WO2014059052A1 - Méthodes et compositions destinées au diagnostic et au traitement de la maladie de parkinson et au parkinsonisme - Google Patents

Méthodes et compositions destinées au diagnostic et au traitement de la maladie de parkinson et au parkinsonisme Download PDF

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WO2014059052A1
WO2014059052A1 PCT/US2013/064183 US2013064183W WO2014059052A1 WO 2014059052 A1 WO2014059052 A1 WO 2014059052A1 US 2013064183 W US2013064183 W US 2013064183W WO 2014059052 A1 WO2014059052 A1 WO 2014059052A1
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protein
lrrk2
activity
subject
sample
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Andrew West
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Uab Research Foundation
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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
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a 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
    • G01N2800/52—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 is related to methods and compositions for the diagnosis and treatment of Parkinson's disease (PD) and/or parkinsonism in a subject.
  • PD Parkinson's disease
  • the present invention overcomes previous shortcomings in the art by providing methods and compositions for diagnosing and treating PD and/or parkinsonism in a subject, Summ ary of the Inventio n
  • the present invention provides a method of diagnosing Parkinson's disease (PD) or parkinsonism in a subject, comprising; obtaining a sample (e.g., a urine sample or
  • this method can further comprise administering to the subject a treatment that decreases the amount of LRRK2 protein and/or inhibits LRRK2 activity in the subject.
  • Also provided herein is a method of identifying a subject as having an increased likelihood of having or developing Parkinson's disease (PD) or parkinsonism, comprising; obtaining a sample from the subject; detecting the amount of or activity of LRRK2 protein in the sample from the subject; and comparing the amount of or activity of LRRK2 protein in the sample from the subject to the amount of or activity of L RK2 protein in a sample of a control, wherein an increase in the amount of or activity of LRRK2 protein as compared to a control identifies the subject as having an increased likelihood of having or developing PD or parkinsonism.
  • PD Parkinson's disease
  • the present invention also provides a method of identifying a subject as a suitable candidate for treatment that decreases the amount of LRRK2 protein and/or inhibits or reduces LRRK2 protein activity in a subject, comprising: obtaining a sample from the subject; detecting the amount of or activity of LRRK2 protein in the sample from the subject; and comparing the amount of or activity of LRRK2 protein in the sample from the subject to the amount of or activity of LRRK2 protein in a sample of a control, wherein an increase in the amount of or activity of LRRK2 protein as compared to a control indicates that the subject is a suitable candidate for treatment that decreases the amount of LRRK2 protein and/or inhibits or reduces LRR 2 protein activity.
  • a method of determining the efficacy in a subject of a treatment for Parkinson's disease (PD) or parkinsonism comprising: a) measuring the amount or activity of LRRK2 protein in a sample collected from the subject before the treatment; and b) measuring the amount of activity of LRRK2 protein in a sample collected from the subject after the treatment, wherein a decrease in the amount or activity of LRRK2 protein measured after the treatment as compared to before the treatment indicates efficacy of the treatment for PD or parkinsonism.
  • the treatment can be administration of a substance that decreases the amount o LRRK2 protein and/or inhibits LRRK2 activity in the subject.
  • the methods of this invention can further comprise the step(s) of treating the subject, e.g., by administering to the subject identified or diagnosed as described above, a substance that decreases LRRK2 protein and/or inhibits LRRK2 activity (e.g., sunitinib).
  • a substance that decreases LRRK2 protein and/or inhibits LRRK2 activity e.g., sunitinib
  • the methods of this invention can further comprise the steps of determining efficacy of the treatment by measuring the amount of LRR 2 protein in a sample from a subject before and after administration of a substance that decreases LRR 2 protein and/or inhibits LRRK2 activity, wherein a decrease in the amount of LRRK2 protein measured after administration as compared to before administration indicates efficacy of the treatment.
  • FIG. 1 Discovery of LRRK2 protein enriched in urinary exosomes.
  • An exosome pellet was derived from 60 ml of human urine from a healthy young adult, and the pellet was resuspended in PBS and imaged using cryo-E . Pure ⁇ 100 nm vesicles were detected.
  • B) The purified exosomes were digested with trypsin with or without exosorne- permeabilizing TRITON-X 100 to determine the localization of LRRK2 in exosomes.
  • LRRK2 is present on the outside of exosomes, similar to ALIX and other charged multi- vesicular proteins and Rab-type proteins.
  • FIG. 1 Urinary exosome pellets were derived from 6 healthy controls, quantified with BCA analysis, and 1 ug of protein was loaded onto SDS-PAGE gels. The PVDF was split into two, and probed for LRRK2 and TGSIOI (exosome control). B) ECL signal was quantified by an Alpha-innotech HD in replicate runs, and protein content normalized to TGS 101. Large variability in this control population was observed for LRRK2 in contrast with TGSIOI .
  • FIG. 3 A) The cancer drug sunitinib is a potent LRR 2 kinase inhibitor in LRRK2 peptide based kinase assays.
  • Figure 4 Whole-exosome proteomic analysis.
  • A) Commassie gel of purified exosomes compared to a standard of BSA, prior to in-solution mass spectrometry. Abundant band is THP.
  • B) Normalized Spectral Count (NSC) associated with 935 proteins plotted with standard deviations in replicate runs (n 6). The more abundant proteins, as in C) have satisfactory technical error that results in low standard error (indicated).
  • NSC Normalized Spectral Count
  • FIG. 1 Localization of LRRK2 to urinary exosomes.
  • FIG. 6 Localization of LRRK2 in the kidney and proteomic characterization of urinary exosomes.
  • FIG. 7 LRRK2 exosome release is regulated by 14-3-3.
  • LRRK2 was
  • C HE -293T cells expressing LRR 2 were treated with the indicated drug ( ⁇ ) or equivalent DMSO concentrations (0.01%) for 36 hours. Cells were harvested into total cell lysates, LRR 2 immunoprecipitated, and exosomes collected.
  • FIG. 9 Lack of effect of LRRK2 mutations and lack of localization with a- synuclein in exosomes.
  • Exosomes were isolated from urine of PD and control patients, and lysates were analyzed by western blot in quadruplicate independent runs.
  • the "pool" value is a sample comprised of 10% of each exosome lysate of the 20 PD and 15 control individuals mixed together, allowing comparison of samples analyzed on different gels.
  • B-D Plots showing relative LRRK2 expression normalized to TSG101 expression; data points of each subject are the mean of four independent runs, with removal of subject outliers. Error bars are S.E.M. and the horizontal line is the cohort mean.
  • F Clinical summary of the PD affected case analyzed in panel E.
  • G Urinary exosomes collected from a neurological ly normal individual over the course of 1 week, and analyzed for LRRK2 and TSG101 expression.
  • FIG. 11 LRR 2 exosome secretion by macrophages.
  • D E) Confocal images of thioglycollate collected peripheral primary macrophages isolated from LRRK2 KO or LRRK2 BAC-transgenic mice were stained for LRRK2 protein (antibody c41-2) or TSGIOI. LPS (100 ng/ml) was used for 24 hours prior to fixing cells. Representative images are shown; results were typical of three independent experiments. Scale bars are 10 ⁇ for all panels.
  • FIG. 12 LRRK2 exosome secretion by neurons and in the CSF.
  • a cell can mean one cell or a plurality of cells.
  • the term "about,” as used herein when referring to a measurable value such as an amount of a compound or agent of this invention, dose, time, temperature, and the like, is meant to encompass variations of ⁇ 20%, ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1 % of the specified amount.
  • sample or “biological sample” of this invention can be any biological material, such as a biological fluid (e.g., urine, cerebrospinal fluid, blood, serum, plasma, saliva, joint fluid, semen, washings, etc.), an extract from a cell, an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.
  • a biological fluid e.g., urine, cerebrospinal fluid, blood, serum, plasma, saliva, joint fluid, semen, washings, etc.
