Discriminating Parkinson's disease from multiple system atrophy using alpha-synuclein PMCA
A method is provided for distinguishing between and/or diagnosing Parkinson's disease (PD) or multiple system atrophy (MSA) in a subject who is exhibiting symptoms associated with both PD and MSA. The method comprises: (A) contacting a biological sample obtained from the subject and comprising soluble, misfolded alpha-synuclein (αS) protein with a pre-incubation mixture comprising a monomeric αS substrate and an indicator to form an incubation mixture; (B) conducting an incubation cycle two or more times on the incubation mixture to form misfolded αS aggregates; (C) subjecting the incubation mixture to excitation and detecting via indicator fluorescence emission the misfolded αS aggregates; and (D) diagnosing the subject has having PD or MSA depending on the fluorescence emission intensity. In some aspects, the incubation cycles are conducted in the presence of a bead.
1. A method for differentiating a diagnosis of Parkinson's Disease (PD) from a diagnosis of Multiple System Atrophy (MSA) in a human subject, the method comprising:
(A) contacting a cerebrospinal fluid (CSF) or a brain homogenate (BH) sample (i) obtained from the human subject and (ii) comprising soluble, misfolded alpha-synuclein (αS) protein, with a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric αS substrate;
(2) a buffer composition;
(3) sodium chloride (NaCl); and
(4) a fluorescent protein aggregation indicator,
to form an incubation mixture;
(B) incubating and agitating the incubation mixture to form an incubated mixture comprising misfolded αS aggregates;
(C) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator, including:
(1) determining an initial average fluorescence (AF i ) of the incubated mixture prior to the formation of the misfolded αS aggregates;
(2) determining a standard deviation (SD i ) of the AF i ; and
(3) determining a maximum fluorescence (Fmax) of the incubated mixture after the formation of the misfolded αS aggregates; and
(D) diagnosing the human subject as having PD or MSA, where:
(1) if the Fmax is from about 22.2×SD i +AF i to 250×SD i +AF i , the patient is diagnosed as having MSA; and
(2) if the Fmax is above 250×SD i +AF i , the patient is diagnosed as having PD.
2. The method of claim 1 , further comprising contacting the misfolded αS aggregates with a protease after step D(2).
3. The method of claim 1 , further comprising evaluating the structure of the misfolded αS aggregates using circular dichroism after step D(2).
4. The method of claim 1 , further comprising evaluating the structure of the misfolded αS aggregates using cryo-electron tomography after step D(2).
5. The method of claim 1 , wherein the monomeric αS substrate comprises SEQ ID NO: 2.
6. The method of claim 5 , wherein the monomeric αS substrate is present in a concentration of about 1 mg/mL.
7. The method of claim 1 , wherein the buffer composition comprises piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES).
8. The method of claim 1 , wherein the buffer composition has a pH of about 6.5.
9. The method of claim 1 , wherein the NaCl is present in a concentration of about 500 mM.
10. The method of claim 1 , wherein the fluorescent protein aggregation indicator comprises thioflavin-T (ThT).
11. The method of claim 10 , wherein the ThT is present in a concentration of about 5 μM.
12. The method of claim 1 , further comprising contacting the misfolded αS aggregates with an oligothiophene after step D(2).
13. The method of claim 1 , further comprising evaluating the structure of the misfolded αS aggregates using Fourier-transformed infrared spectroscopy after step D(2).
14. The method of claim 1 , further comprising, after step D(2), contacting the misfolded αS aggregates to a cell and evaluating toxicity of the misfolded αS aggregates to the cell.
15. A method for differentiating a diagnosis of Parkinson's Disease (PD) from a diagnosis of Multiple System Atrophy (MSA) in a human subject, the method comprising:
(A) contacting a cerebrospinal fluid (CSF) or a brain homogenate (BH) sample (i) obtained from the human subject and (ii) comprising soluble, misfolded alpha-synuclein (αS) protein, with a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric αS substrate;
(2) a buffer composition;
(3) sodium chloride (NaCl); and
(4) a fluorescent protein aggregation indicator,
to form an incubation mixture;
(B) incubating and agitating the incubation mixture to form an incubated mixture comprising misfolded αS aggregates;
(C) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator, including determining a maximum fluorescence (Fmax) of the incubated mixture; and
(D) diagnosing the human subject as having PD or MSA, where:
(1) if the Fmax is from about 200 to about 1800 relative fluorescence units (RFU), the patient is diagnosed as having MSA; and
(2) if the Fmax is above 2000 RFU, the patient is diagnosed as having PD.
