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WO2007119237A1 - Méthodes et trousses pour diagnostiquer des maladies associées à un stress oxydatif - Google Patents

Méthodes et trousses pour diagnostiquer des maladies associées à un stress oxydatif Download PDF

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WO2007119237A1
WO2007119237A1 PCT/IL2007/000478 IL2007000478W WO2007119237A1 WO 2007119237 A1 WO2007119237 A1 WO 2007119237A1 IL 2007000478 W IL2007000478 W IL 2007000478W WO 2007119237 A1 WO2007119237 A1 WO 2007119237A1
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Prior art keywords
disease
oxidative stress
expression
stress associated
activity
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Inventor
Nirit Lev
Devora Ickowicz
Yael Barhum
Netta R. Blondheim
Eldad Melamed
Daniel Offen
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Ramot at Tel Aviv University Ltd
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Ramot at Tel Aviv University Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • 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
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to methods and kits for diagnosing oxidative stress associated diseases.
  • Free radicals are extremely reactive chemical species that cause significant destruction in biological systems. Indiscriminate reaction of free radicals with biological molecules can lead to the destruction of cells and cellular components (e.g. mitochondria), thereby affecting physiological processes by causing cells to lose their structure and/or function.
  • cellular components e.g. mitochondria
  • ROS reactive oxygen species'
  • Oxidative stress has been implicated as a causative factor in a number of degenerative diseases.
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS and its animal model, experimental autoimmune encephalomyelitis (EAE) are believed to result from autoimmune mediated activated immune cells such as T- and B-lymphocytes as well as macrophages and microglia.
  • EAE experimental autoimmune encephalomyelitis
  • MS is characterized by perivenous infiltration of lymphocytes and macrophages into the CNS parenchyma, resulting in demyelinative lesions termed plaques.
  • plaques which are the hallmark of MS, are associated with oligodendrocytes death, axonal damage and neuronal loss.
  • the view that MS can be considered an inflammatory neurodegenerative disease is supported by studies demonstrating neuronal and axonal injury in regions remote from acute plaques, as well as imaging studies that demonstrated changes in normal appearing white and grey matter.
  • MS The etiology of MS has not yet been fully elucidated, and it is attributed to both genetic and environmental causes. Accumulating data indicate that oxidative stress plays a major role in the pathogenesis of MS.
  • ROS reactive oxygen species
  • the neurotransmitter glutamate is one of the sources of oxidative stress in the
  • MS primarily through activation of its ionotropic receptors. Oligodendrocytes, the myelin-producing cell of the CNS, are also highly vulnerable to glutamate excitotoxicity, mainly via the AMPA/kainate receptors. ROS causes damage to cardinal cellular components such as lipids, proteins and nucleic acids, resulting in cell death. Weakened cellular antioxidant defense systems in the CNS of MS patients resulting in increased vulnerability to ROS effects may increase CNS damage.
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig's disease is a progressive, fatal neurological disease affecting as many as 30,000 Americans with 5,000 new cases occurring in the United States each year.
  • the disorder belongs to a class of disorders known as motor neuron diseases.
  • ALS occurs when specific nerve cells in the brain and spinal cord that control voluntary movement gradually degenerate.
  • Familial amyotrophic lateral sclerosis (FALS) is a form of ALS distinguished from the more common sporadic variant only by its familial background.
  • SODl superoxide dismutase 1
  • oxidative stress occurs when there is an excess of ROS, a decrease in antioxidant levels, or both. Accordingly, agents that interfere with the production of ROS or eliminate ROS may be used to treat neurodegenerative disorders associated with oxidative stress, such as Multiple Sclerosis and ALS disease.
  • DJ-I is a small 189 amino acid protein that is ubiquitously expressed and highly conserved throughout diverse species, which was discovered as a novel oncogene in 1997. Accumulating data revealed its involvement in various cellular processes, especially in oxidative stress. DJ-I is known to have several isoforms with isoelectric points between 5.5 and 7, with dominance of alkaline isoforms in normal conditions.
  • DJ-I is widely distributed and is highly expressed in the brain, and is not confined to a single functional system or anatomical location. DJ-I is expressed in neurons of different neurotransmitter phenotypes and in all glial cell types, such as astrocytes, microglia and oligodendrocytes.
  • DJ-I mutations were discovered to cause familial Parkinson's disease (PD) [Bonifati et al., 2003, Science 299: 256-9, 2003].
  • PD familial Parkinson's disease
  • DJ-I immunoreactivity was detected in other neurodegenerative diseases including multi-system atrophy, Alzheimer's disease, progressive supranuclear palsy, fronto-temporal dementia with parkinsonism linked to chromosome 17, and Pick's disease [Bandopadhyay R, et al., Brain 127: 420-430, 2004; Neumann N. et al., Acta Neuropathol (Berl) 107: 489-496, 2004; Rizzu P. et al., Ann Neurol 55: 113-118, 2004].
  • DJ-I has not been suggested as a potential diagnostic marker for oxidative-stress related disorders.
  • a method of diagnosing an oxidative stress associated disease comprising determining in a biological sample of a subject activity and/or expression of DJ-I, wherein an alteration in activity and/or expression of the DJ-I compared to a control sample is indicative of the oxidative stress associated disease.
  • a method of monitoring effectiveness of a therapeutic agent towards an oxidative stress associated disease comprising determining in a biological sample of a subject activity and/or expression of DJ-I prior to, concomitant with and/or following administering of the therapeutic agent to the subject, wherein an alteration in the activity and/or expression of the DJ-I following the therapeutic agent administration is indicative of the effectiveness of the therapeutic agent.
