WO2019161302A1

WO2019161302A1 - Biomarkers for parkinson's disease

Info

Publication number: WO2019161302A1
Application number: PCT/US2019/018350
Authority: WO
Inventors: Sarah F. HAMM-ALVAREZ; Curtis OKAMOTO; Mihir Shah; Maria EDMAN-WOOLCOTT; Srikanth R. JANGA; Mark LEW; Wendy MACK; Danielle FEIGENBAUM
Original assignee: University Of Southern California
Priority date: 2018-02-16
Filing date: 2019-02-15
Publication date: 2019-08-22

Abstract

Applicant demonstrates for the first time that PD-dependent alterations in cholinergic stimulation and/or in the trophic effects of cholinergic stimulation on the function of lacrimal gland acinar cells (LGAC) themselves result in a characteristic or diagnostic profile in the composition of tear fluid, which can be measured relatively easily, cost-effectively, and non- invasively. These biomarkers can aid in early proper identification of PD patients. Thus, provided herein are novel methods of determining whether a subject is likely to have or develop Parkinson's disease prior to the onset of motor symptoms, methods of identifying a subject at risk of developing Parkinson's disease, methods of detecting at least one biomarker of Parkinson's disease in a tear sample, methods of treating a subject with Parkinson's disease prior to the onset of motor symptoms, and kits to diagnose Parkinson's disease.

Description

BIOMARKERS FOR PARKINSON’S DISEASE

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. provisional application Serial Nos.: 62/710,292; 62/655,092; and 62/664,877, filed February 16, 2018; April 9, 2018; and April 30, 2018, the contents of each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Parkinson’s disease (PD) is characterized by its hallmark central nervous system effects and Lewy body pathology. However, PD is also a multiple system disorder. For example, the gastrointestinal tract ( GI tract) of PD patients is predominantly innervated by acetylcholine-secreting (cholinergic) neurons and is affected very early in the disease. These changes are responsible for delayed gastric emptying together with delayed intestinal transit time as well as constipation, and typically occur prior to the manifestation of the classic motor deficits in PD patients. This phase is known as pre-motor PD. The tear-producing lacrimal gland is an organ that is also highly innervated by cholinergic neurons, and tear fluid secreted by lacrimal gland acinar cells is greatly stimulated by cholinergic neurons.

[0003] The ability to diagnose PD accurately and cost-effectively in the pre-motor phase, prior to the extensive destruction of central nervous system neurons and classic motor manifestations, is essential for the search of effective disease-modifying and neuroprotective interventions for people with PD or at risk of developing PD. Thus, there exists a need in the art for accurate and cost-effective diagnosis of PD and related disorders. This disclosure satisfies this need and provides related advantages as well.

SUMMARY

[0004] Applicant demonstrates for the first time that PD-dependent alterations in cholinergic stimulation and/or in the trophic effects of cholinergic stimulation on the function of lacrimal gland acinar cells (LGAC) themselves result in a characteristic or diagnostic profile in the composition of tear fluid, which can be measured relatively easily, cost-effectively, and non- invasively. These biomarkers can aid in early proper identification of PD patients. Thus, provided herein are novel methods of determining whether a subject is likely to have or develop Parkinson’s disease prior to the onset of motor symptoms, methods of identifying a subject at risk of developing Parkinson’s disease, methods of detecting at least one biomarker of

Parkinson’s disease in a tear or saliva sample, methods of treating a subject with Parkinson’s disease prior to the onset of motor symptoms, and kits to diagnose Parkinson’s disease.

[0005] In some aspects, provided herein are methods of determining whether a subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder, prior to the onset of motor symptoms, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from the subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) determining that the subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0006] In other aspects, provided herein are methods of identifying a subject at risk of developing Parkinson’s disease or a Parkinson’s related disorder, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from the subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) identifying the subj ect as at risk of developing Parkinson’ s disease or a Parkinson’ s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0007] In some aspects, provided herein are methods of determining whether a subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder prior to the onset of motor symptoms, the methods comprising, consisting of, or consisting essentially of: (a) comparing a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder measured in a sample isolated from a subject to a reference level or reference activity; and (c) determining that the subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0008] In other aspects, provided herein are methods of identifying a subject at risk of developing Parkinson’s disease, the methods comprising, consisting of, or consisting essentially of: (a) comparing a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder measured in a sample isolated from a subject to a reference level or reference activity; and (b) identifying the subject as at risk of developing Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0009] In some aspects, provided herein are methods for detecting at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear s or saliva ample isolated from a subject suspected of having or developing Parkinson’s disease, the methods comprising, consisting of, or consisting essentially of: (a) collecting a tear or saliva sample from the subject; and (b) measuring a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample. In some embodiments, the methods further comprise, consist of, or consist essentially of comparing the level or activity of the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder to a reference level or reference activity.

[0010] In a further aspect, provided herein are methods for detecting at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from a subject, the methods comprising, consisting of, or consisting essentially of: (a) collecting a tear or saliva sample from the subject; and (b) measuring a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample. In some embodiments, the methods further comprise, consist of, or consist essentially of comparing the level or activity of the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder to a reference level or reference activity.

[0011] In other aspects, provided herein are methods of treating a subject with Parkinson’s disease or a Parkinson’s related disorder prior to the onset of motor symptoms, the methods comprising, consisting of, or consisting essentially of: (a) screening a tear or saliva sample isolated from a subject for a level or activity of at least one biomarker of Parkinson’s disease; and (b) treating the subject screened in step (a) by administering a Parkinson’s disease or a Parkinson’s related disorder therapy. [0012] In some aspects, provided herein are methods of treating a subject with Parkinson’s disease or a Parkinson’s related disorder prior to the onset of motor symptoms, the method comprising, consisting essentially of, or consisting of administering a Parkinson’s disease or a Parkinson’s related disorder therapy to a subject; wherein a tear or saliva sample isolated from a subject has been screened for a level or activity of at least one biomarker of Parkinson’s disease.

[0013] In some embodiments of the disclosed methods, the reference level or reference activity is selected from: (a) the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in a subject or population that does not have Parkinson’s disease;

(b) a normal reference standard; and (c) a normal reference cutoff value.

[0014] In some embodiments of the disclosed methods, the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder is elevated relative to the reference level or reference activity. In some embodiments, the elevated level or activity is about 1.5 times, or alternatively, about 2.0 times, or alternatively, about 2.0 times, or alternatively, about 3.0 times, or alternatively, about 5 times, or alternatively, about 10 times, or alternatively about 50 times, or yet further alternatively more than about 100 times higher than the reference level or activity. In other embodiments, the level or activity of the biomarker of Parkinson’s disease or a

Parkinson’s related disorder is decreased relative to the reference level or reference activity. In some embodiments, the elevated level or activity is about 1.5 times, or alternatively, about 2.0 times, or alternatively, about 2.0 times, or alternatively, about 3.0 times, or alternatively, about 5 times, or alternatively, about 10 times, or alternatively about 50 times, or yet further alternatively more than about 100 times lower than the reference level or activity. In some embodiments, the biomarker is referred to as“over expressed” or“under expressed.” In other embodiments, the biomarker may also be referred to as“up regulated” or“down regulated.”

[0015] In some embodiments of the disclosed methods, the level or activity of the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder is measured by one or more methods of the group of: fluorometric analysis, Western blot, gel electrophoresis, enzyme- linked immunosorbent assay (ELISA), multiplex ELISA, enzymatic assay, mass spectrometry, protein array, Luminex assay, quantitative PCR, nucleic acid sequencing, nanoparticle tracking analysis (NTA), flow cytometry, dot blot analysis, immunohistochemistry,

immunofluorescence, real-time quaking-induced conversion and immunocytochemistry. In particular embodiments, the level of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder is measured by ELISA or multiplex ELISA.

[0016] In one aspect, the biomarker is oligomeric synuclein. In another aspect, the methods are combined with the measurement of the C-C motif chemokine ligand (CCL2) biomarker and/or the Schirmer’s test for increased sensitivity (see Table 7). In another aspect, they further comprise CCL2. In a further aspect, CCL2 and tear volume is assessed (see, e.g., Table 7 below).

[0017] In some embodiments of the disclosed methods, a tear or saliva sample is isolated from the subject using one or more of: a solid substrate, a filter substrate, capillary tube, polyester fiber rod, microfluidic device, chip device, and impression cytology. In particular embodiments, the filter substrate is a Schirmer’s test strip. In some embodiments, the tear sample is isolated from one eye of the subject. In other embodiments, the tear sample is a pool of samples isolated from both eyes of the subject. In some embodiments, the tear sample is isolated by means of a functionalized eyewear device, e.g., a contact or similar device that collects a tear sample.

[0018] In some embodiments of the disclosed methods, the tear sample comprises one or more exosomes. In some embodiments, the methods further comprise performing one or more of column chromatography, differential centrifugation, and nanoparticle tracking analysis on the tear sample to enrich or isolate exosomes. In some embodiments, enrichment or isolation of exosomes is performed prior to measuring the level or activity of at the least one biomarker of Parkinson’s disease. In some embodiments, measuring the level or activity of at least one biomarker of Parkinson’s disease is performed on exosomes isolated from the tear or saliva sample.

[0019] In some embodiments of the disclosed methods, the tear sample is an anesthetized tear sample. In other embodiments, the tear sample is an unanesthetized tear sample. In other embodiments, the tear sample is an anesthetized or unanesthetized tear sample.

[0020] In some embodiments of the disclosed methods, the methods further comprise, consist of, or consist essentially of measuring a total protein content in the tear or saliva sample isolated from the subject. In some embodiments, increased protein content relative to a reference sample is indicative of increased risk of having or developing Parkinson’s disease or a Parkinson’s related disorder. In one aspect, the tear sample is an anesthetized tear sample. In other aspects, the tear sample is an unanesthetized tear sample. In further aspects, the tear sample is an anesthetized or unanesthetized tear sample. In another aspect, the tear sample is isolated using a Schirmer’s strip, capillary flow device or equivalent detection method marked as to measure tear flow. In some embodiments, decreased tear flow relative to a reference sample is indicative of increased risk of having or developing Parkinson’s disease or a Parkinson’s related disorder. In one aspect the tear sample is an anesthetized tear sample. In other aspects, the tear sample is an unanesthetized tear sample. In particular embodiments of the disclosed methods, at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder comprises a-synuclein. In some embodiments, at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder comprises total a-synuclein, oligomerized a-synuclein, and/or phosphorylated a- synuclein. In particular embodiments, the biomarker of Parkinson’ s disease or a Parkinson’ s related disorder is total a-synuclein. In some embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is oligomerized a-synuclein. In some embodiments, the ratio of oligomerized a-synuclein to total a-synuclein is increased in tear samples isolated from subjects likely to have or develop Parkinson’s or a Parkinson’s related disorder prior to the onset of motor symptoms. In some embodiments, the ratio of oligomerized a-synuclein to total a-synuclein is increased in tear or saliva samples isolated from subjects at risk of having or developing Parkinson’s or a Parkinson’s related disorder. In some embodiments, the increase in the ratio of oligomerized a-synuclein to total a-synuclein is an increase relative to a reference values according to healthy patients. In another aspect, the biomarker is oligomeric synuclein.

In another aspect, the biomarker also comprises C-C motif chemokine ligand 2 (CCL2). In a further aspect, the methods further comprise the use of a Schirmer’s test (see, e.g. Table 7).

[0021] In some embodiments of the disclosed methods, the subject is a mammal. In particular embodiments, the subject is a human.