  • an extract from a cell e.g., an extracellular matrix isolated from a cell, a cell (in solution or bound to a solid support), a tissue, a tissue homogenate, and the like as are well known in the art.
  • one or more can mean one, two, three, four, five, six, seven, eight, nine, ten or more, up to any number.
  • the term “subject” and “patient” are used interchangeably herein and refer to both human and nonhuman animals.
  • the term “nonhuman animals” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dog, pig, cat, horse, cow, chickens, amphibians, reptiles, rodents (e.g., mice, rats, etc.) and the like.
  • the subject of this invention is a human subject.
  • a "subject in need thereof or "a subject in need of is a subject known to have, or is suspected of having or developing PD and/or parkinsonism and/or a PD-associated disorder or PD-associated condition of this invention or is at risk of having or developing PD and/or parkinsomism and/or a PD-associated disorder or PD-associated condition as described herein.
  • Parkinson's disease refers to a well known and well characterized neurodegenerative disorder.
  • Parkinson's disease refers to a well known and well characterized neurodegenerative disorder.
  • Parkinson's disease refers to a well known and well characterized neurodegenerative disorder.
  • Parkinson's disease refers to a well known and well characterized neurodegenerative disorder.
  • Parkinson's disease refers to a well known and well characterized neurodegenerative disorder.
  • Parkinsononism refers to a condition that results in a
  • administering or “administered” as used herein is meant to include topical, parenteral and/or oral administration, all of which are described herein.
  • Parenteral administration includes, without limitation, intravenous, subcutaneous and/or intramuscular administration (e.g., skeletal muscle or cardiac muscle administration).
  • the compound or composition of this invention may be administered alone and/or simultaneously with one or more other compounds.
  • the compounds may be administered sequentially, in any order. It will be appreciated that the actual method and order of administration will vary according to, inter alia, the particular preparation of compound(s) being utilized, and the particular formulation(s) of the one or more other compounds being utilized.
  • the optimal method and order of administration of the compounds of the invention for a given set of conditions can be ascertained by those skilled in the art using conventional techniques and in view of the information set out herein.
  • administering also refers, without limitation, to oral, sublingual, buccal, transnasal, transdermal, rectal, intramuscular, intravenous, intraarterial (intracoronary), intraventricular, intrathecal, and subcutaneous routes.
  • the instant compounds can be administered at a dose that will produce effective beneficial effects without causing undue harmful or untoward side effects, i.e., the benefits associated with administration outweigh the detrimental effects.
  • the terms “treat,” “treating” or “treatment” refer to any type of action that imparts a modulating effect, which, for example, can be a beneficial and/or therapeutic effect, to a subject afflicted with a condition, disorder, disease or illness, including, for example, improvement in the condition of the subject (e.g., in one or more symptoms), delay in the progression of the disorder, disease or illness, delay of the onset of the disease, disorder, or illness, and/or change in clinical parameters of the condition, disorder, disease or illness, etc., as would be well known in the art.
  • an “effective amount” or “therapeutically effective amount” refers to an amount of a compound or composition of this invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect.
  • the effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art.
  • an effective amount or therapeutically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation. (See, for example, Remington, The Science and Practice of Pharmacy (latest edition)).
  • the term “ameliorate” refers to the ability to make better, or more tolerable, a condition, disorders and/or symptom.
  • the term “prevent” refers to the ability to keep a condition, a reaction, a disorder and/or symptom from happening or existing or developing.
  • LRRK2 refers to the Leucine-Reach Repeat Kinase 2 protein (NCBI Accession. No. NP_490980.3 in Homo sapiens, NCBI Accession No. NPJ ) 01178718.1 in Rattus norvegicus, NCBI Accession No. NP_080006.3 in Mus musculus). Missense mutations in the LRRK2 gene cause late-onset Parkinson's disease (PD) in both autosomal-dominant disease transmitting families, and in sporadic late-onset disease populations. In addition to linkage to PD through genome-wide association studies, LRRK2 genetic variants impose susceptibility risks to inflammation-linked diseases that include Crohn's disease and mycobacterial infection.
  • PD Parkinson's disease
  • the LRRK2 gene encodes a protein with a unique multi-domain composition, including functional GTPase and protein kinase domains, and missense mutations that cause PD alter these enzymatic activities. Understanding the function of LRRK2 can provide insight into pathogenic mechanisms as well as determine particular targets for disease- modifying therapeutics.
  • the present invention is based on the unexpected discovery that proteins linked to or associated with PD and parkinsonism can be detected and quantified from exosomes derived from human urine samples that can be obtained, e.g., in clinical settings.
  • CSF proteins linked to or associated with PD and parkinsonism
  • one aspect of the present invention is based on the discovery that proteins linked to PD, such as, e.g., LRRK2 and DJ-1 (linked to a recessive form of Parkinsonism) can be detected and quantitatively measured in exosomes derived from human urine fractions. These exosomes can be easily obtained in clinical settings, as only urine samples are required, and in the present invention, protocols have been developed to measure LRRK2 and DJ-1 expression from urine samples. In addition, technology has been developed to quantitatively measure 935 other proteins using an innovative multi-plex mass spectrometry proteomic platform that can scale to large clinical populations.
  • proteins linked to PD such as, e.g., LRRK2 and DJ-1 (linked to a recessive form of Parkinsonism) can be detected and quantitatively measured in exosomes derived from human urine fractions.
  • LRRK2 and DJ-1 linked to a recessive form of Parkinsonism
  • LRRK2 expression in mammals is widely distributed in many cell types but particularly enriched in the kidney and in activated macrophages of the innate immune system.
  • LRRK2 expression is relatively modest and includes medium spiny neurons in the striatum that form striosomes.
  • LRRK2 associates with a number of vesicle types and to intraluminal vesicles within multivesicular bodies (MVBs). Assignment of LRRK2 function within the endocytic pathway has been suggested, with recent evidence for action in retrograde vesicle trafficking from endosomes to the trans-Golgi network, and for modifying chaperone-mediated autophagy.
  • Elevated LRR 2 kinase activity is linked to PD susceptibility since the most common pathogenic LRRK2 mutation G2019S in humans increases kinase activity approximately 3- fold in most assays, and overall LRR.K2 subcellular localization has been described as extraordinarly sensitive to acute LRRK2 kinase inhibition.
  • LRRK2 interaction with 14-3-3 proteins appears to be one of the main modulators of LRRK2 subcellular localization, consistent with the notion that 14-3-3 isoforms principally function by modulating cell localization of binding partners.
  • 14-3-3 LRRK2 interaction or impact that 14-3-3 mediated localization may have on LRR 2 functionality, particularly within the endocytic pathway.
  • LRRK2 is secreted from intra-luminal vesicles from MVBs (i.e., exosomes) from a variety of cells where LRRK2 is natively expressed, including cells in the kidney, brain, and immune system. LRRK2 can be readily detected through purification of exosomes from urine or cerebral spinal fluid (CSF) in clinical populations, or through exosomes from cell culture media. It has also been found that a major role for the 14-3-3/L RK2 interaction may be the regulation of LRRK2 association with late-endosomes and uptake into MVBs with subsequent extracellular release of LRRK2 protein via exosome secretion.
  • MVBs i.e., exosomes
  • the present invention provides a method of diagnosing PD in a subject, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from the subject as compared to a control, thereby diagnosing PD in the subject.
  • one embodiment of the method comprises detection of LRRK2 protein from a sample obtained from urine or CSF.
  • the urine sample is collected fresh, i.e., has not been frozen and is processed the same day as collection. For example, if collected in the morning, the urine sample is processed in the afternoon.
  • the sample prior to processing, the sample is stored at 4°C for no longer than about two hours following collection.
  • processing comprises isolation of exosomes from the sample.
  • the method comprises detection of LRRK2 protein found in exosomes isolated from the sample,
  • the cellular source of the exosomes in the sample is not particularly limited, in one embodiment, the exosomes isolated from a urine sample are derived from the kidney.
  • Isolation of exosomes from a sample for analysis may take place according to any protocol for isolating exosomes that will be understood by one of skill in the art.