  • the oxidative stress associated disease is a neurodegenerative disease.
  • the neurodegenerative disease is Multiple Sclerosis (MS) or Amyotrophic Lateral Sclerosis (ALS).
  • MS Multiple Sclerosis
  • ALS Amyotrophic Lateral Sclerosis
  • the neurodegenerative disease is selected from the group consisting of Multiple Sclerosis,
  • ALS Alzheimer's disease
  • progressive supranuclear palsy progressive supranuclear palsy
  • the expression of the DJ-I is selected from the group consisting of:
  • the determining is effected at the JANA level.
  • the determining is effected at the protein level.
  • the diagnosing comprises determining a severity of the oxidative stress associated disease.
  • control sample is obtained from a subject diagnosed as not suffering from the oxidative stress associated disease.
  • the alteration is an up- regulation.
  • the alteration comprises an up-regulation.
  • the alteration comprises a down-regulation.
  • the alteration comprises an increase in expression and/or activity of DJ-I
  • a low efficacy of the therapeutic agent is indicated.
  • the alteration comprises a decrease in expression and/or activity of DJ-I
  • a high efficacy of the therapeutic agent is indicated.
  • the biological sample is a body fluid.
  • the body fluid is selected from the group consisting of serum, plasma, whole blood, cerebrospinal fluid, amniotic fluid, and synovial fluid.
  • the present invention successfully addresses the shortcomings of the presently known configurations by providing a novel method of diagnosing oxidative stress related disorders based on the expression and/or activity of DJ-I.
  • FIG. 1 is a bar graph illustrating that the myelin oligodendrocyte glycoprotein (MOG) mRNA levels in brains of EAE mice decline as disease severity increase.
  • MOG myelin oligodendrocyte glycoprotein
  • FIG. 2 is a bar graph illustrating the mRNA levels of DJ-I in brains of EAE mice.
  • Female C3H.SW mice (6-8 weeks old) were immunized twice by subcutaneous injection with MOG. Induced EAE mice were sacrificed at different clinical disease severities. Total RNA was isolated from mice brain tissues and Real-time quantitative PCR of DJl was performed as described in Materials and Methods. An increase in disease severity up regulated DJl mRNA levels while in terminal stages, mRNA levels were down regulated.
  • FIGs. 3A-B are Western blots and bar graphs illustrating DJ-I protein expression levels in brains of EAE mice. Induced EAE mice were sacrificed at different clinical disease severities. Proteins were extracted from brain tissues and DJ- 1 expression was determined by Western blot analysis as described in Materials and Methods.
  • Figure 3A The membranes of Western blot were probed with rabbit anti- DJ-I (1:5000) and mouse anti actin (1:10000) antibodies.
  • Figure 3B is a graphical representation of the results. Increased disease severity upregulated DJ-I expression levels, while in terminal stages DJ-I levels were down regulated.
  • FIGs. 4A-B depict the multiple isoforms of DJ-I in brains of EAE mice.
  • Induced EAE mice were sacrificed at different clinical disease severities. Proteins were extracted from brain tissues and DJ-I pi was determined by isoelectric focusing (IEF) as described in Materials and Methods. IEF demonstrated a shift of DJ-I from alkaline isoforms in control towards acidic isoforms in severe disease.
  • Figure 4A is a blot of IEF gel.
  • Figure 4B is a bar graph quantifying the DJ-I isoforms.
  • FIG. 5 is a bar graph illustrating the effect of 3-(4-morpholinyl)-Sydnonimine (SIN-I) on glioma U-87 cell viability.
  • SIN-I 3-(4-morpholinyl)-Sydnonimine
  • FIG. 6 is a bar graph illustrating that SIN-I upregulates DJ-I mRNA levels in U-87 glioma cells.
  • U-87 glioma cells were treated for Ih with SIN-I (0.5mM).
  • FIGs. 7A-C illustrate that SIN-I can induce upregulation of DJ-I expression in U-87 glioma cells. Proteins were extracted from cells treated with 0.5 niM SIN-I for 24h. DJ-I expression levels were determined by Western blot analysis as described in Materials and Methods.
  • Figure 7A is a representative membrane following a Western blot probed with rabbit anti-DJ-1 (1:5000) and mouse anti actin (1:10000) antibodies.
  • Figure 7B is a bar graph illustrating the quantity of DJ-I levels per actinlevels. Actin was used as an internal protein control. The data presented here are froma representative experiment repeated three times with similar results.
  • Figure 7C is a photomicrograph illustrating that DJ-I is present both in the cytoplasm and the nuclei of U-87 glioma cells, as shown by immunocytochemistry. In control cells, in which anti-DJ-1 antibody was withdrawn from the samples, there was no staining.
  • Original magnification X 20 In control cells, in which anti-DJ-1 antibody was withdrawn from the samples, there was no staining.
  • FIG. 8 is a graph illustrating the motor performance of mutant SODl transgenic mice as compared to age matched controls. Accelerating rotarod test was used as a quantitative tool in order to measure motor function of mutant SODl mice. Time (measured in seconds, up to 120 sec) until fall from an accelerating rotarod was measured for mutant SODl mice and age-matched wild type littermates. Each mouse was measured 3 times at each time point.
  • FIG. 9 is a bar graph illustrating the mRNA levels of DJl in brains of mutant SODl transgenic mice.
