Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation
View Patent ↗A method of diagnosing Alzheimer's disease in a patient comprises determining whether the phosphorylation level of an indicator protein in cells of the patient after stimulus with an activator compound is abnormally elevated as compared to a basal phosphorylation level, the indicator protein being e.g. Erk1/2 and the activator compound being e.g. bradykinin.
1. A method of diagnosing Alzheimer's disease in an individual, the method comprising:
(a) contacting cells from the individual and from a non-Alzheimer's control subject with at least one inhibitor chosen from BiSM-1, PP1, 2-aminoethoxydiphenyl borate, and LY-294002;
(b) contacting cells from the individual and from the non-Alzheimer's control subject with at least one agent that triggers intracellular calcium release via the inositol 1,4,5-trisphosphate (IP-3) receptor, wherein the at least one agent is chosen from bradykinin, a bradykinin receptor agonist, and bombesin;
(c) measuring the amount of phosphorylation of at least one mitogen activated protein kinase (MAPK) protein in the individual's cells and in the non-Alzheimer's control subject's cells at one or more time points, and comparing the amount of phosphorylation of the at least one MAPK protein in the individual's cells with the amount of phosphorylation of the MAPK protein in the cells from the non-Alzheimer's control subject at the same one or more time points;
wherein increased phosphorylation of the at least one MAPK protein in the individual's cells compared to the non-Alzheimer's control subject's cells is indicative of Alzheimer's disease,
wherein the one or more time points are chosen from about 0.5 minutes, about 1 minute, about 2 minutes, about 2.5 minutes, about 5 minutes, about 10 minutes, about 20 minutes, and about 30 minutes after contacting the individual's cells and the non-Alzheimer's control subject's cells with the at least one agent.
2. The method of claim 1 , wherein the at least one MAPK protein is Erk1/2.
3. The method of claim 1 , wherein step (c) comprises measuring the amount of phosphorylation of the at least one MAPK protein at a single time point.
4. The method of claim 1 , wherein step (c) comprises measuring the amount of phosphorylation of the at least one MAPK protein in a first aliquot of the individual's cells and the non-Alzheimer's control subject's cells at a first time point and measuring the amount of phosphorylation in a second aliquot of the individual's cells and the non-Alzheimer's control subject's cells at a second time point.
5. The method of claim 1 , wherein the cells are from peripheral tissue.
6. The method of claim 1 , wherein the cells are skin fibroblasts.
7. The method of claim 1 , wherein step (c) comprises detecting phosphorylation in a lysate of the individual's cells and of the non-Alzheimer's control subject's cells.
8. The method of claim 1 , wherein step (c) is carried out in vitro.
9. The method of claim 1 , wherein step (c) comprises measuring with gel electrophoresis.
10. The method of claim 1 , wherein step (c) comprises measuring with Western blotting.
11. The method of claim 10 , wherein the Western blotting comprises using at least one anti-phospho-MAP kinase antibody.
12. The method of claim 1 , wherein the increased phosphorylation is determined by detecting an elevation in the amount of phosphorylated protein at a single time point.
13. The method of claim 1 , wherein the increased phosphorylation is determined by detecting an increase in duration of the phosphorylated protein.
14. The method of claim 1 , wherein the individual lacks clinical manifestations of Alzheimer's disease.
15. The method of claim 1 , wherein the increased phosphorylation is inhibited by contacting the individual's cells with the at least one inhibitor.
16. A method of diagnosing Alzheimer's disease in an individual, the method comprising:
(a) contacting skin fibroblast cells from the individual and from a non-Alzheimer's control subject with at least one inhibitor chosen from BiSM-1, PP1, 2-aminoethoxydiphenyl borate, and LY-294002;
(b) contacting skin fibroblast cells from the individual and from the non-Alzheimer's control subject with an amount of bradykinin effective for stimulating phosphorylation;
(c) measuring the amount of phosphorylated Erk1/2 in the individual's cells at one or more time points chosen from about 2 minutes, about 5 minutes, about 10 minutes, about 20 minutes, and about 30 minutes after contacting the individual's cells with the bradykinin by Western blotting using an antibody specific for phospho-Erk1/2;
(d) measuring the amount of phosphorylated Erk1/2 in cells from the non-Alzheimer's control subject at the same one or more time points as in step (c) after contacting the non-Alzheimer's disease subject's cells with the bradykinin by Western blotting using an antibody specific for phospho-Erk1/2;
wherein each amount of phosphorylated Erk1/2 in steps (c) and (d) is normalized to the total amount of protein present in the individual's cells and the non-Alzheimer's control subject's cells, respectively; and
(e) comparing the amount of phosphorylated Erk1/2 in the individual's cells with the amount of phosphorylated Erk1/2 in the non-Alzheimer's control subject's cells at the one or more time points;
wherein an increased amount of phosphorylated Erk1/2 in the individual's cells compared to the non-Alzheimer's control subject's cells is indicative of Alzheimer's disease; and
wherein the bradykinin induces an increase in the amount of phosphorylated Erk1/2 in the individual's cells compared to the non-Alzheimers control subject's cells that is reduced by the at least one inhibitor.