worse prognosis as far as disease- progression and life-expectancy are concerned [5]. Therefore, early treatment of dementia is critical since early therapy of the cognitive deficits is considered to be crucial to its success [6]·
Parkinson's disease dementia (FDD) is neuropathologically characterized by the presence of cortical Lewy bodies which also occur in patients with Lewy-body-dementia [7- 10], These Lewy bodies contain alpha-synuclein, a presynaptic filament protein which is expressed i high amounts in the terminal ends of neurons. The fact that these inclusions are detected mostly in living cells rather than in apoptotic cells suggests that the inclusions play a protective role by sequestering toxic molecules [11]. A possible link between aggregation, neurotoxicity and disease-propagation might be that neurotoxic oligomers of alpha-synuclein can be transformed to non-toxic oligomers which have a higher aggregation- tendency [12, 13]. Several studies to improve the early diagnosis of FDD in cerebrospinal fluid and serum have been undertaken [14]. However the results are of limited use for the prognosis of disease progression.
To date, diagnosis and/or prognosis of a neurodegenerative disease such as PD or PDD and the differential diagnosis and/or prognosis between the neurodegenerative diseases PD and PDD is still performed by physicians on the basis of the patient's medical history and neurological examination..
Thus, there is a need in the art for laboratory-based tests for the diagnosis and/or prognosis of a neurodegenerative disease such as PD or PDD and for the differential diagnosis and/or prognosis between the neurodegenerative diseases PD and PDD.
Using a gel-free proteomic mass-spectrometry approach with isotope-labelled samples
(iTRAQ) optimised for cerebrospinal fluid (CSF), the present inventors investigated clinically well-characterized patients with PD, PDD and controls (NDC's) in order to find a diagnostic marker which allows diagnosis of PD and PDD and which allow differentiation between demented and non-demented persons suffering from Parkinson disease. With this approach, the present inventors found differentially regulated proteins. They verified them in a larger group of patients using a m ass-spec trometry-based technique, the so-called multiple-reaction- monitoring (MRM), which uses specific, synthetic peptides to quantify proteins in a complex mixture. The MRM-approach itself has been used for some time in the field of pharmacokinetics, but has only very recently been applied as a quantitative proteomic tool [15-21] due to the challenging development of the method. MRM or the smglc-analyte analogue selected-reaction-monitoring (SUM) are highly specific methods as exclusive peptides of the protein(s) of interest are first selected via their intact mass and then further fragmented and characterized via their specific fragments. Therefore, at least two correct ion- masses are required for a single measurement, thus, enhancing sensitivity by reducing cross- reactivity and background (for a detailed review see: [22]). The use of MEM as a tool to validate biomarker in CSF generally allows quantitation of proteins for which no antibodies are available and also the quantitative analysis of posttranslational modifications is possible.
With the afore-mentioned techniques, the inventors of the present invention found that patients having a neurodegenerative disease such as PD or PDD can be identified on the basis of the expression level of at least one biomarker selected from the group consisting of Netrin G l . Tyrosin-protein phosphatase non -receptor-type 13, Apohpoprotein B-100 and Golgin- i 60 in a biological sample such as a cerebrospinal fluid (CSF) sample. SUMMARY OF THE INVENTION
In a first aspect, the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of:
(i) determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease or being prone thereto, if the expression level of said biomarker is altered in the biological sample from the subject compared to a control.
In a second aspect, the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160 for diagnosing and/or prognosing a neurodegenerative disease.
In a third aspect, the present invention relates to' means for diagnosing and/or prognosing a neurodegenerative disease comprising at least one molecule according to the second aspect.
In a fourth aspect, the present invention relates to a kit for diagnosing and/or prognosis a neurodegenerative disease comprising
(i) means for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160, and optionally
(ii) a data carrier, and/or
(iii) a container.
This summary of the invention does not necessarily describe all features of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).
Several, documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in. any number to create additional embodiments. The variously described, examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the excl usion of any other integer or step or group f integer or step.
As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.
The inventors of the present invention surprisingly found that a subject experiencing a neurodegenerative disease such as PD or FDD can be identified on the basis of the expression level of at least one biomarker selected from the group consisting of etrin Gl , Tyrosin- protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample such as a cerebrospinal fluid (CSF) sample from said subject. Thus, in a first aspect, the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of:
(i) determining the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B- I QO and Golgin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease or being prone thereto, if the expression level of said biomarker is altered, preferably increased, or decreased, in the biological sample from the subject compared to a control.
Preferably, the present invention relates to a method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of;
(i) determining the expression level of
(ia) the biomarker Netrin Gl , (ib) the biomarker Tyrosin-protein phosphatase non- receptor-type 13, (ic) the biomarker Apolipoprotein B- 100, (id) the biomarker Golgin- 160, (ie) the biomarkers Netrin Gl and Tyrosin-protein phosphatase non-receptor-type 13, (if) the biomarkers Netrin Gl and Apolipoprotein B-100, (ig) the biomarkers Netrin Gl and Golgin-160, (ih) the biomarkers Tyrosin-protein phosphatase non- receptor-type 13 and Apolipoprotein B-100, (ij) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160, (ik) the biomarkers Apolipoprotein B-100 and Golgin-160, (il) the biomarkers Netrin. Gl , Tyrosin-protein phosphatase non-receptor- type 13 and Apolipoprotein B-100, (im) the biomarkers Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13 and Golgin-160, (in) the biomarkers Tyrosin-protein. phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, (io) the biomarkers Apolipoprotein B-100, Netrin Gl and Golgin- 160, or (ip) the biomarkers Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160
in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease or being prone thereto, if the expression level of said biomarker(s) is altered, preferably increased or decreased, in the biological sample from the subject compared, to a control.
The term "neurodegenerative disease", as used herein, refers to a range of conditions which primarily affect the neurons in the brain. Neurons are the building blocks of the nervous system which includes the brain and. spinal cord. They normally don't reproduce or replace themselves, so when they become damaged or die they cannot be replaced by the body. Thus, particularly, the term "neurodegenerative disease" refers to a range of conditions which leads to a progressive loss of structure or function of neurons, including death of neurons. This causes problems with, movement (called ataxias), or mental functioning (called dementias). Examples of neurodegenerative diseases include, but are not limited to, Parkinson's, Alzheimer's, and Huntington's disease.
The term "diagnosing a neurodegenerative disease", as used herein, means determining whether a subject shows signs of or suffers from a neurodegenerative disease. The term "prognosing a neurodegenerative disease", as used herein, means predicting whether a subject will show signs of or suffer from a neurodegenerative disease in the future, but preferably also means predicting the course of a neurodegenerative disease of a subject already showing signs of or suffering from a neurodegenerative disease.
The biomarker Netrin Gl belongs to a family of proteins which are involved in the process of axonal guiding through which growing axons are guided towards their correct targets. Netrin Gl was first described in the year 2000 as a glycosyl phosphatidylinositol (GPi) anchored member of that family. Its expression in the brain reaches a maximum at perinatal stages in most regions. However in the cerebellum, Netrin Gl is only expressed in certain regions which are implicated in controlling motor activity. The term "Netrin Gl" encompasses Netrin Gl variants, e.g. all non-naturally or naturally occurring variants such as Netrin Gl homologues. particularly orthologues or paralogues. Preferably, the Netrin Gl variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 1 (human Netrin Gl , Accession number: AAH30220.1). For example, the Netrin Gl variants have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66. 67. 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 1. Preferably, the sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 420, or more amino acids, preferably over the whole length of the Netrin Gl amino acid sequence.
The biomarker Tyrosin-protein phosphatase non-receptor-type 13 (PTPN13) belongs to the protein tyrosine phosphatase (PTP) family. PTPs are known to be signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPN13 is a large protein that possesses a PTP domain at C- terminus, and multiple non-catalytic domains, which include a domain with similarity to band 4.1 superfamily of cytoskeletal-associated proteins, a region consisting of five PDZ domains, and a leucine zipper motif. PTPN13 was found to interact with, and dephosphorylate Fas receptor, as well as IkappaBalpha through the PDZ domains, which suggested its role in Fas mediated programmed cell death, PTPN13 was also shown to interact with GTPase- activating protein, and, thus, may function as a regulator of Rho signaling pathway. The term "Tyro sin-protein phosphatase non-receptor-type 13" encompasses Tyrosin-protei phosphatase non-receptor-type 13 variants, e.g. all non-naturally or naturally occurring variants such as Tyrosin-protein phosphatase non-receptor-type 13 homologues, particularly orthologues or paralogues. Preferably, the Tyrosin-protein phosphatase non-receptor-type 13 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 2 (human PTPN 13, Accession number: AAH39610.1). For example, the Tyrosin-protein phosphatase non-receptor-type 13 variants have an amino acid sequence which is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 2. Preferably, the sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, or more amino acids, preferably over the whole length of the Tyrosin-protein phosphatase non- receptor-type 13 amino acid sequence.
The biomarker Apolipoprotein B-100 is the primary Apolipoprotein of low-density lipoproteins (LDL or "bad cholesterol"), which is responsible for carrying cholesterol to tissues. In the brain, Apolipoprotein B-100 can lead to neurod egen erati on through increasing the levels of serum lipids thus leading to cerebrovascular lesions. The term "Apolipoprotein B-100" encompasses Apolipoprotein B-100 variants, e.g. all non-naturally or naturally occurring variants such as Apolipoprotein B-100 homologues, particularly orthologues or paralogues. Preferably, the Apolipoprotein B-100 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 3 (human Apolipoprotein B-100 precursor, Accession number: P_000375.2) or SEQ ID NO: 4 (human Apolipoprotein B- 100, Accession number: A A A51758.1). For example, the Apolipoprotein B-100 variants have an amino acid sequence which is at least 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or more amino acids, preferably over the whole length of the above Apolipoprotein B-100 amino acid sequences.
Golgins are a large family of proteins which have different functions in connection with the Golgi-apparatus. The biomarker Golgin A3 protein also known as Golgin-160, because of its molecular weight, is involved in intra-Golgi vesicle mediated transport. The term "Golgin-160" encompasses Golgin-160 variants, e.g. all non-naturally or naturally occurring variants such as Golgin-160 homologues, particularly orthologues or paralogues. Preferably, the Golgin-160 variants have an amino acid sequence which is at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 5 (human Golgin subfamily A member 3 isoform 1, Accession number: NP 005886.2). For example, the Golgin-160 variants have an amino acid sequence which is at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to SEQ ID NO: 5. Preferably, the sequence identity is over a continuous stretch of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or more amino acids, preferably over the whole length of the Golgin-160 amino acid sequence. The above-mentioned terms encompass all types of isoforms of said biomarkers.
Alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.iik/emboss/align index.htrril) using standard settings, preferably for Align EMBOSS ::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.
Gene expression is the process by which information from a gene is used for the synthesis of a functional gene product. Gene expression occurs in two major stages. The first is transcription. In this process, the gene is copied to produce a R A molecule (a primary transcript) with essentially the same sequence as the gene. Most eukaryotic genes are divided into exons and introns, and only the exons carry information required for protein synthesis. Most primary transcripts are, therefore, processed by splicing to remove intron sequences and. generate a mature transcript or messenger RNA (mRNA) that only contains exons. In the second step, the final protein encoded by said mRNA is produced. This stage is also known as translation.
In the context of the present invention, the term "expression level" of Netrin Gl, Tyro sin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160 refers to the expression level of the gene of Netrin Gl , Tyro sin-protein phosphatase non- receptor-type 13, Apolipoprotein B- 100 or Golgin-160. The expression level of the gene of Netrin G l , Tyrosin-protein phosphatase non-rcceptor-type 13, Apolipoprotein B-100 or Golgin-160 may be determined on the mRNA level (transcriptional level) or protein level (translationai level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via immunohistochemistry) or mRNA (e.g. via in situ hybridization). Preferably, the amount of the Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160 mRNA or protein is determined.
As afore-mentioned, the subject is identified as experiencing a neurodegenerative disease or being prone thereto, if the expression level of the above-mentioned biomarker is altered compared to a control. Preferably, an altered expression level is an expression level changed to a higher (increased) level or lower (decreased) level in a subject experiencing a neurodegenerative disease or being prone thereto compared to a control.
