Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1997-07-18
2001-10-09
Duffy, Patricia A. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C436S063000, C436S172000, C436S811000
Reexamination Certificate
active
06300085
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for diagnosing Alzheimer's disease. The technique utilizes newly discovered differences between cells from healthy donors and those with Alzheimer's disease. In one method, differences in the existence of functional potassium channels are assessed. In another method, differences in intracellular calcium levels in response to depolarization by a potassium channel blocker are assessed. In yet another method, differences in intracellular calcium levels in response to a chemical known to increase intracellular calcium levels by releasing calcium from intracellular stores are assessed. In another method, differences in the levels of a memory associated GTP-binding protein (Cp20) between cells from healthy donors and Alzheimer's patients are assessed. This invention also relates to the amino acid sequence for the Cp20 protein. In addition diagnostic indexes that distinguish Alzheimer's patients' cells from control cells are also provided.
BACKGROUND OF THE INVENTION
Alzheimer's disease is associated with extensive loss of specific neuronal subpopulations in the brain (Sims, N. R., et al. (1987)
Annals of Neurology
21:451), with memory loss being the most universal symptom. (Katzman, R. (1986)
New England Journal of Medicine
314:964). Alzheimer's disease has been linked to a genetic origin. (Schellenberg, G. D., et al. (1992)
Science
258:668; Li, G., et al. (1991)
Psychiatric Clinics of North America
14:267; St. George-Hyslop, P. H., et al. (1989)
Neurobiology of Aging
10:417; St. George-Hyslop, P. H., et al. (1987)
Science
235:885). Early-onset familial forms of the disease exhibit a genetic defect on chromosome 21. (St. George-Hyslop, P. H., et al. (1987)).
Cellular changes, leading to neuronal loss and the underlying etiology of the disease, remain unknown. Proposed causes include environmental factors, (Perl, D. P. (1985)
Environmental Health Perspective
63:149; Katzman, R. (1986)), including metal toxicity, (Perl, D. P., et al. (1980)
Science
208:297), defects in &bgr;-amyloid protein metabolism, (Shoji, M., et al. (1992)
Science
258:126; Joachim, C. L. and Selkoe, D. J. (1992)
Alzheimer Disease Assoc. Disord.
6:7; Kosik, K. S. (1992)
Science
256:780; Selkoe, D. J. (1991)
Neuron
6:487; Hardy, H. and Allsop, D. (1991)
Trends in Pharmacological Science
12:383), and abnormal calcium homeostasis and/or calcium activated kinases. (Mattson, M. P., et al. (1992)
Journal of Neuroscience
12:376; Borden, L. A., et al. (1991)
Neurobiology of Aging
13:33; Peterson, E., et al. (1989)
Annals of New York Academy of Science
568:262; Peterson, C., et al. (1988)
Neurobiology of Aging
9:261; Peterson, C., et al. (1986)
Proceedings of the National Academy of Science
83:7999).
Alzheimer's disease is well characterized with regard to neuropathological changes. However, abnormalities have been reported in peripheral tissue supporting the possibility that Alzheimer's disease is a systemic disorder with pathology of the central nervous system being the most prominent. (Rizopoulos, E., et al. (1989)
Neurobiology of Aging
10:717; Peterson (1986)).
Potassium channels have been found to change during memory storage. (Etcheberrigaray, R., et al. (1992)
Proceeding of the National Academy of Science
89:7184; Sánchez-Andrés, J. V. and Alkon, D. L. (1991)
Journal of Neurobiology
65:796; Collin, C., et al. (1988)
Biophysics Journal
55:955; Alkon, D. L., et al. (1985)
Behavioral and Neural Biology
44:278; Alkon, D. L. (1984)
Science
226:1037). This observation, coupled with the almost universal symptom of memory loss in Alzheimer's patients, led to the investigation of potassium channel function as a possible site of Alzheimer's disease pathology and to the current invention.
The so-called patch clamp technique and improvements thereof, have been developed to study electrical currents in cells. The method is used to study ion transfer through channels. To measure these currents, the membrane of the cell is closely attached to the opening of the patch micropipette so that a very tight seal is achieved. This seal prevents current from leaking outside of the patch micropipette. The resulting high electrical resistance across the seal can be exploited to perform high resolution current measurements and apply voltages across the membrane. Different configurations of the patch clamp technique can be used. (Sakmann, B. and Neker, E. (1984)
Annual Review of Physiology
46:455).
Currently, there is no laboratory diagnostic test for Alzheimer's disease. Therefore, there is a great need for a method to rapidly and clearly distinguish between Alzheimer's patients, normal aged people, and people suffering from other neurodegenerative diseases, such as Parkinson's, Huntington's chorea, Wernicke-Korsakoff or schizophrenia. Although some investigators have suggested that calcium imaging measurements in fibroblasts were of potential clinical use in diagnosing Alzheimer's disease (Peterson et al. 1986, 1988, supra), other researchers using similar cell lines and techniques, have shown no difference in calcium levels in Alzheimer's and normal control fibroblasts. (Borden et al. 1991, supra). Thus, the latter work refutes the findings of the former work.
The two proteins most consistently identified in the brains of patients with Alzheimer's disease have been &bgr;-amyloid and tau, whose roles in the physiology or pathophysiology of brain cells are not fully understood. However, there has been no diagnostic nor prognostic laboratory tests for Alzheimer's disease involving these or other proteins. Further, few other proteins have been identified which have physiological implications for Alzheimer's disease.
The methods for diagnosing Alzheimer's disease of the present invention using cells isolated from patients are needed and will greatly improve the now very complicated clinical diagnostic process for Alzheimer's disease. These methods are especially important because they are able to distinguish patients with Alzheimer's disease from patients with other neurodegenerative diseases.
SUMMARY OF THE INVENTION
The invention provides a method for assaying for Alzheimer's disease using cells isolated from patients. In one embodiment of the invention, the presence or absence of a particular potassium channel is measured. In a cell from a healthy control, potassium channels with slope conductances of 113 pS (picosiemens) and 166 pS are present and functional. In Alzheimer's cells, the 113 pS potassium channel is missing or nonfunctional.
In a second embodiment of the present invention, the effect of potassium channel blockers specific for the 113 pS potassium channel on intracellular calcium levels is assessed. In this method, intracellular calcium levels are found to be elevated in response to potassium channel blockers in normal cells, but not in cells from donors with Alzheimer's disease. The preferred potassium channel blocker is tetraethylammonium (“TEA”) at a final extracellular concentration of 100 mM. However, other potassium channel blockers which specifically block the 113 pS potassium channel may also be used. Furthermore, when TEA is used, other final concentrations of TEA may be used as long as the level of TEA causes intracellular calcium levels to be elevated in normal cells, but not in cells from donors with Alzheimer's disease.
In a third embodiment of the invention, sample cells from a patient are contacted with an activator of intracellular calcium release, in an amount sufficient to release calcium from intracellular storage sites, and the resulting increase in intracellular calcium levels is measured. In this embodiment, both normal cells and cells from Alzheimer's patients exhibit an increase in intracellular calcium; however, the increase in Alzheimer's patients is much greater. When an inositol-1,4,5,-trisphosphate (IP
3
) activator is used to increase intracellu
Duffy Patricia A.
Morgan & Finnegan , LLP
The United States of America as represented by the Department of
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