WO1994001579A1

WO1994001579A1 - Assay for non-familial alzheimer's disease

Info

Publication number: WO1994001579A1
Application number: PCT/US1993/005881
Authority: WO
Inventors: Elizabeth R. Simons; Theresa A. Davies
Original assignee: Boston University
Priority date: 1992-07-09
Filing date: 1993-06-18
Publication date: 1994-01-20
Also published as: EP0649474A1; EP0649474A4

Abstract

A method of assaying for non-familial Alzheimer's disease in a live patient, by preparing washed platelets from the blood of the patient; inserting a pH probe in the platelets in the form of a fluorescent dye such as 2',7'-bis (carboxyethyl)- 5(6)-carboxyfluorescein; inhibiting alkalinization of platelet response by blocking Na+/H+ transfer across the platelet membranes with dimethyl amiloride; stimulating the platelets with α-thrombin so that the cytoplasm of the stimulated platelets shows a characteristic acidification response or change in cytoplasmic pH; measuring the cytoplasmic pH of the platelets following stimulation; and comparing the measured pH with the corresponding stimulated cytoplasmic pH in platelets from normal individuals.

Description

ASSAY FOR NON-FAMILIAL ALZHEIMER'S DISEASE

This invention was made during the course of work supported in part by the U.S. Government, and the government has certain rights in the invention. This invention relates to an assay, and more particularly to an assay for detecting the presence of Alzheimer's disease in living patients. Alzheimer's disease (hereinafter AD), the most common form of dementia in the elderly, is a neurodegenerative disease of the central nervous system, presently affecting an estimated four million people in the United States alone. Two types of AD are recognized. A first is Familial Alzheimer' s Disease (FAD) which is genetically linked, generally exhibits an early onset and may be caused by a faulty gene for the Alzheimer's precursor protein, βAPP, on chromosome 21 leading to an altered βAPP sequence. The other type of AD has a later onset, is more common, does not appear to be familial, and the βAPP is normal. AD is distinguished and defined by the presence of mature, senile plaques with a central core containing the 4.5 kDa amyloid β -protein (β/A4) which is obtained by proteolytic processing of its amyloid precursor protein (βAPP). The latter is synthesized in neural as well as non-neural mammalian cells and has a highly conserved sequence. The detection, in autopsy, of β/A4 plaques and of neurofibrillary tangles in the brain has, to date, been the primary definitive diagnosis of the disease. In living patients, diagnosis has only been approached by using a set of behavioral and psychological criteria, or made by assay that detects depletion of Alzheimer's precursor protein in spinal fluid as described by Van Nostrand et al., (1992) Proc. Nat. Acad. Sciences 89, 2551. It is known that relatively large quantities of amyloid precursor protein exist in the α- granules of human platelets together with growth factors and other proteins. While it has been shown that the platelet βAPP in non-familial Alzheimer's diseased patients has exactly the same sequence as that in normal controls, some abnormalities in platelets from such patients have been reported: altered membrane fluidity specific to platelets and attributed to altered internal membranes but not to abnormal phospholipid synthesis, and a report that platelets from such patients exhibit an enhanced Ca⁺⁺ response to thrombin, tentatively attributed to a large storage pool, a report with which the data presented in this specification do not agree. Platelets are one of several cellular components of blood and when appropriately stimulated, exhibit a response that includes, inter alia, a change in the membrane potential of the platelet, changes in the intra-cellular Ca^{+ +} and a secretion or release of granules and the granules' contents. For example, platelet activation in response to physiological stimuli such as α-thrombin (less than 4.5nM) has been extensively studied. A thrombin-induced increase in cytosolic [Ca⁺⁺], a membrane depolarization which co-occurs temporally with a rapid cytosolic acidification, followed by a slower alkalinization and an eventual degranulation, have all been documented. Cf. for example, Davies et al, (1987) Analyt. Biochem. 167, 118-123; Davies et al, (1988) Cytometry 9, 138. The products released from each of the three platelet granule types, dense granules, α- granules and lysosomal granules, have all been identified. The degranulation of platelets appears to be controlled by cytoplasmic pH as well as by [K⁺] concentrations. Cf. Greenberg-Sepersky and Simons, (1984) J. Biol. Chem. 259, 1502-1508. A principal object of the present invention is to provide an assay for Alzheimer' s disease in live patients. It is now been found that the response of platelets in blood from living individuals with AD, when stimulated by a physiological agonist, shows a characteristic abnormally high acidification response leading to a lower cytoplasmic pH. A quantitative and significant difference can be demonstrated between that cytoplasmic pH response and the platelet cytoplasmic pH response found in normal individuals. These differences are the basis for the present invention, a diagnostic assay for AD, since, as will be shown, such differences are not age related and do not appear to exist in platelets from a patient with FAD. The invention herein described and claimed generally then is a method of assaying for non-familial Alzheimer' s disease in a live patient, comprising the steps of collecting platelets from the blood of said patient and stimulating those platelets with a physiological agonist, typically α-thrombin, in order to invoke a characteristic acidification response in the platelet cytoplasm. Other known agonists include, but are not limited to, collagen, adenosine disphosphate, epinephrin, ristocetin and the like. The extent of that response immediately following stimulation is determined, preferably with a pH- sensitive probe such as fluorescent material which can be emplaced within the platelet cytoplasm. That measured extent of response is then compared with the corresponding stimulated acidification response in platelets from normal individuals, i.e- those apparently not suffering from non-familial Alzheimer's disease. Stimulation of human platelets by thrombin is known to be accompanied by, inter alia, a rapid thrombin-dose-dependent cytoplasmic acidification or change in the pH;., within a few seconds (e.g. 3 or 4 sec.), followed by a cytoplasmic alkalinization that maximizes quickly. There is a dose-dependent time until the maximal change occurs, that time varying