Preferably, an altered, more preferably an increased or a decreased, expression level is considered to be present for the purpose of the present invention when the expression profile of the tested subject differs by at least 1%, 5% or 10%, preferably by at least 20% or 30%, more preferably by at least 50% or 70%, most preferably by at least 80%, 90%, 100%, or more than 100%, e.g. by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, from a control.
The term "subject", as mentioned in the method above, may mean a subject suspected to experience a neurodegenerative disease such as PD or PDD. The subject may be diagnosed to experience a neurodegenerative disease such as PD or PDD, i.e. be diseased. The term "subject", as mentioned in the method above, may also mean a subject which already experiences a neurodegenerative disease such as PD or PDD. Particularly, the subject may be retested for experiencing a neurodegenerative disease such as PD or PDD and may be diagnosed to still experience a neurodegenerative disease such as PD or PDD, e.g. a more severe or pronounced form, level or stage of a neurodegenerative disease such as PD or PDD. The term "subject", as mentioned in the method above, may further mean a subject suspected to develop a neurodegenerative disease such as PD or PDD. The subject may be prognosed to develop a neurodegenerative disease such as PD or PDD in the future. Particularly, the subject may be a subject which already suffers from PD but which is suspected to have developed a dementia in the course of the disease. The "subject", as mentioned in the method above, may further be a human or another mammal, e.g. a rodent (e.g. rat, hamster, or mouse) or monkey, or may be another animal than a mammal, e.g. an avian. In a preferred embodiment, the subject is a human or another mammal. The subject to be diagnosed and/or prognosed with the method of the present invention may also be designated as "test subject" herein.
The term "control (value/data)", as mentioned in the method above, may refer to a value/data of a (control) subject known to experience a neurodegenerative disease such as PD or PDD (positive control), i.e. be diseased. The term "control", as mentioned in the method above, may also refer to a value/data of a (control) subject known to not experience a neurodegenerative disease such as PD and/or PDD (negative control), i.e. be healthy. Said (control) subject may be a human or another mammal, e.g. a rodent (e.g. rat, hamster, or mouse) or monkey, or may be another animal than a mammal, e.g. an avian. In a preferred embodiment, said (control) subject is a human or another mammal. The subject known to experience a neurodegenerative disease or known to not experience a neurodegenerative disease may also be designated as "control subject" herein. In this respect, it should be noted that a "(control) subject" that is known to be healthy, i.e. not suffering from a neurodegenerative disease such as PD and/or PDD, may possibly suffer from another disease not known/tested, e.g. migraine.
It is preferred that in a method for diagnosing and/or progtiosing a neurodegenerative disease, the control (value) is
(i) the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarkcr(s)) selected from the group consisting of Netrin Gl , Tyrosin- protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Goigin-160 of a healthy subject, and/or
(ii) the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl , Tyro sin -protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience a neurodegenerative disease (e.g. a specific form, level or stage of a neurodegenerative disease).
As to the definition of the term "expression level" it is referred, to the explanations given above.
Preferably, the control (value) is an average control (value). It is particularly preferred that the control (value) is an. average control (value) of at least 2 to 40 (control) subjects, more preferably of at least 10 to 40 (control) subjects, and most preferably of at least 15 to 40 (control) subjects, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 (control) subjects. To assure the comparability of the results (values) determined by analyzing the biological sample from a test subject, with the control results (values) determined by analyzing the biological sample from a control subject, both results (values) are preferably achieved with the same methods, more preferably carried out under the same method/process conditions. Thus, preferably, only results (values) achieved with the same methods and more preferably under comparable, most preferably identical method/process conditions, are compared to identify a subject experiencing a neurodegenerative disease.
It is preferred that the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). It is also preferred that the method of the present invention relates to the differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
Thus, in a preferred embodiment, the present invention relates to a method for diagnosing and/or prognosing Parkinson's disease (PD). Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD) comprising the steps of:
(i) determining the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing Parkinson's disease (PD) or being prone thereto, or as experiencing Parkinson's disease dementia (PDD) or being prone thereto, if the expression level of said biomarker is altered, preferably decreased or increased, in the biological sample from the subject compared to a control.
As to the preferred percentages regarding the alterations between the expression profiles, it is referred to the above.
The terms "differential diagnosing" and "differential prognosing" between PD and PDD relate to the discrimination between both disease states based on observations made with respect to the expression level of at least one of the biomarkers selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160.
The term "Parkinson.' s disease (PD)", as used herein, refers to a chronic (persistent) disorder of part of the brain. It is named after the person who first described it. It mainly affects the way the brain co-ordinates the movements of the muscles in various parts of the body. The main symptoms of Parkinson's disease are, for example, stiffness, shaking (tremor), and slowness of movement. Symptoms typically become gradually worse over time.
The term "Parkinson's disease dementia (PDD)", as used herein, denotes the impairment of one or more cognitive processes, particularly related to memory, in subjects showing signs of or suffering from Parkinson's disease, also known as Parkinson disease, Parkinson's, idiopathic parkinsonism, primary parkinsonism, PD or paralysis agitans.
Accordingly, it is particularly preferred that in a method for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD), the control (value) is
(i) the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a healthy subject,
(ii) the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protem phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience Parkinson's disease (PD), and/or
(iii) the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience Parkinson's disease dementia (PDD).
It is more preferred that the method of the present invention is for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD) comprising the steps of:
(i) determining the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 in a biological sample from a subject, and
(ii) identifying the subject as experiencing Parkinson's disease dementia (PDD) or being prone thereto, if the expression level of the biomarker Netrin Gl and/or Tyrosin- protein phosphatase non-receptor-type 13 is increased in the biological sample from the subject compared to a control. Particularly, said control is
(iia) the expression level of the biomarker Netrin Gl and/or Tyrosin-protem phosphatase non-receptor- type 13 known to be present in a healthy subject, and/or (lib) the expression level of the biomarker Netrin Gl and/or Tyrosin-protein. phosphatase non-receptor- type 13 known to be present in a subject experiencing Parkinson's disease (PD).
If, for example, the expression level of the biomarker Netrin Gl and/or Tyrosin- protein phosphatase non-receptor-type 13 in a biological sample from a subject, determined with the above-mentioned method, is increased compared to a control which is the expression level of the biomarker Netrin Gl and/or Tyrosin-protem phosphatase non-receptor-type 13 known to be present in a healthy subject, the subject is identified as experiencing FDD or being prone thereto. Further, if, for example, the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-rcceptor-type 13 in a biological sample from a subject, determined with the above-mentioned method, is increased compared to a control which is the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a subject experiencing Parkinson's disease (PD), the subject is identified as experiencing FDD or being prone thereto.
It is particularly more preferred that the method of the present invention is for diagnosing and or prognosing Parkinson's disease dementia (FDD) and that the control is
(iia) the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a healthy subject, and
(iib) the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-reccptor-type 13 known to be present in a subject experiencing Parkinson's disease (PD).
In a preferred embodiment, the subject is identified as experiencing Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), if the expression level of the biomarker Netrin Gl is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g. is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above- mentioned respective control. In a particularly preferred embodiment, the expression level of the biomarkcr Netrin Gl is increased by at least 5%, at least 10%, at least 15%, or at least
20%.
In another preferred embodiment, the subject is identified as experiencing Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), if the expression level of the biomarkcr Tyrosin-protein phosphatase non-receptor-type 13 is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g. is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 105, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned respective control. In a particularly preferred embodiment, the expression level of the biomarkcr Tyrosin-protein phosphatase non-receptor- type 13 is increased by at least 5%, at least 8%, at least 10%, at least 15%, or at least 20%.
To increase the significance of the determined data as to the diagnosis and/or prognosis of PDD, it is particularly preferred to use Netrin Gl as well as Tyrosin-protein phosphatase non-receptor-type 13 as biomarkers.
Thus, in a more preferred embodiment, the subject is identified as experiencing
Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing Parkinson's disease dementia (PDD), (i) if the expression level of the biomarker Netrin Gl is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g. is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%. or more than 100%, in the biological sample from the subject compaxed to the above-mentioned respective control and (ii) if the expression level of the biomarker Tyrosin- protein phosphatase non-receptor-type 13 is increased by at least 1%, at least 5%, or at least 10 %, more preferably by at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably by at least 60 % , at least 70%, at least 80%, and most preferably by at least 90 %, 95 %, 99%, 100% or more than 100%, e.g. Is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13. 14, 15, 16. 17, 18, 19, 20. 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned respective control.
In case of retesting for experiencing PDD, e.g. in order to evaluate whether the test subject still experiences PDD, e.g. a more severe or pronounced form, level or stage of PDD, the (average) expression level of the biomarker Netrin Gl and or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a subject experiencing Parkinson's disease dementia (PDD) is preferably used as a control.
It is also more preferred that the method of the present invention is for diagnosing and/or prognosing Parkinson's disease (PD) comprising the steps of:
(i) determining the expression level of the biomarker Apoiipoprotem B-100 in a biological sample from a subject, and
(ii) identifying the subject as experiencing Parkinson's disease (PD) or being prone thereto, if the expression level of the biomarker Apolipoprotein B-100 is decreased in the biological sample from the subject compared to a control. Particularly, said control is
(iia) the expression level of the biomarker Apoiipoprotem B-100 known to be present in a healthy subject, and/or
(iib) the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD).
If, for example, the expression level of the biomarker Apolipoprotein B-100 in a biological sample from a subject, determined with the above-mentioned method, is decreased compared to a control which is the expression level of the biomarker Apolipoprotein B-100 known to be present in a healthy subject, the subject is identified as experiencing PD or being prone thereto. Further, if, for example, the expression level of the biomarker Apolipoprotein B-100 in a biological sample from a subject, determined with the above-mentioned method, is decreased compared to a control which is the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD), the subject is identified as experiencing PD or being prone thereto.
It is particularly more preferred that the method of the present invention is for diagnosing and/or prognosing Parkinson's disease (PD) and that the control is (iia) the expression level of the biomarker Apolipoprotein B-100 known to be present in a healthy subject, and
(iib) the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD).
In a preferred embodiment, . the subject is identified as experiencing Parkinson's disease (PD) or being prone thereto, particularly as experiencing Parkinson's disease (PD), if the expression level of the above-mentioned biomarker is decreased by at least 1%, at least 5%, or at least 10 %, more preferably at least 20 %, at least 30%, at least 40% or at least 50 %, still more preferably at least 60 % , at least 70%, at least 80%, and most preferably at least 90 %, 95 %, 99%, 100% or more, e.g. is decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned control In a particularly preferred embodiment, the expression level of the biomarker Apolipoprotein B-100 is decreased by at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%.
hi case of retesting for experiencing PD, e.g. in order to evaluate whether the test subject still experiences PD, e.g. a more severe or pronounced form, level or stage of PD, the (average) expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease (PD) is preferably used as a control.
It is further more preferred that the method of the present invention is for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) comprising the steps of:
(i) determining the expression level of the biomarker Goigin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto, if the expression level of the biomarker Golgin-1 60 is decreased in the biological sample from the subject compared to a control. Particularly, said control is the expression level of the biomarker Golgin-160 known to be present in a healthy subject.
If, for example, the expression level of the biomarker Golgin-160 in a biological sample from a subject, determined with the above-mentioned method, is decreased compared to a control which is the expression level of the hiomarker Golgin- 160 known to be present in a healthy subject, the subject is identified as experiencing Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto.
In a preferred embodiment, the subject is identified as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto, particularly as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD), if the expression level of the above-mentioned hiomarker is decreased by at least 1%, at least 5%, or at least 10 %, more preferably at least 20 %, at least 30%. at least 40% or at least 50 %, still more preferably at least 60 % , at least 70%, at least 80%, and most preferably at least 90 %, 95 %, 99%, 100% or more, e.g. is decreased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, or more than 100%, in the biological sample from the subject compared to the above-mentioned control. In a particularly preferred embodiment, the expression level of the hiomarker Golgin-160 is decreased by at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%.