[0022] Also provided herein are kits to diagnose Parkinson’s disease, comprising, consisting of, or consisting essentially of one or more reagents for isolating a tear or saliva sample from a subject and at least one detection reagent that is specific for a biomarker of Parkinson’s disease. In some embodiments, the reagents for isolating a tear or saliva sample comprise one or more of: a filter substrate, capillary tube, polyester fiber rod, lateral flow immunoassay, microfluidic device, chip device, and impression cytology; and optionally an anesthetic suitable for use in an eye. In some embodiments, the kits further comprise a reference standard. BRIEF DESCRIPTION OF DRAWINGS

[0023] FIG. 1 : Schematic diagram showing the positioning of the human main lacrimal gland relative to the ocular surface and the organization of several of the cell types within the gland, as well as the neural reflex arc that connects the ocular surface to the lacrimal gland. Activation of the afferent sensory nerves in the cornea and conjunctiva leads to activation of efferent parasympathetic and sympathetic nerves that signal the lacrimal gland to secrete proteins, electrolytes and water. AM, apical membrane; BM, basolateral membrane; CNS, central, nervous system; D, duct; L, lumen; LG, lacrimal gland; ME, myoepithelial cell; N, nucleus; NE, nerve ending; OS, ocular surface; SV, secretory vesicle.

[0024] FIG. 2 : The various regulated secretory pathways in lacrimal gland acinar cells. For simplicity, only the population of regulated secretory granules and secretory lysosomes are shown; the transcytotic pathway is not shown.

[0025] FIG. 3 : Median cathepsin S (CTSS) activity in tears from patients with Sjogren’s Syndrome (SS), rheumatoid arthritis (RA), other autoimmune conditions (OTHERS), or healthy controls (HC), illustrating the potential for tear CTSS activity to be used as a biomarker for SS.

[0026] FIG. 4 : Western blot of lactoferrin (arrow) from tears demonstrates its substantial decrease in primary Sjogren’s Syndrome (pSS) patients as compared to patients with no autoimmune dry eye, systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). Human recombinant lactoferrin was used as a positive control. Demonstrating the feasibility of detecting differences in tear fluid protein abundance between patient groups with standard biochemical assays.

[0027] FIG. 5: Stability against proteolytic degradation was tested on tear proteins of interest stored“dry” on Schirmer’s strips (e.g., without eluting proteins with elution buffer). After storing the strips for 1- and 2- weeks at -80 °C, all proteins were incubated with elution buffer and assayed with ELISA or biochemical assays as appropriate. Proteins stored on Schirmer’s strips remained stable with levels comparable to those analyzed on day 0. Data are represented as percentages of the concentration measured on day 0.

[0028] FIG. 6 : Race distribution of PD patients and Healthy controls (HC) recruited during the study described in Example 4.

[0029] FIG. 7: a-synucleinTotal levels in tears of PD patients was significantly lower when compared to those in tears of HC (p<0.00l) (FIG. 7 A). a-synucleinoligo levels in PD patient tears was significantly higher compared to those in HC (p=0.0l) (FIG. 7B). The ratio of a- synoligo/a-synTotal was significantly increased in PD patients compared to HC (p=0.002)

(FIG. 7C). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11.

An unpaired t-test with Welch’s correction was used to measure significance.

[0030] FIG. 8 : Total protein concentration was significantly increased in tears of PD patients when compared to that of healthy controls (HC) (p=0.005). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11 An unpaired t-test with Welch’s correction was used to measure significance.

[0031] FIG. 9 : Lactoferrin (LF) and matrix metalloproteinase-9 (MMP-9), markers of lacrimal acinar secretory function and ocular surface inflammation, respectively, and CCL2 and DJ-l, two putative PD biomarker proteins that are implicated in disease development and progression, were measured in tears of healthy controls (HC) and PD patients. These proteins remained unchanged in PD patients when compared to healthy controls (HC) (p=0.46, p=0.56, p=0.44 and p=0.64, respectively). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11. An unpaired t-test with Welch’s correction was used to measure significance.

[0032] FIG. 10: Amount of tear wetting measured with the Schirmer’s strip (strip values were pooled from both eyes) which did not show significant differences in PD and healthy control (HC) subjects (p=0.65), suggesting that tear fluid production was not affected significantly in this cohort of PD patients. Data are presented as mean ± SEM and are from the cohort in FIG. 11. An unpaired t-test with Welch’s correction was used to measure significance.

[0033] FIG. 11: Summary of demographics in PD patients and healthy controls (HC) for collection of anesthetized tear samples used for measurements in FIGS. 7-10.

[0034] FIGS. 12A to 12H graphically depicts the data depicted in Table 3. Each point on an ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. The area under the ROC curve is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normal). An area of 1 represents a perfect test that can identify a disease individual; an area of 0.5 represents a test that is unable to distinguish a diseased individual. FIG. 12A shows data for CCL2, FIG. 12B shows DJ-l, FIG. 12C shows MMP9, FIG. 12D shows lactoferrin, FIG. 12E shows a-synuclein total, FIG. 12F shows a- synuclein oligo, FIG. 12G shows a-synuclein oligo/a-synuclein total, and FIG. 12H shows Schirmer’s.

[0035] FIG. 13A-13D graphically depicts a-synuclein total is decreased (FIG. 13A), whereas a-synuclein oligo (FIG. 13B) and the ratio of a-synuclein oligo/a-synuclein total (FIG. 13C) are increased in PD patients tears compared to healthy controls. Levels of a-synuclein total, a-synuclein oligo and the ratio of a-synuclein oligo/a-synuclein total in tears of healthy controls (n=60) and PD patients (n=94) (FIG. 13D). a-synuclein levels were normalized to total tear protein, log transformed. Transversal line indicates median, bars indicate max and min values. Medians, interquartile ranges and p values are listed in the table. P value is calculated by Wilcoxon rank sum test.

[0036] FIG. 14 graphically depicts a receiver operating characteristic analysis for gender corrected ratio of a-synuclein oligo/a-synuclein total.

[0037] FIG. 15A-15B show tear lactoferrin (FIG. 15A) and MMP-9 (FIG. 15), indicators of dry eye and ocular inflammation, are not altered in PD patients compared to healthy controls. Levels of tear lactoferrin and MMP9 in tears of healthy controls and PD patients were unchanged. Marker proteins were normalized to mg of total tear protein. Data was normalized to total protein and log transformed. Transversal line indicates median, bars indicate minimum and maximum values.

[0038] FIG. 16A-16B show tear protein concentration is increased (FIG. 16A) while Schirmer’s strip test values are unaffected (FIG. 16B) in PD patients relative to healthy controls. Tear protein concentration in tears pooled from both eyes of each subject in healthy controls (n=60) and PD patients (n=94). Schirmer’s strip values from both eyes of each subject in healthy controls (n=l20) and PD (n=l88). Transversal line indicates median, bars indicate Min and Max values. * p < 0.05 as determined by Wilcoxon rank sum test.

[0039] FIG. 17A-17F shown tear levels. Normalized levels (FIGS. 17A-17C) and area under the ROC of a-synuclein oligo (FIGS. 17D-17F) in reflex tears of all HC (n=84) and PD patients (n=84), in male HC (n=45) and male PD patients (n=48) and in female HC (n=39) and female PD patients (n=36). a-synuclein oligo levels were normalized to total tear protein and log transformed. The transverse line indicates the median, boxes the 25th-75th percentiles and whiskers the 5th and 95th percentiles. The p values were calculated by Wilcoxon rank sum test. [0040] FIG. 18A-18B show normalized levels of CCL2 in reflex tears of all HC (n=84) and PD patients (n=84) (FIG. 18A), and male HC (n=45) and male PD patients (n=48) (FIG. 18B). CCL2 levels were normalized to total tear protein and log transformed. The transverse line indicates the median, boxes the 25th-75th percentiles and whiskers the 5th and 95th percentiles. The p-values were calculated by Wilcoxon rank sum test.

DETAILED DESCRIPTION

[0041] Embodiments according to the present disclosure will be described more fully hereinafter. Aspects of the disclosure may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. While not explicitly defined below, such terms should be interpreted according to their common meaning.

[0043] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0044] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0045] Unless explicitly indicated otherwise, all specified embodiments, features, and terms intend to include both the recited embodiment, feature, or term and biological equivalents thereof.

Definitions

[0046] As used herein, the singular forms“a,”“an,” and“the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0047] It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term“about.” The term“about” means that the number comprehended is not limited to the exact number set forth herein, and is intended to refer to numbers substantially around the recited number while not departing from the scope of the invention. As used herein,“about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used,“about” will mean up to plus or minus 15%, 10%, 5%, 1%, or 0.1% of the particular term.

[0048] Also as used herein,“and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0049] As used herein, the term“comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method.“Consisting of’ shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or

compositions (consisting of).

[0050] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as“up to,”“at least,” “greater than,”“less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above.

[0051] As used herein, an“antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term“antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein, any of which can be incorporated into an antibody of the present invention.

[0052] In one aspect, the“biological activity” means the ability of the antibody to selectively bind its epitope protein or fragment thereof as measured by ELISA or other suitable methods. Biologically equivalent antibodies, include but are not limited to those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody.

[0053] “Biomarkers” are biological characteristics used to indicate, detect, measure, or predict disease risk, disease presence, disease outcome, response to treatment, and/or disease progression. Biomarkers include but are not limited to behavioral characteristics, physiological characteristics, peptides, lipids, carbohydrates, metabolites, proteins, enzymes or enzymatic activity, cells, cellular fragments, cellular vesicles, exosomes, nucleic acids, RNA, DNA, genes, mRNAs, miRNAs, and cell-free DNAs. In some embodiments, a biomarker for Parkinson’s disease or a Parkinson’s related disorder is precise, reliable, inexpensive, and/or reflects the pathophysiological mechanisms of Parkinson’s disease or related disorders. Furthermore, biomarkers are most useful when they can detect at an early or even preclinical stages of diseases. In some embodiments, the biomarker of PD or a PD related disorder detects PD prior to the onset of one or more motor symptoms of PD.

[0054] The terms“protein”,“polypeptide” and“peptide” are used interchangeably herein when referring to a gene product. [0055] Tear flow” is a measure of the output of liquid from the eye. Tear flow may be measured in units not limited to pL/min.

[0056] “Parkinson’s disease therapy” or“regime” may refer to Levodopa and carbidopa (Sinemet), Levodopa (also called L-dopa), safmamide, dopamine agonists (for example, ropinirole, pramipexole, or rotigotine), amantadine, trihexyphenidyl, benztropine, selegiline, rasagiline, tolcapone, entacapone, deep brain stimulation (electrode implantation into the brain), and occupational or speech therapy. Further included are any drugs or therapies known to the skilled worker in the treatment of Parkinson’s disease.

[0057] Schirmer's” test determines whether the eye produces enough tears to keep it moist. This test is used when a person experiences very dry eyes or excessive watering of the eyes. It poses no risk to the subject. A value exceeding more than 10 mm of moisture on the filter paper within 5 minutes is a normal test result for most healthy individuals. Both eyes will normally secrete about the same amount of tears. Schirmer's test uses paper strips inserted into the eye for several minutes to measure the production of tears. The exact procedure may vary somewhat. Both eyes are tested at the same time. Most often, this test consists of placing a small strip of filter paper inside the lower eyelid (inferior fornix). The eyes are closed for 5 minutes. The paper is then removed and the amount of moisture is measured. Sometimes a topical anesthetic is placed into the eye before the filter paper to prevent tearing due to the irritation from the paper. The use of the anesthetic ensures that only basal tear secretion is being measured. What follows is how to read results of the Schirmer's test: 1. Normal which is >15 mm wetting of the paper after 5 minutes. 2. Mild dryness which is 14-9 mm wetting of the paper after 5 minutes. 3. Moderate dryness which is 8-4 mm wetting of the paper after 5 minutes. 4. Severe dryness which is <4 mm wetting of the paper after 5 minutes.

[0058] “Parkinson’s disease” is the second most common neurodegenerative disease after Alzheimer's disease and is hallmarked by the dopaminergic neurons of the substantia nigra (SN) and by a-synuclein protein containing inclusion bodies (Lewy pathology; LP) in the surviving neurons, resulting in the characteristic motor impairment.

[0059] Proxymetacaine (INN) or“proparacaine” (USAN) is a topical anesthetic drug of the aminoester group. Other examples of topical anesthetics that may be used in the present invention include benzocaine, butamben, dibucaine, lidocaine, oxybuprocaine, pramoxine, proparacaine, proxymetacaine, and tetracaine (also named amethocaine).