  • Female mutant SODl transgenic mice (5-18 weeks old) were sacrificed at different clinical disease severities, and compared to wild type age matched littermates.
  • Total RNA was isolated from mice brain tissues and Real-time quantitative PCR of DJl was performed as described in Example 6. Quantitative calculations of DJl versus GAPDH were done using the ddCT method. Upregulation of DJl mRNA levels were detected in mutant SODl mice as compared to controls.
  • FIGs. 10A-B depict DJl protein expression levels in brains of mutant SODl transgenic mice.
  • the present invention is of methods and kits for diagnosing oxidative stress related diseases.
  • the present invention can be used to monitor the severity of oxidative stress related diseases and the effectiveness of drug treatments.
  • DJ-I has been shown to be associated with a variety of brain oxidative stress related disorders including Parkinson's, Alzheimer's disease, progressive supranuclear palsy and Pick's disease. However, no correlation has been done to date between DJ-I expression and presence or state of oxidative stress associated diseases in general and neurodegenerative diseases in particular.
  • DJ-I is up-regulated in Multiple Sclerosis and that it may be used as a marker to determine the severity of the disease. This is reflected by the levels of both DJ-I niRNA ( Figure 2) and DJ-I protein ( Figures 3 A-B). Furthermore, the present inventors have shown that the acidity of DJ-I may also serve as a marker for MS, wherein an alteration in the ratio of the acidic isoform: alkali isoform is indicative of the disease ( Figures 4A-B). In addition, the present inventors have shown that DJ-I is up-regulated in ALS, both on the level of RNA ( Figure 9) and protein ( Figures 10A- B).
  • a method of diagnosing an oxidative stress associated disease comprising determining in a biological sample of a subject activity and/or expression of DJ-I, wherein an alteration in activity and/or expression of the DJ-I compared to a control sample is indicative of the oxidative stress associated disease.
  • diagnosis refers to classifying a disease or a symptom as an oxidative stress related disease, determining a predisposition to an oxidative stress related disease, determining a severity of an oxidative stress related disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
  • oxidative stress refers to an undesirable imbalance where oxidants outnumber antioxidants. This situation can arise if the rate of ROS production overwhelms existing antioxidant defenses. In such circumstances, a series of cellular responses can occur that can lead to an even greater increase in ROS production. Excessive ROS production and its otherwise ineffective regulation can be detrimental to cells and tissue, inducing cellular damage that ultimately can lead to cell death (apoptosis). Oxidative stress-associated damage also can cause undesirable changes to the structural and functional integrities of cells that can lead to the propagation of cells instead of apoptosis. Additionally, oxidatively-damaged cellular macromolecules can trigger immune responses that can lead to disease. See generally, D. G. Lindsay et al. (2002) MoI. Aspects of Med. 23:1-38, incorporated herein by reference. While oxidative stress may not be responsible for initiating or otherwise causing disease, the progression of the disease can be affected by any resultant oxidative stress.
  • oxidative stress related disease refers to a disease or medical condition (including syndromes) wherein the onset or progression thereof is promoted by oxidative stress. Since oxidative stress is believed to be responsible for the pathogenesis of many neurological, heart, malignant and age- associated diseases, the present invention contemplates all such diseases including for example, atherosclerosis, autoimmune diseases, cancer, cardiovascular disease, cataract, dementia, diabetes and diabetic vasculopathy, and neurodegenerative diseases.
  • the method of the present invention may be used to diagnose any one or more of these disease states in a subject. It has previously been shown that subjects can be under severe oxidative stress for long periods of time before these illnesses become evident, and therefore the method of the present invention may be used in the early diagnosis of any one or more of these disease states.
  • alteration refers to an up-regulation or a down- regulation.
  • oxidative stress animal models can be used to indicate whether there is an up regulation or a down regulation of DJ-I.
  • exemplary oxidative stress animal models include, but are not limited to Experimental Allergic Encephalomyelitis, Diabetic Retinopathy, SODl transgenic mice and phenylketonuria animal model.
  • the oxidative stress related disease is a neurodegenerative disease.
  • exemplary neurodegenerative diseases which may be diagnosed using the method of the present invention include, but are not limited to Multiple Sclerosis, ALS, Parkinson's, dementia, multi-system atrophy, Alzheimer's disease, progressive supranuclear palsy, fronto-temporal dementia with parkinsonism linked to chromosome 17 and Pick's disease.
  • the method of the present invention comprises the step of detecting DJ-I in a biological sample retrieved from a subject and comparing the measured level of DJ-I with the level from a non-diseased subject.
  • the level of DJ-I will typically be higher compared to a non-diseased subject.
  • the level of DJ-I will generally be higher compared to a subject suffering from a non-severe form.
  • DJ-I refers to the polypeptide as set forth in GenBank Accession No: AB073864, and derivatives, orthologues and homologues thereof.
  • the biological sample may be any biological fluid in the subject in which DJ-I may be found including, but not limited to, serum, plasma, whole blood, cerebrospinal fluid, amniotic fluid, and synovial fluid - see for e.g. Waragai et al., Biochem Biophys Res Commun. 2006 JuI 7;345(3):967-72.
  • the biological sample may be any tissue that is retrieved from the subject in which DJ-I may be found including, but not limited to, brain tissue.
  • control sample may also be of the same subject from a healthy tissue, prior to disease progression or following disease remission.
  • Detection of DJ-I may be effected by analyzing expression thereof, either on the RNA level or the protein level.