  • Detection of the amount of LRRK2 protein in a sample can be carried out by any technique using any standard protocol that will be appreciated by one of skill in the art.
  • detection of LRRK2 protein may take place using immunohistochemistry or immunofluorescence.
  • detection of LRRK2 protein can be achieved using cryo-electron microscopy (cryo-EM) or standard emission depletion (STED) microscopy.
  • detection of LRRK2 protein may employ western blotting assays.
  • the protocol for detecting the activity of LRRK2 protein in a sample can be carried out by any known procedure to assay for LR-RK2 activity that is known to one of skill in the art.
  • LRRK2 is a multi -domain protein with multiple activities.
  • activity of LRRK2 protein is detected by measuring kinase activity of LRRK2 protein.
  • Kinase assays of LRRK2 protein may be performed according to any standard protocol for measuring kinase activity that is known to one of skill in the art.
  • Determining whether the subject has PD is made by comparing the amount or activity of LRRK2 protein of the sample to the amount or activity of LRRK2 protein detected in a control sample, wherein an increased or greater amount or activity of LR K2 protein in the sample from the subject as compared to the amount or activity of LRRK2 protein in the control sample is indicative of the subject having PD.
  • a control is, for example, a sample from a normal subject, or subjects, who does not or do not have PD.
  • the significance of the comparison between subject sample and control sample may be determined through statistical analysis.
  • General analyses may be accomplished, for example, using SAS version 9.3, R, StatXact, GeneSring, Partek, and Ingenuity Pathway Analysis (IP A) software packages, depending on the test.
  • categorical variables e.g., PD affected/unaffected, sunitinib treated/untreated, LRRK2 high/low expression, etc.
  • estimates of proportions and confidence intervals can be calculated.
  • the Chi-square or Fisher's exact test can be used as appropriate.
  • a paired comparison of proportions can be analyzed using the McNemar's test.
  • the logistic regression model can be used for group comparisons of proportions when covariate adjustments are desired. Model building using different selection procedures can be performed to determine the significant factors in the model. Methods of cross-validation can be employed when feasible.
  • Likelihood ratio tests, R 2 and analysis of residuals can be performed to assess the adequacy of the resulting model.
  • the present invention provides a method of identifying a subject as having an increased likelihood of having or developing PD, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from the subject as compared to a control.
  • the protocols for obtaining a sample, detecting the amount of and/or detecting the activity of LRRK2 protein can be the same as the protocols set forth above for the diagnosis of PD according to the present invention,
  • a determination of whether the subject has an increased likelihood of having or developing PD is made by comparing the amount or activity of LRRK2 protein of the sample from the subject to the amount or activity of LRR 2 protein detected in a control sample, wherein an increased or greater amount or activity of LRRK2 protein in the sample from the subject as compared to the amount or activity of L K2 protein in the control sample is indicative of the subject having an increased likelihood of having or developing PD.
  • a control is, for example, a sample from a normal subject or subjects, who does not or do not have PD. The significance of the comparison between subject sample and control sample can be determined through statistical analysis as set forth above for the diagnosis of PD according to the present invention.
  • the present invention provides a method of identifying a subject as a suitable candidate for treatment with a substance that decreases the amount of L RK2 protein in the subject and/or inhibits or reduces LPJRK2 activity in the subject, comprising detecting an increase in the amount or activity of LRRK2 protein in a sample from a subject as compared to a control sample.
  • the substance that decreases the amount of LR K2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is a kinase inhibitor.
  • the kinase inhibitor can be sunitinib.
  • the protocols for obtaining a sample, detection of the amount of and/or the activity of LRRK2 protein can be done according to the same protocols set forth above for diagnosing PD or parkinsonism or for determining the likelihood of having or developing PD or parkinsonism according to the present invention.
  • a determination of whether the subject is a candidate for treatment with a substance that decreases the amount of L RK2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is made by comparing the amount of or activity of LRRK2 protein of the sample from the subject to the amount of or activity of LRRK2 protein detected in a control sample, wherein an increased or greater amount or activity of LRJRK2 protein in the sample from the subject as compared to the amount or activity of LRRK2 protein in the control sample identifies the subject as a candidate for treatment with a substance that decreases the amount of LRR 2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject.
  • a control sample is, for example, a sample from a normal subject or subjects, who does not or do not have PD.
  • the significance of the comparison between subject sample and control sample can be determined through statistical analyses as set forth above for the diagnosis of PD or for determining the likelihood for having or developing PD according to the present invention.
  • the present invention provides a method of treating a subject having PD, such as a subject identified by the method described above, comprising to the subject an effective amount of a substance that decreases the amount of LRRK2 protein and/or inhibits LRRK2 activity in the subject.
  • the substance that decreases the amount of LRR 2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject is a kinase inhibitor.
  • the kinase inhibitor is sunitinib.
  • the efficacy of the treatment is monitored by measuring the amount or activity of LRRK2 protein in a sample from the subject before administration and after administration of the substance that decreases the amount of LRRK2 protein in the subject and/or inhibits or reduces LRRK2 activity in the subject, wherein a decrease in the amount and/or of LRRK2 protein in the subject after administration of the substance indicates efficacy of the treatment.
  • the protocols for obtaining, detecting the amount of and/or detecting the activity of LRRK2 protein can be the same as the protocols set forth above for diagnosing PD, determining the likelihood of having or developing PD, or identifying whether a subject is a candidate for treatment with a substance that decreases the amount of LRRK2 protein in the subject and/or inhibits or reduces L K2 protein activity in the subject according to the present invention.
  • LRRK2 total LRRK2 as well as L RK2 phosphorylated at amino acid 1292 of the LRRK2 protein will be reduced in individuals taking a LRR 2 kinase inhibitor, or other therapeutics used to treat Parkinson's disease relevant to LRRK2.
  • the test can be used for individualized treatment, such that some patients may respond to low levels of drug with complete LRRK2 removal from exosome, and thus have minimal off-target and drug side effects from taking lower amounts of drug, whereas some patients may need higher amounts of drug to inhibit LRRK2 and remove LRRK2 signal from the assay, and these higher amounts would be needed to see therapeutic benefit (i.e., neuroprotection in Parkinson's disease, or other related diseases).
  • the methods of this invention can include additional steps such as modulating (i.e., increasing or decreasing) the dose of a kinase inhibitor or other therapeutic on the basis of a change in the amount of LRRK2 detected in a sample from a subject.
  • the pi 292 LRRK2 levels, and/or total LRRK2 protein levels can identify individuals at increased risk of developing Parkinson's disease. These individuals would be suitable candidates to receive neuroprotective therapeutics to stave off the onset of Parkinson's disease. Mutations in the LRR 2 protein cause PD, but only in about 50% of patients with any given LRRK2 mutation. Measurement of pi 292 and/or total LRRK2 in urinary exosomes can show that the affected (manifesting) patients have higher levels of signal in the assay.
  • known LRRK2 mutation carriers may want to monitor their levels of active LRR 2 ( i 292 lrrk2) and total LRRK2 to assess risk of development of PD, and possibly enroll in clinical trials or take other pro-active measures, should the test reveal heightened risk for PD.
  • Detection of LRRK2 in urinary exosomes can correlate well with other markers in blood and urine in disease, and provide a biochemical link and/or the discovery of additional biomarkers that are easier to assay and quantify than LRRK2 is; post-LRR 2 detection, levels of hundreds of other proteins and chemicals will be evaluated to shed light on mechanisms important in PD and other diseases or physiological processes like aging.
  • the present invention provides a method of screening for substances/molecules/proteins related to PD.
  • the analysis is performed on exosomes, wherein the exosomes have been isolated from either urine or cerebral spinal fluid (CSF).
  • CSF cerebral spinal fluid
  • the analysis examines modulators of the activity of
  • LRRK2 protein LRRK2 protein.
