METHODS OF DIAGNOSING A SYNUCLEINOPATHY

1 . A method of diagnosing a synucleinopathy in a subject, the method comprising:

(a) determining the expression level of at least one nitrated alpha-synuclein protein selected from the group consisting of an alpha-synuclein protein nitrated at tyrosine residue 39 (nY39), nY125, nY133, and nY136 in an alpha-synuclein protein in a cerebrospinal fluid (CSF) sample obtained from the subject; and

(b) comparing the expression level of the at least one nitrated alpha-synuclein protein to a reference level of the at least one nitrated alpha-synuclein protein, wherein an increase in the expression level of the at least one nitrated alpha-synuclein protein in the CSF sample relative to the reference expression level of the at least one nitrated alpha-synuclein protein identifies a subject having a synucleinopathy.

2 . The method of claim 1 , wherein the expression level of at least two nitrated alpha-synuclein proteins selected from the group consisting of nY39, nY125, nY133, and nY136 in a CSF sample obtained from the subject are determined.

3 . The method of claim 1 or 2 , wherein a statistically significant increase in the expression level of the at least one nitrated alpha-synuclein protein in the CSF sample relative to the reference expression level of the at least one nitrated alpha-synuclein protein identifies a subject having a synucleinopathy.

4 . The method of any one of claims 1-3 , wherein the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the concentration of the at least one nitrated alpha-synuclein protein in the CSF relative to the reference level.

5 . The method of any one of claims 1-4 , wherein the expression level of the nitrated alpha-synuclein protein is calculated as the concentration of the nitrated alpha-synuclein protein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample.

6 . The method of any one of claims 1-5 , wherein the reference expression level is calculated as the concentration of the nitrated alpha-synuclein protein in a reference sample divided by the concentration of total alpha-synuclein in the reference sample.

7 . The method of claim 6 , wherein the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

8 . The method of claim 2 , wherein:

(a) the expression level of at least two nitrated alpha-synuclein proteins is an average of the at least two nitrated alpha-synuclein protein of the CSF sample;

(b) the reference expression level of the at least two nitrated alpha-synuclein proteins are an average of the at least two nitrated alpha-synuclein proteins of the reference; and

(c) the average of the at least two nitrated alpha-synuclein proteins of the CSF sample are compared to the average of the at least two nitrated alpha-synuclein proteins of the reference.

9 . The method of claim 2 , wherein the at least two nitrated alpha-synuclein proteins are nY39 and nY136.

10 . The method of claim 1 , wherein the nitrated alpha-synuclein is nY39.

11 . The method of claim 1 , wherein the nitrated alpha-synuclein is nY136.

12 . The method of claim 1 , wherein the one nitrated alpha-synuclein protein is nY39 and nY136.

13 . The method of any one of claims 1-12 , wherein the synucleinopathy is Parkinson's disease (PD) or a subtype of the disease thereof, dementia with Lewy Bodies (DLB), or Multiple-System Atrophy (MSA)

14 . The method of claim 13 , wherein the subtype of the disease thereof is a prodromal stage of PD.

15 . The method of any one of claims 1-14 , wherein the method further comprises administering to the subject an effective amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, and/or a neuroprotective agent.

16 . The method of any one of claims 1-6 and 8-15 , wherein the reference expression level is the mean level of expression of the at least one nitrated alpha-synuclein protein in a population of subjects.

17 . The method of any one of claims 1-16 , wherein the expression level is a protein expression level.

18 . The method of claim 17 , wherein the protein expression level is determined by single molecule detection, western blot, enzyme-linked immunoassay (ELISA), immunohistochemistry, or mass spectrometry.

19 . A method of identifying a subject having a synucleinopathy, the method comprising determining the protein level of alpha-synuclein comprising a nitration of Y39, Y125, Y133, and/or Y136 in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein the presence of nY39, nY125, nY133, and/or nY136 identifies the subject as having a synucleinopathy.

20 . The method of claim 19 , wherein the protein expression level is determined by single molecule detection, western blot, ELISA, immunohistochemistry, or mass spectrometry.

21 . A method of diagnosing a synucleinopathy in a subject, the method comprising determining the protein level of alpha-synuclein comprising a nitration of Y39, Y125, Y133, and/or Y136 in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein the presence of nY39, nY125, nY133, and/or nY136 identifies the subject as having a synucleinopathy.

22 . The method of claim 21 , wherein the protein expression level is determined by single molecule detection, western blot, ELISA, immunohistochemistry, or mass spectrometry.

23 . The method of any one of claims 19-22 , wherein the presence of nY39 identifies the subject as having a synucleinopathy.

24 . The method of any one of claims 19-23 , wherein the presence of nY136 identifies the subject as having a synucleinopathy.

25 . The method of any one of claims 19-24 , wherein the presence of nY39 and nY136 identifies the subject as having a synucleinopathy.

26 . The method of any one of claims 19-25 , further comprising administering to the subject a therapeutically effect amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, and/or a neuroprotective agent.

