Journal of Nenrochemislry Raven Press, Ltd., New York 0 1992 International Society for Neurochemistry
Rapid Communication
Brain Levels of Microtubule-Associated Protein T Are Elevated in Alzheimer's Disease: A Radioimmuno-Slot-Blot Assay for Nanograms of the Protein Sabiha Khatoon, Inge Grundke-Iqbal, and Khalid Iqbal New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, U.S.A.
Abstract: The microtubule-associated protein 7,which stimulates the assembly of a-P tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of 7 and abnormally phosphorylated 7 were determined in brain homogenates of AD and age-matched control cases. A radioimmuno-slot-blot assay was developed, using a primary monoclonal antibody, Tau-I, and a secondary antibody, antimouse '251-immunoglobulin G. To assay the abnormally phosphorylated 7, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total T were about eightfold higher in AD (7.3 ? 2.7 ng/pg of protein) than in control cases (0.9 +- 0.2 ng/pg), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation-not a decrease in the level of 7-is a likely cause of neurofibrillary degeneration in AD. Key Words: Radioimmunoslot-blot assay-Microtubule-associated protein 7-Abnormal phosphorylation-Neurofibrillary tangles-Paired helical filaments. Khatoon S. et al. Brain levels of microtubule-associated protein 7 are elevated in Alzheimer's disease: A radioimmuno-slot-blot assay for nanograms of the protein. J. Neurochem. 59, 750-753 ( 1992).
1986; Nieto et al., 1990). However, the levels of normal and abnormally phosphorylated T in AD brain are not known. In the present study, we report (a) a radioimmuno-slot-blot assay, using anti-mouse '251-immunoglobulinG (IgG) as a secondary antibody, for measuring levels of normal and abnormally phosphorylated T in brain homogenates and (b) levels of T in AD and control brains.
MATERIALS AND METHODS Tissue source and brain homogenate preparations Frontal cortex from five histopathologically confirmed AD cases, two neurological control cases, and six normal aged cases obtained between 2 and 8 h postmortem and stored frozen at -75°C was used for this study. Approximately 1-2 g of the frozen tissue was chiseled 06thawed, and cleaned free of meninges and white matter. Gray matter was homogenized with a Dounce homogenizer at 4°C in 20 mM Tris (pH 7.4) containing EDTA and EGTA at 2.5 mM each (10 ml/g of tissue). The homogenate was stored in aliquots at -75°C until use. Smears of the homogenates ( 1 pl) stained with Congo red (Iqbal et al., 1984) revealed six to 16 tangles in AD and none to one tangle in control cases. The protein concentration of the homogenates was determined by the method of Bensadoun and Weinstein (1 976).
In Alzheimer's disease (AD), the most characteristic lesion is the accumulation of paired helical filaments (PHFs). The major protein subunit of PHFs is abnormally phosphorylated T (Grundke-Iqbal et al., 1986a,b; Iqbal et al., 1986, 1989; Lee et al., 1991). In a normal neuron, T promotes assembly, coassembles with tubulin into microtubules, and helps maintain the structure of microtubules (Weingartenet al., 1975;Drubin and Kirschner, 1986).This role of r is probably regulated by its phosphorylation because hyperphosphorylated T does not promote the assembly of microtubules in vitro (Lindwall and Cole, 1984). Lack ofmicrotubule assembly in AD brain in vitro indicates that abnormal phosphorylation of T is a cause of the breakdown of the microtubule system in this disease (Iqbal et al., ~~
Radioimmuno-slot-blot assay for normal and abnormally phosphorylated 7 Levels of normal and abnormally phosphorylated T in brain homogenates were determined with the monoclonal antibody (mAb) Tau-1 (Binder et al., 1985) with and without prior dephosphorylation (Grundke-Iqbal et al., 1986b). Antigens were applied to a nitrocellulose membrane (pore size, 0.22 pm), using a slot-blot unit (both from Schleicher and Schuell, Keene, NH, U.S.A.) that was attached to the house vacuum. This device allows the consecutive applica-
~~
Abbreviationsused; AD, Alzheimer's disease; BSA, bovine serum albumin; IgG, immunoglobulin G, mAb, monoclonal antibody; PBS, phosphate-buffered saline; PHF, paired helical filament; TBS, Tris-buffered saline.
Resubmitted manuscript received April 10, 1992; accepted April 22, 1992. Address correspondence and reprint requests to Dr. K. Iqbal at New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A.
