Brain Research, 507 (1989) 273-280 Elsevier
273
BRES 15141
Aberrant casein kinase II in Alzheimer's disease Devin S. Iimoto, Eliezer Masliah, Richard DeTeresa, Robert D. Terry and Tsunao Saitoh University of California, San Diego, School of Medicine, Department of Neurosciences, M-024, and The (?enterfi)r Molecular Genetics, La Jolla, CA 92093 (U.S.A.)
(Accepted 20 June 1989) Key words: Alzheimer's disease; Casein kinase; Neuronal degeneration; Phosphorylation
Abnormal protein phosphorylation has been identified in Alzheimer's disease (AD) for several proteins including a M~ 60,000 protein, a M r 86,000 protein and a microtubule-associated protein r. The M~ 86,000 protein is phosphorylated by protein kinase C, whereas protein kinases responsible for other aberrant phosphorylation reactions are not known. In addition to protein kinase C, another kinase, casein kinase II (CK-II), has now been shown to be aberrant in AD. The spermine-dependent CK-II activity is reduced by 84% in AD and the amount of CK-II as determined by its immunoreactivity on a Western blot is reduced by 63%. Furthermore, the distribution of CK-II in AD is altered. Although the neuronal cell body reacts well with CK-II antisera in the normal cortex, the non-tangle-bearing neurons in the AD cortex showed a 15-30% decrease in anti-CK-I1 immunoreactivity. The neurofibrillary tangles, on the other hand, stain very strongly with rabbit anti-CK-II and indicates that CK-II may be involved in the pathology of AD. The study of CK-II immunoreactivity for dementing diseases other than AD revealed a similar reduction, suggesting the CK-II involvement in the common process of neurodegeneration.
INTRODUCTION A l z h e i m e r ' s disease ( A D ) is a m a j o r cause of d e m e n t i a which affects 10-15% of the people over the age of 65 years 26. In A D , there is a characteristic loss of neurons from several cortical and subcortical areas of the brain, Terry and his colleagues have shown that there is an extensive loss of neocortical large neurons, while the small neurons and glial cells are relatively unaffected 27. Some of the remaining neurons contain neurofibrillary tangles ( N F T ) and some neuroterminals form neuritic plaques (NP) which characterize the pathology of this disease 2s. The causal relationship between neuronal death and these pathologies is yet to be d e t e r m i n e d . It is known that protein phosphorylation and dephosphorylation are i m p o r t a n t in regulating the activity of various enzymes 3"~'ls. The cascade of protein phosphorylation, often initiated by the binding of growth factors and n e u r o t r a n s m i t t e r s to their receptors and m e d i a t e d through the second messengers like Ca 2+, cAMP, and lipid metabolites, is also involved in the regulation of expression of certain genes. A compromise in these cascade reactions can adversely affect cellular functions and possibly lead to cell death as found in A D . Several investigators have o b s e r v e d an alteration in protein p h o s p h o r y l a t i o n in brain tissue of people with A D ~'4'~'25.
We have d e t e r m i n e d that in A D h o m o g e n a t e s , a M r 60,000 protein (P60) can be p h o s p h o r y l a t e d to a greater extent than in the control samples 23, while the activity of protein kinase C and a M r 86,000 protein (P86) phosphorylation are decreased 4. F u r t h e r m o r e , r, a protein which p r o m o t e s the polymerization of tubulin into microtubules, is o v e r p h o s p h o r y l a t e d in A D ~''~, and this form of r is also found associated with paired helical filaments ( P H F ) in both the N F T and the dystrophic neurites of senile plaques L3. Thus, the a b n o r m a l phosphorylation not only affects the proteins involved in cellular homeostasis, but also o t h e r proteins which are directly linked to the A D pathology, To further u n d e r s t a n d how a b e r r a n t phosphorylation events may be related to A D pathology, it is necessary to study the protein kinases and/or p h o s p h a t a s e s that may be responsible for the a b n o r m a l p h o s p h o r y l a t i o n . Because r is o v e r p h o s p h o r y l a t e d in A D and the human brain r sequence showed possible casein kinase phosphorylation sites 7, we l o o k e d at casein kinase (CK) activities in A D . We found that while the activity of CK-I is unaffected, the s p e r m i n e - d e p e n d e n t activity of CK-II and the concentration of CK-II as m e a s u r e d by immunoreactivity decreased in A D . F u r t h e r m o r e , the distribution of CK-II immunoreactivity on brain sections was altered suggesting that this kinase is affected in A D .