  • the analysis examines modulators of the activity of DJ-1. These screening methods can be carried out according to the protocols described herein. EXAMPLE 1
  • Exosomes in neurodegeneration and PD are relatively recently discovered structures that are small -100 nm average vesicles encapsulated intra-cellularly in the cytosolic of numerous cell types within multi-vesicular bodies (MVB). Upon MVB binding to the outer membrane, some or all of the exosomes are released to the extracellular environment, and these exosomes contain a rich array of proteins, lipids, and RNA. Released exosomes can be readily purified and visualized in native conformation (aqueous, without fixation or processing) by a technique known as Cryo-Electron Microscopy (Figure 1A). It is believed that exosomes are an evolutionarily conserved structure for intercellular
  • exosomes are able to endocytize exosomes and functionally incorporate the cargo.
  • the protein composition of exosomes is speculated to generally reflect much of the protein constituency of the originating cell's cytosol, based on the thousands of proteins identified from a number of highly purified exosome sources in humans (see www.exocarta.org). Due to the accessibility of exosomes in human fluids and early-linkages to biological processes, it is considered that exosomes may hold tremendous value as biomarkers for human disease.
  • exosomes In neurodegeneration, exosomes have been proposed as the primary vehicles for transfer of toxic proteins. Exosomes can transmit prion diseases through the trafficking of PrP-Sc. In models of Alzheimer's disease, trafficking of ⁇ and components of APP are in part accomplished via exosomes. In cell models of ALS, mutant SOD-1 can traffic through exosomes and aggregate in the host cell. In PD, primary neuronal cultures secrete both tau and alpha-synuclein monomers and aggregates in exosomes.
  • alpha-synuclein is thought to be expressed solely in neurons but aggregates can be found in glia cell types in MSA and other neurological diseases, and exosome transport of alpha-synuclein is the likely process for this to occur.
  • Mutations in VPS35 cause late-onset PD and VPS35 is involved in retrograde transport of endosomes to the Golgi network, and possibly endosomes to MVB critical for exosome formation.
  • the proteins believed to be at the heart of most neurodegenerative disorders are associated with exosomes in some manner. However, the specific role of exosomes in disease etiology is not clear.
  • LRRK2 and PD-proteins in urinary exosomes originated from localization work with the LRRK2 protein.
  • LRRK2 was discovered in late-onset PD kindreds and subsequently identified as the most common genetic cause of late-onset PD, where pathogenic mutations are causing disease in 20-30% of late-onset cases in some populations. In most Caucasian populations, pathogenic L RK2 mutations are present at ⁇ 1- 5%, and common variation in the L RK2 gene associates with LRR 2 susceptibility in large GWAS meta-analyses.
  • LRRK2 expression or activity is abnormal in PD without LRPvK2 mutations (e.g., the vast majority) is unknown.
  • L RK2 and other proteins of interest in PD have been difficult since the neurodegenerative condition irreversibly alters cell composition and proportions in affected areas, leaving behind cells that are resistant, or sick, or reactive to the inflammatory process (e.g., L RK2 -filled microglia), and no easy way to distinguish between these cells for most biochemical approaches.
  • post-mortem brain material inherently has variables such as pH, delay to collection (PMI), and time spent in storage, that greatly influence most metrics associated with proteins.
  • PMI delay to collection
  • R 2 localizes to vesicular structures in cells that normally express LRRK2.
  • the LRRK2 GTPase domain is most similar to human Rab-vesicular sorting proteins (BLAST analysis), and Rab proteins are among the more abundant species on exosomes and directly participate in vesicular trafficking in cells.
  • EM studies have also revealed LRRK2 association with MVBs (i.e., exosome precursors).
  • a database search www.exocarta.com
  • LRRK.2 has been detected in exosomes in mass spectrometry studies.
  • several other PD proteins in addition to LRR 2 were detected in exosomes isolated from human urine.
  • VPS35 protein and DJ-1 protein are present.
  • Exosome analysis in PD While it would be ideal to directly analyze susceptible brain tissue in PD cases early in disease, later in disease, and longitudinally during a clinical trial for neuroprotective therapeutics, brain tissue specimens are obviously limited to postmortem collection. Novel imaging approaches are very promising non-invasive procedures, but these techniques have not yet been able to directly measure proteins or biochemical pathways that may underlie neurochemical changes. Emphasis has been placed in recent years on analyzing CSF, which has been challenging because of limited patient participation (especially controls), the expense of the procedure, and that only small amounts of CSF can be drawn and the resultant fluid is very dilute in proteins (including exosomes) and other markers of interest, and CSF is susceptible to variable contamination of blood cells during the procedure.
  • exosomes quantitatively measured from these exosomes. It is reasonable to consider that peripheral expression in exosomes might correlate to expression in PD-susceptible tissue. It is also possible that urinary exosomes directly sample proteins from a variety of organs, including the brain.
  • LRRK2 and alpha-synuclein- interacting proteins 14-3-3, HSP90, and HSP70, ERM/Radixin are detectable.
  • Each of these can be quantified using very standardized and controlled conditions of collection and processing afforded by the non-invasiveness of the urine collection procedure.
  • Proteins can be measured in a longitudinal manner in clinical populations, and can potentially be used to assess the effects of treatments on the intended targets. For example, during a clinical trial of LRRJ .2 inhibitors, it would be possible to directly assay changes in LRRK2 expression and phosphorylation. This analysis would provide vital information. Also, we have the opportunity to be well powered to detect changes associated with early PD (Hoehn and Yahr stage 1 -2), which is not easily done with post-mortem tissue.
  • the payoff of a PD biomarker in urinary exosomes would be incredibly large, and the biomarker could be widely implemented by most centers. With the finding of protein(s) of interest, cheap ELISA and western blot approaches can be developed to follow up on mass spectrometry results. Based on the data included, at a minimum the development of urinary exosomes as a biomarker for LRRK2 target engagement in LRRK2 clinical trials is very likely to succeed, and this is essential for de-risking LRRK2 inhibitors for pharmaceutical development and phase experiments. The identification of urinary exosome biomarkers related to the state or progression of the disease has potential for very high reward. Exosome analysis in PD cases and controls.
  • Multi-plex whole-exosome proteomic approaches A relatively low cost technique for rapidly and quantitatively assessing nearly a thousand proteins from exosome preparations has been developed.
  • TMT-MudPIT tumor necrosis plasminogen activator-based technology
  • This approach can be widely implemented in a number of human diseases in the search for biomarkers.
  • LRRK2 and PD-linked genes in late-onset PD Missense mutations in the LRRK2 gene that are predicted to enhance GTP-binding (e.g., R1441C, Y1699C) or kinase activity (e.g., G2019S) in the encoded protein may induce gain-of-function activity.
  • GTP-binding e.g., R1441C, Y1699C
  • G2019S kinase activity
  • gain or loss of function mutations associate with PD in LRRK2 it is a possibility that enhanced LRRK2 expression may represent a risk factor for PD susceptibility.
  • reduced levels of DJ-1 may be a risk factor, potentially in concert with LRRK2 expression. This hypothesis has been extremely difficult to test in clinical populations for the reasons mentioned above concerning post-mortem tissue.
  • the embodiments of this invention have the developed technology and sample availability to quantitatively measure the abundance of these proteins in PD versus control samples, and therefore can test, in relevant samples sizes, whether there is
  • LRRK2 knockout animals are resistant to the neurodegenerative effects of alpha-synuclein over-expression, and mutations may induce increases in kinase activity that cause iate-onset PD
  • inhibition of LR K2 kinase activity may provide neuroprotective benefit.
  • This invention provides a plan to develop and validate technology to test target engagement in LRRK2 protein isolated from kidney exosomes. Specifically, we expect to see vastly diminished levels of LRRK2 phosphorylation and lower LRR 2 expression in urinary exosomes when LRRK2 is inhibited by sunitinib exposure.
  • Inclusion criteria will be based on UK Brain Bank criteria for the clinical diagnosis of PD, and will require: 1) the presence of bradykinesia and either rest tremor or rigidity; 2) asymmetric onset; 3) progressive motor symptoms 4) age at onset 35-85 years.