[0060] “Reference” as used herein, may refer to a reference sample of known concentration (level) and constitution of a specific biomarker. The reference biomarker may be acquired from a commercial source or isolated under a variety of techniques such as chromatography or extraction. In some embodiments, the reference concentration and constitution is representative of the healthy non-Parkinson’s disease having population, or representative of a population that will not develop Parkinson’s disease in the future. “Reference activity” may refer to biochemical or chemical reactivity of a sample representative of a normal healthy population that will not develop Parkinson’s disease. The difference in activity or level of the tested concentration vs the reference activity or level may be a quantitative difference or a qualitative difference. Both reference levels or activities and tested levels or activities may be measured using any of the analytical methods described herein.“Screening” refers to testing for an analyte, for example, a protein, using any of the methods disclosed herein.

[0061] “Likely” as used herein to refer to the likelihood of the subject developing

Parkinson’s disease (PD) may be selected from 1-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50- 75%, 75-100%, 100-250%, 250-500%, 500-1,000%, or greater than 1,000% more likely to develop PD than a subject whose level or activity does not differ from the reference level or activity.

[0062] The chemokine (C-C motif) ligand 2 (CCL2) is also referred to as monocyte chemoattractant protein l(MCPl) and small inducible cytokine A2. CCL2 is a

small cytokine that belongs to the CC chemokine family. CCL2 recruits monocytes, memory T cells, and dendritic cells to the sites of inflammation produced by either tissue injury or infection. The gene and polynucleotide sequence are known in the art, see

genecards.org/cgi-bin/carddisp. pl?gene=CCL2 (last accessed on February 15, 2019).

Antibodies to the protein are commercially available, seemdsystems.com/target/ccl2-je-mcp-l (last accessed on February 15, 2019).

[0063] “Lactoferrin” or“LTF” is a member of the transferrin family of genes and its protein product is found in the secondary granules of neutrophils. The protein is a major iron-binding protein in milk and body secretions with an antimicrobial activity, making it an important component of the non-specific immune system. The protein demonstrates a broad spectrum of properties, including regulation of iron homeostasis, host defense against a broad range of microbial infections, anti-inflammatory activity, regulation of cellular growth and differentiation and protection against cancer development and metastasis. Representative sequences include UniProtKB: P02788 and Entrez Gene: 4057.

[0064] As used herein,“a-synuclein” or“alpha-synuclein” refers to either soluble, total, and/or oligomerized alpha synuclein. The human alpha-synuclein protein is encoded by the SNCA gene (Entrez gene: 6622; RefSeq mRNA: NM_000345, NM_00l 146054,

NM_00l 146055, NM_007308; RefSeq Protein: NP_000336, NP_00l 139526, NP_00l 139527, NP 009292). Although the function of a-synuclein is not well understood, studies suggest that it plays a role in maintaining a supply of synaptic vesicles in presynaptic terminals by clustering synaptic vesicles. Adverse intra- and extracellular effects of a-synuclein are believed to be central to the pathogenesis in Parkinson's disease and other disorders with Lewy body pathology in the nervous system. One of the physiological roles of a-synuclein relates to the regulation of neurotransmitter release at the presynapse, although it is still unclear whether this mechanism depends on the action of monomers or smaller oligomers. Oligomers or protofibrils of a- synuclein have been shown to impair protein degradation as well as the function of several organelles, such as the mitochondria and the endoplasmic reticulum. These oligomers may herein be referred to as“a-synuclein oligo.”“a-synuclein total” refers to the total a-synuclein content in the sample.

[0065] A“subject” intends, animals, mammals and humans. A mammal is a class of vertebrate animals whose females are characterized by the possession of mammary glands while both males and females are characterized by sweat glands, hair, three middle ear bones used in hearing, and a neocortex region in the brain. Non-limiting examples of a mammal include a simian, a murine, a bovine, an equine, a porcine or an ovine. In one aspect, a mammal is a mouse. In another aspect, a mammal is a rat. In yet another aspect, a mammal is a rabbit. In yet another aspect, a mammal is a human.

[0066] “Expression” as applied to a gene or a protein, refers to the production of the mRNA transcribed from the gene or the protein product encoded by the gene. In one aspect,

“expression” level is determined by measuring the expression level of a gene of interest for a given patient population, determining the median expression level of that gene for the population, and comparing the expression level of the same gene for a single patient to the median expression level for the given patient population. For example, if the expression level of a gene of interest for the single patient is determined to be above the median expression level of the patient population, that patient is determined to have high expression of the gene of interest. Alternatively, if the expression level of a gene of interest for the single patient is determined to be below the median expression level of the patient population, that patient is determined to have low expression of the gene of interest.

[0067] The term“interact” as used herein is meant to include detectable interactions between molecules, such as can be detected using, for example, a hybridization assay. The term interact is also meant to include“binding” interactions between molecules. Interactions may be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature.

[0068] The term“isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an“isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term“isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and

recombinant polypeptides.

[0069] As used herein, the term“nucleic acid” refers to polynucleotides such as

deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides. Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. For purposes of clarity, when referring herein to a nucleotide of a nucleic acid, which can be DNA or an RNA, the terms “adenosine,”“cytidine,”“guanosine,” and“thymidine” are used. It is understood that if the nucleic acid is RNA, a nucleotide having a uracil base is uridine.

[0070] The terms“oligonucleotide” or“polynucleotide,” or“portion,” or“segment” thereof refer to a stretch of polynucleotide residues which is long enough to use in PCR or various hybridization procedures to identify or amplify identical or related parts of mRNA or DNA molecules. The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

[0071] When gene or protein expression level“is used as a basis” for selecting a patient for a treatment described herein, the gene or protein expression level is measured before and/or during treatment, and the values obtained are used by a clinician in assessing any of the following: (a) probable or likely suitability of an individual to initially receive treatment(s); (b) responsiveness to treatment; (c) probable or likely suitability of an individual to continue to receive treatment(s); (d) adjusting dosage; (e) predicting likelihood of clinical benefits. As would be well understood by one in the art, measurement of the gene expression level in a clinical setting is a clear indication that this parameter was used as a basis for initiating, continuing, adjusting and/or ceasing administration of the treatments described herein.

[0072] The term“treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease. As used herein, to“treat” further includes systemic amelioration of the symptoms associated with the pathology and/or a delay in onset of symptoms. Clinical and sub-clinical evidence of“treatment” will vary with the pathology, the subject and the treatment.

[0073] “An effective amount” intends to indicate the amount of a compound or agent administered or delivered to the patient which is most likely to result in the desired treatment outcome. The amount is empirically determined by the patient’s clinical parameters including, but not limited to the stage of disease, age, gender, histology, and likelihood for recurrence.

[0074] “Administration” can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the agents are known in the art. Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non limiting examples of route of administration include oral administration, nasal administration, injection, topical application, intraperitoneal, intravenous and by inhalation. An agent of the present disclosure can be administered for therapy by any suitable route of administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0075] As used herein,“Parkinson’s disease” or“PD” refers to Parkinson’s disease and related disorders. PD is a progressive and degenerative brain disorder that ultimately affects an individual’s muscle movement. A hallmark feature of PD is the degeneration of the dopamine neurons in the substantia nigra pars compacta and the consequent striatal dopamine deficiency. PD affects an estimated 1 million Americans and the prevalence of PD increases with age.

Parkinson’s related disorders include but are not limited to symptoms of Parkinson’s disease, young onset Parkinson’s disease, supranuclear palsy, multiple system atrophy, lewy bodies disease or dementia with lewy bodies, and corticobasal degeneration. Symptoms of Parkinson’s disease and related disorders include but are not limited to tremor, bradykinesia (slowness of movement), rigidity, postural instability, problems with speech and voice, difficulty swallowing, freezing, excessive sweating, constipation, dry skin, mood changes, anxiety, depression, cognitive changes, loss of balance, intellectual changes, dementia, urinary and/or bowel incontinence, sleep disturbances, hyper sexuality, low sex drive, and frequent urination. Motor symptoms include but are not limited to tremors, rigidity, bradykinesia, postural instability, walking or gait difficulties, and vocal symptoms. [0076] Disease models for PD include but are not limited to toxin-based models such as MPTP mice, MPTP monkeys, 6-OHDA rats, Rotenone, Paraquat/maneb, and MET/MDMA; genetic models such as animals with altered a-synuclein, LRKK2, PINK1, PARKIN, DJ-l, ATP13A2, SHH, Nurrl, Engrailed 1, Pitx3, C-Rel-NFKB, MitoPark, Atg7, and/or VMAT2 genes.

[0077] Schirmer’ s test strips have traditionally been used for quantitative measurement of tear production. The standardized Schirmer test strip consists of a 5x35 mm strip of Whatman #41 filter paper; the paper has a notch located 5 mm from one end of the strip. The strips are commercially available from vendors such as Alcon Manufacturing, Ltd. The notched end of the strip can be rounded. As described in ET.S. Patent No. 5,006,310 a Schirmer tear test is performed by bending the strip at the notch (-120° bend). The rounded end of the Schirmer tear test strip is then inserted into the lower conjunctival sac of each eye. The eyes are then closed and the strip is progressively wetted by capillary action drawing up tears as they are produced. The distance the tear migration front has moved is measured after 5 minutes. The migration distance of the tears is measured from the notch of the strip as the zero point. Reading the test involves removing the strip from the eye and placing it against a scale graduated in millimeters.

15 mm of wetting in 5 minutes is considered normal. For tear production levels, it has been recommended that the tear migration front be measured as close to the 5-minute time mark as possible because the tear front will continue to migrate up the strip after the strip is removed from the eye. Thus, late readings give rise to results that are artificially high.

[0078] As used herein, the term "detectable label" intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the composition to be detected, e.g., polynucleotide or protein such as an antibody so as to generate a "labeled" composition. The term also includes sequences conjugated to the polynucleotide that will provide a signal upon expression of the inserted sequences, such as green fluorescent protein (GFP) and the like. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable. The labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified. A response that is simply detected generally comprises a response whose existence merely is confirmed, whereas a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property. In luminescence or fluoresecence assays, the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0079] Examples of luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research

Chemicals (6^ ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.

[0080] Examples of suitable fluorometric labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red. Other suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6^ ed.).

Descriptive Embodiments

[0081] It has been discovered herein that the expression level or activity of certain peptides and other types of biomarkers and tear flow are altered in the tears of patients having a

Parkinson’s related disorder. The altered expression level or activity of these biomarkers and tear flow can be used for diagnosis or prediction of Parkinson’s related disorders, methods of restoring the expression level or activity, and therefore, can be used to treat the Parkinson’s related disorder. Like the lacrimal gland, the salivary gland is an exocrine gland. It is responsible for producing fluid and protein for the oral cavity to aid in mastication and digestion. It is stimulated by a variety of neural pathways including cholinergic pathways. The secretion of specific proteins into saliva by changes in cholinergic stimulation may likewise be affected in Parkinson’s disease. The samples used in the methods described below can comprise either or both of tear and saliva.

Biomarkers

[0082] In some aspects, the methods described herein are based, at least in part, on determination of the expression or activity levels of a biomarker in a tear or saliva sample isolated from a subject, wherein the biomarker is selected from the group of a peptide, lipid, carbohydrate, metabolite, protein, enzyme or enzymatic activity, cell, cellular fragment, cellular vesicle, exosome, nucleic acid, RNA, DNA, gene, mRNA, miRNA, and cell-free DNA. In one aspect at least one biomarker level is determined or measured, in another at least two, or alternatively at least three, or alternatively at least 4, or alternatively at least 5, or alternatively at least 6, or alternatively at least 7, or alternatively at least 8, or alternatively at least 9, or alternatively at least 10, or alternatively at least 11, or alternatively at least 12, or alternatively at least 13, or alternatively at least 14, or alternatively at least 15 biomarker expression levels are determined or measured.