Correspondence: T. Saitoh, Department of Neurosciences, School of Medicine (M-024), University of California. San Diego, Ira Jolla, CA 92(193, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
274 MATERIALS AND METHODS Human brains, AD and control, were obtained from autopsy. The post-mortem times, 11.2 _+_6.1 (controls) and 12.8 + 7.9 (AD), were not different. Furthermore, the chronological age of the control samples, 64.8 + 7.4. was not significantly different from the AD sample, 68.5 _+ 6.0 years. The error is one standard deviation from the mean. The AD brains were confirmed by the presence of NP and NFT. Other brains used in the study were from Parkinson's disease confirmed by the presence of Lewy bodies either in the substantia nigra or the substantia innominata, Pick's disease confirmed by the presence of Pick bodies, Multi-infarct dementia confirmed by the presence of multiple infarcts, and Binswanger's disease (confirmed by the cerebrovascular lesions in the white matter). Brains from non-AD cases did not contain an appreciable amount of NP and NFT. The brains were homogenized essentially as described by Saitoh and Dobkins 24. Briefly, 0.5 g of frozen frontal cortex was sliced and placed in a centrifuge tube. All the following procedures were at 4°C. Homogenization buffer (5 ml, 10x volume, pH 8.0), consisting of 0.32 M sucrose, 5 mM HEPES, 5 mM benzamidine, 2 mM fl-mercaptoethanol, 3 mM EGTA, 0.5 mM MgSO 4, 10~uM sodium vanadate (meta), 0.01 mg/ml leupeptin, 0.005 mg/ml pepstatin A, and 0.1 mg/ml aprotinin, was added. The tissue was homogenized with a Polytron, using three 5-s bursts. The homogenate was centrifuged for 8 rain at 50(10 g. The supernatant was poured off and 2.5 ml (5x volume) of homogenization buffer was added to the pellet. The pellet was rehomogenized, two 5-s bursts, and centrifuged for 8 min at 5000 g. The two supernatants were combined and were ultracentrifuged 1 h at 100,000 g using a Ti 50 rotor. The supernatant, which contained the cytosolic fraction, was poured off while the pellet containing the membrane-cytoskeletal fraction was reconstituted into the homogenization buffer. The protein concentration was determined by a Lowry assay ~6. The samples were assayed for casein kinase activity as follows: in each tube were placed 5.0 I*g casein, 10 !,M ATP, 1.0 ~Ci [7--~2p]ATP, and assay buffer (0.1 M HEPES, pH 7.4, 10 mM MgSO4, 5 mM fl-mercaptoethanol). To this was added 6.5 gg of homogenate. The assay was either in the presence or absence of 1 mM spermine at 30 °C for 12 rain. In some assays, heparin, 150 units/ml, was added as an inhibitor of CK-II activity. The assay was terminated using 6.3 ul of 5x sample buffer. The 5x sample buffer consisted of 20% glycerol, 10% sodium dodecyl sulfate (SDS), 5% fl-mercaptoethanol, 0.31 M Tris, pH 6.8, 25 mM EDTA, and 0.19; bromophenol blue. The samples were analyzed on a 6.5-12.5% SDS-polyacrylamide gel eleetrophoresis (PAGE). The gel was run until the Bromophenol blue dye front was 0.5 cm from the bottom of the gel. The gel was then fixed in 10% acetic acid, 15~ isopropanol for 1 h and stained with 0.1% Coomassie blue R-250 in 10% acetic acid, 40% isopropanol. The gel was destained in 10c'i acetic acid, 15% isopropanol and then washed 1 h with distilled water. The gel was dried on a slab gel dryer (Bio-Rad) and then exposed to a X-Omat AR or RP film for 3-20 h at -70 °C. The film was developed on