750
T
751
LEVELS IN ALZHEIMER'S DISEASE
tion of 72 samples in 1- x 9-mm slots to the membrane. To keep both the concentration and the amount of protein constant for binding to the nitrocellulose membrane (see below), both standards and the test samples were diluted in bovine serum albumin (BSA) to a total protein concentration of 1 pg/250 pl of 50 mMTris (pH 7.6) and 200 mM NaCl [Tris-buffered saline (TBS)] and a cocktail of protease inhibitors (1 mMphenylmethylsulfonyl fluoride, 0.015 pM aprotinin, 1 pM leupeptin, and 1 pM pepstatin) and phosphatase inhibitors (1 mMNa3V04and 5 mM KF). These samples were then applied, with a slight vacuum, to the nitrocellulose membrane, which had been preequilibrated with TBS. For calibration curves, different amounts of isolated bovine T (Grundke-Iqbal et al., 19864, ranging from 0 to 10 ng, plus 1 pg of BSA were applied. After the samples had completely seeped through (- 3 rnin), the vacuum was turned up for 3 min. The unit was then disassembled,the membrane was allowed to air-dry at room temperature for 30 min, and the positions of the slots were marked on the reverse side of the membrane. The remaining protein-binding sites of the membrane were then blocked in 150 ml of freshly prepared 3% (wt/vol) BSA in TBS and 0.1% NaN, at room temperature on an oscillating shaker for 1 h. After two consecutive washes in 100 ml of TBS for 10 rnin each, all traces of buffer were carefully removed from the membrane by suction with a Pasteur pipette. The membrane was cut in half, with each half containing the same samples in triplicate. One-half was incubated for 18 h at 37°C with 15 ml of alkaline phosphatase (226 units/ml; calf intestine; Boehringer Mannheim, Indianapolis, IN, U.S.A.) in 100 mM Tris (pH 8.0) and protease inhibitors (see above). The other half ofthe membrane was incubated identically but with buffer lacking alkaline phosphatase. At the end of the incubation period, the membranes were each washed separately three times in 100 ml of 10 mM
0.2
0.4
0.6
0.8
pg Homogenate Protein
FIG. 2. Effect of alkaline phosphatase treatment of an AD brain homogenate on the linearity of the assay. Aliquots of frontal cortex homogenate from an AD brain containing 0.1,0.5, and 1.O jig of protein brought to 1 jig with BSA/250 jiI were used per slot. One curve (0-U)represents samples treated with alkaline phosphatase before incubation with mAb Tau-1; samples in the other curve (0-0)were untreated. All other details are the same as in Fig. 1. Each point is the average of three assays.
phosphate-buffered saline (PBS; pH 7.0) and blocked in 150 ml of 5% (wt/vol) BSA and 0.05% (vol/vol) Tween-20 in PBS for 1 h. The membrane strips were then suction-dried, overlaid with a 1:25,000 dilution of mAb Tau-1 (generously supplied by Dr. L. I. Binder) in blocking buffer, and incubated in a humid chamber at room temperature for 18 h. The membranes were washed three times in 200 ml of PBS containing 0.05% Tween-20 and overlaid, as above, with 15 ml per 2.5- X 9-inch blot of 'Z51-labeledantibody to mouse IgG (Amersham, Arlington Heights, IL, U.S.A.) at 0.05 pg/ ml ( 13-20 pCi/pg). After incubation for 2 hat room temperature, the secondary antibody was sucked off, and the blots were washed with PBS containing 0.05% Tween-20 (twice with 20 ml, 1 rnin each; three times with 200 ml, 30 rnin each; and three times with 200 ml, 1 h each). The blots were then air-dried, cut into individual strips with scissors, and counted for radioactivity in a Packard liquid scintillation counter, using 7.5 ml of Gammascint (National Diagnostics, Somerville, NJ, U.S.A.) per strip. Background counts were obtained by counting strips for radioactivity in triplicate from areas of the blot without any antigen.