  • RNA in the cells of the present invention can be determined using methods known in the arts.
  • RNA sample is denatured by treatment with an agent (e.g., formaldehyde) that prevents hydrogen bonding between base pairs, ensuring that all the RNA molecules have an unfolded, linear conformation.
  • agent e.g., formaldehyde
  • the individual RNA molecules are then separated according to size by gel electrophoresis and transferred to a nitrocellulose or a nylon-based membrane to which the denatured RNAs adhere.
  • the membrane is then exposed to labeled DNA probes.
  • Probes may be labeled using radio-isotopes or enzyme linked nucleotides. Detection may be using autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of particular RNA molecules and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the gel during electrophoresis.
  • RNA molecules are purified from the cells and converted into complementary DNA (cDNA) using a reverse transcriptase enzyme (such as an MMLV-RT) and primers such as, oligo dT, random hexamers or gene specific primers.
  • a reverse transcriptase enzyme such as an MMLV-RT
  • primers such as, oligo dT, random hexamers or gene specific primers.
  • Exemplary primers that may be used to identify DJ-I are described in Example 2 and Example 6 herein below.
  • a PCR amplification reaction is carried out in a PCR machine.
  • RNA molecules Those of skills in the art are capable of selecting the length and sequence of the gene specific primers and the PCR conditions (i.e., annealing temperatures, number of cycles and the like) which are suitable for detecting specific RNA molecules. It will be appreciated that a semi-quantitative RT-PCR reaction can be employed by adjusting the number of PCR cycles and comparing the amplification product to known controls.
  • RNA in situ hybridization stain DNA or RNA probes are attached to the DJ-I RNA molecules present in the cells.
  • the cells are first fixed to microscopic slides to preserve the cellular structure and to prevent the RNA molecules from being degraded and then are subjected to hybridization buffer containing the labeled probe.
  • the hybridization buffer includes reagents such as formamide and salts (e.g., sodium chloride and sodium citrate) which enable specific hybridization of the DNA or RNA probes with their target mRNA molecules in situ while avoiding non-specific binding of probe.
  • formamide and salts e.g., sodium chloride and sodium citrate
  • oligonucleotide microarray oligonucleotide probes capable of specifically hybridizing with the polynucleotides of the present invention are attached to a solid surface (e.g., a glass wafer). Each oligonucleotide probe is of approximately 20-25 nucleic acids in length.
  • a specific cell sample e.g., blood cells
  • RNA is extracted from the cell sample using methods known in the art (using e.g., a TRIZOL solution, Gibco BRL, USA). Hybridization can take place using either labeled oligonucleotide probes (e.g., 5'-biotinylated probes) or labeled fragments of complementary DNA (cDNA) or RNA (cRNA). Briefly, double stranded cDNA is prepared from the RNA using reverse transcriptase (RT) (e.g., Superscript II RT), DNA ligase and DNA polymerase I, all according to manufacturer's instructions (Invitrogen Life Technologies, Frederick, MD, USA).
  • RT reverse transcriptase
  • DNA ligase DNA polymerase I
  • the double stranded cDNA is subjected to an in vitro transcription reaction in the presence of biotinylated nucleotides using e.g., the BioArray High Yield RNA Transcript Labeling Kit (Enzo, Diagnostics, Affymetix Santa Clara CA).
  • the labeled cRNA can be fragmented by incubating the RNA in 40 mM Tris Acetate (pH 8.1), 100 mM potassium acetate and 30 mM magnesium acetate for 35 minutes at 94 °C.
  • the microarray is washed and the hybridization signal is scanned using a confocal laser fluorescence scanner which measures fluorescence intensity emitted by the labeled cRNA bound to the probe arrays.
  • each gene on the array is represented by a series of different oligonucleotide probes, of which, each probe pair consists of a perfect match oligonucleotide and a mismatch oligonucleotide. While the perfect match probe has a sequence exactly complimentary to the particular gene, thus enabling the measurement of the level of expression of the particular gene, the mismatch probe differs from the perfect match probe by a single base substitution at the center base position.
  • the hybridization signal is scanned using the Agilent scanner, and the Microarray Suite software subtracts the non-specific signal resulting from the mismatch probe from the signal resulting from the perfect match probe.
  • DJ-I proteins expressed in the ceils of the cultures of the present invention can be determined using methods known in the arts. Typically detection of DJ-I is effected using an antibody.
  • the antibodies may be monoclonal or polyclonal.
  • DJ-I specific antibodies may be generated according to methods known in the art. DJ-I specific antibodies are also widely commercially available, for example from AMS Biotechnology, Cat# KAM-SAl 00E;Chemicon,
  • Enzyme linked immunosorbent assay This method involves fixation of a sample (e.g., fixed cells or a proteinaceous solution) containing a protein substrate to a surface such as a well of a microtiter plate. A DJ-I specific antibody coupled (either directly or indirectly) to an enzyme is applied and allowed to bind to the substrate.
  • a sample e.g., fixed cells or a proteinaceous solution
  • a DJ-I specific antibody coupled (either directly or indirectly) to an enzyme is applied and allowed to bind to the substrate.
  • Presence of the antibody is then detected and quantitated by a colorinietric reaction employing the enzyme coupled to the antibody.
  • Enzymes commonly employed in this method include horseradish peroxidase and alkaline phosphatase. If well calibrated and within the linear range of response, the amount of substrate present in the sample is proportional to the amount of color produced. A substrate standard is generally employed to improve quantitative accuracy.