[0083] In some embodiments, at least one, or two, or three, or four, or five or six or more biomarkers of Parkinson’s disease or a Parkinson’s related disorder in the tear or saliva sample is selected from a-synuclein (oligomerized and/or total), DJ-l, CCL-2, uric acid, ST13, epidermal growth factor, apolipoprotein-Al, LRRK2 (leucine-rich repeat kinase 2), NURR1 (nuclear receptor-related 1 protein), Neuromelanin antibodiy, glutathione-SH, parkin, ubiquitin, BDNF, salslinol, homocysteine, osteopontin, MAOB, monamines, cytokines, platelet complex-l activity, 8-hydroxy-2’deoxyguanosine, tau, phosphor-tau, b-amyloid peptide 1-42, prostate specific antigen, VEGF, EGF, glucose, thyroid hormones, neurotransmitters and small molecules such as dopamine, serotonin, and homocysteine, inflammatory cytokines such as TNF-alpha, IL-l beta, IL-6, IL-8, IL-l 2, and IFN-gamma, lysozyme, lactoglobin, EGF, lipocalin-l, cystatin S100, MCP-l, lipocalin-l, heat shock protein, and complement proteins. In another aspect, the methods comprise the measurement of a-synuclein (oligomerized and/or total) and CCL2. In a further aspect, the methods further comprise the use of a Schirmer’s test to measure tear flow (see, e.g. Table 7).

[0084] In particular embodiments of the disclosed methods, the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder comprises a-synuclein. In some embodiments, the at least one biomarker is a ratio of oligomerized a-synuclein to total a- synuclein. In particular embodiments, the at least one biomarker is DJ-l, protein deglycase DJ-l, also known as Parkinson disease protein 7, is a protein which in humans is encoded by the PARK7 gene (Entrez gene: 11315, refSeq protein: NP 001116849, NP 009193). In particular embodiments, the at least one biomarker is CCL2. The chemokine (C-C motif) ligand 2 (CCL2) is also referred to as monocyte chemoattractant protein 1 (MCP1) (Entrez gene: 6347; Refseq Prot: NP 002973). In another aspect, the methods comprise the measurement of a-synuclein (oligomerized and/or total) and CCL2. In a further aspect, the methods further comprise the use of a Schirmer’s test to measure tear flow (see, e.g. Table 7).

[0085] In some embodiments, a-synuclein is detected or measured by ELISA. For example, the a-synuclein human ELISA kit is available from ThermoFisher Scientific (Cat. KHB0061) and Human Synuclein OLIGO kit (ajRoboscreen, Germany) may be used. In some

embodiments, levels of oligomeric and total a-synuclein can be measured by means of antibody detection. Antibodies specific for oligomeric a-synuclein include, for example, Millipore Sigma’s Anti-a-synuclein, anti-CCL2, antibody, oligomer-specific Syn33 (cat # ABN2265) and Agrisera Mouse anti -human a-synuclein| oligomer-specific (cat# AS 132718). Antibodies specific for total a-synuclein include, for example, Abeam’ s anti-a-synuclein antibody [MJFR1] (Cat. abl3850l).

[0086] In some embodiments of the disclosed methods, the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder is measured or determined by one or more methods of the group of: fluorometric analysis, Western blot, gel electrophoresis, ELISA, multiplex ELISA, enzymatic assay, mass spectrometry, protein array, Luminex assay, quantitative PCR, nucleic acid sequencing, nanoparticle tracking analysis (NTA), flow cytometry, dot blot analysis, immunohistochemistry, immunofluorescence, real time quaking- induced conversion and immunocytochemistry. In particular embodiments, the level of the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder is measured by ELISA or multiplex ELISA.

Diagnostic and Prognostic Methods

[0087] Provided herein are methods of analyzing a tear or saliva sample, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from a subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) determining that the subj ect is likely to have or develop Parkinson’ s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0088] Provided herein are methods of determining whether a subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder prior to the onset of motor symptoms, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear sample isolated from the subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) determining that the subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0089] In other aspects, provided herein are methods of identifying a subject at risk of developing Parkinson’s disease or a Parkinson’s related disorder, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from the subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) identifying the subj ect as at risk of developing Parkinson’ s disease or a Parkinson’ s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0090] In some aspects, provided herein are methods of determining whether a subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder prior to the onset of motor symptoms, the methods comprising, consisting of, or consisting essentially of: (a) comparing a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder measured in a sample isolated from a subject to a reference level or reference activity; and (b) determining that the subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity. The sample comprises a tear or saliva sample.

[0091] In other aspects, provided herein are methods of identifying a subject at risk of developing Parkinson’s disease, the methods comprising, consisting of, or consisting essentially of: (a) comparing a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder measured in a tear or saliva sample isolated from a subject to a reference level or reference activity; and (b) identifying the subject as at risk of developing Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0092] In some aspects, provided herein are methods for detecting at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from a subject suspected of having or developing Parkinson’s disease, the methods comprising, consisting of, or consisting essentially of: (a) collecting a tear or saliva sample from the subject; and (b) measuring a level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample. In some embodiments, the methods further comprise, consist of, or consist essentially of comparing the level or activity of the at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder to a reference level or reference activity.

[0093] Also provided herein are methods of monitoring the progression of Parkinson’s disease or a Parkinson’s related disorder, the methods comprising, consisting of, or consisting essentially of: (a) measuring the level or activity of at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder in a tear or saliva sample isolated from the subject; (b) comparing the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample to a reference level or reference activity; and (c) determining that the subject is likely to have or develop Parkinson’s disease or a Parkinson’s related disorder if the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in the sample differs from the reference level or reference activity.

[0094] In some embodiments of the disclosed methods, a tear or saliva sample is isolated from the subject using one or more of: a filter substrate, capillary tube, polyester fiber rod, microfluidic device, chip device, and impression cytology. In particular embodiments, the filter substrate is a Schirmer’s test strip. In some embodiments, the tear sample is isolated from one eye of the subject. In other embodiments, the tear sample is a pool of samples isolated from both eyes of the subject.

[0095] Collection of samples of tear can be done with methods known in the art and described briefly herein. Stimulation can be applied when needed, for example by eye-wash prior to sample collection. For the purpose of illustration only, tears can be collected onto a Schirmer’s test strip containing or embedded with a quantitatively-labeled substrate, e.g., an antibody coupled to a detectable marker such as a fluorometric label or substrate. The polypeptide or protein in the tear will react with the detectably labeled substrate allowing measurement of the expression level or activity level of the one first and/or second polypeptide. A saliva sample can be isolated using the same techniques.

[0096] In some embodiments of the disclosed methods, a tear sample is isolated using impression cytology. Aqueous tears come largely from the lacrimal gland but some materials are shed from the surface of the eye including the conjunctiva and corneal tissues. Without being bound by theory, biomarkers may reach the tears by shedding from conjunctiva and cornea if these tissues are damaged in Parkinson’s disease, for instance by formation of Lewy bodies in corneal nerves. To evaluate proteins in superficial cells of the ocular surface, impression cytology or gently pressing a cellulose acetate filter to the ocular surface to capture superficial cells which can then be evaluated on the filter by histological, immunological or molecular analyses.

[0097] In some embodiments of the disclosed methods, a tear or saliva sample is isolated or processed using a method suitable to isolate or enrich exosomes. Exosomes are small extracellular vesicles that are secreted by fusion of multivesicular intermediate endosomes carrying luminal vesicles by exocytosis, and are thought to be involved in intercellular communication. Exosomes have been identified in many extracellular fluids including tears, cerebrospinal fluid and saliva and are just now being characterized. Methods suitable to isolate or enrich exosomes include performing one or more of column chromatography, differential centrifugation, and nanoparticle tracking analysis prior to measuring the level of the one or more biomarkers in the tear sample. In some embodiments, the level of biomarker in the isolated exosomes is determined by dot blotting or western blotting. In some embodiments, one or more exosomes in the tear sample comprise a-synuclein. [0098] In some embodiments of the disclosed methods, the tear sample is an anesthetized tear sample. In other embodiments, the tear sample is an unaesthetized tear sample. Suitable agents for anesthetizing the eye to obtain an anesthetized tear sample include but are not limited to topical anesthetics. Nonlimiting examples of topical anesthetics include poparacaine, tetracaine, and lidocaine. In some embodiments, the tear sample is a reflex tear. Reflex tears are obtained without anesthetization.

[0099] In some embodiments of the disclosed methods, the methods further comprise, consist of, or consist essentially of measuring a total protein content in the tear or saliva sample isolated from the subject. In some embodiments, increased protein content relative to a reference sample is indicative of increase risk of having or developing Parkinson’s disease or a Parkinson’s related disorder.

[0100] Methods of measuring the expression level of polypeptide are known in the art.

Non- limiting examples include Western blot, gel electrophoresis, ELISA, multiplex ELISA, luminex assay, fluorometric analysis mass spectrometry, or protein array.

[0101] “Activity level” as applied to a protein, refers to the enzymatic activity level of the protein. Determination of the activity level can be made based on the capability of the protein to catalyze a chemical or biological reaction using one or more substrates. In one aspect, activity level is protease activity level which can be determined by the protein’s capability to hydrolyze a peptide sequence at a specific location.

[0102] Methods of measuring the activity level of a polypeptide are known in the art. For example, for polypeptides that have protease activities, their activities can be measured by their capability to hydrolyze a substrate peptide. Protease activity measuring kits for various proteases are commercially available from vendors such as Sigma-Aldrich (St. Louis, MO) and BioVision Inc. (Mountain View, CA).

[0103] Measurement of biomarker level or activity level can be made in comparison to reference levels. In some embodiments, reference levels include internal and/or external controls. Suitable internal controls can be a protein or other agent that is constantly present in the same sample from different mammals. Suitable internal controls can also be the total volume of samples collected, such as the total volume of tear fluid. Suitable external controls can also be used for determination of the protein expression level or activity level. Suitable external controls can be a subject that does not have, or does not appear to have PD or a related disorder. Suitable external controls can also be historical samples collected that have been proven to be from subjects that do not have the disease.

[0104] In some embodiments of the disclosed methods, the reference level or reference activity is selected from: (a) the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder in a subject or population that does not have Parkinson’s disease;

(b) a normal reference standard; and (c) a normal reference cutoff value.

[0105] In some embodiments of the disclosed methods, the level or activity of the biomarker of Parkinson’s disease or a Parkinson’s related disorder is elevated relative to the reference level or reference activity. In other embodiments, the level or activity of the biomarker of

Parkinson’s disease or a Parkinson’s related disorder is decreased relative to the reference level or reference activity.

[0106] As used herein, a subject that is“likely to have or develop Parkinson’s disease” is a subject that is more likely than not to develop Parkinson’s disease or a related disorder. In some embodiments, the subject is likely to have or develop Parkinson’s disease or a related disorder prior to the onset of motor symptoms.

[0107] As used herein, a subject that is“at risk of developing Parkinson’s disease” is a subject that has an elevated risk of developing Parkinson’s disease or a related disorder relative to a reference population. The reference population comprises at least one subject that is not the test subject. In some embodiments, the reference population is of a similar age, sex, weight ethnicity, and/or geographic origin as the subject. In some embodiments, the reference population is representative of the average risk of developing PD or a related disorder.

[0108] In one aspect, aiding in the diagnosis refers to providing confirmation to existing diagnosis. In another aspect, aiding in the diagnosis refers to using the diagnosis method in a panel of diagnosis methods, each method of the panel contributing to a final diagnosis. In yet another aspect, aiding in the diagnosis refers to that more than one of the markers recited herein are used in combination to make a diagnosis.

[0109] It is to be intended, although not always explicitly stated, that the methods of this invention can be further modified by measuring or determining the expression level or activity level of at least two, or alternatively at least three, or alternatively at least four, or alternatively at least five, or alternatively at least six, or alternatively at least seven, or alternatively at least eight of biomarkers of PD that are measured and compared to suitable controls, and a diagnosis can be made based on their overall expression level or activity level changes.

[0110] In another aspect of the above embodiments, the method further comprises diagnosing the mammal with a test selected from the group of Schirmer test, a slit-lamp examination, a radiological test, or a blood test. The results from this additional test can be combined with the methods provided in the above embodiment to assist diagnosis. In this aspect, the methods as disclosed herein aid in the diagnosis of autoimmune disease when combined with other known or yet to be developed diagnostic methods.