  • Control inclusion criteria Ages of between 35-85 years, a lack of PD in first-degree blood relatives, and a lack of positive responses on more than 3 items on our PD Screening Questionnaire.
  • Parkinson-Plus disorder Progressive Supranuclear Palsy (PSP), Multiple System Atrophy (MSA), Corticobasal Degeneration (CBD)
  • PSP Progressive Supranuclear Palsy
  • MSA Multiple System Atrophy
  • CBD Corticobasal Degeneration
  • neuroleptic treatment at time of onset of parkinsonism active treatment with a neuroleptic at time of study entry
  • history of repeated strokes with stepwise progression of parkinsonism history of repeated head injury
  • history of definite encephalitis prominent gait imbalance early in the course ( ⁇ 5 years)
  • dementia known severe anemia (hematocrit ⁇ 30), history of kidney disease and/or current or past glomerular filtration rate (GFR ⁇ 60) possibly indicative of kidney disease, or a serious comorbidity that may interfere with participation in the study.
  • GFR ⁇ 60 current or past glomerular filtration rate
  • Phenofypes Databasing Clinical Phenofypes. Clinical assessments are conducted by board certified neurologists with subspecialty training in movement disorders, and nurse-clinicians with specialized training in PD. Phenotype collection meets core and relevant requirements of the PDBP clinical data elements. Cases and controls have all phenotype data uploaded into a database (into a Microsoft Access platform run on a SQL server available to this project). All individuals associated with the study have access to these data. Data on all subjects further conform to the NINDS Common Data Elements toolkit to ensure uniformity of data collection, optimize data analyses and data mining
  • Demographics include gender, age, race, ethnicity, education, PD Medical History Onset and nature of PD symptoms, degree of diagnostic certainty, PD Family History Relatives and family members with PD, RFQ-U Height and Weight Height and weight data, for use in covariate analyses, non-prescribed drug use, vitamins and nutriceuticals for covariate analyses, Movement Disorder Society- Unified Parkinson's disease Rating Scale, the Hoehn and Yahr rating scale, and Parkinson's disease Medication Log Estimated dose and duration of therapy with levodopa and other PD medication, Non-Parkinson's disease Medication, Log Estimate dose and duration for non-PD medications. Notably, there will be no permanent sample banking in this study as all clinical samples are completely consumed in the proposed analyses.
  • Sample Collection 4-6 samples on average are be collected per week. Urine samples are collected between 8:00AM and 10:00AM during routine clinic hours to avoid possible diurnal variations, and these samples are processed into exosome fractions that afternoon. In general, control samples are collected from spouses or significant others. PD is more common in males, and we anticipate that the study population will have a male:female ratio of 2:1, reflecting the gender specific prevalence of the disorder. Control recruitment is monitored to achieve matching of ages and genders. Based on our estimations of ⁇ 0.5 ug of exosome protein per mL of urine (this number is also well-established in the literature), we require at least 70 mL (i.e., 2.3 oz) of urine for enrollment.
  • the stabilization buffer has been optimized so that no adverse effects on exosome stability are observed up to our longest time point analyzed of 8 hours post aliquoting. Protocols to normalize the effects of variable pH, ionic concentration, active proteases and phosphatases have been previously created, and each sample stored at 4°C for a maximum of 2 hours before processing.
  • the composition of the 20x supplement is 1M Tris pH 7.4, 20mM AEBSF, 40mM EDTA, 1 OOmM b-glycerol-phosphate, 20uM pepstatin A, 20uM E-64.
  • Exosome isolation protocols have been compared including ultracentrifugation, ultrafiltration, and immunoprecipitation with magnetic beads.
  • the most reliable method is the ultracentrifugation method, where urine is centrifuged at 500 xG for 10 min, supernatant removed and centrifuged at 17,000 xG for 15 min, and
  • LRRK2 can be efficiently detected via western blot from exosome preparations, but mass spectrometry detection was variable presumably due to low abundance. Because LRRK2 is prioritized, a targeted approach specifically for detection of LR K2 is used. Exosome pellets are first resuspended in PBS, sonicated, and protein quantified by BCA analysis. 50 ⁇ g of exosomes are set aside and frozen at -80°C for whole-proteomic analysis. 4 ⁇ g of exosome proteins are combined into Ix Laemmli buffer with 5% BME and heated at 70°C for 10 min prior to loading samples onto a 10% TBX 12 well gel (Bio-rad).
  • the LRRK2 antibody has been validated in both rat and mouse KO tissue, and in human brain samples (single band) and human cell lines. Ratios for signals generated by LRRK2 compared to TGS101 will be determined by chemiluminesence on an Alpha-Innotech Fluorchem HD machine. This approach has been found to have a greater linear range of protein detection than comparable technology by Li -Cor using fluorescence. Ratios for most experiments will be normalized between gels using a reference exosome sample that is arbitrarily defined as 1 ,0, thus multiple western blots can be integrated together with full detection of technical error for each sample.
  • Isobaric labeled peptides from each sample are combined, purified, and loaded onto a custom packed tandem long-flow SCX/C18 column, and MuDPIT analysis is accomplished using a nanp-HPLC (nLC) with a 14 fraction salt bump gradient carried out in duplicate.
  • Peptides are eluted from the analytical column after each salt bump with optimized 105-min reverse-phase solvent gradient.
  • the peptides are directly electrosprayed into a LTQ Orbitrap Velos mass spectrometer operated in a data-dependent mode with dynamic exclusion to enable identification of less abundant ions.
  • Tandem mass spectra are analyzed using a suite of custom developed bioinformatics tools. The combination of XCorr and DeltaCN is used initially for score optimization, and results are filtered to a 2% peptide false discovery rate (FDR) using concatenated (forward and reverse db's) with the requirement of a minimum of two quantifiable peptides per reported protein.
  • FDR 2% peptide false discovery rate
  • Exosome pellets are glass-like in appearance (similar to isopropanol R A pellets), and exosome pellets that are discolored or otherwise unusual in visual composition will be noted and marked for possible study exclusion.
  • wet exosome pellets will be resuspended and sonicated in PBS and stored at a standard concentration of 0,5 mg/mL, as determined by microtiter BCA assays, and samples maintained at -80°C. Samples that do not meet study criteria of normal urinalysis and kidney function will be discarded once those test results are made available in the study database.
  • Yahr stage 1-2 and 50 later-stage PD cases will be collected, with matching of age and gender distribution between cases and controls. Gender distribution will match that of clinical distribution, roughly 2:1 male to female.
  • Exosome fractions will be produced and 20 g of exosomes from each case and control will be combined together to create a standard exosome pool consisting of ⁇ 4 mg of exosomes. This exosome pool will provide an ample supply of protein to normalize between both targeted and unbiased runs as described herein.
  • the reference pool will be used to perform an initial statistical analysis of clinical samples to determine our power and to measure statistically significant changes between clinical phenotypes and particular protein targets of interest.
  • the described immunoblot approach will be used to quantify LR K2 relative to the ubiquitous exosome marker TSG101, and ratios normalized to the pooled exosome standard.
  • groups sizes of 5 (plus the exosome pool control) will be analyzed in individual multi-plex MudPIT runs (x6 isobaric labels are included in the TMT labeling approach), and we have previously established our power to detect at least 935 proteins with high confidence in this multi-plex technique.
  • Exosome structures have never been formally analyzed in PD, yet these molecules contain many of the proteins linked to PD pathology and genetics.
  • age and gender matched samples are continued to be collected from early-stage PD (Hoehn and Yahr 1-2) and late-stage PD (Hoehn and Yahr 3-5) together with control patients.
  • exosome samples will be derived from patients treated with sunitinib, together with control patients, from the Comprehensive Cancer Center.
  • exosome preparations will be analyzed by whole-proteomic screening and targeted protein detection.
  • Fourth clinical phenotypes are correlated and effect size determined for targets of interest for PD progression, susceptibility, and sunitinib treatment.