  • Western blot This method involves separation of DJ-I from other proteins by means of an acrylamide gel followed by transfer of the substrate to a membrane (e.g., nylon or PVDF). Presence of the DJ-I is then detected by antibodies specific to the substrate, which are in turn detected by antibody binding reagents.
  • Antibody binding reagents may be, for example, protein A, or other antibodies. Antibody binding reagents may be radiolabeled or enzyme linked as described hereinabove. Detection may be by autoradiography, colorimetric reaction or chemiluminescence. This method allows both quantitation of an amount of substrate and determination of its identity by a relative position on the membrane which is indicative of a migration distance in the acrylamide gel during electrophoresis.
  • Radio-immunoassay In one version, this method involves precipitation of the desired protein (i.e., DJ-I) with a specific antibody and radiolabeled antibody binding protein (e.g., protein A labeled with I 125 ) immobilized on a precipitable carrier such as agarose beads. The number of counts in the precipitated pellet is proportional to the amount of substrate.
  • a labeled substrate and an unlabelled antibody binding protein are employed. A sample containing an unknown amount of substrate is added in varying amounts. The decrease in precipitated counts from the labeled substrate is proportional to the amount of substrate in the added sample.
  • Fluorescence activated cell sorting This method involves detection of DJ-I in situ in cells by DJ-I specific antibodies.
  • the DJ-I specific antibodies are linked to fluorophores. Detection is by means of a cell sorting machine which reads the wavelength of light emitted from each cell as it passes through a light beam. This method may employ two or more antibodies simultaneously.
  • DJ-I specific antibodies may be enzyme linked or linked to fluorophores. Detection is by microscopy and subjective or automatic evaluation. If enzyme linked antibodies are employed, a colorimetric reaction may be required. It will be appreciated that immunohistochemistry is often followed by counterstaining of the cell nuclei using for example Hematoxyline or Giemsa stain.
  • diagnosis of oxidative stress related diseases may be determined on the basis of the acidity of DJ-I.
  • an abundance of the acidic isoform of DJ-I is indicative of the disease.
  • a level of acidic DJ-I may be measured, a level of alkali DJ-I may be measured or a level of both acidic and alkali DJ-I may be measured and a ratio of acidic: alkali may be deduced.
  • Such measurements may be effected using isoelectric focusing as described in Example 3 herein below. It will be appreciated that DJ-I is known to comprise neuroprotecting activity
  • DJ-I may also be assayed on the basis of its neuroprotecting activity.
  • the components for assaying the level or activity of DJ-I may be provided in a kit e.g. an FDA approved kit and be accompanied by instructions for use. Since the present inventors have shown that DJ-I levels reflect the severity of an oxidative stress related disease, the present inventors also contemplate monitoring the effectiveness of a therapeutic agent towards an oxidative stress associated disease by analyzing the level and/or activity of DJ-I in a biological sample of the subject. For example, an effective therapeutic agent for treating MS or ALS is one which decreases the level of DJ-I in the biological sample.
  • the therapeutic agent may be administered prior to, concomitant with and or/following detection of DJ-I .
  • the optimal dose and optimal treatment regimen may also be identified according to the method of the present invention. In this way a therapeutically effective amount of an agent may be determined.
  • the subject may be treated according to the therapeutic agent selected with the aid of the method of the present invention and optionally retested after a suitable time period. In this way a patient's response may be continually monitored whilst undergoing therapy.
  • the analyzing DJ-I levels and administering steps may be repeated a number of times during the course of a treatment. For instance the DJ-I levels may be analyzed one day following administration of the agent. If the DJ-I levels are lower than those compared with a control, the dose of the agent may be increased. If the DJ-I levels are higher than those compared with a control, the dose of the agent may be increased.
  • the method of the present invention may also be useful for determining whether a pre-diagnosed subject is in need of treatment.
  • the levels of DJ-I in an MS patient suffering a relapse may indicate the necessity to begin a new treatment course or alternatively to change a treatment course.
  • Reagents used were as follows: Tri-reagent (Sigma, St Louis, MO, USA); rabbit anti-DJ-1 (Chemicon, Temecula, CA); mouse anti-actin (Sigma, St Louis, MO, USA); Alexa 568-conjugated goat anti-rabbit (Molecular probes, Invitrogen, Eugene, OR, USA); horseradish peroxidase conjugated goat anti mouse and goat anti rabbit (Sigma, St Louis, MO, USA); 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, MO, USA); 3-(4-morpholinyl)-sydnonimine (SIN- 1) (Sigma, St Louis, MO, USA); Super Signal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA); BCA protein assay kit (Pierce Biotechnology, Rockford, IL 5 USA); myelin oligodend
  • Chronic EAE was induced according to previously described procedures [Gilgun-Sherki Y, Melamed E, and Offen D. J Neurol 251: 261-268, 2004]. Briefly, female C3H.SW mice (6-8 weeks old) were immunized twice, at day 1 and day 8, by subcutaneous injection with an emulsion containing myelin oligodendrocyte glycoprotein (pMOG35-55), in complete Freund's adjuvant (CFA) containing 200 ⁇ g heat-activated Mycobacterium tuberculosis in a total volume of 0.2ml.