[0111] Schirmer’ s test determines whether the eye produces enough tears to keep it moist. This test is used when a person experiences very dry eyes or excessive watering of the eyes. Schirmer’ s test uses paper strips inserted into the eye for several minutes to measure the production of tears. This technique measures basic tear function. Applicants have adapted these filter paper strips in common usage to collect human tears for measurement of protein activities.

[0112] A slit-lamp examination uses an instrument, slit-lamp, to provide a magnified, three- dimensional view of the different parts of the eye. The slit lamp is an instrument consisting of a high-intensity light source that can be focused to shine a thin sheet of light into the eye. It is used in conjunction with a biomicroscope. The lamp facilitates an examination of the anterior segment, or frontal structures and posterior segment, of the human eye, which includes the eyelid, sclera, conjunctiva, iris, natural crystalline lens, and cornea. The binocular slit-lamp examination provides stereoscopic magnified view of the eye structures in detail, enabling anatomical diagnoses to be made for a variety of eye conditions.

[0113] In some embodiments, at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder comprises total a-synuclein, oligomerized a-synuclein, and/or phosphorylated a-synuclein. In particular embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is total a-synuclein. In some embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is oligomerized a-synuclein. In some embodiments, the biomarker is a ratio of oligomerized a-synuclein to total a-synuclein. In particular embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is phosphorylated a-synuclein. In some embodiments, the biomarker is a ratio of phosphorylated a-synuclein to total a-synuclein. In some embodiments, the ratio of oligomerized a-synuclein to total a-synuclein in increased in tear or saliva samples isolated from subjects likely to have or develop Parkinson’s or a Parkinson’s related disorder prior to the onset of motor symptoms. In some embodiments, the ratio of oligomerized a-synuclein to total a-synuclein in increased in tear or saliva samples isolated from subjects at risk of having or developing Parkinson’s or a Parkinson’s related disorder.

[0114] In some embodiments, at least one biomarker of Parkinson’s disease or a Parkinson’s related disorder comprises total CCL2. In particular embodiments, the biomarker of

Parkinson’s disease or a Parkinson’s related disorder is total a-synuclein in combination with CCL2, and further optionally in combination with a Schirmer’s test strip analysis. In some embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is oligomerized a-synuclein. In some embodiments, the biomarker is a ratio of oligomerized a- synuclein to total a-synuclein and CCL2. In particular embodiments, the biomarker of Parkinson’s disease or a Parkinson’s related disorder is phosphorylated a-synuclein and CCL2. In some embodiments, the biomarker is a ratio of phosphorylated a-synuclein to total a- synuclein and CCL2. In some embodiments, the ratio of oligomerized a-synuclein to total a- synuclein in increased in tear or saliva samples, as well as CCL2 isolated from subjects likely to have or develop Parkinson’s or a Parkinson’s related disorder prior to the onset of motor symptoms. In some embodiments, the ratio of oligomerized a-synuclein to total a-synuclein and CCL2 in increased in tear samples or saliva isolated from subjects at risk of having or developing Parkinson’s or a Parkinson’s related disorder.

[0115] In some embodiments of the disclosed methods, the ratio of oligomerized a-synuclein to total a-synuclein in subjects likely to have or develop Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5:1, about 2: l, about 2.5: 1, about 3: l, about 3.5: 1, about 4: 1, about 4.5: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 100: 1, or greater. In some embodiments of the disclosed methods, the ratio of oligomerized a-synuclein to total a-synuclein in subjects likely to have or develop Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 100, or about 1000 fold greater than a reference ratio.

[0116] In some embodiments of the disclosed methods, the ratio of oligomerized a- synuclein to total a-synuclein in subjects at risk of developing Parkinson’s disease or a related disorder is about 1.5: 1, about 2: l, about 2.5: l, about 3 : l, about 3.5: l, about 4: 1, about 4.5: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 100: 1, or greater. In some embodiments of the disclosed methods, the ratio of oligomerized a-synuclein to total a-synuclein in subjects at risk of developing Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 100, or about 1000 fold greater than a reference ratio.

[0117] In some embodiments of the disclosed methods, the ratio of phosphorylated a- synuclein to total a-synuclein in subjects likely to have or develop Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5: 1, about 2: 1, about 2.5: 1, about 3 : 1, about 3.5: 1, about 4: 1, about 4.5: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 100: 1, or greater. In some embodiments of the disclosed methods, the ratio of phosphorylated a-synuclein to total a-synuclein in subjects likely to have or develop Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 100, or about 1000 fold greater than a reference ratio.

[0118] In some embodiments of the disclosed methods, the ratio of phosphorylated a- synuclein to total a-synuclein in subjects at risk of developing Parkinson’s disease or a related disorder is about 1.5: 1, about 2: l, about 2.5: l, about 3 : l, about 3.5: l, about 4: 1, about 4.5: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 100: 1, or greater. In some embodiments of the disclosed methods, the ratio of phosphorylated a-synuclein to total a-synuclein in subjects at risk of developing Parkinson’s disease or a related disorder prior to the onset of motor symptoms is about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 100, or about 1000 fold greater than a reference ratio.

[0119] The methods are useful in the diagnosis of a mammal, an animal, or yet further a human patient. For the purpose of illustration only, a mammal includes but is not limited to a human, a simian, a murine, a bovine, an equine, a porcine or an ovine.

[0120] In some embodiments, the methods further consist of administering an effective amount of a suitable therapy to the subject, thereby treating the subject. Methods of Treatment

[0121] Provided herein are methods of treating a subject with Parkinson’s disease or a Parkinson’s related disorder, the methods comprising, consisting of, or consisting essentially of: (a) screening a tear or saliva sample isolated from a subject for a level or activity of at least one biomarker of Parkinson’s disease; and (b) treating the subject screened in step (a) by

administering a Parkinson’s disease or a Parkinson’s related disorder therapy. In some embodiments, the treatment is administered prior to the onset of motor symptoms in the subject.

[0122] In some aspects, provided herein are methods of treating a subject with Parkinson’s disease or a Parkinson’s related disorder, the method comprising, consisting essentially of, or consisting of administering a Parkinson’s disease or a Parkinson’s related disorder therapy to a subject; wherein a tear or saliva sample isolated from a subject has been screened for a level or activity of at least one biomarker of Parkinson’s disease. In some embodiments, the treatment is administered prior to the onset of motor symptoms in the subject.

[0123] In some embodiments of the disclosed methods, a subject whose tear or saliva sample was screened for the ratio of oligomerized a-synuclein to total a-synuclein is administered a Parkinson’s disease therapy if the ratio is about 1.5: 1, about 2: l, about 2.5: l, about 3: l, about 5: 1, about 4: 1, about 4.5: 1, about 5: 1, about 10: 1, about 15: 1, about 20: 1, about 30: 1, about 40: 1, about 50: 1, about 100: 1, or greater. In some embodiments of the disclosed methods, the ratio of oligomerized a-synuclein to total a-synuclein in the screened subject subjects is about 1.5, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 50, about 100, or about 1000 fold greater than a reference ratio. **

[0124] Non-limiting examples of suitable therapies for PD, PD related disorders, and symptoms thereof include but are not limited to administration of therapeutic medications and invasive and non-invasive surgical procedures. Surgical procedures to treat PD, PD related disorders, and symptoms thereof include but are not limited to deep brain stimulation, lesioning, thalamotomy, pallidotomy, and sub-thalamotomy. These therapies can be used separately or in combination to treat, or alternatively ameliorate the symptoms of autoimmune disease.

[0125] In some embodiments of the methods, treatment comprises treating dry eye symptoms in the subject. Moisture replacement therapies such as artificial tears may ease the symptoms of dry eyes (some patients with more severe problems use goggles to increase local humidity or have punctal plugs inserted to help retain tears on the ocular surface for a longer time). Cyclosporin (Restasis) is available by prescription to help treat chronic dry eye by suppressing the inflammation that disrupts tear secretion. Prescription drugs are also available that help to stimulate salivary flow, such as cevimeline and pilocarpine. Nonsteroidal anti inflammatory drugs can be used to treat musculoskeletal symptoms. Corticosteroids or immunosuppressive drugs can be prescribed to ameliorate symptoms. Disease-modifying antirheumatic drugs (DMARDs) such as methotrexate can also be helpful to relieve the patient of the symptoms. Multiple monoclonal antibodies are currently under investigation (Meijer et al. (2007) Clin Rev Allergy Immunol 32 (3):292-7). For patients with severe symptoms, punctal plugs can be inserted into the lower or upper tear drainage canals of the eyes.

[0126] Success of treatment can be determined or assessed by detecting improvement, alleviation, ablation, or amelioration of PD, PD related disorders, or one or more symptoms of each thereof. For example, success can be determined by detecting an improvement in one or more of: tremor, bradykinesia (slowness of movement), rigidity, postural instability, problems with speech and voice, difficulty swallowing, freezing, excessive sweating, constipation, dry skin, mood changes, anxiety, depression, cognitive changes, loss of balance, intellectual changes, dementia, urinary and/or bowel incontinence, sleep disturbances, hyper sexuality, low sex drive, and frequent urination. In some embodiments, success does not depend on whether or not PD and/or the PD related disorder is considered to be“cured” or“healed” and whether or not all symptoms are resolved.

Kits

[0127] As set forth herein, the disclosure provides diagnostic methods for Parkinson’s disease and Parkinson’s related disorders. Accordingly, described herein are kits for performing these methods as well as instructions for carrying out the methods of this invention such as collecting tear or saliva and/or performing the screen, and/or analyzing the results, and/or administration of an effective amount of the suitable therapy.

[0128] Thus, in some aspects, provided herein are kits to diagnose Parkinson’s disease, determine whether a subject is likely to have or develop Parkinson’s disease prior to the onset of motor symptoms, identify a subject at risk of developing Parkinson’s disease, detect at least one biomarker of Parkinson’s disease, or treat a subject with Parkinson’s disease. The kits comprise, consist of, or consist essentially of one or more reagents for isolating a tear or saliva sample from a subject and at least one detection reagent that is specific for a biomarker of Parkinson’s disease. In some embodiments, the one or more reagents for isolating a tear sample comprise a filter substrate or a collection modality chip. In some embodiments, the reagents for isolating a tear sample comprise one or more of: a filter substrate, capillary tube, polyester fiber rod, microfluidic device, chip device, and impression cytology; and optionally an anesthetic suitable for use in an eye. In some embodiments, the kits further comprise a reference standard.

[0129] In some embodiments, at least one detection reagent that is specific for a biomarker of Parkinson’s disease is an antibody, antibody fragment, or nucleic acid.

[0130] In some embodiments, the kits further comprise an anesthetic suitable for use in an eye. In some embodiments, the anesthetic is a topical anesthetic. Non-limiting examples of topical anesthetics include poparacaine, tetracaine, and lidocaine.

[0131] In some embodiments, the kits comprise reference samples. The reference samples used in the diagnostic kits can be tears. Methods for preparing protein extracts are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized. A non-limiting illustrative example is the Schirmer’s test strip discussed above. In one embodiment, the test strip contains or is embedded with a quantitative substrate that is detectably labeled, such as a fluorometrically labeled antibody, for quantitative detection of the one or more polypeptides identified above. In some embodiments, the tear sample or saliva is eluted from the test strip.

[0132] The kits can include all or some of the reference standards, pre-determined cutoff values, positive controls, negative controls, reagents, probes and antibodies described herein for determining the protein expression level or activity level in the subject.

[0133] As amenable, these suggested kit components may be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components may be provided in solution or as a liquid dispersion or the like.

[0134] The methods and kits now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the disclosed methods, and are not intended to limit the invention.