A comprehensive diagnosis and/or prognosis of a neurodegenerative disease preferably involves the analysis, whether the test subject suffers from PD or PDD. Therefore, the expression profile of at least one hiomarker indicative for PD (e.g. Apoiipoprotein B- 100) and at least one biomarker indicative for PDD (e.g. Netrin Gl or Tyrosin-protein phosphatase non-receptor-type 13) is preferably determined. Accordingly, in a preferred embodiment, the expression profile of (i) Netrin Gl and Apoiipoprotein 13- 100, (ii) Tyrosin-protein phosphatase non-receptor-type 13 and Apoiipoprotein B-100, or (iii) Netrin Gl , Tyrosin- protein phosphatase non-receptor-type 13 and Apoiipoprotein B-100 is determined.
In the context of the present invention, the term "increased compared to a control" may also mean increased compared to an expression level of zero. This may be the case where a specific biomarker is not present/detectable in the biological sample from a control subject, e.g. subject known to be healthy or known to suffer from a neurodegenerative disease such as PD or PDD, but present/detectable in the biological sample from the test subject, e.g. subject which is suspected to experience or develop a neurodegenerative disease such as PD or PDD.
The term "biological sample", as used herein, refers to any biological sample comprising the above-mentioned biomarkers. The biological sample may be any sample comprising cells or the products of cells derived from a subject. It may be a body fluid sample, a tissue sample (e.g. explant or section), or a cell sample (e.g. ccll(s) or cell colonies). For example, said biological sample may be an. explant sample, a section sample, a single cell sample, a cell colony sample, a ceil culture sample, a blood sample, a urine sample, or a sample from another peripheral source. The biological samples may be mixed or pooled, e.g. a biological sample may be a mixture of a blood sample and a urine sample. The biological sample may be provided by removing cell colonies, an explant, or a section from a subject, but may also be provided by using a previously isolated sample. For example, a tissue sample may be removed from a subject by conventional biopsy techniques or a blood sample may be taken from a subject by conventional blood collection techniques.
Preferably, the biological sample is a body fluid sample, a tissue sample, a cell colony sample, a single cell sample or a cell culture sample. More preferably, the tissue sample is a section or an explant sample.
It is preferred that the tissue sample from a subject has a weight of between 0.1 and 500 mg, more preferably of between 0.5 and 250 mg, and most preferably of between 1 and 50 mg, i.e. 0.1, 0.2, 03, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg.
It is further preferred that the cell sample (e.g. cell colony sample or cell culture sample) from a subject consists of between 102 and 1010 cells, more preferably of between 103 and 107 cells, and most preferably of between 104 and 106 cells, i.e. 102, 103, 1 4, 10\ 106, 107, 10*, 109, or 1010 cells.
The term "body fluid sample", as used herein, refers to a liquid sample derived from the body of a subject, e.g. human or animal. Said body fluid sample may be a blood, urine, sputum, breast milk, cerebrospinal fluid (CSF), cerumen (earwax), endolymph, perilymph, gastric juice, mucus, peritoneal fluid, pleural fluid, saliva, sebum (skin oil), or a sweat sample including components or fractions thereof. Preferably, it is a cerebrospinal fluid (CSF) sample, a blood sample, more preferably a whole blood sample or serum sample, an urine sample, or a saliva sample including components or fractions thereof. A "body fluid sample" may be provided by removing a body liquid from a subject, but may also be provided by using previously isolated body fluid sample material. In the context of the present invention said "body fluid sample" may allow for a non-invasive diagnosis/and or prognosis of a subject. It is preferred that the body fluid sample from a subject has a volume of between 0.1 and 20 ml, more preferably of between 0.2 and 10 ml, more preferably between 0.4 and 8 ml and most preferably between 1 and 5 ml, I.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ml.
In a preferred embodiment of the method of the first aspect of the present invention, the expression level is determined with an immunoassay, gel electrophoresis, spectrometry, chromatography, in situ hybridization, or a combination thereof. Preferably, the immunoassay is an enzyme immunoassay, more preferably an ELISA, or a Western Blot (also designated as immunoblot). The gel electrophoresis preferably is ID (One-dimensional) or 2D (Two- dimensional) gel electrophoresis. The spectrometry preferably is mass spectrometry (MS), more preferably tandem mass spectrometry (MS/MS). The chromatography preferably is liquid chromatography (LC, or alternative HPLC) or affinity chromatography, e.g. protein affinity chromatography. The in situ hybridization preferably is a silver in situ hybridization (SISH), chromogenic in situ hybridization (CISH), or fluorescence in situ hybridization (FISH), The chromatography is preferably combined with spectrometry, more preferably mass spectrometry (MS), and is even more preferably liquid chromatography-mass spectrometry (LC-MS, or alternative HPLC-MS) and most preferably liquid chrornatography- tandem mass spectrometry (LC-MS/MS, or alternative HPLC -MS/MS). Liquid chromatography-mass spectrometry is an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (LC, or alternatively HPLC) with the mass analysis capabilities of mass spectrometry (MS). LC-MS is a powerful technique as it has very high sensitivity and selectivity. Generally its application is oriented towards the specific detection and potential identification of molecules in the presence of other molecules, e.g. in a complex mixture. The gel electrophoresis is preferably combined with an immunoassay and is more preferably a 2D immunoblot.
If ID gel electrophoresis is used, the biological sample is preferably beforehand purified, e.g. with affinity chromatography. The term "ID (One-dimensional) gel electrophoresis", as used herein, includes protein separation techniques such as Sodium dodecyl sulfate polyacryl amide gel electrophoresis (SDS-PAGE), native gel electrophoresis and isoelectric focusing. The SDS-PAGE, for example, is a technique for separating proteins based on their ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. The addition of the SDS detergent to these samples gives the proteins the same electrical charge. SDS-PAGE allows for separation of proteins from a wide range of samples including cells, tissues and whole blood. Native Gel Electrophoresis, for example, is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio. The main types of native gels used in protein electrophoresis are polyacrylamide gels and agarose gels. It should be noted that unlike SDS-PAGE type electrophoreses, native gel electrophoresis does not use a charged denaturing agent. The proteins being separated, therefore, differ in molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Since the proteins remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining. Further, isoelectric focusing (IEF) (also known as electrofocusing), for example, is a technique to separate the proteins by isoelectric point. Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If the proteins are positively charged, they will be pulled towards the more negative end of the gel and if the proteins are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the IEF will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge). IEF is preferably carried out using immobilized pH gradient (lPG) gels, or iPG strips, more preferably dry and rehydratabie iPG strips. Microfluidic chip based isoelectric focusing may also be used (Sommer and Hatch, Electrophoresis. 2009 Mar;30(5):742-5.). Preferably, the ID gel electrophoresis is isoelectric focusing (IEF) or SDS-PAGE as first dimension to separate the proteins according to their isoelectric point (pi).
The term "2D (Two-dimensional) gel electrophoresis", refers to a form of gel electrophoresis commonly used to analyze proteins in two dimensions. 2D gel electrophoresis, for example, begins with ID electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first. In ID electrophoresis, proteins are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a 2D gel. The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass. Preferably, the first dimension is isoelectric focusing (IEF) and the second dimension is SDS-PAGE. As to the separation of proteins by their isoelectric point (isoelectric focusing, IEF) and as to the separation of proteins by their mass (SDS-PAGE) it is referred to the explanations made above. Preferably, the 2D gel electrophoresis is isoelectric focusing (IEF) as first dimension and SDS-PAGE as second dimension to separate the proteins according to their isoelectric point (pi) and according to their protein mass.
The proteins separated with gel electrophoresis can then be detected by a variety of means known to the person skilled in the art. Preferably, silver and Coomassie Blue staining are used. In the case of silver staining, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The darkness of the silver can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes.
Western blotting, for example, is a technique which allows the detection of specific proteins (native or denatured) from extracts made from cells or tissues or body liquid samples, before or after any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred to a synthetic membrane (typically nitrocellulose or PVDF) via dry, semi-dry, or wet blotting methods. The membrane can then be probed using antibodies using methods similar to imrnunohistochemistry, but without a need for fixation. Detection is typically performed using reporter enzyme linked antibodies, e.g. peroxidase linked antibodies to catalyze a ehemiluminescent reaction or alkaline phosphatase linked antibodies to catalyze a colori metric reaction. Western blotting is a routine molecular biology method that can be used to semiquantitatively or quantitatively compare protein levels between extracts. The size separation prior to blotting allows the protein molecular weight to be gauged as compared with known molecular weight markers. Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/ non-denaturing conditions). The col ri metric detection method may depend on incubation of the Western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot may be then stopped by washing away the soluble dye. Protein levels may be evaluated through densitometry (how intense the stain is) or spectrophotometry. Further, ehemiluminescent detection methods may depend on incubation of the Western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which capture a digital image of the Western blot. The image may be analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. The enzyme-linked immunosorbent assay or ELISA, for example, is a method for quantitatively or semi-quantitatively determining protein concentrations from, blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 6-wclls per plate). Broadly, proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.
The term "mass spectrometry (MS)", as used herein, refers to the use of an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer. The term "laser desorption mass spectrometry" refers to the use of a laser as an ionization source to generate gas phase ions from a sample on a surface and detecting the gas phase ions with a mass spectrometer. The mass spectrometry may be a matrix-assisted laser desorption ionization mass spectrometry or MALDI. In MALDI, the analyte is typically mixed with a matrix material that, upon drying, co-crystallizes with the analyte. The matrix material absorbs energy from the energy source which otherwise would fragment the labile biomolecuies or analytes. The mass spectrometry may also be a surface- enhanced laser desorption/ionization mass spectrometry or SELDI. In SELDI, the surface on which the analyte is applied plays an active role in the analyte capture and/or desorption. The biological sample used in the method of the first aspect of present invention may have undergone chromatographic or other chemical processing. In mass spectrometry the "apparent molecular mass" refers to the molecular mass (in Daltons)-to- charge value, m/z, of the detected ions. How the apparent molecular mass is derived is dependent upon the type of mass spectrometer used. With a time of-ttight mass spectrometer, the apparent molecular mass is a function of the time from ionization to detection. The term "signal" refers to any response generated by a biomolecule such as protein under investigation. For example, the term signal refers to the response generated by a biomolecule hitting the detector of a mass spectrometer. The signal intensity correlates with the amount or concentration of the biomolecule. The signal is defined by two values: an apparent molecular mass value and an intensity value generated as described. The mass value is an elemental characteristic of the biomolecule, whereas the intensity value accords to a certain amount or concentration of the biomolecule with the corresponding apparent molecular mass value. Thus, the "signal" always refers to the properties of the biomolecule.
The term "tandem mass spectrometry (MS/MS)", as used herein, refers to multiple rounds of mass spectrometry, usually separated by some form of molecule fragmentation. For example, one mass analyzer can isolate one peptide from many entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation (CID). A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiative dissociation (BIRD), electron-detachment dissociation (EDD) and surface-induced, dissociation (SID). An important application using tandem mass spectrometry is in protein identification.
Further, Tandem mass spectrometry enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine- sequences as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), and precursor ion scan. In SRM, the first analyzer allows only a single mass through and the second analyzer monitors for a single user defined fragment ion. MRM allows for multiple user defined fragment ions. SRM and MRM are most often used with scanning instruments where the second mass analysis event is duty cycle limited. These experiments are used to increase specificity of detection of known molecules, notably in pharmacokinetic studies. Precursor ion scan refers to monitoring for a specific loss from, the precursor ion. The first and second mass analyzers scan across the spectrum as partitioned by a user defined m/z value. This experiment is used to detect specific motifs within unknown molecules.
In a preferred embodiment, the mass spectrometry is an electrospray ionization mass spectrometry (ESI-MS), a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), or an electron capture dissociation mass spectrometry (ECD-MS).
In another preferred embodiment, the mass spectrometry employs tandem mass tags
(TMT), isobaric tags for relative and absolute quantitation (lTRAQ), or isotope-eoded affinity tags (ICATs). Isobaric tags for relative and absolute quantitation (i'T'RAQ) axe particularly preferred. ITRAQ is a non-gel-based technique used to quantify proteins from different sources in a single experiment. It uses isotope-coded covalent tags. The method is based on the covalent labeling of the N-terminus and side-chain amines of peptides from protein digestions with tags of varying mass. There are currently two mainly used reagents: 4-plex and 8-plex, which can be used to label all peptides from different samples/treatments. These samples are then pooled and usually fractionated by nano liquid chromatography and analyzed by tandem mass spectrometry (MS/MS). A database search is then performed using the fragmentation data to identify the labeled peptides and hence the corresponding proteins. The fragmentation of the attached tag generates a low molecular mass reporter ion that can be used to relatively quantify the peptides and the proteins from which they originated, using software such as the freely available i-Tracker.