  • CFA complete Freund's adjuvant
  • mice were followed for clinical disease severity daily, using a score scale: 0, no disease signs; 1, loss of tail tonicity; 2, mild hind limb weakness; 3, complete hind limb paralysis; 4, paralysis of four limbs; 5, moribund; 6, death (32). Mild disease severity was defined as clinical scores ⁇ 2, moderate disease severity was defined as clinical score of 3 and severe disease was defined as scores >4. 30 control and MOG induced EAE mice were sacrificed at different clinical disease severities. Tissues were frozen in liquid nitrogen and stored at -80 0 C until protein and RNA extractions.
  • RNA isolation Total RNA was isolated from mice brain tissues by the single- step method (Chomczynski and Sacchi, 1987), using a commercial reagent TriReagentTM (Sigma) and the manufacturer's recommended procedure. The amount of RNA was determined spectrophotometrically using the ND- 1000 spectrophotometer (Nano-drop). RNA quality was verified by measuring OD260/OD280 ratio. RNA was stored at -80 0 C until used.
  • cDNA synthesis was carried out in a final reaction volume of 20 ⁇ l containing 1 ⁇ g of the total RNA, random primer (1.3 ⁇ M, Invitrogen UK) in diethyl pyrocarbonate (DEPC) water at a total volume of 10 ⁇ l. After incubation at 70 °C for 10 min and cooling at 4 0 C for 10 min, the following reagents were added to a final concentration of: lxBuffer supplied by the manufacturer, 10 mM DTT, 20 ⁇ M dNTPs, 20 U of RNase inhibitor (RNAguard, Amersham Pharmacia biotech) and 10 U of the enzyme RT-superscript II (Invitrogen) reverse transcriptase. RT reaction was performed at 25 0 C for 10 min, 42 0 C for 2 hours followed by 70 0 C for 15 min and 95 0 C for 15 min. Samples were stored at -20 °C until used.
  • Real-time quantitative reverse transcription polymerase chain reaction Real-time quantitative PCR of the desired genes was performed in an ABI Prism 7700 sequence detection system (Applied biosystems) using Sybr green PCR master mix (Applied biosystems) and the following primers: GAPDH sense CGA CAG TCA GCC GCA TCT T (SEQ ID NO: 1), GAPDH antisense CCA ATA CGA CCA AAT CCG TTG (SEQ ID NO: 2); MOG sense CCT GGT TGC CTT GAT CAT CTG CTA C (SEQ ID NO: 3), MOG antisense TCT ACT CGG TAT CCA GAA TGT GTC TG (SEQ ID NO: 4); GAPDH gene, which served as an internal control, is a valid reference 'housekeeping' gene for transcription profiling, which is also used for qPCR experiments in previous studies. EAE induction and clinical severity did not affect the constitutive expression of the reference gene, GAPDH.
  • qPCR real time quantitative PCR
  • the PCR was performed in a total volume of 20 ⁇ l containing l ⁇ l of the above-described cDNA, l ⁇ l each of the 3' and 5' primers (final concentration of 500 nmol/L each), lO ⁇ l of AbsoluteTM QPCR SYBR® Green ROX Mix and 8 ⁇ l of DEPC water.
  • the amplification protocol was 40 cycles of 95 0 C for 15 sec followed by 60 0 C for 1 min each. Quantitative calculations of MOG versus GAPDH was done using the ddCT method, as instructed in the user bulletin #2 ABI prism 7700 sequence detection system (updated 10/2001).
  • MOG mRNA levels were found in EAE brains. Moreover, there was an inverse relation between MOG mRNA levels and the clinical disease severity, i.e. as the clinical score of EAE increased the levels of MOG mRNA declined ( Figure
  • RNA Extraction; cDNA synthesis and real-time PCR Performed as described for Example 1.
  • Primers for DJ-I sense CAT GAG GCG AGC TGG GAT TA (SEQ ID NO: 5), DJ-I antisense GCT GGC ATC AGG ACA AAT GAC (SEQ ID NO: 6). RESULTS
  • DJ-I protein levels were assessed using Western blot.
  • Proteins were extracted from brain tissue by grinding in lysis buffer containing 250 mM sucrose, 10 rnM KCl, 1.5 mM MgCh, 2 mM EDTA 5 20 mM Hepes and protease inhibitors cocktail (Roche). Protein concentration was determined by the BCA method (Pierce). Twenty-five micrograms of total protein from brain samples or glioma cells lysate were separated by 12 % SDS-PAGE gels and transferred to a nitrocellulose membrane.
  • the membranes were probed with rabbit anti-DJ-1 (1:5000; Chemicon Laboratories), and mouse anti actin antibodies (1:10000; Sigma), followed by horseradish peroxidase conjugated secondary antibody (1:10000; Sigma) and developed with the Super Signal West Pico Chemiluminescent substrate (Pierce).
  • Isoelectric focusing (IEF) Proteins from EAE brain extracts were separated in pH 5-8 ranges of isoelectric focusing phoresis gel (Bio-Rad), transferred onto nitrocellulose membranes, and blotted with rabbit anti-DJ-1 antibodies (Chemicon).
  • SIN-I 3-(4-morpholinyl)-Sydnonimine
  • Example 3 In order to prepare whole-cell lysate from the glioma U-87 cells, cells were trypsynized, centrifuged and resuspended in lysis buffer. Cell debris was removed by centrifugation at 20,000 xg for 15 min at 4 °C.
  • RNA Extraction; cDNA synthesis and real-time PCR Performed as described for Example L EAE induction and clinical severity did not affect the constitutive expression of the reference gene, GAPDH, nor did SIN-I treatment of glioma U-87 cells.