Example 1 [0135] The Applicant tested the hypothesis that tear secretion is altered in PD patients to exhibit a characteristic or diagnostic profile, reflected in changes in the protein composition of tear fluid, which can be measured relatively easily, cost-effectively, and non-invasively. If these changes occur early in disease, these biomarkers may aid in early proper identification of PD patients. The ability to diagnose PD accurately in the pre-motor phase, prior to the extensive destruction of central nervous system neurons and classic motor manifestations, is essential for the search of effective disease-modifying and neuroprotective interventions for people with PD or at risk of developing PD. The types of biomarkers that Applicant screened are primarily the major proteins secreted into tear fluid by LGAC. In addition, Applicant will screen other proteins in tear fluid that are currently either being investigated as candidate biomarkers of PD, such as a-synuclein and its oligomers, or are indicative of a pro-inflammatory state, such as matrix metalloproteinase-9 (MMP-9) (4, 5).

Cell biology of tear secretion

[0136] Applicant has extensive expertise in characterizing the mechanisms of tear secretion from LGAC (6-12). LGAC are exocrine secretory cells that are the predominant source of proteins and fluid secreted in tears. The volume and composition of tears is under neural and hormonal control. Tear proteins, thought to serve protective and trophic functions for ocular surfaces such as the cornea, are secreted through a variety of intracellular secretory pathways. These intracellular secretory pathways are comprised of multiple types of regulated secretory vesicles (granules), the transcytotic pathway, and regulated lysosomal -like secretory granules (FIG. 2). Secretory proteins are sorted to regulated secretory granules from the trans-Golgi network likely via canonical secretory pathways. However, there is documented heterogeneity of secretory proteins within the population of regulated secretory granules (11, 12), consistent with the notion that LGAC may fine-tune their secretory profile of proteins in response to physiological or pathological stimuli. In addition, protein cargo may be secreted into tears via a vesicle-mediated transcytotic pathway, which also seems to be regulated by neurotransmitters that stimulate secretion, known as secretagogues (10, 11). Finally, LGAC appear to possess a population of lysosome-like secretory granules containing more lytic components that can be mobilized for secretion by secretagogues in a process that is not yet well -understood (9, 12).

[0137] These secretory pathways are differentially regulated mainly by parasympathetic cholinergic and sympathetic adrenergic stimulation via innervation of the lacrimal gland by central and peripheral nerves (FIG. 1) and can be mimicked in vitro by cholinergic and adrenergic agents (3). The volume of tear secretion can vary from a low basal rate (i.e., little cholinergic stimulation), to maximal output (i.e., maximal cholinergic stimulation). Thus, the profile of proteins secreted into tears may change under differing circumstances, in a way that would reflect the secretory pathways that are stimulated selectively in a physiological fashion, as well as those pathways that may be selectively inhibited or stimulated by disease, such as PD.

The goal of this proposal is to exploit these differences in the identification and characterization of protein biomarkers in tears of PD patients.

[0138] Tears as a novel fluid for biomarker identification in PD: Tear samples can be collected relatively easily, non-invasively, and relatively cost-effectively. In addition, of all of the biological fluids, tears are among the least complex with respect to their proteome (13).

[0139] Finally, PD patients exhibit symptoms that might influence the protein composition of tear fluid in a characteristic fashion: dry eye and altered blink rates (14, 15). However, tears have essentially not been extensively explored for PD biomarkers (13, 16). Tear flow

measurement and tear fluid sample collection will be made through collection of tears from both eyes through an anesthetized and an unanaesthetized Schirmer’ s test from each patient, as described below. The anesthetized Schirmer’ s test represents basal tear secretion, while the unanesthetized Schirmer’s test represents stimulated tear secretion (17). It is possible that changes in protein profile of PD patient tears will occur in either type of tear (18, 19). The process of collection with a Schirmer’s test strip engages the corneal nerves in a way that may provide additional stimulation of the lacrimal gland, evoking additional mobilization of secretory pathways.

Experimental

Example 1

[0140] Approaches used in studies on the characterization of the differential secretion of proteins from the lacrimal gland in health and disease to identify potential biomarkers for PD in patient tears were applied. Applicant has demonstrated such experience in the identification of the lysosomal protease, cathepsin S, as a biomarker in tears of Sjogren’s syndrome (SS) (20), an autoimmune disease of unknown etiology with ocular involvement that is not easily diagnosed in its early stages. [0141] The characterization of tear biomarkers for PD focuses initially in a directed fashion on secreted proteins in normal tear fluid, which are thought to reflect the overall health and function of the lacrimal gland. Applicant has previously characterized many of these proteins in tears from experimental animals as well as from patients, as they are the most straightforward to detect and measure biochemically by enzyme assays (FIG. 3), western blot (FIG. 4), or enzyme- linked immunosorbent assays (ELISA). In addition, the statistical analysis of power as well as the amount of tear protein available from each patient limits applicant to the characterization of a limited number of tear proteins for initial screening. The tear proteins that will be assayed are: beta-hexosaminidase; lactoferrin (FIG. 4); MMP-9; a-synuclein, a-synuclein oligomer, DJ-l and CCL-2.

[0142] Lactoferrin is a marker of overall lacrimal gland health and secretory function.

Lactoferrin is a secretory protein with antimicrobial and anti-inflammatory activities. It is a significant protein component of normal tears, with its abundance also indicative of general lacrimal gland function (18, 19, 22-24). Lactoferrin was one of the first proteins suggested to exhibit characteristic changes in tears of patients with dry eye, and is reduced in patients with different forms of dry eye (18, 23, 24), including mild evaporative dry eye (25) and autoimmune- mediated dry eye associated with SS (26, 27). The utility of a clinical test for tear lactoferrin was suggested in 1983 (22), while recently a point of care test for lactoferrin has been released to the market by Advanced Tear diagnostics®. Although low tear lactoferrin is a good predictor of dry eye of both non-SS and SS origin, it has been reported that tear LF is also reduced with age in subjects without dry eye (24). Lactoferrin will be measured using ELISA or Western blotting (FIG. 4)

[0143] MMP-9 is a marker for lacrimal gland inflammation. Proteins indicative of a local or systemic inflammation, such as MMP-9, may be in parallel with changes in innervation or inflammation of the lacrimal gland (4, 5). These proteins are also representatives of the multiple secretory pathways mentioned above that appear to be differentially stimulated by

parasympathetic cholinergic and sympathetic adrenergic neural inputs, as well as reflective of inflammatory changes in the gland associated with functional quiescence or decreased output, which may also occur in pre-motor PD patients. MMP-9 will be measured using ELISA.

[0144] a-synuclein is the most prominent protein found in the Lewy body and Lewy neurites which are the pathologic hallmarks of PD (28). Evidence suggesting that a-synuclein deposition occurs early in the course of PD has provided the rationale for the use of a-synuclein as a biomarker in PD (29, 30). Multiple studies have evaluated potential biopsable neural tissues in attempts to find new biomarkers for PD (31). While there has been success finding a- synuclein in the colon, the invasiveness of this biopsy site limits its practical use to a small patient cohort, typically retrospective research studies (32). More recently, less invasive sites have shown promise including the skin and salivary submandibular gland (33, 34). Preliminary studies in the literature suggest that saliva is a non-invasively acquired bio-fluid containing a- synuclein, and that lower a-synuclein levels might correlate with the severity of motor symptoms in PD, although these results did not meet the standard of statistical significance (35, 36). A recent study presented at the 2015 International Congress of Parkinson’s Disease and Movement Disorders confirmed that total a-synuclein is reduced in the saliva of PD patients; further this study reported that the oligomeric a-synuclein and the ratio of oligomeric a- synuclein to total a-synuclein was significantly increased in the saliva of PD patients. The total a-synuclein concentration was negatively correlated with oligomeric a-synuclein concentration, suggesting that monomeric a-synuclein may be consumed by the formation of a-synuclein aggregates (personal communication). Studies on cerebrospinal fluid have shown that a- synuclein total concentration is reduced in PD patients when compared to healthy controls, but oligomeric a-synuclein and the ratio of oligomeric a-synuclein to total a-synuclein is increased in PD patients (37-39).

[0145] a-synuclein has not been assayed in tears, representing a further novel feature of this example. Its appearance in saliva suggests that it may also be present in the tears of PD patients, given that the secretory apparatus of salivary glands is highly homologous to that of the lacrimal gland. Also, collection of tears may be advantageous relative to saliva for biomarker characterization, since tears lack the acute contamination intrinsic to saliva associated with eating, drinking, and smoking as well as the complications of a commensal flora that complicate the analysis of saliva. Applicant can measure total and oligomeric a-synuclein by commercially available ELISA-based kits. With the validity of detection of oligomeric a-synuclein currently under debate, the interpreted the results from assays for oligomeric a-synuclein can be evaluated with caution.

[0146] Power analysis, determination of sample size, and tear collection. Possible effect sizes to compare biomarkers between persons with and without PD were derived from: (1) published data (35, 36) comparisons of average salivary a-synuclein levels between PD patients and healthy controls and (2) comparison of average lactoferrin levels from this data comparing patients with SS versus other autoimmune disease. From these studies, the average effect size (mean difference between groups, divided by SD) was 0.76. The sample size required to detect this effect size, testing at a two-sided alpha level of 0.05 with 80% power, is 29 subjects per group (total sample size = 58).

[0147] Tears can be obtained from PD patients with established disease (early, moderate and advanced) recruited from the Parkinson’s Disease and Movement Disorder Center at the University of Southern California (USC). Prior to the formal analysis, tear samples from 10-15 patients and healthy control subjects can be obtained to optimize collection and processing of tear samples, and to also maximize the ability to measure all 4 potential biomarkers of interest in a single sample. Both male and female patients with PD can be tested. Although there is no strong gender preference affecting PD patients, relative to the overwhelming predominance of ocular symptoms associated with dry eye and autoimmune disorders such as SS in female patients, male and female patient tears are collected and analyzed separately for the best study rigor. Based on current studies of additional tear biomarkers in SS and rheumatic disease patients as well as the power analysis described above, it is anticipated that recruitment of 58 (29 female, 29 male) PD patients and 58 age- and gender-matched healthy controls will enable the identification of changes in tear proteins characteristic of the PD population as well as to identify potential combinations for further investigation.

[0148] For statistical analysis, values from each of the four potential biomarkers are natural log-transformed to achieve normality for statistical analyses involving correlations and comparisons of means. As tear biomarkers are measured from each eye, subjects contribute two values for each marker. To account for the correlated data, statistical comparisons of biomarker means among study groups are generalized estimating equations for correlated outcomes; an identity link function and an exchangeable correlation structure can also be used. Patient group comparisons on the biomarkers are adjusted for age. To evaluate the ability of each biomarker to discriminate between subjects with and without PD, logistic regression analyses can be performed; each subject will contribute one observation, using the average of each biomarker value across the two eyes. Each biomarker is first tested individually in a logistic regression model. Logistic regression results are summarized as odds ratios, with 95% confidence intervals; area under the receiver operating characteristic (ROC) curves are computed and tested for differences among models.

[0149] For the anesthetized Schirmer’s test, one drop of 0.5% Proparacaine (Bausch &

Lomb, Rochester, NY) is added to each eye and residual fluid will removed before Schirmer's strips are placed in the inferior fornix of each eye. Schirmer's strips are removed after 5 minutes, and the length of tear saturation can be recorded in mm (20). Proteins are eluted from the strips according to established methods, and the markers described above are analyzed.

[0150] Since the tear collection process should only be conducted once within a window of a few hours to avoid ocular irritation, patients recruited to the study can contribute the anesthetized Schirmer’s strips during the first visit and the unanesthetized Schirmer’s strips during the second visit, with an attempt to time the repeat visit and collection for each patient at the same time of day, making a single collection from a patient complete within a 3-4 month time frame.

Applicant anticipates no difficulty in securing two independent tear collections from patients in sequential visits, since Applicant has a highly stable repeating patient population who follow a regular quarterly visit schedule. Applicant can exclude PD patients under treatment with anticholinergics.

[0151] Other candidate biomarkers characterized in cerebrospinal fluid are neurofilament light chain, clusterin, and granins (proteins that are co-packaged into dopaminergic secretory granules) (13, 40). As well, observed changes in any of the initial biomarkers in PD patients would prompt further evaluation of related proteins in tears to expand knowledge of the characteristic profile; for instance, changes in MMP-9 suggest evaluation of additional pro- inflammatory cytokines while changes in beta-hexosaminidase or lactoferrin suggest evaluation of other tear proteins responsive to cholinergic stimulation. Applicant can stratify and expand the PD patient groups into those with early, intermediate and advanced disease to further understand the changes indicative of each stage. Identification of changes associated with early disease is most important for diagnostic purposes, increasing the potential for earlier diagnosis of patients with pre-motor and early stage disease.