  • samples from early stage PD H&Y 1-2
  • later stage PD H&Y 3-5
  • matched control individuals samples from early stage PD (H&Y 1-2), later stage PD(H&Y 3-5), and matched control individuals, are derived as described above, for both targeted and unbiased proteomic analysis.
  • the sample number based on the above analysis is adjusted to maximize the possible study power, for example by increasing sample replicate runs and decreasing new sample recruitment.
  • the results of power calculations are prioritized, particularly for proteins of interest such as LRRK2 in the exosome samples.
  • samples collected in the study are processed using unbiased whole-proteomics and targeted measurements of LRRK2 as samples are continued to be collected, using the pooled exosome fraction as a reference sample for cross-run normalization.
  • the pooled exosome fraction serves as the best possible measure of quality control during individual runs, and runs where the pooled standard is not correlated with past runs of the pooled standard will be re-performed.
  • a critical component to biomarker discovery is replication of targets of interest in naive cohorts. Targets of interest identified and prioritized above are thus defined in a new group of patients and controls. In addition, a larger combined dataset can reveal other significant exosome/phenotype associations that can be followed upon in future studies.
  • Urine specimens were collected from the Movement Disorder Clinic at the University of Alabama at Birmingham, and CSF and brain tissue samples were obtained from the NICHD Brain and Tissue Bank at the University of Maryland. Samples were supplemented with 5GmM Tris pH 7.4, ImM AEBSF, 2mM EGTA, 5mM ⁇ -Glycerolphosphate, ⁇ pepstatin A, and 1 ⁇ E-64 before exosome purifications.
  • mice Male WT and LRRK2 KO rats were obtained from Sigma and used at 10-12 weeks of age.
  • Primary neurons and macrophages were prepared from C57BL/6J WT or LRRK2-BAC mice (J AX strain 012467).
  • NeuN staining was used to confirm cultures that were comprised of >95% NeuN positive cells.
  • LRRK2 was immunoprecipitated using Dynal ProtG Dynabeads together with anti-Myc antibody (Roche) following manufacturer's instructions.
  • HE -293T cells and macrophage Raw264.7 cells were maintained in exosome-free media composed of DMEM with 10% fetal-bovine serum (FBS).
  • FBS fetal-bovine serum
  • the small molecule inhibitor HG-10-102 was synthesized in-house according to the method previously described, and L2inl compound was a gift from Dr. Dario Alessi.
  • Antibodies to LRRK2 (Epitomics c41-2 and UDD3, and NeuroMab N241 A/34), pS935 (Epitomics UDD2), TSG101 and 14-3-3pan (Abeam), Alix and CD9 (Santa Cruz), tubulin and FLAG (Sigma), myc (Roche), V5 (Invitrogen), and secondary donkey anti-mouse and anti -rabbit conjugated to HRP, Cy2, Cy3, Cy5, and Biotin (Jackson immunoResearch) were used.
  • Antibody ATTO 647N Goat anti-Rabbit (Active Motif) was used for super resolution imaging.
  • Vesicle marker antibodies EEA1 , Rab7,9,l 1 , and AAPL1 were from Cell
  • Lysotracker was from invitrogen.
  • Dissociated cells were cultured on glass coverslips and fixed with 4% PFA in PBS for 10 min. at room temperature. Cells were stained for LRRK2 and other targets as previously described. In all experiments, cells from LR K2 KO animals were used to confirm LRRK2 staining. Exosome preparations were resuspended into 0.5% PFA in PBS and incubated for 5 min at room temperature. Glycine was supplemented to 0.1M concentration, and the exosomes pelleted at 500 kG for 15 min. Exosome pellets were resuspended in a blocking buffer (5% exosome-free donkey serum, 0.3% TRITON X-100 in PBS) and incubated for 30 min on a rotating wheel.
  • a blocking buffer 5% exosome-free donkey serum, 0.3% TRITON X-100 in PBS
  • exosomes were resuspended in water, applied to Quantifoil holey film (Quantifoil MicroTools, Jena, Germany) and plunge frozen using an FEI Vitrobot.
  • Quantifoil holey film Quantifoil MicroTools, Jena, Germany
  • the samples were observed in an FEI Tecnai F20 200kV field-emission gun microscope equipped with a high-sensitivity Gatan Ultrascan 4000 CCD camera.
  • Sonicated exosomes suspended in PBS were supplemented to 50% trifiuoroethanol and reduced by addition of tris(2-carboxyethyl)phosphine and incubated for 45 min at room temperature, then alkylated by adding iodoacetimide, followed by digestion with trypsin gold (Promega). Tandem mass tag (TMT) labeling reagents (Thermo-Pierce) were added to the samples, incubated, and then supplemented with 5% hydroxylamine.
  • TMT Tandem mass tag
  • Isobaric labeled peptides from each sample were combined, purified, and loaded onto a tandem long-flow SCX C18 column, and MuDPIT analysis was accomplished using a nano-HPLC (nLC) with a 14 fraction salt bump gradient carried out in duplicate.
  • Peptides were eluted from the analytical column after each salt bump with optimized 105-min reverse-phase solvent gradient.
  • the peptides were directly electrosprayed into a LTQ Orbitrap Velos mass spectrometer operated in a data-dependent mode with dynamic exclusion to enable identification of less abundant ions. Resultant spectra were analyzed using a suite of custom developed bioinformatics tools.
  • LRRK2 ( ⁇ 1-970) was purchased from Invitrogen and full-length LRRK2 protein was isolated from HEK-293T cells over-expressing LRRK2 protein. Kinase reactions were performed as previously described. Autophosphorylation was monitored using an antibody directed to pi 503 of LRRK2 using the Alpha-Screen kit following manufacturer instructions (Perkin Elmer). Recombinant proteins were assessed for equal purity (>95% by Coomassie SDS-PAGE) and protein concentration as determined by BCA assay (Pierce). In some experiments, kinase activity was determined by coomassie stain and autoradiography film exposure.
  • LRRK2 action in the endocytic pathway may include aspects of lysosomal and autophagy mediated degradation, receptor recycling, and vesicle budding.
  • PI 00 100k x g pellet
  • LRRK2 was present at an approximate concentration of 1 picogram per milliliter of urine, and all of the LRRK2 protein localized to the PI 00 (exosome) fraction. Exosomes are organelles that maintain the orientation of the proteins from the parental cells, so that extracellular proteins or extracellular protein domains remain extra-exosomal, whereas cell cytosolic proteins are encapsulated within the exosome, A trypsin-sensitivity assay demonstrated that the addition of T ITON-X 100 detergent to break the vesicles was required for proteolytic digestion of LRRK2, suggesting that LRRK2 is localized within exosomes (Figure 5B). The LRRK2 binding partner 14-3-3 was also sensitive to trypsin degradation in the presence of detergent. In contrast, CD9 is known to be present in a large trans-lummal protein complex within the plasma membrane that was resistant to trypsin digestion whether detergent was added or not.
  • CD9 is a transmembrane protein that is abundant in plasma-membrane derived vesicles that traffic to MVBs. We found that CD9 diffusely spreads across the high density fractions, with the highest concentration in the exosome-containing fraction.
  • CD 9 has been previously used in fluorescence and immunoaffinity approaches to purify exosomes, but we found complete exclusion of LRRK2 immunoreactivity in CD9 positive exosomes via immunofluorescence in dilute exosome preparations ( Figure 5F).
  • LRRK2 partially co-localized to TSG101 enriched exosomes, although examples of TSG101 positive exosomes with weak or no detectable LRRK2 expression could also be observed.
  • CD9 is a ubiquitously expressed plasma membrane protein, this could indicate that the intracellular source of LRRK2-positive exosomes could be distinct from plasma membrane-derived exosomes.
  • LRR 2 dimerization and phosphorylation are activities that have been linked to LRRK2 kinase activity.
  • urinary exosomes were resuspended in buffer containing the non-ionic detergent TRITON X-100 and lysates analyzed by native- PAGE (Figure 5G).