RESULTS 0 0
2
6
8
10
ng tau
FIG. 1. Calibration curve for levels of T by radioimmuno-slot-blot assay. On each slot was applied bovine T , 0-10 ng/l pg of BSA, in 250 pl of TBS containing protease and phosphatase inhibitors. The compositionof the inhibitor cocktail was 0.015 p M aprotonin, 1 jiM leupeptin,1 jiM pepstatin,1 mM Na3V04,and 5 mM KF. The blots were blocked with TBS containing BSA and developed with mAb Tau-1 (diluted 1:25,000) as the primary antibody and 1251-labeled anti-mouse IgG as the secondary antibody (1 jig/20 ml; 13-20 PCi/jig). Bound 'z51-lgGwas quantified by liquid scintillation counting. The curve was obtained from three to five values at each concentration. One set of blots was treated overnight at 37°C with alkaline phosphatase [-226 units/ml of 100 mM Tris buffer (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride] before incubation with mAb Tau-1. No difference in amount of bound 1251-lgG'was seen, indicating that the bovine T was free of abnormally phosphorylated T .
A standard curve obtained with bovine T dilutions in TBS was linear up to 10 ng ofantigen11 pg of BSA tested (Fig. 1). To determine the amount of brain homogenate appropriate for the assay, 0.1,0.5, and 1 pg of an AD brain homogenate brought to 1 pg of protein with BSA were assayed. Before assaying, both protease and phosphatase inhibitors were added to the homogenate. The alkaline phosphatase-treated sample followed the linear curve up to 0.5 pg of homogenate protein, whereas the untreated sample revealed a concentration dependence up to 1.O pg of protein (Fig. 2). To determine the incubation time for the maximal dephosphorylation of abnormallyphosphorylated T with alkaline phosphatase, the time kinetics of the dephosphorylation were investigated (Fig. 3). Homogenate of an AD brain was prepared with the protease and phosphatase inhibitors, and slots were loaded with 250-p1 aliquots containing 0.35 pg of homogenate protein. After blocking with BSA, strips were incubated at 37°C for 0.0,0.5,2.5,6, and 18 h with alkaline J. Neurochem.. Vol. 59, No.2, 1992
"
S. KI-IATOON ET AL.
752
-0.1% (wt/wt) of total tissue protein. However, total T levels in AD brain were severalfold greater than in normal brain because abnormally phosphorylated T was present at high levels in the former but was not detected (
Rapid Communication
Brain Levels of Microtubule-Associated Protein T Are Elevated in Alzheimer's Disease: A Radioimmuno-Slot-Blot Assay for Nanograms of the Protein Sabiha Khatoon, Inge Grundke-Iqbal, and Khalid Iqbal New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York, U.S.A.
Abstract: The microtubule-associated protein 7,which stimulates the assembly of a-P tubulin heterodimers into microtubules, is abnormally phosphorylated in Alzheimer's disease (AD) brain and is the major component of paired helical filaments. In the present study, the levels of 7 and abnormally phosphorylated 7 were determined in brain homogenates of AD and age-matched control cases. A radioimmuno-slot-blot assay was developed, using a primary monoclonal antibody, Tau-I, and a secondary antibody, antimouse '251-immunoglobulin G. To assay the abnormally phosphorylated 7, the blots were treated with alkaline phosphatase before immunolabeling. The levels of total T were about eightfold higher in AD (7.3 ? 2.7 ng/pg of protein) than in control cases (0.9 +- 0.2 ng/pg), and this increase was in the form of the abnormally phosphorylated protein. These studies indicate that the abnormal phosphorylation-not a decrease in the level of 7-is a likely cause of neurofibrillary degeneration in AD. Key Words: Radioimmunoslot-blot assay-Microtubule-associated protein 7-Abnormal phosphorylation-Neurofibrillary tangles-Paired helical filaments. Khatoon S. et al. Brain levels of microtubule-associated protein 7 are elevated in Alzheimer's disease: A radioimmuno-slot-blot assay for nanograms of the protein. J. Neurochem. 59, 750-753 ( 1992).
1986; Nieto et al., 1990). However, the levels of normal and abnormally phosphorylated T in AD brain are not known. In the present study, we report (a) a radioimmuno-slot-blot assay, using anti-mouse '251-immunoglobulinG (IgG) as a secondary antibody, for measuring levels of normal and abnormally phosphorylated T in brain homogenates and (b) levels of T in AD and control brains.