from 8 seconds at 9.0 nM thrombin to 30 seconds at 0.45 nM thrombin. The thrombin dose dependence of such platelet stimulation has been described in Davies et al, (1987) Analyt. Biochem., 167, 118-123. Because the change in the cytoplasmic pR_m may be too fast to be readily quantified, one can prevent alkalinization by pretreatment of the platelets just before stimulation with an agent to block Na⁺/H⁺ exchange across the membranes of said platelets and thus inhibit or arrest alkalinization. Suitable agents or diuretics for this purpose include amiloride and derivatives thereof such as dimethyl amiloride. Cf. Davies et al, (1990), J. Biol. Chem. , 265, 11522-11526. The extent of cytoplasmic acidification can then be determined using any of a number of pH sensitive techniques such as with fluorescent probes including 6-carboxyfluorescein (hereinafter 6-CF), 2^*,7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (hereinafter BCECF) and the like. The esters of these fluorescein derivatives are non-fluorescent and, in this form, pass freely across the platelet plasma membrane. Inside the cell, the ester group is believed to be cleaved by nonspecific cytoplasmic esterases, generating an in situ pH-sensitive fluorescein probe. For purposes of the present invention, BCECF is preferred as a probe over 6-CF inasmuch as the former has a higher pK (6.9) and is pH sensitive to 7.8 or 7.9. Thrombin-induced activation of platelets was measured by signal transduction and degranulation for younger individuals (hereinafter Control) and age-matched older normal (AM) and Alzheimer's diseased patients (AD) as identified by the criteria developed by the Alzheimer's Disease and Related Dementia Association in conjunction with the National Institute of Aging (McKhann et al (1984) Neural. 34, 939). Neutrophils isolated from the same blood samples were used as controls. The neutrophils from all three groups exhibited identical responses (depolarization and oxidative burst) to the chemotactic peptide formyl methionine leucine phenylalanine, indicating that the platelet difference in AD patients are not attributable to any artifact in the patient's blood per se. Additionally, the platelet resting parameters, pH.-,^ and [Ca⁺⁺]_rejting, in the three groups were not significantly different. However, the maximum change in cytoplasmic thrombin-induced acidification, usually reached within the first few seconds (e.g. ca. 5 sec.) following stimulation, was significantly different (p<0.05) for platelets from AD patients than the corresponding change in cytoplasmic pH in platelets from normal individuals, but no differences were found in some of the other signal transduction components such as cytoplasmic Δ[Ca⁺⁺], depolarization or alkalinization. The diagnosis of AD was confirmed by autopsy in a number of patients studied that subsequently died. These data were obtained from the following examples. For the following examples, the materials and methods used were as follows: MATERIALS: Crude bovine topical thrombin was obtained from Parke-Davis division of Warner-Lambert Company, Morris Plains, N.J., and purified according the method described in Lundblad et al, (1975) Biochem. Biophys. Res. Commun. 66, 482-489. Sepharose 2B, a sugar polymer used in column chromatography and serving as a selective filter that permits the larger particles to pass through while retaining the smaller particles, was purchased from Pharmacia, Inc., Piscataway, N.J. The fluorescent probes, such as 2',7'-bis (carboxyethyl)- 5(6)- carboxyfluorescein-acetoxymethyl ester (hereinafter BCECF-AM) and 5(6)-carboxyfluorescein diacetate (6-CF), as well as the ionophore nigericin, were obtained from Molecular Probes, Inc. of Eugene, Oregon. 4-(2 hydroxyethyl)-l-piperazine ethane-sulfonic acid (hereinafter HEPES), was obtained from Sigma Chemical Co., St. Louis, Missouri. Apyrase was prepared according to the method described in Molnar, J. et al, (1961) Arch. Biochem. Biophys., 93, 353-363. Chemicals not specifically identified here were reagent grade. BUFFERS: Hepes buffer: 0.137 M NaCl, 0.0038 M HEPES, 0.0055 M D-glucose, 0.0033 M monobasic sodium phosphate. 0.0027 M KC1, 0.001 M MgCl₂-6H₂0, 0.15U/liter apyrase, pH 7.0 and 7.4. K⁺ -Hepes buffer: 0.140 M KC1, 0.0038 M HEPES, 0.0055 M D-glucose, 0.0033 M monobasic sodium phosphate, 0.001 M MgCl₂-6H₂0, 0.15U/liter apyrase, pH 6.6 - 7.6. PLATELETS: Fresh whole blood from volunteers was anticoagulated with 0.38% sodium citrate. Platelet-enriched plasma was obtained by centrifugation, and the platelets separated by passage over a Sepharose 2B column equilibrated with a Hepes buffer (pH 7.4) as described in Home et al (1981) Eur. J. Biochem. 120, 295-302. EXAMPLE I. Separate samples of washed platelets, pH 7.4, taken from the three groups, Control, AD and AM, were each incubated for 12 minutes at 37° C with 1 μM BCECF-AM prepared in dimethyl sulfoxide with final dimethyl sulfoxide concentration <0.1 %. After incubation, 200 mM ethylenediaminetetraacetic acid (hereinafter EDTA) was added to a final concentration of 3mM and the samples of platelets centrifuged for 10 minutes at 25° C at 3000 rpm. The supernatant was discarded and the resulting pellets were resuspended in 0.2 ml Hepes buffer, pH 7.4, with 3 mM EDTA. The resuspended pellets were then diluted to a final concentration of about 30xl0⁶ platelets/ml with either Hepes buffer, pH 7.4, for thrombin response measurements, or K⁺-Hepes buffer at varying pHs, immediately before use, for calibration curves. One minute prior to stimulation, an aliquot of platelets was treated with lO^M dimethylamiloride, α-thrombin (0.0025-0.05 U/ml, 0.45-9 nM) was then added, and the fluorescence at a constant emission wavelength (λ_^ = 530 nm) was initially recorded immediately for excitations of 450 and 500 nM. The fluorescence was continuously recorded for 30 seconds at 500 nm. All fluorescence measurements were taken on a Perkin-Elmer 650/1 OS spectrofluorometer equipped with a thermostated cuvette holder and stirrer. After maximal stimulation was achieved, emission fluorescence at 530 nm was recorded for excitation at 500 nm, the wavelength at which there is maximal pH dependence and at 450 nm, the isobestic wavelength at which there are no pH-induced changes, so that little or no change in 450 nm excited fluorescence accompanies stimulation. An aliquot of platelets was taken immediately before, and 30-60 seconds after, thrombin stimulation and each was centrifuged through silicone oil at 12,000g. The fluorescence of the supernatant was measured at each excitation wavelength and subtracted from that of the cell suspension to correct for the presence of extracellular dye. The ratio of fluorescence