  • Cell viability was determined by the MTT (3-(4,5- dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) reduction assay. Cells were plated in 96 well plates and viability after SIN-I treatment was analyzed by adding MTT solution to each well (reaching a final concentration of 0.5 mg/ml) followed by incubation at 37 °C for 3 hours. The medium was then removed and the formazan crystals were dissolved in DMSO. Absorbance was determined at 564 nm in a microplate reader. Cell viability was evaluated in triplicates for each treatment. All experiments were repeated at least 3 times. RESULTS
  • Immunocytochemistry Cells were fixed with 4 % paraformaldehyde and permeabilized with 0.5 % Triton X-100. The cells were then incubated in a blocking solution followed by 1-hour incubation at room temperature with rabbit anti-DJ-1 antibodies (1:1000; Chemicon). After washing with PBS, the cells were incubated with fluorescent Alexa 568-conjugated goat anti-rabbit antibodies (1 :4000; Molecular probes) for 1 hour at room temperature. RESULTS
  • Reagents used were as follows: Tri-reagent (Sigma, St Louis, MO, USA); rabbit anti-DJ-1 (Chemicon, Temecula, CA); mouse anti-actin (Sigma, St Louis, MO, USA); horseradish peroxidase conjugated goat anti mouse and goat anti rabbit (Sigma, St Louis, MO, USA); Super Signal West Pico Chemiluminescent substrate (Pierce Biotechnology, Rockford, IL, USA); BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA); random primer (Invitrogen, Carlsbad, CA); Sybr green PCR master mix (Applied Biosystems, Warrington, UK); RNase inhibitor (RNAguard, Amersham Pharmacia biotech); Super Script II RNase H-reverse transcriptase (Invitrogen, Carlsbad, CA); Ready gel for polyacrylamide electrophoresis IEF pH 5-8 (Bio-Rad laboratories, Hercules, CA, USA); IEF 1Ox anode buffer (Bio-
  • mice were housed in standard conditions: constant temperature (22 ⁇ 1 0 C), humidity (relative, 40 %) and a 12-h light/dark cycle, and were allowed free access to food and water. The animals and protocol procedures were approved and supervised by the Animal Care Committee at the Rabin Medical Center. 30 wild type and mSOD transgenic mice were sacrificed at different clinical disease severities. Tissues were frozen in liquid nitrogen and stored at -80 0 C until protein and RNA extractions.
  • Protein extraction and Western blotting Proteins were extracted from brain tissue by grinding in lysis buffer containing 25OmM sucrose, 1OmM KCl, 1.5mM MgCl 2 , 2mM EDTA, 2OmM Hepes and protease inhibitors cocktail (Roche). Cell debris was removed by centrifugation at 20,000xg for 15 min at 4 °C. Protein concentration was determined by the BCA method (Pierce). Twenty-five micrograms of total protein from brain samples or glioma cells lysate were separated by 12 % SDS-PAGE gels and transferred to a nitrocellulose membrane.
  • the membranes were probed with rabbit anti-DJ-1 (1:5000; Chemicon Laboratories), and mouse anti actin antibodies (1:10000; Sigma), followed by horseradish peroxidase conjugated secondary antibody (1:10000; Sigma) and developed with the Super Signal West Pico Chemiluminescent substrate (Pierce).
  • Isoelectric focusing Proteins from EAE brain extracts were separated in pH 5—8 ranges of isoelectric focusing phoresis gel (Bio-Rad), transferred onto nitrocellulose membranes, and blotted with rabbit anti-DJ-1 antibodies (Chemicon).
  • RNA isolation Total RNA was isolated from cultured glioma U-87 cells and mice brain tissues using a commercial reagent TriReagentTM (Sigma) and the manufacturer's recommended procedure. The amount of RNA was determined spectrophotometrically using the ND- 1000 spectrophotometer (Nano-drop). RNA quality was verified by measuring OD260/OD280 ratio. RNA was stored at -80°C until used.
  • cDNA synthesis First-strand cDNA synthesis was carried out in a final reaction volume of 20 ⁇ l containing 1 ⁇ g of the total RNA, random primer (1.3 ⁇ M, Invitrogen UK) in diethyl pyrocarbonate (DEPC) water at a total volume of 10 ⁇ l.
  • DEPC diethyl pyrocarbonate
  • RT reaction was performed at 25 0 C for 10 min, 42 °C for 2 hours followed by 70 0 C for 15 min and 95 0 C for 15 min. Samples were stored at -20 0 C until used.
  • PCR Real-time quantitative PCR of the desired genes was performed in an ABI Prism 7700 sequence detection system (Applied biosystems) using Sybr green PCR master mix (Applied biosystems) and the following primers: GAPDH sense CGA CAG TCA GCC GCA TCT T (SEQ ID NO: 1), GAPDH antisense CCA ATA CGA CCA AAT CCG TTG (SEQ ID NO: 2); MOG sense CCT GGT TGC CTT GAT CAT CTG CTA C (SEQ ID NO: 3), MOG antisense TCT ACT CGG TAT CCA GAA TGT GTC TG (SEQ ID NO: 4); DJ-I sense: CAT GAG GCG AGC TGG GAT TA (SEQ ID NO: 5), DJ-I antisense GCT GGC ATC AGG ACA AAT GAC (SEQ ID NO: 6).
  • GAPDH gene which served as an internal control, is a valid reference 'housekeeping' gene for transcription profiling, which was also used for real
  • DJ-I For quantification of DJ-I, MOG arid GAPDH mRNA, real time quantitative PCR was performed in duplicates.