Further, it is preferred that the diagnosis comprises (i) determining the presence or occurrence of a neurodegenerative disease such as PD or PDD, (ii) monitoring the course of a neurodegenerative disease such as PD or PDD, (iii) staging of a neurodegenerative disease such as PD or PDD, (iv) measuring the response of a subject with a neurodegenerative disease such as PD or PDD to therapeutic intervention, and/or (v) classification of a subject with a neurodegenerative disease such as PD or PDD. It is further preferred that the prognosis comprises (i) predicting or estimating the occurrence, preferably the severity of occurrence, of a neurodegenerative disease such as PD or PDD, and/or (ii) predicting or estimating the response of a subject with a neurodegenerative disease such as PD or PDD to therapeutic intervention.
In a second aspect, the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl , Ί yrosin-protem phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing a neurodegenerative disease, preferably in a biological sample from a subject.
With respect to the meaning of the terms "biological sample" and "subject", it is referred to the definitions provided above.
It is preferred that the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). It is also preferred that the molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160 is for the differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
Thus, in a preferred embodiment, the present invention relates to a molecule for detecting a biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD), or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
In a more preferred embodiment, the present invention relates to a molecule for detecting the biomarker Netrin Gl or Tyrosin-protein phosphatase non-receptor-type 13 for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (FDD),
In another more preferred embodiment, the present invention relates to a molecule for detecting the biomarker Apolipoprotein B-100 for diagnosing and/or prognosing Parkinson's disease (PD).
In a further more preferred embodiment, the present invention relates to a molecule for detecting the biomarker Golgin-160 for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
As mentioned above, the molecule is able to detect or detects the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determinant(s)) of said biomarker. Said molecule may be a protein such as an antibody, a polypeptide such as an antibody fragment, a peptide such as a mass spectrometry probe, a polynucleotide, or a small molecule.
It is preferred that the molecule is able to bind or binds the biomarker Netrin Gl,
Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 or Golgin-160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determmant(s)) of said biomarker. Said molecule may be a protein such as an antibody, a polypeptide such as an antibody fragment, a polynucleotide, or a small molecule.
The term "peptide", as used herein, refers to a short polymer of amino acids linked by peptide bonds. It has the same peptide bonds as those in proteins, but is commonly shorter in length. The shortest peptide is a dipeptide, consisting of two amino acids joined by a single peptide bond. There can also be a tripeptide, tetrapeptide, pentapeptide, etc. A peptide has an amino end and a carboxyl end, unless it is a cyclic peptide. Preferably, a peptide has a length of between 2 to 20 ammo acids, more preferably of between 5 to 20 amino acids and most preferably of between 7 to 15 amino acids, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.
The term "polypeptide", as used herein, refers to a part of a protein which is composed of a single linear chain of amino acids bonded together by peptide bonds. Preferably, the polypeptide has a length of more than 20 amino acids, more than 30 amino acids, or more than 40 amino acids. More preferably, the polypeptide has a length of between 21 and 200 amino acids, most preferably of between 50 and 100 amino acids, e.g. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 1 10, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 amino acids.
The term "protein", as used herein, may refer to a protein which comprises one or more polypeptides that resume a secondary and tertiary structure and additionally refers to a protein that is made up of several amino acid chains, i.e. several subimits, forming quaternary structures. The protein has sometimes non-peptide groups attached, which can be called prosthetic groups or co factors.
The term "polynucleotide", as used herein, may refer to a molecule which comprises at least 10 nucleotides and not more than 80 nucleotides, wherein the nucleotides are covalentiy linked together. Preferably, said polynucleotide is a molecule of 10 to 70 nucleotides or 15 to 60 nucleotides in length, more preferably of 20 to 40 nucleotides or 25 to 35 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length.
The term "small molecule", as used herein, refers to a low molecular weight organic compound which is by definition not a polymer. A small molecule may bind with high affinity to a biopolymer such as a protein and, thus, may allow the detection of said biopolymer. The upper molecular weight limit for a small molecule is usually about 800 Daltons.
It is particularly preferred that the molecule, e.g. protein or polypeptide, which is able to bind or binds the biomarker Netrin Gl , Apolipoprotein B 100, TyTosin-protein phosphatase non-receptor-type 13 or Go!gm- 160, e.g. the amino acid sequence (e.g. the epitope(s), also known as antigenic determinant(s)) of said biomarker, is an antibody or a fragment thereof, a synthetic polypeptide, a recombinant pol peptide, preferably a darpin or an anticalin, or a polynucleotide. Said molecule is specific for said biomarker.
The term "synthetic peptide or polypeptide", as used herein, refers to a synthetically produced peptide or polypeptide. Generally, a peptide or polypeptide is synthetically produced by adding the amino acid from, the carboxylate groups forward, as opposed to ribosomal production, wherein, synthesizing starts with the amino group. For example, a synthetic polypeptide or peptide may be produced using liquid-phase synthesis or solid-phase peptide synthesis (SPPS), preferably Fmoc or Boc. The term "recombinant peptide or polypeptide", as used herein, refers to a genetically engineered polypeptide or peptide, i.e. a polypeptide or peptide with a sequence manipulated by man. Usually a recombinant polypeptide or peptide is produced from recombinant DNA (e.g. DNA coding for said polypeptide or peptide comprised in a vector such as an expression vector), for example, in a host organism such as a bacterial cell or yeast cell.
The term "darpin", as used herein, refers to a genetically engineered antibody mimetic protein typically exhibiting highly specific and high-affinity target protein binding. It is derived from natural ankyrin proteins and consists of at least three, usually four or five repeat motifs of these proteins. Its molecular mass is about 14 or 18 kDa for four- or five-repeat DARPins, respectively.
The term "anticalin", as used herein, refers to an artificial protein that is able to bind to antigens, e.g. to proteins or to small molecules, and is a type of antibody mimetic. It is derived from human lipocalins which are a family of naturally binding proteins. The size is about 180 amino acids and the mass is about 20 kDa.
As mentioned above, said polynucleotide may be a molecule of at least 10 nucleotides and of not more than 80 nucleotides covalently linked together. Preferably, said polynucleotide is a molecule of 10 to 70 nucleotides or 15 to 60 nucleotides in length, more preferably of 20 to 40 nucleotides or 25 to 35 nucleotides in length, i.e. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length. The depiction of a single strand of said polynucleotide also defines the sequence of the complementary strand. Said polynucleotide may be single or double stranded, or may contain portions of both double and single stranded sequences. Said polynucleotide may be a polymer of deoxyribormcleotide or ribonucleotide bases, including DNA (e.g. cDNA and genomic DNA) and R A (e.g. mRNA or cRNA) molecules, both sense and anti-sense strands. Said polynucleotide may be obtained by chemical synthesis methods. Said polynucleotide as a single polynucleotide strand may provide a probe that is capable of binding to or hybridizing with and, thus, detecting a target of complementary sequence, such as the nucleotide sequence of the mRNA of the above-mentioned biomarkers, e.g. through one or more types of chemical bonds, usually through complementary base pairing and hydrogen bond formation. Said polynucleotide as a probe may be unlabeled, directly labeled, or indirectly labeled, such as with biotin to which a streptavidin complex may later bind. Said label may be a molecule detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, suitable labels include 32P, fluorescent dyes, electron- dense reagents, enzymes (e.g., as commonly used in an ELBA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable. A label may be incorporated into nucleic acids at any position, e.g. at the 3' end, at the 5' end or internally.
It is preferred that the polynucleotide is selected from the group consisting of a polynucleotide probe, a peptide nucleic acid (PNA), a locked nucleic acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), a microR A (miR A), and a small interfering RNA (si RNA). Polynucleotides as probes have preferably a length of between 15 and 35 nucleotides and more preferably of between 18 and 30 nucleotides. The above - mentioned peptide nucleic acids (PNAs), locked nucleic acids (LNAs), glycol nucleic acids (GNAs) and threose nucleic acids (TNAs) are artificial polynucleotides which can also be used as probes. Each of these is distinguished from naturally-occurring DNA or RNA by changes to the backbone of the molecule. MiRNAs and siRNAs can also be used as probes.
It is clear to the person skilled in the art that in some preferred embodiments more than one polynucleotide, e.g. two polynucleotides such as a primer pair, is required to determine the expression level of the above-mentioned biomarkers. Preferably, the one or more polynucleotide(s) is (are) selected from the group consisting of a primer(s) (e.g. a primer pair), preferably a primer(s) for polymerase chain reaction (PCR), reverse transcription (RT) reaction, or DNA sequencing. More preferably, the primers (e.g. a primer pair) are for real time polymerase chain reaction (RT-PCR), and most preferably for quantitative real time polymerase chain reaction (qRT-PCR).
Polynucleotides as primers have preferably a length of between 15 and 35 nucleotides and more preferably of between 18 and 30 nucleotides.
The synthetic polypeptides or peptides, recombinant polypeptides or peptides. preferably darpins or anticalins, lectins or small molecules according to the invention may be selected by routine screening of existing libraries, e.g. small molecule libraries. Suitable standard screening methods, e.g. phage display for polypeptides or peptides, are well known to the person skilled in the art. That said molecules are able to detect/determine the above- mentioned biomarkers, e.g. by binding said biomarkers, can easily be tested by the person skilled in the art with methods known to the person skilled in the art, e.g. by fluorescence resonance energy transfer (FRET), co-immunoprecipitarion or an Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA).
In one embodiment, the binding of the above-mentioned molecule to the biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin- 160 may be analyzed in form of an enzyme-linked immunosorbent assay (ELISA)-based experiment. Therefore, the biomarker selected from the group consisting of Netrin G l , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160 may e immobilized on the surface of an ELISA plate and contacted with the above-mentioned molecule. Binding of the molecule may be verified, for example, for proteins, polypeptides, peptides, and epitope-tagged compounds, by antibodies specific for said molecule or the epitope-tag. These antibodies might be directly coupled to an enzyme or detected with a secondary antibody coupled to said enzyme that - in combination with the appropriate substrates - carries out chemiiuminescent reactions (e.g. horseradish peroxidase) or colorimetric reactions (e.g. alkaline phosphatase). In another embodiment, binding of molecules that cannot be detected by antibodies might be verified by labels directly coupled to the molecules. Such labels may include enzymatic labels, radioisotope or radioactive compounds or elements, fluorescent compounds or metals, chemiiuminescent compounds and bioluminescent compounds. In another embodiment, the above-mentioned molecule might be immobilized on the ELISA plate and contacted with the the biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin- 160. Binding of said biomarker may be verified by an antibody specific for said biomarker and chem i I urn i n escence or colorimetric reactions as described above.
The term "antibody or fragment thereof, as used herein, 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. Also comprised are ii nunoglobu!in-!ike proteins that are selected through techniques including, for example, phage display to specifically bind to a target molecule or target protein. The immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl , IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. The "antibodies and fragments thereof include, but are not limited to, polyclonal, monoclonal, monovalent, bispecific, heteroconjugate, multispecific, human, humanized (in particular CDR-grafted), deimmunized, or chimeric antibodies, single chain antibodies (e.g. scFv), Fab fragments, F(ab¾ fragments, fragments produced by a Fab expression library, diabodies or tetrabodies (Holliger P. et al., 1993), nanobodies, anti-idiotypic (anti-Id) antibodies, and epitopc-binding fragments of any of the above.
In some embodiments, the antibody fragments are mammalian, preferably human antigen-binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab¾, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (dsFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable domain(s) alone or in combination with the entirety or a portion of the following: hinge region, CL, CH 1 , CH2, and CH3 domains. The antigen-binding fragments may also comprise any combination of variable domain(s) with a hinge region, CL, CHI, CH2, and CH3 domains.
Antibodies usable in the invention, may be from any animal origin including birds and mammals. Preferably, the antibodies are human, simian (e.g. chimpanzee, bonobo, macaque), rodent (e.g. mouse and rat), donkey, sheep rabbit, goat, guinea pig, camel, horse, or chicken. It is particularly preferred that the antibodies are of human or murine origin. As used herein, "human antibodies" include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins.