before and after thrombin stimulation was determined. The change in ratio was calculated as follows:

F<_^(sus) - F^(SUD isoi (sus) - F^fsup)] = ΔR

LF₄₅₀(sus) - F₄₅₀(sup)Jthrom. LF₄₅₀(sus) - F₄₅₀(sup)I Emniitt

Abbreviations are as follows: sus for suspension, sup for supernatant, throm. for thrombin and ink. for initial. Intracellular pH was determined from a calibration curve prepared using the same ratio procedure as described by Thomas et al, (1979) Biochemistry 18, 2210-2218, correlating the difference at set pH values (pH 6.6 to 7.6) with ratios obtained when cells, incubated in K⁺- Hepes buffer are measured before and after the addition of nigericin (Davies et al, (1987) Analyt. Biochem. 167, 118-123). A relative rate of cytoplasmic pH change can also be determined by measuring the initial slope of the thrombin induced fluorescence change (cm/min), divided by the initial F₄₅₀ (i.e. initial pH-insensitive ) value for the probe (Davies et al (1990) J. Biol. Chem. 265, 11522). The rate and extent of cytoplasmic acidification for the three groups for different doses of thrombin is given in the following Table in which the rate of cytoplasmic acidification was ΔpH/Δt in reciprocal seconds, and the extent of acidification was measured as ΔpH. All data are presented as mean ±SEM(n) where n is the number of donors, and each (n) represents a single donor and includes 1-3 samplings per experiment. TABLE I (nM Thrombin) 0.45 0J) 8 9J) Rate of Cytoplasmic Acidification Control 0.029± .005(7) 0.044± .005(8) 0.054± .005(9) 0.070± .009(8) AD 0.040± .005(9) 0.047 ± .005(9) 0.070±.009(9) 0.068 ± .010(9) AM 0.032 ± .005(8) 0.043 ±.004(9) 0.054 ± .006(9) 0.069 ±.007(9) Extent of Cytoplasmic Acidification Control 0.037± .007(7) 0.038± .007(8) 0.046± .010(8) 0.071 ± .013(9) AD 0.065± .013(9) 0.069± .011(9) 0.083 ± .009(9) 0.121 ± .009(9) AM 0.046±.006(8) 0.052± .009(9) 0.052±.013(8) 0.091 ± .013(9) EXAMPLE II. Washed platelets, pH 7.4, taken from a normal patient, were incubated for 12 minutes at 37° C with 5 μM 5(6) carboxyfluorescein diacetate prepared in dimethyl sulfoxide with final dimethyl sulfoxide concentration <0.1 %. The incubated platelets were then processed as described in Example I and stimulated with the same dose of thrombin added to an aliquot of platelets. The fluorescence at a constant emission wavelength (λ_em = 518 nm) was initially recorded immediately for excitations of 464 and 492 nM. The fluorescence was continuously recorded for 30 seconds at 492 nm. After maximal stimulation was achieved, the excitation wavelength was recorded at the isobestic wavelength of 464 nm. The remainder of the procedure of Example I was then followed. The ratio of fluorescence (F^/F^) before and after thrombin stimulation was determined. The change in ratio was calculated as follows:

[F^sus) - F^-,(sup)1 - [F^fsus - F^_w(sup)l = ΔR LF^sus) - F₄₆₄(sup)Jthrom. LF^sus) - F^sup)].--!!.

Abbreviations have the same meaning as described in Example I. Intracellular pH was determined from a calibration curve prepared as described in Example III, and the data substantiated the data given in Table I for controls. EXAMPLE III. The method described in Example I was repeated, however, 2 μM nigericin, instead of thrombin was added to the platelets in order to prepare a pH calibration curve, and platelets were diluted in K⁺-Hepes at varying pH (6.6 to 7.6) instead of normal Hepes. The change in ratio was calculated as follows:

Fjoolsusi Fsoolsuβ) -500 .!(sus) - F v.(sup)1 = ΔR LF₄₅₀(sus) - F₄₅₀(sup) ige ≥rr.. L IFF₄.₅o(sus) - F₄₅₀(sup)init. Abbreviations have the same meaning as those described in Example I. The results were used to convert the ΔR values obtained in Examples I and II into actual pH values. Since certain changes may be made in the present invention without departing from the scope of the invention herein involved, it is intended that all matter contained in the above description be construed in an illustrative and not in a limiting sense.

Claims

What is claimed is: 1. Method of assaying for non-familial Alzheimer's disease in a live patient, comprising the steps of: collecting platelets from the blood of said patient; stimulating said platelets with a physiological agonist so that the cytoplasm of said platelets shows a characteristic acidification response; measuring the maximum extent of said response immediately following said stimulation; and comparing the measured maximum extent of said response with the corresponding stimulated acidification response in platelets from individuals not suffering from non-familial Alzheimer's disease.

2. Method of assaying as defined in claim 1 wherein said agonist is α-thrombin.

3. Method of assaying as defined in claim 1 wherein said agonist is selected from the group consisting of thrombin, collagen, adenosine diphosphate, epinephrin, ristocetin and mixtures thereof.

4. Method of assaying as defined in claim 1 wherein said step of measuring comprises so using a pH-sensitive probe that the change in cytosolic acidification upon stimulation can be determined by said probe.

5. Method of assaying as defined in claim 4 wherein said probe is a fluorescent material which can be emplaced within the platelet cytoplasm.

6. Method of assaying as defined in claim 5 wherein said material is 2' ,7'-bis (carboxyethyl)- 5(6)-carboxyfluorescein.

7. Method of assaying as defined in claim 5 wherein said material is 5(6)- carboxyfluorescein.

8. Method of assaying as defined in claim 5 wherein said platelets are incubated with an ester of a fluorescein derivative that can pass freely across the platelet plasma membrane and will be cleaved in situ by cytoplasmic esterases to generate said fluorescent material.

9. Method of assaying as defined in claim 8 wherein said ester is 2' ,7'-bis (carboxyethyl)- 5(6)-carboxyfluorescein acetoxymethyl ester.

10. Method of assaying as defined in claim 8 wherein said ester is 5(6)- carboxyfluorescein diacetate.

11. Method of assaying as defined in claim 1 including the step of treating said platelets with an agent to block Na⁺/H⁺ exchange across the membranes of said platelets.

12. Method of assaying as defined in claim 11 wherein said agent is dimethyl amiloride.

13. Method of assaying as defined in claim 11 wherein said agent is amiloride.

14. Method of assaying as defined in claim 11 wherein said agent is an amiloride derivative.