[0152] Applicant can also screen in an unbiased fashion for changes in the tear proteome in

PD patients versus normal subjects through quantitative mass spectrometric analyses of proteins and peptides in tear fluid (41) or by using protein arrays. These approaches work best with binary changes in levels of potential biomarkers, i.e., a biomarker is present in one set of patients but is absent in the other.

Example 2

[0153] Applicant also screen in an unbiased fashion for changes in the tear proteome in PD patients versus normal subjects through quantitative mass spectrometric analyses of proteins and peptides in tear fluid (41) or by using protein arrays. These approaches work best with binary changes in levels of potential biomarkers, i.e., a biomarker is present in one set of patients but is absent in the other, because quantitation of levels of proteins and peptides in biological fluids by mass spectrometry and protein arrays is not very straightforward.

[0154] Non-motor features of PD occur years prior to motor dysfunction, and represent a well- suited platform to investigate for a possible biomarker. Lacrimal glands are highly innervated by cholinergic neurons, and tear fluid secreted by lacrimal glands is greatly stimulated by cholinergic neurons. The production, packaging and secretion of specific proteins into tears may be regulated by changes in nerve function to lacrimal glands. Analysis of any alteration in the secretion of proteins into tears identify a reliable and non-invasive biomarker for PD.

[0155] Tear samples from 55 PD patients of varying severity and 27 age- and gender- matched non-PD controls were collected and pooled from both eyes for analysis of a-synuclein, CC chemokine ligand 2 (CCL-2) and DJ-l (Parkinson’s disease protein 7) using a Human magnetic Luminex assay kit (R&D systems) and analysis of oligomeric a-synuclein using an Human a-synuclein oligo ELISA kit (MyBioSource), respectively.

[0156] Total a-synuclein decreased significantly in PD patients (423.12 ± 52.6 pg/mg tear protein) relative to healthy controls (703.61 ± 136.4 pg/mg tear protein) (p-value=0.05) in tears from patients acquired from Schirmer’s strips taken during an anesthetized Schirmer’ s test.

[0157] Oligomeric a-synuclein increased significantly in PD patients (1.45 ± 0.31 ng/mg tear protein) relative to controls (0.27 ± 0.07 ng/mg tear protein) (p-value= 0.0007). While detectable in tears, neither CCL-2 nor DJ-l varied between PD patients and non-PD controls

[0158] Total a-synuclein and oligomeric synuclein have potential to discriminate between tears of PD patients and healthy controls. To Applicant’s knowledge this is the first report of tear collection and protein analysis as a possible non-invasive, inexpensive and reliable biomarker for PD.

Example 3

[0159] Non-motor features of Parkinson's disease (PD) occur years before motor

dysfunction. Lacrimal glands are highly innervated by cholinergic neurons, while tear fluid secreted by lacrimal glands is greatly stimulated by cholinergic neurons. The production, packaging and secretion of specific proteins into tears may be regulated by early changes in nerve function to lacrimal glands. Analysis of alterations in the secretion of proteins into tears may identify reliable and non-invasive biomarkers for PD at different stages of the disease. The aim of this Example was to evaluate whether tear protein composition differs in individuals with PD versus people without PD.

[0160] Tear samples from 60 diagnosed PD patients of varying severity and 30 age- and gender-matched non-PD controls were collected utilizing an anesthetized Schirmer’s test, eluted, and pooled from both eyes for analysis a-synuclein and matrix metallopeptidase 9 (MMP9) were measured using a human magnetic luminex assay kit (R&D systems) while lactoferrin (LF) was measured using a human lactoferrin ELISA kit (Abeam). Oligomeric a-synuclein was measured using a human a-synuclein oligo ELISA kit (My BioSource). Total protein concentration was measured using the Bio-Rad assay (Bio-Rad). Results are presented as mean ± SEM.

[0161] Total a-synuclein decreased significantly in PD patients (423.81 ± 50.7 pg/mg tear protein) relative to healthy controls (695.71 ± 125.8 pg/mg tear protein) (p-value=0.05) in tears from patients acquired from Schirmer’s strips taken during the anesthetized Schirmer’s test. However, oligomeric a-synuclein increased significantly in PD patients (1.38 ± 0.29 ng/mg tear protein) relative to healthy controls (0.42 ± 0.16 ng/mg tear protein) (p-value= 0.005). While detectable in tears, neither MMP9 nor LF varied significantly between PD patients and controls. Total protein concentration was elevated significantly in PD patients (3.91 ± 0.23 mg/ml) relative to healthy controls (2.96 ± 0.22 mg/ml) (p-value=0.004).

[0162] Total a-synuclein and oligomeric synuclein may have the potential to discriminate between tears of PD patients and healthy controls. To Applicant’s knowledge this is the first report of tear collection and protein analysis as a possible non-invasive, relatively inexpensive and reliable biomarker for PD.

Example 4

[0163] This example explores the identity of potential diagnostic biomarkers of Parkinson’s disease in the tear film with particular focus on anesthetized tears, unanesthetized tears, and longitudinal stability.

[0164] Biomarkers collected include a-synuclein (total and oligomeric form): PD biomarker. MMP-9: marker of ocular surface inflammation. Lactoferrin: marker of lacrimal gland secretory function. Beta-hexosaminidase: marker of lacrimal gland secretory function. CCL-2: marker of inflammation found to be elevated in CSF samples from PD patients. DJ-l : Parkinson’s disease protein 7 (PRK7) associated with early onset PD.

[0165] Assay modality originally proposed as a combination of ELISA, Western blotting, and enzyme activity assays.

[0166] Limitations in material and collection conditions led to development of customized multiplex ELISA plus two stand-alone ELISAs. Tear samples were collected and pooled from both eyes for collection and analysis. Total number of patients/ controls collected:

• 1 st Visits: Total of 105 collections (anesthetized tears)

o 36 controls and 75 patients

• 2^n<^ Visit Follow-Ups (unanesthetized tears) o I^st follow-up patient (#4) on 07/19/17 o Total of 26 follow-ups collected through 01/25/18 (5 controls and 24 patients)

[0167] Stability against proteolytic degradation was tested on tear proteins of interest stored “dry” on Schirmer’s strips (e.g., without eluting proteins with elution buffer). After storing the strips for 1- and 2- weeks at -80 °C, all proteins were incubated with elution buffer and assayed with ELISA or biochemical assays as appropriate. Proteins stored on Schirmer’s strips remained stable with levels comparable to those analyzed on day 0. Data are represented as percentages of the concentration measured on day 0. (FIG. 5).

[0168] Race distribution of PD patients and Healthy controls (HC) recruited during this study is shown in FIG. 6.

[0169] a-synuclein total levels in tears of PD patients was significantly lower when compared to those in tears of HC (p<0.00l). a-synuclein oligo levels in PD patient tears was significantly higher compared to those in HC (p=0.0l). The ratio of a-synuclein oligo/a- synuclein total was significantly increased in PD patients compared to HC (p=0.002) (FIG. 7). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11. An unpaired t- test with Welch’s correction was used to measure significance.

[0170] Total protein concentration was significantly increased in tears of PD patients when compared to that of healthy controls (HC) (p=0.005) (FIG. 8). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11. An unpaired t-test with Welch’s correction was used to measure significance.

[0171] Lactoferrin (LF) and matrix metalloproteinase-9 (MMP-9), markers of lacrimal acinar secretory function and ocular surface inflammation, respectively, and CCL2 and DJ-l, two putative PD biomarker proteins that are implicated in disease development and progression, were measured in tears of healthy controls (HC) and PD patients (FIG. 9). These proteins remained unchanged in PD patients when compared to healthy controls (HC) (p=0.46, p=0.56, p=0.44 and p=0.64, respectively). Data are presented as mean ± SEM and were derived from the cohort in FIG. 11. An unpaired t-test with Welch’s correction was used to measure significance.

[0172] Amount of tear wetting measured with the Schirmer’ s strip is shown in FIG. 10.

Strip values were pooled from both eyes. There were no significant differences in PD and healthy control (HC) subjects (p=0.65), suggesting that tear fluid production was not affected significantly in this cohort of PD patients. Data are presented as mean ± SEM and are from the cohort in FIG. 11. An unpaired t-test with Welch’s correction was used to measure significance. FIG. 11. shows a summary of demographics in PD patients and healthy controls (HC) for collection of anesthetized tear samples used for measurements in FIGS. 7-10.

[0173] Reflex (non-anesthetized) tears were similarly analyzed for a-synuclein oligo in a cohort of HC (n=84) and PD patients (n=84) (FIG. 17A). a-synuclein oligo levels in PD patient reflex tears was significantly higher compared to those in HC (p<0.000l). Male PD patients (n=48) had a-synuclein oligo levels in reflex tears significantly higher than male HD patients (n=45) (r<0.0001) (FIG. 17B). Female PD patients (n=36) had a-synuclein oligo levels in reflex tears significantly higher than female HD patients (n=39) (p<0.000l) (FIG. 17C). CCL2 levels were also analyzed in this cohort. CCL2 levels were normalized to total tear protein and log transformed. PD patients (n=84) had significantly higher CCL2 levels than HC patients (n=4) (p=0.003) (FIG. 18A). PD males (n=48) had significantly higher CCL2 levels than HD males (n=45) (p=0.0008) (FIG. 18B), this increase was greater in males than in the male + female group.

Example 5

[0174] Applicant further analyzed the biomarkers from the previous studies. Group differences of the biomarkers are summarized by median (25^th, 75^th percentiles) and tested for group differences with non-parametric Wilcoxon summed rank test.

Table 1. Demographic comparisons PD vs Control (n=l49 total)

1 p-value for age by independent t-test; p-value for Hispanic ethnicity and race by chi-square test

2 Age missing in 3 control subjects

Table 2. Biomarker comparisons PD vs Control (n=l49 total)

1 p-value by Wilcoxon rank sum test

Table 3. Biomarker comparisons PD vs Control: Area under the ROC (n=l49 total)

0.64

I DJ-1 0.485 (0.389, 0.582) 0.77

LILII»-^ 0.515 (0(417, 0.612) 0.77

! lactoferrin 0.515 ( 0.417, 0.614 ) 0.36

! a-synuclein

T t l

0.654 (0.566, 0.741 ) 0.03

i 0.702 (0.620, 0.785) j 0.001

oligomeric/total i 0.736 (0.656, 0.815)

j 0.002

Schirmer 0.533 (0.439, 0.628) 0.40 1

1 Logistic regression p-value; Note: AROC of 0.5 indicates chance discrimination

Table 4. Tear Flow/Schirmer’s Values and Protein Concentration in PD vs HC in Anesthetized Tears.

Tear flow is unaffected in PD patients relative to HC in anesthetized tears while tear protein is significantly increased.

[0175] FIG. 12 is a graphical depiction of the data of Tables 1-4. Each point on an ROC curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. The area under the ROC curve is a measure of how well a parameter can distinguish between two diagnostic groups (diseased/normal). An area of 1 represents a perfect test that can identify a disease individual; an area of .5 represents a test that is unable to distinguish a diseased individual.