MATERIALS AND METHODS Tissue source and brain homogenate preparations Frontal cortex from five histopathologically confirmed AD cases, two neurological control cases, and six normal aged cases obtained between 2 and 8 h postmortem and stored frozen at -75°C was used for this study. Approximately 1-2 g of the frozen tissue was chiseled 06thawed, and cleaned free of meninges and white matter. Gray matter was homogenized with a Dounce homogenizer at 4°C in 20 mM Tris (pH 7.4) containing EDTA and EGTA at 2.5 mM each (10 ml/g of tissue). The homogenate was stored in aliquots at -75°C until use. Smears of the homogenates ( 1 pl) stained with Congo red (Iqbal et al., 1984) revealed six to 16 tangles in AD and none to one tangle in control cases. The protein concentration of the homogenates was determined by the method of Bensadoun and Weinstein (1 976).
In Alzheimer's disease (AD), the most characteristic lesion is the accumulation of paired helical filaments (PHFs). The major protein subunit of PHFs is abnormally phosphorylated T (Grundke-Iqbal et al., 1986a,b; Iqbal et al., 1986, 1989; Lee et al., 1991). In a normal neuron, T promotes assembly, coassembles with tubulin into microtubules, and helps maintain the structure of microtubules (Weingartenet al., 1975;Drubin and Kirschner, 1986).This role of r is probably regulated by its phosphorylation because hyperphosphorylated T does not promote the assembly of microtubules in vitro (Lindwall and Cole, 1984). Lack ofmicrotubule assembly in AD brain in vitro indicates that abnormal phosphorylation of T is a cause of the breakdown of the microtubule system in this disease (Iqbal et al., ~~
Radioimmuno-slot-blot assay for normal and abnormally phosphorylated 7 Levels of normal and abnormally phosphorylated T in brain homogenates were determined with the monoclonal antibody (mAb) Tau-1 (Binder et al., 1985) with and without prior dephosphorylation (Grundke-Iqbal et al., 1986b). Antigens were applied to a nitrocellulose membrane (pore size, 0.22 pm), using a slot-blot unit (both from Schleicher and Schuell, Keene, NH, U.S.A.) that was attached to the house vacuum. This device allows the consecutive applica-
~~
Abbreviationsused; AD, Alzheimer's disease; BSA, bovine serum albumin; IgG, immunoglobulin G, mAb, monoclonal antibody; PBS, phosphate-buffered saline; PHF, paired helical filament; TBS, Tris-buffered saline.
Resubmitted manuscript received April 10, 1992; accepted April 22, 1992. Address correspondence and reprint requests to Dr. K. Iqbal at New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, NY 10314, U.S.A.
750
T
751
LEVELS IN ALZHEIMER'S DISEASE
tion of 72 samples in 1- x 9-mm slots to the membrane. To keep both the concentration and the amount of protein constant for binding to the nitrocellulose membrane (see below), both standards and the test samples were diluted in bovine serum albumin (BSA) to a total protein concentration of 1 pg/250 pl of 50 mMTris (pH 7.6) and 200 mM NaCl [Tris-buffered saline (TBS)] and a cocktail of protease inhibitors (1 mMphenylmethylsulfonyl fluoride, 0.015 pM aprotinin, 1 pM leupeptin, and 1 pM pepstatin) and phosphatase inhibitors (1 mMNa3V04and 5 mM KF). These samples were then applied, with a slight vacuum, to the nitrocellulose membrane, which had been preequilibrated with TBS. For calibration curves, different amounts of isolated bovine T (Grundke-Iqbal et al., 19864, ranging from 0 to 10 ng, plus 1 pg of BSA were applied. After the samples had completely seeped through (- 3 rnin), the vacuum was turned up for 3 min. The unit was then disassembled,the membrane was allowed to air-dry at room temperature for 30 min, and the positions of the slots were marked on the reverse side of the membrane. The remaining protein-binding sites of the membrane were then blocked in 150 ml of freshly prepared 3% (wt/vol) BSA in TBS and 0.1% NaN, at room temperature on an oscillating shaker for 1 h. After two consecutive washes in 100 ml of TBS for 10 rnin each, all traces of buffer were carefully removed from the membrane by suction with a Pasteur pipette. The membrane was cut in half, with each half containing the same samples in triplicate. One-half was incubated for 18 h at 37°C with 15 ml of alkaline phosphatase (226 units/ml; calf intestine; Boehringer Mannheim, Indianapolis, IN, U.S.A.) in 100 mM Tris (pH 8.0) and protease inhibitors (see above). The other half ofthe membrane was incubated identically but with buffer lacking alkaline phosphatase. At the end of the incubation period, the membranes were each washed separately three times in 100 ml of 10 mM
0.2
0.4
0.6
0.8
pg Homogenate Protein
FIG. 2. Effect of alkaline phosphatase treatment of an AD brain homogenate on the linearity of the assay. Aliquots of frontal cortex homogenate from an AD brain containing 0.1,0.5, and 1.O jig of protein brought to 1 jig with BSA/250 jiI were used per slot. One curve (0-U)represents samples treated with alkaline phosphatase before incubation with mAb Tau-1; samples in the other curve (0-0)were untreated. All other details are the same as in Fig. 1. Each point is the average of three assays.