Antibodies according to the second aspect of the invention may be produced by methods well known, in the art or may simply be ordered to be made commercially. Means of preparing and characterizing antibodies or antibody fragments are also well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by reference).
The antibody that recognizes the target antigen, here the biomarker selected from the group consisting of etrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-! OO and Golgin- i 60, is generally called the "primary antibody". Said antibody may be labeled with a detectable taglabel in order to allow direct detection of the target antigen. Said detectable tag/label may be an enzymatic, fluorescent or radioisotope tag/label. Usually, however, the primary antibody is not labeled for direct detection. Instead a "secondary antibody" that has been labeled with a detectable tag/label (e.g. enzymatic, fluorescent or radioisotope tag/label) is applied in a second step to probe for the primary antibody, which is bound to the target antigen. For example, the primary antibody or the secondary antibody may be labeled with an affinity tag such as biotin.
Tt is preferred that the biomarker specific molecule is able to bind or binds the amino acid sequence according to SEQ ID NO: 1 to 5 or a variant thereof (see above).
As mentioned above, for detection purposes, the molecule of the second aspect may be directly or indirectly labeled, e.g. with biotin to which a streptavidin complex may bind. The term "label", as used herein, means a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which are or can be made detectable.
It is also preferred that the molecule, e.g. peptide, for detecting the biomarker selected, from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-1 0 is a mass spectrometry peptide (probe). The terms mass spectrometry probe or mass spectrometry peptide are interchangeable used herein. Said probe is a synthetic peptide analog to a native peptide of the biomarker Netrin Gl , Tyrosin- protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 which is cleavable with a protease (e.g. trypsin protease). Said probe enables protein identification and absolute protein quantitation of said biomarker with mass spectrometry, preferably with HPLC-MS or HPCL-MS/MS, particularly supported by multiple reaction monitoring (MRM). It preferably incorporates one stable isotope labeled amino acid, creating a slight increase (e.g. 6-10 daltons) in molecular weight. When mixed, the native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 and the synthetic peptide co-elute chromatographically, co-migrate el ectrophoret i cally , and ionize with the same intensity. Nevertheless, by mass spectrometry, the native peptide of the biomarker Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160 and the synthetic peptide can easily be distinguished. In a typical mass spectrometry procedure, preferably HPLC-MS or HPLC- MS/MS procedure, a known amount of the synthetic peptide is added to a sample of a subject. The sample is then digested (e.g. by a protease such as trypsin protease) and analyzed by mass spectrometry, preferably by HPLC-MS or H PLC -MS/MS. Extracted ion chromatograms are generated for the native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin-160 and the synthetic peptide internal standard. Using peak ratios, the quantity of native peptide of the biomarker Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 is calculated. It also allows quantitation of the amount of the biomarker Netrin Gl, Tyrosin- protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in the sample of the subject. The techniques for selecting and preparing the synthetic peptide are well known in the art. For example, the skilled person knows how to select precursor- fragment -ion- transitions under the aspect of optimal selectivity and sensitivity. Preferably, the mass spectrometry peptide (probe) has an amino acid sequence according to SEQ ID NO: 6 to SEQ ID NO: 9.
In a further aspect, the present invention relates to a composition or set comprising a molecule for detecting the bioraarker Netrin Gl, a molecule for detecting the biomarker Tyrosin-protein phosphatase non-receptor-type 13, a molecule for detecting the biomarker Apolipoprotein B-100 and/or a molecule for detecting the biomarker Golgin-160 for diagnosing and'or prognosing a neurodegenerative disease, preferably in a biological sample from a subject. It is preferred that the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). All preferred and particularly preferred embodiments previously described in the context of the molecule of the second aspect of the invention are similarly preferred in the context of this aspect of the invention.
Preferably, the composition or set comprises a molecule(s) for detecting (i) the biomarker Netrin Gl, (ii) the biomarker Tyrosin-protein phosphatase non-receptor-type 13, (iii) the biomarker Apolipoprotein B-100, (iv) the biomarker Golgin-160, (v) the biomarkers Netrin Gl and Tyrosin-protein phosphatase non-receptor-type 13, (vi) the biomarkers Netrin Gl and Apolipoprotem B-100, (vii) the biomarkers Netrin Gl and Golgin-160, (viii) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Apolipoprotein B-100, (ix) the biomarkers Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160, (x) the biomarkers Apolipoprotein B-100 and Golgin-160, (xi) the biomarkers Netrin Gl, Tyrosin- protein phosphatase non - receptor-t ype 13 and Apolipoprotein B-100, (xii) the biomarkers Netrin Gl . Tyrosin-protein phosphatase non-receptor-type 13 and Golgin-160, (xiii) the biomarkers Tyrosin-protein phosphatase non-reee tor-type 13, Apolipoprotein B-100 and Golgin-160, (xiv) the biomarkers Apolipoprotein B-100, Netrin Gl and Golgin-160, or (xv) the biomarkers Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, preferably in a biological sample from a subject.
In a third aspect, the present invention relates to means for diagnosing and/or prognosing a neurodegenerative disease comprising at least one molecule (e.g. 1 , 2, 3, or 4 molecule(s)) according to the second aspect.
It is preferred that said means allows the detection/determination of the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of etrin Gl, Tyrosin-protein phospliatase non-receptor-type 13, Apolipoprotem B-100 and Golgin-160, preferabl in a biological sample from a subject, and, thus, the diagnosis and/or prognosis of a neurodegenerative disease. As to the definitions of the terms "biological sample" and "subject", it is referred to the explanations given above. As to the definition, of the term "expression level", it is also referred to the explanations given above. Particularly, the expression level of the gene of Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13, Apolipoprotein B-100 or Golgin-160 may be determined on the mRNA level (transcriptional level) or protein level (translational level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via tmmunohistochemistry) or mRNA (e.g. via in situ hybridization). Preferably, the amount of the Netrin Gl, Tyrosin-protein phosphatase non -receptor-type 13, Apolipoprotein B-100 or Golgin- 160 mRNA or protein is determined.
It is more preferred that the above-mentioned neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (FDD). Particularly, said means are for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
All preferred and particularly preferred embodiments previously described in the context of the method of the first aspect of the invention and the molecule of the second aspect of the invention are similarly preferred in the context of this aspect of the invention.
Preferably, said means comprise a solid support. It is preferred that said means further comprises means for immobilising the at least one molecule according to the second aspect of the present invention on said solid support or for attaching the at least one molecule according to the second aspect of the present invention to said solid support. The solid support may be made of the following materials: glass (including modified or function ah zed glass), plastics (including acrylics, polystyrene, polypropylene, polyethylene, polybutylcne, polyurethanes, teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials (including silicon and modified silicon), carbon, metals or mixtures/combinations thereof. The solid support may be planar, e.g. a slide, chip, matrix, or array, although also other configurations of the solid support may be possible as well, e.g. tubes, beads, or microspheres.
It is particularly preferred that the at least one molecule according to the second aspect of the present invention is attached to or immobilized on the solid support.
It is more particularly preferred that said means consists of a solid support to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized. More preferably, said means comprise a biochip.' m i croarray or a set of beads. It is preferred that said means further comprises means for immobilising the at least one molecule according to the second aspect of the present invention on the biocMp or beads of said set of beads or for attaching the at least one molecule according to the second aspect of the present invention to the biochip or beads of said set of beads.
It is more particularly preferred that the at least one molecule according to the second aspect of the invention is attached to or immobilized on the biochip/microarray or the at least one molecule according to the second aspect of the invention is attached to or immobilized on the beads of said set of beads.
It is most particularly preferred that said means consists of (i) a biochip to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized, or (ii) beads of a set of beads to which the at least one molecule according to the second aspect of the present invention is attached or on which the at least one molecule according to the second aspect of the invention is immobilized.
It is preferred that said beads or microspheres have a mean diameter of between 2 to 20 microns, preferably 4 to 10 microns, most preferably 5 to 7 microns, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 microns.
The terms "biochip'" and "microarray" are interchangeable used herein. The terms "attached" or "'immobilized"., as used herein, refer to the binding between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, and may mean that the binding between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, is sufficient to be stable under conditions of binding, washing, analysis and removal. The binding may be covalent or non-covalent. Covalent bonds may be formed directly between the molecule according to the second aspect of the present invention and the solid support, e.g. biochip, or may be formed by a cross linker or by inclusion of specific reactive groups on either the solid support, e.g. biochip, or the molecule according to the second aspect of the invention, or both. Non-covalent binding may be electrostatic, hydrophilic arid hydrophobic interactions or combinations thereof. Immobilization or attachment may also involve a combination of covalent and non-covalent interactions.
It is also preferred that the above-mentioned means comprises or consists of the molecule composition or set as described above. In a preferred embodiment, at least one molecule (e.g. 1, 2, 3, or 4 molecule(s)) according to the second aspect of the invention is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
In another preferred embodiment, the above-mentioned molecule composition or set (e.g. composition or set comprising 2, 3, or 4 biomarker(s)) is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least two biornarkers (e.g. 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
In another further preferred embodiment, the means according to the third aspect of the invention is used in step (i) of the method of the first aspect of the invention for determining the expression level of at least one biomarker (e.g. 1. 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject.
In a fourth aspect, the present invention relates to a kit for (use in) the diagnosis and/or prognosis of a neurodegenerative disease comprising
(i) means for determining the expression level of at least one biomarker (e.g. 1, 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, and optionally
(ii) a data carrier, and/or
(hi) a container.
In the context of the present invention, a kit of parts (in short a kit) is understood to be any combination of at least some of the components identified herein, which are combined, coexisting spatially, to a functional unit, and which can contain further components.
It is preferred that the expression level of at least one biomarker (e.g. 1 , 2, 3, or 4 biomarker(s)) selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 is determined in a biological sample from a subject. This may then allow the diagnosis and/or prognosis of a neurodegenerative disease. As to the definition of the terms "biological sample" and "subject", it is referred to the explanations given above. As to the definition of the term "expression level", it is also referred to the explanations given above. Particularly, the expression level of the gene of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoproteiii B-100 or Golgin- 1 60 may be determined on the mR A level (transcriptional level) or protein level, (translational level), for example, by measuring the transcribed mRNA (e.g. via northern blot), the expressed protein (e.g. via Western Blot), or by directly staining the protein (e.g. via immunohistochemistry) or mRNA (e.g. via in situ hybridization). Preferably, the amount of the Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotcin B-100 or Golgin- 160 mRNA or protein is determined.
It is also preferred that the above-mentioned neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD). Particularly, said means are for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
All preferred and particularly preferred embodiments previously described in the context of the method of the first aspect of the invention, the molecule of the second aspect of the invention, and the means of the third aspect of the invention are similarly preferred in the context of the fourth aspect of the invention. Particularly, as to the terms "biological sample" or "subject", it is referred to the definitions mentioned above.
It is further preferred that said means comprises or consists of
(i) at least one molecule (e.g. 1 , 2, 3, or 4 rnolecule(s)) according to the second aspect of the present invention, and/or
(ii) a means according to the third aspect of the present invention.
It is also preferred that the above-mentioned means (alternatively or additionally) comprises or consists of a molecule composition or set as defined above.
It is particularly preferred that the kits comprise materials desirable from a commercial and user standpoint such as a bnffer(s), a reagent(s) and/or a dilnent(s). Said materials may be ■useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein. phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160. It is further particularly preferred that the kits comprise reporter- means such as an affinity tag binding protein, for example, a biotin binding molecule (e.g. avidin or streptavidin) bound to a detectable label, e.g. an enzymatic, fluorescent or radioisotope label and/or a secondary antibody that is labeled with an affinity tag, for example, biotin. Said reporter-means may also be useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin G l , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Goigin- 160.
The above-mentioned data carrier may be a graphically data carrier such as an information leaflet, an information sheet, a bar code or an access code, or an electronically data carrier such as a floppy disk, a compact disk (CD), or a digital versatile disk (DVD), The access code may allow the access to a database, e.g. an internet database, a centralized, or a decentralized database.