a Konica developer. After careful analysis of the film and the gel, the casein bands were excised and placed in 12-ml scintillation vials with 3 ml scintillation fluid and the radioactivity counted on a liquid scintillation counter (Tin Analytic) for 10 rain. The activity of CK-II in the above samples was also analyzed by assaying its activity on a native gel which contained no detergent. Thirty-five/~g of each sample were placed on a 5% native gel and the gel was run until the Bromophenol blue dye front was 0.5 cm from the bottom. The gel was electroblotted onto a piece of nitrocellulose paper. The blotting was done at 300 mA for 2 h. The blot was incubated 2 h with 1% casein (partially dephosphorylated) in phosphate-buffered saline (PBS) at room temperaturc. The blots were then incubated in 25 ml of assay buffer containing 5/*M ATP, 50 !~Ci [?,-32P]ATP, with or without 1 mM sperminc. In some experiments, heparin, 150 units/ml, was also added. The incubation was done at room temperature for 1 h. The blot was washed 3× with Tris-buffered saline (TBS) and then exposed to a X-Omat AR film for 20-24 h. The film was developed and the bands on the fihn werc
anatyzcd by a Laser l)ensitomcter (LKB, I Itroscan XL/. Undc~ these conditions, the intensity of bands ~m auloradiograms was between (I.7 and 3.0 absorbance units falling within the linear mngc of sensitivity of the film. The concentration of CK-II was determined by its immunodetcction on protein blots. Rabbit anti-calf thymus CK-II antiscrum was a generous gift from Dr. Michael E. Dahmus. This antibody detects all CK-II subunits (M~ - 44,000, 40,000, and 26,000) on Western blots and immunolocalizes CK-II in mouse fibroblasts specifically; staining was absorbed by a previous incubation of the antibody with purified CK-II
273
BRES 15141
Aberrant casein kinase II in Alzheimer's disease Devin S. Iimoto, Eliezer Masliah, Richard DeTeresa, Robert D. Terry and Tsunao Saitoh University of California, San Diego, School of Medicine, Department of Neurosciences, M-024, and The (?enterfi)r Molecular Genetics, La Jolla, CA 92093 (U.S.A.)
(Accepted 20 June 1989) Key words: Alzheimer's disease; Casein kinase; Neuronal degeneration; Phosphorylation
Abnormal protein phosphorylation has been identified in Alzheimer's disease (AD) for several proteins including a M~ 60,000 protein, a M r 86,000 protein and a microtubule-associated protein r. The M~ 86,000 protein is phosphorylated by protein kinase C, whereas protein kinases responsible for other aberrant phosphorylation reactions are not known. In addition to protein kinase C, another kinase, casein kinase II (CK-II), has now been shown to be aberrant in AD. The spermine-dependent CK-II activity is reduced by 84% in AD and the amount of CK-II as determined by its immunoreactivity on a Western blot is reduced by 63%. Furthermore, the distribution of CK-II in AD is altered. Although the neuronal cell body reacts well with CK-II antisera in the normal cortex, the non-tangle-bearing neurons in the AD cortex showed a 15-30% decrease in anti-CK-I1 immunoreactivity. The neurofibrillary tangles, on the other hand, stain very strongly with rabbit anti-CK-II and indicates that CK-II may be involved in the pathology of AD. The study of CK-II immunoreactivity for dementing diseases other than AD revealed a similar reduction, suggesting the CK-II involvement in the common process of neurodegeneration.