[0176] The cohort of HC (n=84) and PD patients (n=84) discussed in example 4 had their reflex tears analyzed for the same proteins. The results are summarized in Table 5 (males and females) and Table 6 (males only). CCL2 levels are increased in PD patients as a whole, and more strongly in males. Schirmer’s test measurements (tear flow) are significantly decreased in PD patients, with a stronger effect in males. While the oligomeric synuclein increase alone gives greater specificity and sensitivity, when the use of oligomeric synuclein, CCL2 and the Schirmer’s test is combined (Table 7, last line) biomarker specificity is observed with a combined AUC of 0.85 in males and females and 0.89 in males. Table 5: Biomarker comparisons PD vs Control (n^:=T68 total; males and females)

Biomarker PD (n=84) Control (n=84) p-value ¹ AROC (95% p-value ⁴

Cl)

Concentration 3.30 (2.00, 3.12 (2.18, 0.95

4.56) ² 4.57)

CCL-2 109.68 (59.88, 66.47 (42.58, 0.003 0.632 (0.547, 0.11

214.99) 127.15) 0.717)

DJ-1 62.26 (38.66, 50.02 (34.84, 0.24 0.447 (0.359, 0.53

94.46) 88.56) 0.535)

MM P-9 11.18 (4.10, 10.26 (2.83, 0.23 0.554 (0.467, 0.06

33.99) 21.20) 0.641)

Lactoferrin 196.05 (120.38, 145.84 (50.09, 0.002 0.638 (0.554, 0.007

354.65) 243.74) 0.721)

a-synuclein

Total ³ 211.23 (103.30, 230.71 (141.65, 0.37 0.459 (0.369, 0.34

544.14) 540.61) 0.549)

Oligomeric 2.85 (1.14, 0.65 (0.13, <0.0001 0.800 (0.730, <0.0001

5.37) 1.14) 0.870)

Oligomeric/Total ³ 10.77 (2.80, 1.85 (0.46, <0.0001 0.744 (0.667, <0.0001

31.20) 5.05) 0.822)

Schirmer 18.0 (9.5, 27.0) 27.0 (15.0, 0.001 0.645 (0.562, 0.001

45.5) 0.729)

1 p-value by Wilcoxon rank sum

2 Numbers in table are median (25^th, 75^th percentiles)

3 N = 163

4 Logistic regression p-value

Table 6: Biomarker comparisons PD vs Control (n=93 total; males only)

Biomarker _ PD (n=48) Control (n=45) _ p-value ¹ AROC (95% Cl) p-value ⁴

Concentration 2.84 (1.82, 4.27) ² 3.20 (2.20, 4.50) 0.38

CCL-2 128.38 (76.09, 292.50) 61.65 (36.82, 113.98) 0.0008 0.703 (0.594, 0.07

0.812)

DJ-1 69.84 (37.66, 110.95) 47.94 (34.40, 67.83) 0.058 0.614 (0.498, 0.60

0.731)

MM P-9 14.31 (6.26, 39.33) 16.10 (5.53, 25.08) 0.57 0.534 (0.415, 0.18

0.653)

Lactoferrin 235.76 (140.00, 324.23) 125.19 (44.11, 243.40) 0.005 0.670 (0.559, 0.02

0.782)

Alpha-synuclein

Total ³ 211.23 (113.09, 575.38) 228.46 (112.81, 0.92 0.507 (0.385, 0.33

688.36) 0.628)

Oligomeric 3.65 (1.14, 7.38) 0.66 (0.27, 1.18) <0.0001 0.812 (0.720, <0.0001

0.905)

Oligomeric/Total ³ 12.22 (3.18, 40.70) 2.17 (0.67, 5.12) 0.0002 0.732 (0.627, 0.008

0.838)

Schirmer 18.0 (10.0, 26.5) 27.0 (15.0, 46.0) 0.006 0.665 (0.555, 0.005

0.776)

1 p-value by Wilcoxon rank sum

2 Numbers in table are median (25^th, 75^th percentiles)

3 N = 90

4 Logistic regression p-value

Table 7. PD vs Control: Area under the ROC, added contribution of biomarkers beyond oligomeric a-synuclein

Biomarker _ Total Sample (n=168) Male (n=93) Female (n=75) a-Synoiigo 0.800 (0.730, 0.870) 0.812 (0.720, 0.905) 0.782 (0.673, 0.892)

Added Biomarkers:

CCL2 0.826 (0.762, 0.890) 0.868 (0.795, 0.942) 0.781 (0.669, 0.892) p-value ² 0.13 0.08 0.81

Schirmer 0.831 (0.769, 0.892) 0.862 (0.789, 0.934) 0.788 (0.682, 0.895) p-value ² 0.12 0.09 0.82

CCL2 and Schirmer 0.847 (0.789, 0.905) 0.892 (0.827, 0.956) 0.789 (0.683, 0.896) p-value ² 0.049 0.039 0.80

lumbers in table are AROC (95% confidence interval)

² p-value for difference in two AROCs

Note: AROC of 0.5 indicates chance discrimination

[0177] This example finds a-synuclein in tears and differences in a-synuclein protein comparing PD patients to healthy controls. Without being bound by theory, the a-synuclein oligo/ a-synuclein total ratio in anesthetized tears may distinguish PD patients from Healthy Controls (ROC 0.75). Further, other tear proteins evaluated (DJ-l, CCL2) are unchanged in PD patient tears compared to healthy controls.

Equivalents

[0178] It should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be with the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0179] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0180] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0181] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control. REFERENCES

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Claims

WHAT IS CLAIMED:

1. A method of determining whether a subj ect is likely to have or develop

Parkinson’s disease prior to the onset of motor symptoms, the method comprising:

(a) measuring the level or activity of at least one biomarker of Parkinson’s disease in a tear or saliva sample isolated from the subject;

(b) comparing a level or activity of the biomarker of Parkinson’s disease in the sample to a reference level or reference activity; and

(c) determining that the subject is likely to have or develop Parkinson’s disease if the level or activity of the biomarker of Parkinson’s disease in the sample differs from the reference level or reference activity.

2. A method of identifying a subject at risk of developing Parkinson’s disease, the method comprising:

(b) comparing the level or activity of the biomarker of Parkinson’s disease in the sample to a reference level or reference activity; and

(c) identifying the subject as at risk of developing Parkinson’s disease if the level or activity of the biomarker of Parkinson’s disease in the sample differs from the reference level or reference activity.

3. A method of determining whether a subj ect is likely to have or develop

(a) comparing a level or activity of at least one biomarker of Parkinson’s

disease measured in a sample isolated from the subject to a reference level or reference activity; and (b) determining that the subject is likely to have or develop Parkinson’s disease if the level or activity of the biomarker of Parkinson’s disease in the sample differs from the reference level or reference activity.

4. A method of identifying a subj ect at risk of developing Parkinson’ s disease, the method comprising:

(a) comparing a level or activity of at least one biomarker of Parkinson’s

disease measured in a sample isolated from a subject to a reference level or reference activity; and

(b) identifying the subject as at risk of developing Parkinson’s disease if the level or activity of the biomarker of Parkinson’s disease in the sample differs from the reference level or reference activity.

5. A method for detecting at least one biomarker of Parkinson’s disease or a

Parkinson’s related disorder in a tear or saliva sample isolated from a subject, the method comprising:

(a) collecting the tear or saliva sample from the subject; and

(b) measuring a level or activity of at least one biomarker of Parkinson’s disease in the sample.

6. The method of claim 5, wherein the subject is suspected of having Parkinson’s disease or a Parkinson’s related disorder.

7. The method of claim 5 or 6, further comprising comparing the level or activity of the at least one biomarker of Parkinson’s disease to a reference level or reference activity.

8. A method of treating a subject with Parkinson’s disease prior to the onset of motor symptoms, the method comprising:

(a) screening a tear or saliva sample isolated from the subject for a level or activity of at least one biomarker of Parkinson’s disease; and

(b) treating the subject screened in step (a) by administering a Parkinson’s disease therapy.

9. A method of treating a subj ect with Parkinson’ s disease or a Parkinson’ s related disorder prior to the onset of motor symptoms, the method comprising administering a Parkinson’s disease therapy to a subject; wherein a tear or saliva sample isolated from the subject has been screened for a level or activity of at least one biomarker of Parkinson’s disease.

10. The method of any one of claims 1-4 or 7, wherein the reference level or

reference activity is selected from:

(a) the level or activity of the biomarker of Parkinson’s disease in a subject or population that does not have Parkinson’s disease;

(b) a normal reference standard; and

(c) a normal reference cutoff value.

11. The method of any one of claims 1-4, 7, or 10, wherein the level or activity of the biomarker of Parkinson’s disease is elevated relative to the reference level or reference activity.

12. The method of any one of claims 1-4, 7, or 10, wherein the level or activity of the biomarker of Parkinson’s disease is decreased relative to the reference level or reference activity.

13. The method of any one of the previous claims, wherein the level or activity of the at least one biomarker of Parkinson’s disease is measured by one or more methods of the group of: fluorometric analysis, Western blot, gel electrophoresis, enzyme-linked immunosorbent assay (ELISA), multiplex ELISA, enzymatic assay, mass spectrometry, protein array, Luminex assay, quantitative PCR, nucleic acid sequencing, nanoparticle tracking analysis (NTA), flow cytometry, dot blot analysis, immunohistochemistry, immunofluorescence, and immunocytochemi stry .

14. The method of claim 13, wherein the level of the at least one biomarker of

Parkinson’s disease is measured by ELISA.

15. The method of any one of the previous claims, wherein the tear or saliva sample is isolated from the subject using one or more of: a filter substrate, capillary tube, polyester fiber rod, microfluidic device, chip device, and impression cytology.

16. The method of claim 15, wherein the filter substrate is a Schirmer’s test strip.

17. The method of any one of the previous claims, wherein the level or activity of the at least one biomarker of Parkinson’s disease is measured by contacting the tear sample with a quantitative fluorometric antibody or a quantifiable label contained or embedded within a substrate test strip and measuring the antibody or label bound to the at least one biomarker of Parkinson’s disease.

18. The method of any one of the previous claims, wherein the tear sample is isolated from one eye of the subject.

19. The method of any one of claims 1-18, wherein the tear sample is a pool of samples isolated from both eyes of the subject.

20. The method of any one of the previous claims, wherein the tear sample is an anesthetized or unanesthetized tear sample.

21. The method of any one of the previous claims, wherein the tear or saliva sample comprises one or more exosomes.

22. The method of claim 21, further comprising performing one or more of column chromatography, differential centrifugation, and nanoparticle tracking analysis on the tear sample prior to measuring the level or activity of at the least one biomarker of Parkinson’s disease.

23. The method of any one of the previous claims, further comprising measuring a total protein content in the tear or saliva sample isolated from the subject.

24. The method of any one of the previous claims, wherein the at least one biomarker of Parkinson’s disease or Parkinson’s related disorder is a marker of response to one or more therapeutic regimes for a Parkinson’s disease or Parkinson’s related disorder.

25. The method of any one of the previous claims, wherein the at least one biomarker of Parkinson’s disease or Parkinson’s related disorder is a marker of disease progression.

26. The method of any one of the previous claims, wherein the at least one

biomarker of Parkinson’s disease or Parkinson’s related disorder comprises a- synuclein.

27. The method of claim 26, wherein the at least one biomarker of Parkinson’s disease or Parkinson’s related disorder comprises total a-synuclein, oligomerized a- synuclein, or phosphorylated a-synuclein.

28. The method of claim 27, wherein the biomarker of Parkinson’s disease is total a-synuclein.

29. The method of claim 27, wherein the biomarker of Parkinson’s disease is oligomerized a-synuclein.

30. The method of claim 27, wherein the biomarker of Parkinson’s disease is phosphorylated a-synuclein.

31. The method of any one of the previous claims, wherein the subject is a mammal.

32. The method of claim 31, wherein the subject is a human.

33. A kit to diagnose Parkinson’ s disease, comprising one or more reagents for isolating a tear sample from a subject and at least one detection reagent that is specific for a biomarker of Parkinson’s disease.

34. The kit of claim 33, wherein the one or more reagents for isolating a tear or saliva sample comprise a filter substrate or a collection modality chip.

35. The kit of claim 33 or 34, wherein the at least one detection reagent that is specific for a biomarker of Parkinson’s disease is an antibody, antibody fragment, or nucleic acid.

36. The kit of any one of claims 33 to 35, further comprising an anesthetic suitable for use in an eye.

37. The kit of claim 36, wherein the anesthetic is a topical anesthetic.

38. The kit of claim 37, wherein the topical anesthetic is proparacaine.

39. The kit of any one of claims 33 to 38, further comprising a reference standard.