  • Urinary exosome LRRK2 migration was identical to that of endogenous and kinase-active LRRK2 isolated from cell cytosols. Low amounts of LRRK2 in preparations without detergent treatment were likely due to exosome breakage during processing, but show that detergent did not alter the migration pattern of LRRK2 in native gels.
  • LRRK2 can be readily measured from urinary exosomes and that LRRK2 protein in exosomes derives from the cell cytosol, either captured in vesicles that fuse with MVBs, or packaged during the inward internalization of vesicles fusing with MVBs.
  • LRRK2 protein concentration is appreciable in urinary exosomes, the overall levels are not sufficient to directly assess LRRK2 kinase activity with any described kinase activity assay.
  • LRRK2 protein interactors in exosomes may chaperone and control LRRK2 vesicular association.
  • urinary exosome proteomic studies have been performed, these have been qualitative assessments that have not capitalized on recent and dramatic improvements in proteomic technology.
  • TMT-MudPIT long-column multi -dimensional protein identification
  • LRRK2 may be tightly bound to heat-shock proteins and 14-3-3 chaperones that may control LRRK2 solubility and oligomerization. We sought to test whether interactions with these proteins may control LRRK2 extracellular secretion.
  • HEK-293T cells transfected with LRRK2 actively secrete exosomes into cell culture media (Figure 7A). While knockdown of all 14-3-3 isoforms in HE -293T cells is difficult to accomplish, a short-peptide inhibitor known as difopein has been developed in HEK-293T cells that effectively acts as a pan 14-3-3 inhibitor by blocking 14-3- 3 dimerization. Transfection of difopein in LRRK2-expressing HEK-293T cells resulted in a very efficient ablation of LRRK2 binding to 14-3-3 proteins, as observed through
  • Acute LRRK2 kinase inhibition via small molecules causes a reduction in 14-3-3 binding to LRR 2.
  • LRRK2 kinase inhibition via small molecules causes a reduction in 14-3-3 binding to LRR 2.
  • HG-10-102 that is known to be a selective LRRK2 inhibitor, and the widely utilized L2inl compound, were first defined for potency in a kinase inhibition assay measuring LRRK2 autophosphorylation in vitro ( Figure 7C,D).
  • the cytosolic distribution of LRR 2 may be important for LRRK2 packaging into exosomes.
  • LRRK2 cytoplasmic distribution appears to critically mediate extracellular secretion, we next co-localized LRRK2 with the exosome marker TSGl Ol in HE -293T cells that constitutively secrete exosomes. While TSGlOl had a discrete vesicular like distribution, LRRK2 was more evenly expressed across the cytosol with rarer examples of discrete vesicle-sized puncta, clearly distinguishing LRRK2 from a canonical vesicular protein (Figure 9 A). Some previous evidence suggests that the G2019S mutation may alter this cytoplasmic distribution to LRJ K2 positive intracellular puncta, so we tested whether this mutation affects LRRK2 secretion.
  • Exosomes may be recruited to VBs through plasma membrane derived vesicles enriched in transmembrane plasma proteins such as CD9 in a clathrin-dependent manner, or through the direct uptake of late-endosomal vesicles into MVBs.
  • LR K2 cytoplasmic association with late-endosome vesicles is dependent on 14-3-3 binding.
  • 14-3-3 bound LRRK2 is relatively diffuse and soluble, and this is critical for packaging into MVBs.
  • L RK2 is likely captured inward into invaginating late-endosomal vesicles in the cytosol ( Figure 11).
  • LRRK2-containing exosomes after MVB docking to the plasma membrane in either a tightly controlled manner, such as in macrophages, or a constitutive manner, such as in HEK.-293T cells, or ceils may traffic LRRK2-positive MVBs to other parts of the cell, such as along dendrites of a neuron, for possible release at a physical proximity different than the originating vesicle, and to a cell that does not normally express LRR 2 protein.
  • the present invention provides protocols optimized for purification of LRRK2 protein from a sample (e.g., urine and/or CSF) for utilization as a biomarker for disease state and phenotype.
  • a sample e.g., urine and/or CSF
  • LRRK2 would be otherwise undetectable in standard clinical protocols to measure protein in urine, with the combination of rapid thawing of clinical samples with vortexing, optimized differential centrifugation that removes extraneous protein that would otherwise inhibit detection of LRR 2 in protein assays, optimized lysis of exosome structures that requires both high concentrations of denaturing detergents as well as sonication optimization, and western blot technology using optimized conditions with specific antibodies sensitive to low picogram to ferntogram levels of protein, the present invention provides a protocol for measuring LRRK2 in a urine and/or CSF sample.
  • Routine handling of urine without rapid freeze thaw will destroy exosomes that harbor LRRK2, standard centrifugation approaches would not be sufficient to isolate LRRK2 fractions, standard lysis conditions would inefficiently lyse exosomes and LRRK2 containing exosomes to free the protein for detection, and standard protein detection assays would not be sensitive enough to detect LRRK2 at the low picogram to high femtogram levels of this invention due to problems with electrophoresis, protein transfer, and antibody-based detection methods.
  • steps that are not routine and conventional include:
  • -Samples are processed using differential ultracentrifugation (e.g., multiple spins at increasing G forces) with final centrifugation at speeds of about 500 kG in thick wall tubes optimized for isolation of LRRK2 containing exosomes.
  • -Non routine western blotting conditions which include the use of acrylamide SDS PAGE gels (e.g., commercial pre-cast gels such as 7.5% TGX, Bio-Rad), optimized transfer to nitrocellulose (e.g.,PVDF) membranes, with about 4 hour to about 24 hour transfers (e.g., about!6 hour transfers) in a glycine/10% to 20% methanol buffer, and blocking conditions and antibodies optimized for this assay (e.g., monoclonal antibody C41-2, monoclonal antibody N241/34, monoclonal antibody UDD-03 and/or an antibody to P1292). Incubation times of assays are also optimized for the highest signal to noise ratios.
  • acrylamide SDS PAGE gels e.g., commercial pre-cast gels such as 7.5% TGX, Bio-Rad
  • optimized transfer to nitrocellulose (e.g.,PVDF) membranes with about 4 hour to about 24 hour transfers (e.g., about!6 hour
  • subtilisin/kexin type 9 PCSK9 74.22 1.00 2 10.00 0.98 9.83
  • element-derived protein 7 TIGD7 63.18 0.91 2 10.00 1.1 1 1 1.12
  • Chromodomain-helicase- DNA-binding protein 1 CHD1 196.55 0.75 2 10.00 0.93 9.33
  • Phosphatidyiethanolamine- binding protein 1 PEBP1 21.03 1.00 3 10.00 2.03 20.31
  • Zinc finger protein 326 ZN326 65.60 0.95 2 8.00 1.31 16.40
  • toxin substrate 1 RACI 21.42 1.00 3 6.00 1.07 17.86
  • Ankyrin repeat domain- containing protein 12 ANR12 235.49 1.00 10 6.00 2.10 34.97
  • Rab-3 A- interacting protein RAB3I 52.97 1.00 5 6.00 1.36 22.67
  • VTA1 Vacuolar protein sorting- associated protein VTA1
  • Ficolin-2 FCN2 33.96 1.00 2 5.00 1.36 27.28
  • TFIID subunit 3 TAF3 103.50 0.96 3 5.00 0,61 12, 12
  • RNA -binding protein 44 RBM44 1 17.89 1.00 4 5.00 1.01 20.15
  • Chromodomain-helicase- DNA -bin ding protein 6 CHD6 305.20 1.00 6 5.00 0.70 13.94
  • Insulin-like growth factor 1 Insulin-like growth factor 1
  • Zinc finger protein Rlf RLF 217.80 1.00 6 4.00 0.61 15.36
  • Proline-rich protein 12 PRR12 129.89 0.89 3 4.00 0.71 17.80
  • body protein 2a CHM2A 25.07 1.00 4 4.00 0.95 23.70
  • GTP-binding protein 1 GTPB1 72.39 1.00 4 3.00 0.82 27.23 Total
  • basonuclin-1 BNC1 1 10.88 0.73 2 3.00 0.50 16.58
  • Ankyrin repeat domain- containing protein 1 1 ANR11 297.71 0.99 5 3.00 0.62 20.58
  • Ankyrin repeat domain- containing protein 24 ANR24 124.09 1.00 4 3.00 1.02 34.05
  • Estrogen receptor ESR1 66.16 0.96 3 3.00 0.66 21.86
  • SP-A receptor submit SP- Q5QD0
  • Thymosin beta- 10 TYB10 5.00 0.97 2 3.00 0.99 32.88
  • VDAC1 Voltage-dependent anion- selective channel protein 1 VDAC1 30.74 1 .00 4 3.00 0.93 31.16
  • Zinc finger protein 616 ZN616 90.20 1.00 4 3.00 0.46 15.29
  • guanine nucleotide- exchange protein 2 BIG2 201.89 0.98 4 3.00 1 22 40.70

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Abstract

La présente invention concerne le diagnostic de la maladie de Parkinson ou du parkinsonisme, l'identification de sujets présentant une possibilité accrue de présenter ou de développer la maladie de Parkinson ou le parkinsonisme, et le traitement de la maladie de Parkinson ou du parkinsonisme.