INTRODUCTION A l z h e i m e r ' s disease ( A D ) is a m a j o r cause of d e m e n t i a which affects 10-15% of the people over the age of 65 years 26. In A D , there is a characteristic loss of neurons from several cortical and subcortical areas of the brain, Terry and his colleagues have shown that there is an extensive loss of neocortical large neurons, while the small neurons and glial cells are relatively unaffected 27. Some of the remaining neurons contain neurofibrillary tangles ( N F T ) and some neuroterminals form neuritic plaques (NP) which characterize the pathology of this disease 2s. The causal relationship between neuronal death and these pathologies is yet to be d e t e r m i n e d . It is known that protein phosphorylation and dephosphorylation are i m p o r t a n t in regulating the activity of various enzymes 3"~'ls. The cascade of protein phosphorylation, often initiated by the binding of growth factors and n e u r o t r a n s m i t t e r s to their receptors and m e d i a t e d through the second messengers like Ca 2+, cAMP, and lipid metabolites, is also involved in the regulation of expression of certain genes. A compromise in these cascade reactions can adversely affect cellular functions and possibly lead to cell death as found in A D . Several investigators have o b s e r v e d an alteration in protein p h o s p h o r y l a t i o n in brain tissue of people with A D ~'4'~'25.
We have d e t e r m i n e d that in A D h o m o g e n a t e s , a M r 60,000 protein (P60) can be p h o s p h o r y l a t e d to a greater extent than in the control samples 23, while the activity of protein kinase C and a M r 86,000 protein (P86) phosphorylation are decreased 4. F u r t h e r m o r e , r, a protein which p r o m o t e s the polymerization of tubulin into microtubules, is o v e r p h o s p h o r y l a t e d in A D ~''~, and this form of r is also found associated with paired helical filaments ( P H F ) in both the N F T and the dystrophic neurites of senile plaques L3. Thus, the a b n o r m a l phosphorylation not only affects the proteins involved in cellular homeostasis, but also o t h e r proteins which are directly linked to the A D pathology, To further u n d e r s t a n d how a b e r r a n t phosphorylation events may be related to A D pathology, it is necessary to study the protein kinases and/or p h o s p h a t a s e s that may be responsible for the a b n o r m a l p h o s p h o r y l a t i o n . Because r is o v e r p h o s p h o r y l a t e d in A D and the human brain r sequence showed possible casein kinase phosphorylation sites 7, we l o o k e d at casein kinase (CK) activities in A D . We found that while the activity of CK-I is unaffected, the s p e r m i n e - d e p e n d e n t activity of CK-II and the concentration of CK-II as m e a s u r e d by immunoreactivity decreased in A D . F u r t h e r m o r e , the distribution of CK-II immunoreactivity on brain sections was altered suggesting that this kinase is affected in A D .
Correspondence: T. Saitoh, Department of Neurosciences, School of Medicine (M-024), University of California. San Diego, Ira Jolla, CA 92(193, U.S.A.
0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
274 MATERIALS AND METHODS Human brains, AD and control, were obtained from autopsy. The post-mortem times, 11.2 _+_6.1 (controls) and 12.8 + 7.9 (AD), were not different. Furthermore, the chronological age of the control samples, 64.8 + 7.4. was not significantly different from the AD sample, 68.5 _+ 6.0 years. The error is one standard deviation from the mean. The AD brains were confirmed by the presence of NP and NFT. Other brains used in the study were from Parkinson's disease confirmed by the presence of Lewy bodies either in the substantia nigra or the substantia innominata, Pick's disease confirmed by the presence of Pick bodies, Multi-infarct dementia confirmed by the presence of multiple infarcts, and Binswanger's disease (confirmed by the cerebrovascular lesions in the white matter). Brains from non-AD cases did not contain an