27 . A method of treating a subject having a synucleinopathy, the method comprising administering to the subject a therapeutically effective amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, and/or a neuroprotective agent, wherein the expression level of a nitrated alpha-synuclein protein having at least one of the following tyrosine residues Y39, Y125, Y133, and Y136 in an alpha-synuclein protein nitrated in a CSF sample obtained from the subject has been determined to be increased relative to a reference expression level of the at least one nitrated alpha-synuclein protein.

28 . The method of claim 27 , wherein a nitrated alpha-synuclein protein having at least two of the following tyrosine residues Y39, Y125, Y133, and Y136 in an alpha-synuclein protein nitrated in a CSF sample obtained from the subject has been determined.

29 . The method of claim 27 or 28 , wherein the expression level of a nitrated alpha-synuclein protein having the tyrosine residue Y39 nitrated in a CSF sample obtained from the subject has been determined.

30 . The method of any one of claims 27-29 , wherein the expression level of a nitrated alpha-synuclein protein having the tyrosine residue Y136 nitrated in a CSF sample obtained from the subject has been determined.

31 . The method of any one of claims 28-30 , wherein the expression level of a nitrated alpha-synuclein protein having the tyrosine residues Y39 and Y136 nitrated in a CSF sample obtained from the subject have been determined.

32 . A method of treating a subject having a synucleinopathy, the method comprising:

(a) determining the protein expression level of a nitrated alpha-synuclein protein comprising a nitration of Y39, Y125, Y133, and/or Y136 in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein an increase in the expression level of the nitrated alpha-synuclein protein relative to a reference expression level identifies the subject as having a synucleinopathy; and

(b) administering to the subject a therapeutically effective amount of a cognition-enhancing agent, an antidepressant agent, a dopamine promoter, an anti-tremor agent, and/or a neuroprotective agent.

33 . The method of claim 32 , wherein the nitrated alpha-synuclein protein comprises a nitration of Y39.

34 . The method of claim 32 or 33 , wherein the nitrated alpha-synuclein protein comprises a nitration of Y136.

35 . The method of any one of claims 32-34 , wherein the nitrated alpha-synuclein protein comprises a nitration of Y39 and Y136.

36 . The method of any one of claims 27-35 , wherein an increase is a statistically significant increase, wherein the statistically significant increase is an increase is at least a 10%, 25%, 50%, 100%, 2-fold, or 3-fold increase in the concentration of the at least one nitrated alpha-synuclein protein in the CSF relative to the reference level.

37 . The method of any one of claims 27-36 , wherein the expression level of the nitrated alpha-synuclein protein is calculated as the concentration of the nitrated alpha-synuclein protein in the CSF sample divided by the concentration of total alpha-synuclein in the CSF sample.

38 . The method of any one of claims 27-37 , wherein the reference expression level is calculated as the concentration of the nitrated alpha-synuclein protein in a reference sample divided by the concentration of total alpha-synuclein in the reference sample.

39 . The method of claim 38 , wherein the reference sample is a CSF sample from a normal subject that does not have a synucleinopathy.

40 . The method of any one of claims 19-39 , wherein the synucleinopathy is PD or a subtype of the disease thereof, DLB, or MSA.

41 . The method of claim 40 , wherein the subtype of the disease thereof is a prodromal stage of PD.

42 . The method of any one of claims 27-41 , wherein, after administering, the method further comprises determining the protein expression level of a nitrated alpha-synuclein protein comprising a nitration of Y39, Y125, Y133, and/or Y136 in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein a decrease in the expression level of the nitrated alpha-synuclein protein relative to the expression level before administration identifies the subject as having responded to treatment.

43 . The method of any one of claims 27-41 , wherein, after administering, the method further comprises determining the protein expression level of a nitrated alpha-synuclein protein comprising a nitration of Y39, Y125, Y133, and/or Y136 in an alpha-synuclein protein in a CSF sample obtained from the subject, wherein an increase in the expression level of the nitrated alpha-synuclein protein relative to the expression level before administration identifies the subject as in need of further treatment.

44 . A kit for diagnosing a synucleinopathy in a subject, the kit comprising:

(a) probes or antibodies capable of determining the level of one or more of a nitrated alpha-synuclein protein in a CSF sample from the subject; and optionally

(b) instructions for use of the probes or antibodies to determine the level of one or more nitrated alpha-synuclein protein in the CSF sample from the subject, wherein an increase in the level of one or more of the nitrated alpha-synuclein protein relative to a respective reference level indicates that the subject is likely to have a synucleinopathy.

45 . The kit of claim 44 , wherein the one or more nitrated alpha-synuclein protein is nY39 and nY136.

46 . The kit of claim 45 , wherein the one or more nitrated alpha-synuclein protein is nY39.

47 . The kit of claim 45 , wherein the one or more nitrated alpha-synuclein protein is nY136.

48 . The kit of any one of claims 44-47 , wherein the synucleinopathy is PD or a subtype of the disease thereof, DLB, or MSA.

49 . The kit of claim 48 , wherein the subtype of the disease thereof is a prodromal stage of PD.