Preferably, said data carrier comprises a control (value), particularly to allow the interpretation of information obtained when performing the above-mentioned method for diagnosing and/or prognosing a neurodegenerative disease, preferably for diagnosing and/or prognosing Parkinson's disease (PD), Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD). Thus, said control (value) may allow for the diagnosis and/or prognosis of Parkinson's disease (PD), Parkinson's disease dementia (PDD) or for differential diagnosis and/or prognosis between Parkinson's disease (PD) and Parkinson's disease dementia (PDD), As to the preferred embodiments of said control (value), it is referred to the first aspect of the invention.
Additionally or alternatively, the data carrier comprises instructions for the method according to the first aspect of the present invention, the molecule according to the second aspect of the present invention and/or the means according to the third aspect of the present invention in order to diagnose and/or prognose a neurodegenerative disease, preferably to diagnose and/or prognose Parkinson's disease (PD), Parkinson's disease dementia (PDD) or to differential diagnose and/or prognose between Parkinson's disease (PD) and Parkinson's disease dementia (PDD). Said data carrier may further comprise the following:
(i) instructions for use of the means for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-rcceptor-typc 13, Apolipoprotein B-100 and Golgin-160, and/or instructions for use of the kit,
(ii) instructions with respect to the biological sample which is to be used and/or instructions how said biological sample is to be obtained from the subject ,
(iii) quality information material such as information about the lot/batch number of the means for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160, and/or of the kit, (iv) information concerning the detailed composition of the buffer(s), dilucnt(s) and reagent(s) -useful for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B-100 and Golgin- 160,
(v) warnings about possible miss-interpretations or wrong results when applying a wrong method and/or wrong means, and/or
(vi) warnings about possible miss-interpretations or wrong results when using wrong reagent(s) and/or buffer(s).
In a further aspect, the present invention relates to the use of the molecule according to the second aspect, the means according the third aspect, or the kit according to the fourth aspect in the method of the first aspect.
In another further aspect, the present invention relates to the use of a gel- free mass- spectrometry approach with isotope-labelled samples (iTRAQ) or multiple-reaction- monitoring (MRM) for the determination of a biomarker for diagnosing and/or prognosing a neurodegenerative disease, preferably Parkinson's disease (PD) or Parkinson's disease dementia (FDD), in a cerebrospinal fluid (CSF) sample.
Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention. BRIEF DESCRIPTION OF THE FIGURES
The following Figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way. Fig. 1 : HPLC of CSF-samples. Elution diagrams for the two-dimensional HPLC-pre- fractionation of the iTRAQ-samples. a: Strong-cation-exchange column - individual runs, b-d: reversed-phase chromatography of the fractions collected after SCX-separation. Fig. 2: M M-Measurements. Quantitative analysis of MRM-data in PD, FDD and
NDC with 25th, 50th, 75th, and min/max . a: Proteins with a significant difference between NDC and FDD. b: proteins with a significant regulation between PD and N'DC.
EXAMPLES
The examples given below are for illustrative purposes only and do not limit the invention described above in any way.
Patients and methods
Patients
All CSF samples used for the iTRAQ-measurements were taken from patients attending the general outpatient clinic (University of Ulm, Department of Neurology), CSF-samples were stored at -80°C after analysis of the routine parameters (cell count, lactate, Q-albumine and total protein). For the validation study with MRM-measurements samples from the Department of Neurology, University of Eastern Finland, Kuopio were also used. Collection and analysis of CSF samples was approved by the Ethics Committees and conformed to the requirements of the declaration of Helsinki in 1964.
All persons or in case of demented patients also their relatives gave written informed consent to participate in the study, and they underwent clinical, neurological and ncuroradioiogical examinations as well as a neuropsychological screening to investigate global cognitive functions. Patients were examined n europsy ch ol ogi cal 1 y for classification of their mental status and exclusion of depressive syndromes.
All PD and FDD patients underwent a detailed psychometric test battery [23] covering the following tests: MMSE [24], Geriatric Depression scale [25], Parkinson Neuropsychometric Dementia Assessment [26], Regensburger Wortfluessigkeitstest. (RWT) [27], Doors Test [28], Alertness/ Go/ oG vgetei ite Aufmerkainkeit [29], Boston Naming Test [30], Wcchslcr Memory Scale (WMS-R) [31 ], Melmstaedter, Coloured Progressive Marices [32] , VOSP [33], Clock Test [34].
Patients with PD and FDD
The clinical characteristics such as the minimental status test (MMST), the Hoehn&Yahr stages for PD as well as CSF tan protein concentrations are given in Table 1 (see below). The diagnosis of all patients was done according to the consensus criteria for PD/PDD [35] as well as the DSM-IV criteria and was established in cooperation by neurologists and neuropsychologists, both blinded with respect to the neurochemical outcomes. Table 1: Listing of detailed patient parameters in all groups investigated. Data are
indicated as mean±SD.
Figure imgf000041_0001
Control patients
Data is given in Table 1 (see above). The control patients showed neither e tra yramidal- motor nor cognitive symptoms. iTRAQ measurements
CSF samples were depleted of albumine and immunoglobuline G with the Albumin and Ig G removal kit (GE-Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. Total protein concentration was measured before and after depletion using a BCA-assay. In total 4 samples for each group (PD, PDD and NDC) were analysed, as well as a pool consisting of equal amounts of all samples. The sample compositions of each iTRAQ run were as follows: The pool was labelled with 114, PDD with 115, PD with 116 and NDC with 117. Proteins were pelleted with 6 volumes of ice-cold-actone for Ih at — 20°C, centrifuged for 30 minutes at 13,000 rpm (4°C) and air-dried. Pellets were stored at 80°C until further analysis. Protein Digestion and iTRAQ Labelling
The pellet was resuspended in 30 \xL dissolution buffer containing 2 ,uL denaturant (iTRAQ reagent kit, Applied Biosystems). Equal protein amounts of each disease state were reduced with 3 mM TCEP for 30 mm at 60°C and alkylated with 6 mM iodoacetamide at room temperature for 20 min in the dark. Proteins were digested with 1 μg LysC (Wako Chemicals) at 37°C for 3 h and incubated over night after adding 10 μΐ. water with 1 μg trypsin (Promega) at 37°C. Digested samples were labelled with the iTRAQ reagents according to the manufacturer's instructions (Applied Biosystems). The four iTRAQ samples (114: Pool; 115: FDD; 116: PD; 117: NDC) were combined and dried by vacuum oentrif ligation. Two-dimensional Peptide Separation
Peptides were fractionated by strong cation exchange liquid chromatography (SCX) using a PolySulfoethyl A 2.1x100 mm, 5 μιη, 200 A column (PolyLC). The dried sample was dissolved in 1 ml of SCX buffer A (10 mM K¾P04, H 2.9, 25% v/v Acetonitrile [ACN]). Peptides were eluted with a gradient of 0 5% buffer B (10 mM KH2P04! pH 2.9, 1 M KC1, 25% v/v ACN) over 17 min and from 15% to 50% buffer B for 8 min at a flow rate of 200 μΙΛηΐη. Fractions were collected at 1 min intervals, pooled depending on peak intensity monitored at 214 nm and dried by vacuum centrifugation.
Each fraction was resuspended in buffer A (2% ACN, 0.1% Trifluoracetic-Acid [TFA]), injected onto a 0.3x5 mm C18 trap column (Dionex) and separated on a 0.1x250 mm Reprosi! Pur C18-AQ, 3 μιη (Dr. Maisch GmbH) analytical column at a flow rate of 500 ni/rnin and 50°C applying a 85 min gradient from 5 to 30% buffer B (85% ACN, 5% isopropanol, 0.1% TFA) and over 35 min from 30 to 50%. Column eluent was mixed with 3 mg ml CHCA at 1.3 μΐ/min and spotted in 13 s intervals on a MALD1 target plate using a Probot (Dionex). MS and MS/MS spectra were acquired on a 4800 TOF/TOF Analyzer (Applied Biosystems). A maximum of 16 unique precursors with a minimum signal/noise of 40 were selected per spot for MS/MS with 1 kV acceleration.
Mass Spectrometry and Data Analysis
MS/MS data were analyzed using ProteinPilot 2.0.1 (Applied Biosystems) which uses the Paragon algorithm to perform database matching for protein identification, protein grouping to remove redundant hits and comparative quantitation. The Swiss-Pro t Homo sapiens protein database was used for ail searches. Data were normalized for loading error by bias corrections calculated using ProteinPilot. All reported data were based on 95% confidence for protein identification as determined by ProteinPilot.
All protein iTRAQ ratios were transformed to base 2 logarithm values, a 2-fold change in levels is reported as -1 or 1 for down- and up-regulated, respectively. Since it is useful for CSF analysis to not compare identical protein concentration but identical volumes of CSF samples [36] the relative ratios were normalised according to CSF volume. 16 regulated proteins between FDD and PD were found. The proteins Netrin Gl, Tyrosin-protein phosphatatase non-receptor-type 13, Golgin- 160 and Apolipoprotein B-100 were significantly regulated.
MRM-measurements
Peptide selection and measurement protocol. For each of the above-mentioned proteins, a peptide which was present in as many iT AQ runs as possible with high intensities was chosen and synthezised (Thermo Scientific, Ulm, Germany) from the iTRAQ measurements since those peptides promised the highest success rate. The peptides are given in Table 2. Peptides were reconstituted to a concentration of 0.5 pg iil with double-distilled water and stored at -80°C until further use.
Table 2: Regulated proteins in the iTRAQ-measurement with, selected peptides.
Figure imgf000043_0001
Charge state, fragmentation pattern as well as optimized measurement conditions for each peptide were determined on a Triple-Quad mass-spectrometer (API 4000 triple quadrupolc mass spectrometer (AB Sciex, Ontario, Canada)) with autosampler (HTS PAL autosampler (CTC Analytics AG, Zwingen, Switzerland)) using the Analyst™- Software (Applied Biosystems). For multiplexed analysis of all peptides as well as CSF, the samples were prefractionated using an HPLC-system (Agilent 1200 Binary Pump, Agilent 1200 Micro Vacuum Degasser and Agilent 1200 Thermostatted Column Compartment (Agilent Technologies, Morges, Switzerland) with a CI S-column (Symmetry CI 8, 5μηι, NanoEase Trap Column (Waters Corporation, Massachusetts, USA)) coupled online to the mass- spectrometer. Peptides were eluted using an Acetonitrile gradient (3 minutes 5%, gradient to 50% within 1 minute, 11 minutes 50%, gradient to 100% within 1 minute and 100% for 3 minutes) containing 0.1 % formic acid with a flow rate of 20 μΐ per minute. Peptide-counts were calculated with Analyst™ as areas under the curve. For standard curves, the peptides were diluted 1 :1000 with 50% Acetonitrile in 0.1% formic acid and further dilutions were made down to approximately 1 n.M per peptide. All measurements were carried out in duplicates and linear regressions were used to calculate the standard-curves. Sample concentrations are given in Table 3.
Table 3: Protein concentrations determined in the MRM-measurements (values are indicated as ng/ml).
Figure imgf000044_0001
Statistics
Analysis for significant differences between all groups or two groups was carried out using Kruskal-Wallis- or Mann- Whitney-tests for each parameter (sigma stat software). P-values below 0.05 were considered to be significant. Results
The characteristics of all patients included in the study are given in Table 1 (see above). iTRAQ - measurement for candidate identification
For iT AQ-measurements 4 patients of each group (PDD. PD and NDC) were analysed. Hereby, an internal standard consisting of a mixture of all 12 samples was used to guarantee compatibility of the individual runs. Samples were subjected to a two-dimensional column- chrom atography in order to reduce sample-complexity thus increasing sensitivity of the mass- spectrometric analysis. In a first step, peptides were separated using a strong cation- exchange column. After peak-dependant pooling, twelve peptide fractions were obtained for each ran. These were then further separated by a reversed-phase (CI 8) column. The elution diagrams are given in Figure 1, Fractions were collected in a time dependant manner. Each reversed- phase fraction was then spotted onto a Maldi target and analysed via mass-spectrometry. With this approach more than 1,000 different proteins could be identified in each of the four independent runs. Primary iTRAQ results which are based on equal protein concentrations were normalised to equal sample volumes. Only proteins which were identified in each of the 4 runs were considered for further analysis. 16 of these proteins were found to be regulated similarly (PD compared to PDD) in each ran. The proteins Netrin Gl, Tyrosin-protein phosphatatase non-receptor-type 13, Golgin- 160 and Apoiipoprotein B- 100 were significantly regulated and were chosen for further validation (Table 2).