PCT/US2013/064183 2012-10-09 2013-10-09 Méthodes et compositions destinées au diagnostic et au traitement de la maladie de parkinson et au parkinsonisme WO2014059052A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015200851A1 (fr) * 2014-06-27 2015-12-30 Inarian Neurodiagnostics, Llc Procédé pour enrichir des exosomes dérivés du système nerveux central
WO2017072335A1 (fr) * 2015-10-28 2017-05-04 Ab Science Utilisation de masitinib et d'autres inhibiteurs de mastocyte pour le traitement de la maladie de parkinson
WO2019147934A1 (fr) * 2018-01-29 2019-08-01 Sackner Bernstein Jonathan Méthodes de modulation de la dopamine dans des maladies neurologiques humaines
WO2020018461A1 (fr) * 2018-07-16 2020-01-23 The University Of Virginia Patent Foundation Compositions et méthodes pour le diagnostic et le traitement de maladies neurologiques
CN111537738A (zh) * 2020-05-18 2020-08-14 南通大学附属医院 用于超早期帕金森病检测的试剂盒
US10914748B2 (en) 2016-09-08 2021-02-09 UNIVERSITé LAVAL Erythrocyte-derived extracellular vesicles as a biomarker for clinically assessing Parkinson's disease
WO2021094751A1 (fr) * 2019-11-11 2021-05-20 Oxford University Innovation Limited Biomarqueurs pour la prédiction et l'identification de la maladie de parkinson
CN113398244A (zh) * 2021-05-28 2021-09-17 南方医科大学 一种治疗帕金森病的制剂及其应用
KR20220047124A (ko) * 2020-10-08 2022-04-15 상지대학교산학협력단 Trdn을 포함하거나 조절하는 파킨슨병용 약제학적 조성물 및 그 치료제
CN114736298A (zh) * 2022-04-08 2022-07-12 陕西脉元生物科技有限公司 用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用
WO2022090804A3 (fr) * 2020-10-30 2022-08-11 Rideout Hardy Méthodes et compositions se rapportant à des biomarqueurs pour des maladies neurodégénératives
WO2022232487A1 (fr) * 2021-04-30 2022-11-03 Denali Therapeutics Inc. Méthodes de traitement et de surveillance de la maladie de parkinson
WO2023174946A1 (fr) * 2022-03-15 2023-09-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthode précoce et non invasive d'évaluation du risque de maladie de parkinson chez un sujet

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012004566A1 (fr) * 2010-07-06 2012-01-12 United Arab Emirates University Procédé de diagnostic
WO2012075046A1 (fr) * 2010-11-30 2012-06-07 Genentech, Inc. Tests et biomarqueurs destinés au lrrk2

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012004566A1 (fr) * 2010-07-06 2012-01-12 United Arab Emirates University Procédé de diagnostic
WO2012075046A1 (fr) * 2010-11-30 2012-06-07 Genentech, Inc. Tests et biomarqueurs destinés au lrrk2

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
LUCKING ET AL.: "'Association between early-onset Parkinson's disease and mutations in the parkin gene'", THE NEW ENGLAND JOURNAL OF MEDICINE, vol. 342, no. 21, 2000, pages 1560 - 1567 *
SHAW ET AL.: "Biomarkers of neurodegeneration for diagnosis and monitoring therapeutics", NATURE REVIEWS DRUG DISCOVERY, vol. 6, no. 4, 2007, pages 295 - 303 *
SINCLAIR ET AL.: "NMR-based metabolomic analysis of cerebrospinal fluid and serum in neurological diseases-a diagnostic tool?", NMR IN BIOMEDICINE, vol. 23, no. ISSUE, 2010, pages 123 - 132 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015200851A1 (fr) * 2014-06-27 2015-12-30 Inarian Neurodiagnostics, Llc Procédé pour enrichir des exosomes dérivés du système nerveux central
WO2017072335A1 (fr) * 2015-10-28 2017-05-04 Ab Science Utilisation de masitinib et d'autres inhibiteurs de mastocyte pour le traitement de la maladie de parkinson
US10914748B2 (en) 2016-09-08 2021-02-09 UNIVERSITé LAVAL Erythrocyte-derived extracellular vesicles as a biomarker for clinically assessing Parkinson's disease
WO2019147934A1 (fr) * 2018-01-29 2019-08-01 Sackner Bernstein Jonathan Méthodes de modulation de la dopamine dans des maladies neurologiques humaines
US11806326B2 (en) 2018-01-29 2023-11-07 Jonathan Sackner-Bernstein Methods for dopamine modulation in human neurologic diseases
WO2020018461A1 (fr) * 2018-07-16 2020-01-23 The University Of Virginia Patent Foundation Compositions et méthodes pour le diagnostic et le traitement de maladies neurologiques
WO2021094751A1 (fr) * 2019-11-11 2021-05-20 Oxford University Innovation Limited Biomarqueurs pour la prédiction et l'identification de la maladie de parkinson
CN111537738A (zh) * 2020-05-18 2020-08-14 南通大学附属医院 用于超早期帕金森病检测的试剂盒
KR20220047124A (ko) * 2020-10-08 2022-04-15 상지대학교산학협력단 Trdn을 포함하거나 조절하는 파킨슨병용 약제학적 조성물 및 그 치료제
KR102663668B1 (ko) 2020-10-08 2024-05-08 상지대학교산학협력단 Trdn을 포함하거나 조절하는 파킨슨병용 약제학적 조성물 및 그 치료제
WO2022090804A3 (fr) * 2020-10-30 2022-08-11 Rideout Hardy Méthodes et compositions se rapportant à des biomarqueurs pour des maladies neurodégénératives
WO2022232487A1 (fr) * 2021-04-30 2022-11-03 Denali Therapeutics Inc. Méthodes de traitement et de surveillance de la maladie de parkinson
CN113398244B (zh) * 2021-05-28 2023-07-07 南方医科大学 一种治疗帕金森病的制剂及其应用
CN113398244A (zh) * 2021-05-28 2021-09-17 南方医科大学 一种治疗帕金森病的制剂及其应用
WO2023174946A1 (fr) * 2022-03-15 2023-09-21 INSERM (Institut National de la Santé et de la Recherche Médicale) Méthode précoce et non invasive d'évaluation du risque de maladie de parkinson chez un sujet
CN114736298A (zh) * 2022-04-08 2022-07-12 陕西脉元生物科技有限公司 用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用
CN114736298B (zh) * 2022-04-08 2023-12-12 陕西脉元生物科技有限公司 用于检测自身免疫系统疾病的抗Shroom2自身抗体及其产品和应用

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