16. The method of claim 15 , further comprising contacting the misfolded αS aggregates with a protease after step D(2).
17. The method of claim 15 , further comprising evaluating the structure of the misfolded αS aggregates using circular dichroism after step D(2).
18. The method of claim 15 , further comprising evaluating the structure of the misfolded αS aggregates using cryo-electron tomography after step D(2).
19. The method of claim 15 , wherein the monomeric αS substrate comprises-SEQ ID NO: 2.
20. The method of claim 19 , wherein the monomeric αS substrate is present in a concentration of about 1 mg/mL.
21. The method of claim 15 , wherein the buffer composition comprises piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES).
22. The method of claim 15 , wherein the buffer composition has a pH of about 6.5.
23. The method of claim 15 , wherein the NaCl is present in a concentration of about 500 mM.
24. The method of claim 15 , wherein the fluorescent protein aggregation indicator comprises thioflavin-T (ThT).
25. The method of claim 24 , wherein the ThT is present in a concentration of about 5 μM.
26. The method of claim 15 , further comprising contacting the misfolded αS aggregates with an oligothiophene after step D(2).
27. The method of claim 15 , further comprising evaluating the structure of the misfolded αS aggregates using Fourier-transformed infrared spectroscopy after step D(2).
28. The method of claim 15 , further comprising, after step D(2), contacting the misfolded αS aggregates to a cell and evaluating toxicity of the misfolded αS aggregates to the cell.
29. A method for differential diagnosis of Parkinson's Disease (PD) from Multiple System Atrophy (MSA) in a human subject, the method comprising:
(A) contacting a cerebrospinal fluid (CSF) or a brain homogenate (BH) sample (i) obtained from the human subject and (ii) comprising soluble, misfolded alpha-synuclein (αS) protein, with a pre-incubation mixture, the pre-incubation mixture comprising:
(1) a monomeric αS substrate;
(2) a buffer composition;
(3) sodium chloride (NaCl);
(4) a fluorescent protein aggregation indicator; and
(5) a bead comprising Si 3 N 4 or borosilicate glass, to form an incubation mixture;
(B) incubating and agitating the incubation mixture to form an incubated mixture comprising misfolded αS aggregates;
(C) illuminating the incubated mixture with a wavelength of light that excites the fluorescent protein aggregation indicator, including:
(1) determining an average fluorescence (AF i ) of the incubated mixture prior to the formation of the misfolded αS aggregates;
(2) determining a standard deviation (SD i ) of the AF i ; and
(3) determining a maximum fluorescence (Fmax) of the incubated mixture after the formation of the misfolded αS aggregates; and
(D) diagnosing the human subject as having PD or MSA, where:
(1) if the Fmax is from about 9.6×SD i +AF i to 180×SD i +AF i , the patient is diagnosed as having MSA; and
(2) if the Fmax is above 180×SD i +AF i , the patient is diagnosed as having PD.
30. The method of claim 29 , wherein the bead is a Si 3 N 4 bead having a diameter of from about 2.3 mm to about 5 mm.
31. The method of claim 29 , further comprising contacting the misfolded αS aggregates with a protease after step D(2).
32. The method of claim 29 , further comprising evaluating the structure of the misfolded αS aggregates using circular dichroism after step D(2).
33. The method of claim 29 , further comprising evaluating the structure of the misfolded αS aggregates using cryo-electron tomography after step D(2).
34. The method of claim 29 , wherein the monomeric αS substrate comprises SEQ ID NO: 2.
35. The method of claim 34 , wherein the monomeric αS substrate is present in a concentration of about 0.3 mg/mL.
36. The method of claim 29 , wherein the buffer composition comprises piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES).
37. The method of claim 29 , wherein the buffer composition has a pH of about 6.5.
38. The method of claim 29 , wherein the NaCl is present in a concentration of about 500 mM.
39. The method of claim 29 , wherein the fluorescent protein aggregation indicator comprises thioflavin-T (ThT).
40. The method of claim 39 , wherein the ThT is present in a concentration of less than or equal to about 10 μM.
41. The method of claim 29 , further comprising contacting the misfolded αS aggregates with an oligothiophene after step D(2).
42. The method of claim 29 , further comprising evaluating the structure of the misfolded αS aggregates using Fourier-transformed infrared spectroscopy after step D(2).
43. The method of claim 29 , further comprising, after step D(2), contacting the misfolded αS aggregates to a cell and evaluating toxicity of the misfolded αS aggregates to the cell.