MRMfor candidate verification
For candidate verification, a mass-spectrometric approach was chosen which utilises synthetic peptides as a means of absolute quantitation.
Peptides for each protein were chosen according to iTR AQ results. An overview of the peptides is shown in Table 2. For each peptide an optimised measurement protocol was established and specificity of the protocols was checked using different mixtures of the synthetic peptides to exclude false positive measurements (data not shown). A ten point standard curve was calculated for each peptide using 2 independent measurements. The coefficient of variation was also calculated separately for each peptide using 4 independent measurements of an identical CSF-sample. These coefficients were well below 20% (data not shown).
For biological analysis, 10 samples per group were chosen and analysed in duplicate. Two of these samples were also analysed in the iTRAQ-experiments. Individual concentrations for each peptide/protein are given in Table 3 and for the regulated proteins also shown in Figure 2.
For the 4 proteins, mentioned above, a fast and reproducible measurement could be achieved. Protein concentrations were calculated based on their unmodified molecular weight.
For Tyrosine-kinase non-receptor-type 13 and Netrin G l , significant differences between PDD and NDC could be detected. In addition, significant differences between PD and PDD could be detected. For Golgin and Apoiipoprotein B-100, a statistically significant difference between PD and NDC was measured.
Discussion
Parkinson's dementia (PDD) is so far diagnosed only according to clinical criteria [37]. However cognitive decline and even dementia is often neglected since usually in the early stages of the disease the classical Parkinson symptoms dominate the clinical picture [38],
Therefore, a biochemical marker for an early and reliable laboratory diagnosis was desired in order to allow an early therapeutic intervention. In this study a gel-free proteomic approach, the iTRAQ-technique, was used in order to identify such a possible marker. With this m ass-spec trometry based technique the inventors were able to reproducibly identify and quantify more than 1,000 proteins in human CSF- samples of PD and PDD patients. With this approach 16 proteins which were different between PD and PDD were identified. Among those proteins, two proteins (Netrin Gl and Tyrosine kinase non-receptor type 13) could be verified in a larger group of patients using multiple-reaction monitoring (MRM). Two further proteins, Apolipoprotein B-100 and Golgin-160 turned also out to be significantly changed.
The inventors found that multiple reaction monitoring in the form applied here is a very promising tool for disease monitoring and diagnosis in CSF since it is highly specific, sensitive and due to the possibility to multiplex, it reduces sample consumption to a minimum.
The proteins identified and verified in this study are interesting candidates. Said candidates are Tyrosinc-protein phosphatase non-receptor-type 13 (PTPN 13), Netrin Gl , Apolipoprotein B- 100 and Golgin- 160.
Netrin Gl belongs to a family of proteins which are involved in the process of axonal guiding through which growing axons are guided towards their correct targets. Netrin Gl was first described in the year 2000 as a glycosyl phosphatidyiinositol (GPI) anchored member of that family. Its expression in the brain reaches a maximum at perinatal stages in most regions. However in the cerebellum, Netrin Gl is only expressed in certain regions which are implicated in controlling motor activity.
Golgins are a large family of proteins which have different functions in connection with the Golgi-apparatus. The Golgin A3 protein also known as Golgin-160 because of its molecular weight is thought to be involved in intra-Golgi vesicle mediated transport.
The results of the present inventors show that gel-free labelling approaches are very promising tools for proteomic projects and that it represents a powerful method for diagnostic validation of biomarkers in connection with multiple-reaction monitoring which has been optimised for CSF. REFERENCES
1 , M. Di Napoli, I, M. Shah, and D. A. Stewart, Molecular pathways and genetic aspects of Parkinson's disease: from bench to bedside, Expert Rev Neurother 7 (12), 1693 (2007).
2, A. Rongve and D. Aarsland, Management of Parkinson's disease dementia : practical considerations, Drugs Aging 23 (10), 807 (2006).
3, N. Cabailol, M. J, Marti, and E. Tolosa, Cognitive dysfunction and dementia in Parkinson disease, Mov Disord 22 Suppl 17, S358 (2007).
4. B. Dubois and B. Pillon, Cognitive deficits in Parkinson's disease, J Neurol 244 (1), 2 (1997).
5. E. D. Louis et al., Mortality from Parkinson disease, Arch Neurol 54 (3), 260 (1997).
6, B. Singh and J. T, O'Brien, When should drug treatment be started for people with dementia?, Maturitas 62 (3), 230 (2009).
7, M. Goedert and M. G. Spillantini, Lewy body diseases and multiple system atrophy as alpha-synucleinopathies, Mol Psychiatry 3 (6), 462 (1998).
8. K. A. Jellinger, A critical evaluation of current staging of alpha-synuclein pathology in Lewy body disorders, Biochim Biophys Acta 1792 (7), 730 (2009).
9. EL A. Jellinger and J. Attems, Prevalence and impact of vascular and Alzheimer pathologies in Lewy body disease, Acta Neurop thol 115 (4), 427 (2008).
10. E. B. Mukaetova- Ladinska and I. G. McKeith. Pathophysiology of synuclein aggregation in Lewy body disease, Mech Ageing Dev 127 (2), 188 (2006).
11. M. Tanaka et al., Aggresomes formed by alpha-synuclein and. synphiiin-1 are cytoprotective, J Biol Chem 279 (6), 4625 (2004).
12. K. M. Danzer et al, Different species of alpha-synuclein oligomers induce calcium influx and seeding, JNeurosci 27 (34), 9220 (2007).
13. C. Schnack, K. M. Danzer, B. Hengerer, and F. Gillardon, Protein array analysis of oligomerization-induced changes in alpha-synuclein protein-protein interactions points to an interference with Cdc42 effector proteins, Neuroscience 154 (4), 1450 (2008). 14. S. Jesse et al., Neurochemical approaches in the laboratory diagnosis of Parkinson and
Parkinson dementia syndromes: a review, CNS Neurosci Ther 15 (2), 157 (2009).
15. L. Anderson and C. L. Hunter, Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins, Mol Cell Proteomics 5 (4), 573 (2006).
16. D. J. Janecki et al., A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1 C1 isoenzyme, Anal Biochem 369
(1), 18 (2007).
17. E. Kuhn et al., Quantification of C-reactive protein in the serum of patients with rheumatoid arthritis using multiple reaction monitoring mass spectrometry and DC- labeled peptide standards, Proteomics 4 (4), 1175 (2004).
18. S. Lin, T. A. Shaler, and C. H. Becker, Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single- quadrapoie ion trap, Anal Chem 78 (16), 5762 (2006).
19. V. Mayya et al, Absolute quantification of multisite phosphorylation by selective reaction monitoring mass spectrometry: determination of inhibitory phosphorylation status of cyclin-dependent kinases, Mol Cell Proteomics 5 (6), 1 146 (2006).
0. A. Wolf-Yadlin, S. Hautaniemi, D. A. Lauffeoburger, and F. M. White, Multiple reaction monitoring for robust quantitative proteomic analysis of cellular signaling networks, Proc Natl Acad Sci USA 104 (14), 5860 (2007).
1. F. Zappacosta, T. S. Collingwood, M. J. Muddiest on, and R. S. Annan, A quantitative results-driven approach to analyzing multisite protein phosphorylation: the phosphate- dependent phosphorylation profile of the transcription factor Pho4, Mol Cell Proteomics 5 (11), 2019 (2006).
22. A, K. Yocam and A. M. Chinnaiyan, Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry, Brief Funct Genomic Proteomic S {2), 145 (2009).
23. M. R. Bothe, 1. Uttner, and M. Otto, Sharpening the boundaries of Parkinson- associated dementia: recommendation for a neuropsychological diagnostic procedure, J Neural Transm 117 (3), 353,
24. J. C. Anthony et al., Limits of the 'Mini-Mental State' as a screening test for dementia and delirium among hospital patients, Psychol Med 12 (2), 397 (1982).
25. J. A. Yesavage et al., Development and validation of a geriatric depression screening scale: a preliminary report, J Psychiatr Res 17 (1), 37 (1982).
26. E. Kalbe et ai, Screening for cognitive deficits in Parkinson's disease with the Parkinson neuropsychometric dementia assessment (PANDA) instrument, Parkinsonism Relat Disord 14 (2), 93 (2008).
27. O. Tucha, S. Aschenbrenner, and K. W. Lange, Mirror writing and handedness, Brain Lang 73 (3), 432 (2000).
28. A. Sunderland, . Watts, A. D. Baddeley, and J. E. Harris, Subjective memory assessment and test performance in elderly adults, J Gerontol 41 (3), 376 (1986). 29. B. Fimm, G. Bartl, P. Zimmermann, and C. W. Wallesch, Different mechanisms under! y shifting set on external and internal cues in Parkinson's disease, Brain Cogn 25 (2), 287 (1994).
30. R. C. Mohs, W. G. Rosen, and K. L. Davis, The Alzheimer's disease assessment scale: an instrument for assessing treatment efficacy, Psychopharmacol Bull 19 (3), 448 (1983).
31. K. Sullivan, Estimates of intcrrater reliability for the Logical Memory subtest of the Wechsler Memory Scale-Revised, J Clin Exp Neuropsychol 18 (5), 707 (1996).
32. J. E. Orme, The Coloured Progressive Matrices as a measure of intellectual subnormality, Br J Med Psychol 34, 291 (1961).
33. L. J. Rapport, S. R. Millis, and P. J. Bonello, Validation of the Warrington theory of visual processing and the Visual Object and Space Perception Battery, J Clin Exp Neuropsychol 2% (2), 211 (1998).
34. H. Tuokko, T. Hadjistavropoulos, S. Rae, and N. O'Rourke, A comparison of alternative approaches to the scoring of clock drawing, Arch Clin Neuropsychol 15 (2), 137 (2000).
35. I. G. McKeith et ai, Consensus guidelines for the clinical and pathologic diagnosis of dementia with Lewy bodies (DLB): report of the consortium on DLB international workshop, Neurology 47 (5), 1 i 13 (1996).
36. P. Brechlin et al., Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease, Proteomics 8 (20), 4357 (2008).
37. D. D. Truong and E. C. Wolters, Recognition and managanent of Parkinson's disease during the premotor (prodromal) phase, Expert Rev Neur other 9 (6), 847 (2009).
8. W. Poewe et al., Diagnosis and management of Parkinson's disease dementia, Int J Clin Pract 62 (10), 1581 (2008).

Claims

1. A method for diagnosing and/or prognosing a neurodegenerative disease comprising the steps of:
(i) determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyro sin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease or being prone thereto, if the expression level of said biomarker is altered in. the biological sample from the subject compared to a control.
2. The method of claim 1, wherein the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
3. The method of claims 1 or 2, wherein the control is
(i) the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a healthy subject,
(ii) the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience Parkinson's disease (PD), and/or
(iii) the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non-receptor-type 13, Apolipoprotein B-100 and Golgin-160 of a subject known to experience Parkinson's disease dementia (PDD).
4. The method of claims 1 to 3 for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD) comprising the steps of:
(i) determining the expression level of the biomarker Netrin Gl and/or Tyrosin- protein phosphatase non-receptor-type 13 in a biological sample from a subject, and identifying the subject as experiencing Parkinson's disease dementia (PDD) or being prone thereto, if the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 is increased in the biological sample from the subject compared to a control which is
(iia) the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a healthy subject, and/or
(iib) the expression level of the biomarker Netrin Gl and/or Tyrosin-protein phosphatase non-receptor-type 13 known to be present in a subject experiencing Parkinson's disease (PD).
The method of claims 1 to 4 for diagnosing and/or prognosing Parkinson's disease (PD) comprising the steps of:
(i) determining the expression level of the biomarker Apolipoprotein B- 100 in a biological sample from a subject, and
(ii) identifying the subject as experiencing Parkinson's disease (PD) or being prone thereto, if the expression level of the biomarker Apolipoprotein B-100 is decreased in the biological sample from the subject compared to a control which is
(iia) the expression level of the biomarker Apolipoprotein B-100 known to be present in a healthy subject, and/or
(iib) the expression level of the biomarker Apolipoprotein B-100 known to be present in a subject experiencing Parkinson's disease dementia (PDD).