phosphate-buffered saline (PBS; pH 7.0) and blocked in 150 ml of 5% (wt/vol) BSA and 0.05% (vol/vol) Tween-20 in PBS for 1 h. The membrane strips were then suction-dried, overlaid with a 1:25,000 dilution of mAb Tau-1 (generously supplied by Dr. L. I. Binder) in blocking buffer, and incubated in a humid chamber at room temperature for 18 h. The membranes were washed three times in 200 ml of PBS containing 0.05% Tween-20 and overlaid, as above, with 15 ml per 2.5- X 9-inch blot of 'Z51-labeledantibody to mouse IgG (Amersham, Arlington Heights, IL, U.S.A.) at 0.05 pg/ ml ( 13-20 pCi/pg). After incubation for 2 hat room temperature, the secondary antibody was sucked off, and the blots were washed with PBS containing 0.05% Tween-20 (twice with 20 ml, 1 rnin each; three times with 200 ml, 30 rnin each; and three times with 200 ml, 1 h each). The blots were then air-dried, cut into individual strips with scissors, and counted for radioactivity in a Packard liquid scintillation counter, using 7.5 ml of Gammascint (National Diagnostics, Somerville, NJ, U.S.A.) per strip. Background counts were obtained by counting strips for radioactivity in triplicate from areas of the blot without any antigen.
RESULTS 0 0
2
6
8
10
ng tau
FIG. 1. Calibration curve for levels of T by radioimmuno-slot-blot assay. On each slot was applied bovine T , 0-10 ng/l pg of BSA, in 250 pl of TBS containing protease and phosphatase inhibitors. The compositionof the inhibitor cocktail was 0.015 p M aprotonin, 1 jiM leupeptin,1 jiM pepstatin,1 mM Na3V04,and 5 mM KF. The blots were blocked with TBS containing BSA and developed with mAb Tau-1 (diluted 1:25,000) as the primary antibody and 1251-labeled anti-mouse IgG as the secondary antibody (1 jig/20 ml; 13-20 PCi/jig). Bound 'z51-lgGwas quantified by liquid scintillation counting. The curve was obtained from three to five values at each concentration. One set of blots was treated overnight at 37°C with alkaline phosphatase [-226 units/ml of 100 mM Tris buffer (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride] before incubation with mAb Tau-1. No difference in amount of bound 1251-lgG'was seen, indicating that the bovine T was free of abnormally phosphorylated T .
A standard curve obtained with bovine T dilutions in TBS was linear up to 10 ng ofantigen11 pg of BSA tested (Fig. 1). To determine the amount of brain homogenate appropriate for the assay, 0.1,0.5, and 1 pg of an AD brain homogenate brought to 1 pg of protein with BSA were assayed. Before assaying, both protease and phosphatase inhibitors were added to the homogenate. The alkaline phosphatase-treated sample followed the linear curve up to 0.5 pg of homogenate protein, whereas the untreated sample revealed a concentration dependence up to 1.O pg of protein (Fig. 2). To determine the incubation time for the maximal dephosphorylation of abnormallyphosphorylated T with alkaline phosphatase, the time kinetics of the dephosphorylation were investigated (Fig. 3). Homogenate of an AD brain was prepared with the protease and phosphatase inhibitors, and slots were loaded with 250-p1 aliquots containing 0.35 pg of homogenate protein. After blocking with BSA, strips were incubated at 37°C for 0.0,0.5,2.5,6, and 18 h with alkaline J. Neurochem.. Vol. 59, No.2, 1992
"
S. KI-IATOON ET AL.
752
-0.1% (wt/wt) of total tissue protein. However, total T levels in AD brain were severalfold greater than in normal brain because abnormally phosphorylated T was present at high levels in the former but was not detected (