  • Optimal experimental parameters for each primer pair. For each gene, verifying a single peak in melting curve analysis assessed the specificity of the PCR product.
  • the PCR was performed in a total volume of 20 ⁇ l containing l ⁇ l of the above-described cDNA, l ⁇ l each of the 3' and 5' primers (final concentration of 500 nmol/L each), lO ⁇ l of Sybr Green Mix and 8 ⁇ l of DEPC water.
  • the amplification protocol was 40 cycles of 95 0 C for 15 sec followed by 60 0 C for 1 min each.
  • Quantitative calculations of the gene of interest (DJ-I or MOG) versus GAPDH was done using the ⁇ CT method, as instructed in the user bulletin #2 ABI prism 7700 sequence detection system (updated 10/2001).
  • RESULTS In order to evaluate possible involvement of DJ-I in the motor neuron disease process, DJ-I mRNA and protein levels were evaluated in mutant SODl transgenic mice as compared to wild type littermates. Disease severity was evaluated using a functional motor test - rotarod examination. Mutant SODl mice were sacrificed at different disease stages (from asymptomatic to end stage disease) and wild type mice were sacrificed at the same ages as controls.

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Abstract

L'invention concerne des méthodes et des trousses pour diagnostiquer des maladies associées à un stress oxydatif. La méthode de diagnostic consiste à déterminer, dans un échantillon biologique d'un sujet, une activité et/ou une expression de DJ-1, une modification de l'activité et/ou de l'expression de DJ-1 par rapport à un échantillon témoin indiquant une maladie associée à un stress oxydatif. L'invention concerne également une méthode pour déterminer l'efficacité d'un agent thérapeutique envers une maladie associée à un stress oxydatif.
PCT/IL2007/000478 2006-04-13 2007-04-15 Méthodes et trousses pour diagnostiquer des maladies associées à un stress oxydatif Ceased WO2007119237A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011033511A1 (fr) 2009-09-17 2011-03-24 Ramot At Tel-Aviv University Ltd. Peptides pour le traitement de troubles liés au stress oxydatif
US8378070B2 (en) 2007-03-12 2013-02-19 Ramot At Tel-Aviv University Ltd. Peptides for the regulation of neurotransmitter sequestration and release

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003081201A2 (fr) * 2002-03-21 2003-10-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Marqueurs de cellules sanguines peripheriques utiles pour le diagnostic de la sclerose en plaques et procedes et trousses comprenant de tels marqueurs
WO2005045034A2 (fr) * 2003-10-23 2005-05-19 Sirna Therapeutics, Inc. Traitement medie par interference arn de la maladie de parkinson au moyen d'un petit acide nucleique interferent (sina)
WO2005075513A1 (fr) * 2004-02-04 2005-08-18 National Institute Of Advanced Industrial Science And Technology Dérivé de dj-1 comportant un résidu de cystéine acidifié

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003081201A2 (fr) * 2002-03-21 2003-10-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Marqueurs de cellules sanguines peripheriques utiles pour le diagnostic de la sclerose en plaques et procedes et trousses comprenant de tels marqueurs
WO2005045034A2 (fr) * 2003-10-23 2005-05-19 Sirna Therapeutics, Inc. Traitement medie par interference arn de la maladie de parkinson au moyen d'un petit acide nucleique interferent (sina)
WO2005075513A1 (fr) * 2004-02-04 2005-08-18 National Institute Of Advanced Industrial Science And Technology Dérivé de dj-1 comportant un résidu de cystéine acidifié

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHOI J ET AL: "Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases", JOURNAL OF BIOLOGICAL CHEMISTRY 21 APR 2006 UNITED STATES, vol. 281, no. 16, 3 March 2006 (2006-03-03), pages 10816 - 10824, XP002444178, ISSN: 0021-9258 1083-351X *
GILGUN-SHERKI YOSSI ET AL: "The role of oxidative stress in the pathogenesis of multiple sclerosis: the need for effective antioxidant therapy.", JOURNAL OF NEUROLOGY MAR 2004, vol. 251, no. 3, March 2004 (2004-03-01), pages 261 - 268, XP002444179, ISSN: 0340-5354 *
LEV N ET AL: "Experimental encephalomyelitis induces changes in DJ-1: Implications for oxidative stress in multiple sclerosis", ANTIOXIDANTS AND REDOX SIGNALING 2006 UNITED STATES, vol. 8, no. 11-12, 2006, pages 1987 - 1995, XP008081691, ISSN: 1523-0864 *
MITSUMOTO A ET AL: "DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin", FREE RADICAL RESEARCH,, YVERDON, CH, vol. 35, no. 6, December 2001 (2001-12-01), pages 885 - 893, XP002989099, ISSN: 1071-5762 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8378070B2 (en) 2007-03-12 2013-02-19 Ramot At Tel-Aviv University Ltd. Peptides for the regulation of neurotransmitter sequestration and release
WO2011033511A1 (fr) 2009-09-17 2011-03-24 Ramot At Tel-Aviv University Ltd. Peptides pour le traitement de troubles liés au stress oxydatif
US8212001B2 (en) 2009-09-17 2012-07-03 Ramot At Tel-Aviv University Ltd. Peptides for the treatment of oxidative stress related disorders
US9023800B2 (en) 2009-09-17 2015-05-05 Ramot At Tel-Aviv University Ltd. Peptides for the treatment of oxidative stress related disorders

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