The method of claims 1 to 5 for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) comprising the steps of:
(i) determining the expression level of the biomarker Golgin-160 in a biological sample from a subject, and
(ii) identifying the subject as experiencing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD) or being prone thereto, if the expression level of the biomarker Golgin-160 is decreased in the biological sample from the subject compared to a control which is the expression level of the biomarker Golgin-160 known to be present in a healthy subject.
7. The method of claims 1 to 6, wherein the biological sample is a body fluid sample or a tissue sample.
8. The method of claim 7, wherein the body fluid sample is a cerebrospinal fluid (CSF) sample, a blood sample, preferably a whole blood sample or a serum sample, a urine sample, or a saliva sample.
9. The method of claims 1 to 8, wherein the subject is a human or another mammal,
10. The method of claims 1 to 9, wherein the expression level is determined with an immunoassay, gel electrophoresis, spectrometry, chromatography, in situ hybridization, or a combination thereof.
11. The method of claim 10, wherein
(i) the immunoassay is an enzyme immunoassay, preferably an ELISA or Western Blot (immunoblot),
(ii) the gel electrophoresis is ID or 2D gel electrophoresis,
(iii) the spectrometry is mass spectrometry (MS), preferably tandem mass spectrometry (MS/MS),
(iv) the chromatography is liquid chromatography (LC) or affinity chromatography,
(v) the in situ hybridization is a silver in situ hybridization (SISH), chroraogcnic in situ hybridization (CISH), or fluorescence in situ hybridization (FISH),
(vi) the chromatography is combined with spectrometry, preferably mass spectrometry (MS), and. is more preferably liquid chromatography-mass spectrometry (LC-MS) and most preferably liquid chromatography-tandcm mass spectrometry (LC-MS/MS), or
(vii) the gel electrophoresis is combined with an immunoassay and is more preferably a 2D immunoblot.
12. The method of claim 11, wherein the mass spectrometry is an electrospray ionization mass spectrometry (ESI-MS), a matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), or an electron capture dissociation mass spectrometry (ECD-MS).
13. The method of claims 11 or 12, wherein the mass spectrometry employs tandem mass tags (TMT), isobaric tags for relative and absolute quantitation (iTRAQ), or isotope- coded affinity tags (ICATs).
14. The method of claims 1 to 13, wherein at least one molecule according to claims 15 to 23 is used in step (i) for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl, Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotein B- 100 and Golgin-160 in a biological sample from a subject.
15. A molecule for detecting a biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non-receptor- type 13, Apolipoprotein B-100 and Golgin- 160 for diagnosing and/or prognosing a neurodegenerative disease.
16. The molecule of claim 15, wherein the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (FDD).
17. The molecule of claims 15 or 16, wherein the molecule is for detecting the biomarker Netrin Gl or Tyrosin-protein phosphatase non-receptor-type 13 for diagnosing and/or prognosing Parkinson's disease dementia (PDD) or for differential diagnosing and/or prognosing between Parkinson's disease (PD) and Parkinson's disease dementia (PDD).
18. The molecule of claims 15 or 16, wherein the molecule is for detecting the biomarker Apolipoprotein B-100 for diagnosing and/or prognosing Parkinson's disease (PD).
1 . The molecule of claims 15 or 16, wherein the molecule is for detecting the biomarker Golgin- 160 for diagnosing and/or prognosing a neurodegenerative disease which is Parkinson's disease (PD) or Parkinson's disease dementia (PDD).
20. The molecule of claims 15 to 19, wherein the molecule is able to bind said biomarkcr.
21. The molecule of claim 20, wherein the molecule which is able to bind said biomarker is an antibody or a fragment thereof, a synthetic polypeptide, a recombinant polypeptide, preferably a darpin or an anticalin, or a polynucleotide.
22. The molecule of claims 15 to 19, wherein the molecule is a mass spectrometry peptide.
23. The molecule of claim 22, wherein the mass spectrometry peptide is a peptide having an amino acid sequence according to SEQ ID NO: 6 to SEQ ID NO: 9.
24. Means for diagnosing and/or prognosing a neurodegenerative disease comprising at least one molecule according to claims 15 to 23.
25. The means of claim 24, wherein said means is
(i) a biochip, or
(if) a set of beads.
26. A kit for diagnosing and/or prognosis a neurodegenerative disease comprising
(i) means for determining the expression level of at least one biomarker selected from the group consisting of Netrin Gl , Tyrosin-protein phosphatase non- receptor-type 13, Apolipoprotcin B-100 and Golgin-160, and optionally
(ii) a data carrier, and/or
(iii) a container.
27. The kit of claim 26, wherein the neurodegenerative disease is Parkinson's disease (PD) or Parkinson's disease dementia (FDD).
28. The kit of claims 26 or 27, wherein said data carrier comprises instructions for the method according to claims 1 to 14.
29. The kit of claims 26 to 28, wherein said means comprises or consists of (ί) at least one molecule according to claims 15 to 23, or
(ii) the means according to claims 24 or 25.
30. Use of the molecule according to claims 15 to 23, the means according to claims 24 or 25, or the kit according to claims 26 to 29 in the method of claims 1 to 14.
31. Use of a gel-free mass-spectrometry approach with isotope-labelled samples (iTRAQ) or multipl e- reacti on-moni tor i ng (MRM) for the determination of a biomarker for diagnosing and/or prognosing a neurodegenerative disease, preferably Parkinson's disease (PD) or Parkinson's disease dementia (FDD), in a cerebrospinal fluid (CSF) sample.
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016032977A1 (en) * 2014-08-24 2016-03-03 Haskins William E Cns-specific biomarkers for cns diseases or injuries
CN108135973A (en) * 2015-05-19 2018-06-08 拉·约拉过敏反应及免疫医学研究所 Human APOB100 epitopes, methods and uses for modulating inflammatory response and treating adverse cardiovascular events, diseases and atherosclerosis
JP2018091735A (en) * 2016-12-05 2018-06-14 磁量生技股▲ふん▼有限公司 How to identify Parkinson's disease dementia from Parkinson's disease
CN112924698A (en) * 2021-03-05 2021-06-08 厦门大学 Biomarker for diagnosing neurodegenerative disease and application thereof
CN119985972A (en) * 2025-01-14 2025-05-13 北京市神经外科研究所 Biomarkers for assisting diagnosis of progressive supranuclear palsy and their application
WO2025120644A1 (en) * 2023-12-08 2025-06-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods and a kit for detecting parkinson's disease with and without dementia

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035826A1 (en) * 2004-08-13 2006-02-16 John Lin Promotion of axonal regeneration
WO2006050475A2 (en) * 2004-11-03 2006-05-11 Brigham And Women's Hospital, Inc. Identification of dysregulated genes in patients with neurological diseases
WO2006134390A2 (en) * 2005-06-17 2006-12-21 Randox Laboratories Ltd Method for diagnosing neurodegenerative disease
DE102007024382A1 (en) * 2007-05-23 2008-11-27 Johann Wolfgang Goethe-Universität Frankfurt am Main Diagnosing a neurodegenerative disease comprises the gene expression level of a specified marker in a biological sample
JP2009112266A (en) * 2007-11-07 2009-05-28 Mitsubishi Tanabe Pharma Corp Diagnostic marker for neuropsychiatric disorder, diagnostic method, and therapeutic drug evaluation method

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060035826A1 (en) * 2004-08-13 2006-02-16 John Lin Promotion of axonal regeneration
WO2006050475A2 (en) * 2004-11-03 2006-05-11 Brigham And Women's Hospital, Inc. Identification of dysregulated genes in patients with neurological diseases
WO2006134390A2 (en) * 2005-06-17 2006-12-21 Randox Laboratories Ltd Method for diagnosing neurodegenerative disease
DE102007024382A1 (en) * 2007-05-23 2008-11-27 Johann Wolfgang Goethe-Universität Frankfurt am Main Diagnosing a neurodegenerative disease comprises the gene expression level of a specified marker in a biological sample
JP2009112266A (en) * 2007-11-07 2009-05-28 Mitsubishi Tanabe Pharma Corp Diagnostic marker for neuropsychiatric disorder, diagnostic method, and therapeutic drug evaluation method

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JEFFREY A LIEBERMAN: "Is schizophrenia a neurodegenerative disorder? a clinical and neurobiological perspective", BIOLOGICAL PSYCHIATRY, vol. 46, no. 6, 1 September 1999 (1999-09-01), pages 729 - 739, XP055035931, ISSN: 0006-3223, DOI: 10.1016/S0006-3223(99)00147-X *
LIN L ET AL: "Axon guidance and synaptic maintenance: preclinical markers for neurodegenerative disease and therapeutics", TRENDS IN NEUROSCIENCE, ELSEVIER, AMSTERDAM, NL, vol. 32, no. 3, 1 March 2009 (2009-03-01), pages 142 - 149, XP025969953, ISSN: 0166-2236, [retrieved on 20090121], DOI: 10.1016/J.TINS.2008.11.006 *
NAKASHIBA T ET AL: "Complementary expression and neurite outgrowth activity of netrin-G subfamily members", MECHANISMS OF DEVELOPMENT, ELSEVIER SCIENCE IRELAND LTD, IE, vol. 111, no. 1-2, 1 February 2002 (2002-02-01), pages 47 - 60, XP002591613, ISSN: 0925-4773, [retrieved on 20011108], DOI: 10.1016/S0925-4773(01)00600-1 *
NIIMI ET AL: "Monoclonal antibodies discriminating netrin-G1 and netrin-G2 neuronal pathways", JOURNAL OF NEUROIMMUNOLOGY, ELSEVIER SCIENCE PUBLISHERS BV, XX, vol. 192, no. 1-2, 22 October 2007 (2007-10-22), pages 99 - 104, XP022378488, ISSN: 0165-5728, DOI: 10.1016/J.JNEUROIM.2007.09.026 *
STEFAN LEHNERT ET AL: "iTRAQ and multiple reaction monitoring as proteomic tools for biomarker search in cerebrospinal fluid of patients with Parkinson's disease dementia", EXPERIMENTAL NEUROLOGY, vol. 234, no. 2, 1 April 2012 (2012-04-01), pages 499 - 505, XP055035852, ISSN: 0014-4886, DOI: 10.1016/j.expneurol.2012.01.024 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016032977A1 (en) * 2014-08-24 2016-03-03 Haskins William E Cns-specific biomarkers for cns diseases or injuries
CN108135973A (en) * 2015-05-19 2018-06-08 拉·约拉过敏反应及免疫医学研究所 Human APOB100 epitopes, methods and uses for modulating inflammatory response and treating adverse cardiovascular events, diseases and atherosclerosis
EP3297652A4 (en) * 2015-05-19 2019-06-05 La Jolla Institute for Allergy and Immunology HUMAN APOB100 EPITOPES, METHODS AND USES FOR MODULATING INFLAMMATORY RESPONSES AND TREATING ADVERSE CARDIOVASCULAR EVENTS, CARDIOVASCULAR DISEASE AND ATHEROSCLEROSIS
US11130798B2 (en) 2015-05-19 2021-09-28 La Jolla Institute For Allergy And Immunology Human APOB100 epitopes, methods and uses for modulating inflammatory responses, and treating adverse cardiovascular events, disease and atherosclerosis
CN108135973B (en) * 2015-05-19 2021-10-19 拉·约拉过敏反应及免疫医学研究所 Human APOB100 epitopes for modulating inflammatory responses and treating adverse cardiovascular events, disease and atherosclerosis, methods and uses
US12448433B2 (en) 2015-05-19 2025-10-21 La Jolla Institute For Allergy And Immunology Human ApoB100 epitopes, methods and uses for modulating inflammatory responses, and treating adverse cardiovascular events, disease and atherosclerosis
JP2018091735A (en) * 2016-12-05 2018-06-14 磁量生技股▲ふん▼有限公司 How to identify Parkinson's disease dementia from Parkinson's disease
CN112924698A (en) * 2021-03-05 2021-06-08 厦门大学 Biomarker for diagnosing neurodegenerative disease and application thereof
WO2025120644A1 (en) * 2023-12-08 2025-06-12 Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. Methods and a kit for detecting parkinson's disease with and without dementia
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