appreciable amount of NP and NFT. The brains were homogenized essentially as described by Saitoh and Dobkins 24. Briefly, 0.5 g of frozen frontal cortex was sliced and placed in a centrifuge tube. All the following procedures were at 4°C. Homogenization buffer (5 ml, 10x volume, pH 8.0), consisting of 0.32 M sucrose, 5 mM HEPES, 5 mM benzamidine, 2 mM fl-mercaptoethanol, 3 mM EGTA, 0.5 mM MgSO 4, 10~uM sodium vanadate (meta), 0.01 mg/ml leupeptin, 0.005 mg/ml pepstatin A, and 0.1 mg/ml aprotinin, was added. The tissue was homogenized with a Polytron, using three 5-s bursts. The homogenate was centrifuged for 8 rain at 50(10 g. The supernatant was poured off and 2.5 ml (5x volume) of homogenization buffer was added to the pellet. The pellet was rehomogenized, two 5-s bursts, and centrifuged for 8 min at 5000 g. The two supernatants were combined and were ultracentrifuged 1 h at 100,000 g using a Ti 50 rotor. The supernatant, which contained the cytosolic fraction, was poured off while the pellet containing the membrane-cytoskeletal fraction was reconstituted into the homogenization buffer. The protein concentration was determined by a Lowry assay ~6. The samples were assayed for casein kinase activity as follows: in each tube were placed 5.0 I*g casein, 10 !,M ATP, 1.0 ~Ci [7--~2p]ATP, and assay buffer (0.1 M HEPES, pH 7.4, 10 mM MgSO4, 5 mM fl-mercaptoethanol). To this was added 6.5 gg of homogenate. The assay was either in the presence or absence of 1 mM spermine at 30 °C for 12 rain. In some assays, heparin, 150 units/ml, was added as an inhibitor of CK-II activity. The assay was terminated using 6.3 ul of 5x sample buffer. The 5x sample buffer consisted of 20% glycerol, 10% sodium dodecyl sulfate (SDS), 5% fl-mercaptoethanol, 0.31 M Tris, pH 6.8, 25 mM EDTA, and 0.19; bromophenol blue. The samples were analyzed on a 6.5-12.5% SDS-polyacrylamide gel eleetrophoresis (PAGE). The gel was run until the Bromophenol blue dye front was 0.5 cm from the bottom of the gel. The gel was then fixed in 10% acetic acid, 15~ isopropanol for 1 h and stained with 0.1% Coomassie blue R-250 in 10% acetic acid, 40% isopropanol. The gel was destained in 10c'i acetic acid, 15% isopropanol and then washed 1 h with distilled water. The gel was dried on a slab gel dryer (Bio-Rad) and then exposed to a X-Omat AR or RP film for 3-20 h at -70 °C. The film was developed on a Konica developer. After careful analysis of the film and the gel, the casein bands were excised and placed in 12-ml scintillation vials with 3 ml scintillation fluid and the radioactivity counted on a liquid scintillation counter (Tin Analytic) for 10 rain. The activity of CK-II in the above samples was also analyzed by assaying its activity on a native gel which contained no detergent. Thirty-five/~g of each sample were placed on a 5% native gel and the gel was run until the Bromophenol blue dye front was 0.5 cm from the bottom. The gel was electroblotted onto a piece of nitrocellulose paper. The blotting was done at 300 mA for 2 h. The blot was incubated 2 h with 1% casein (partially dephosphorylated) in phosphate-buffered saline (PBS) at room temperaturc. The blots were then incubated in 25 ml of assay buffer containing 5/*M ATP, 50 !~Ci [?,-32P]ATP, with or without 1 mM sperminc. In some experiments, heparin, 150 units/ml, was also added. The incubation was done at room temperature for 1 h. The blot was washed 3× with Tris-buffered saline (TBS) and then exposed to a X-Omat AR film for 20-24 h. The film was developed and the bands on the fihn werc
anatyzcd by a Laser l)ensitomcter (LKB, I Itroscan XL/. Undc~ these conditions, the intensity of bands ~m auloradiograms was between (I.7 and 3.0 absorbance units falling within the linear mngc of sensitivity of the film. The concentration of CK-II was determined by its immunodetcction on protein blots. Rabbit anti-calf thymus CK-II antiscrum was a generous gift from Dr. Michael E. Dahmus. This antibody detects all CK-II subunits (M~ - 44,000, 40,000, and 26,000) on Western blots and immunolocalizes CK-II in mouse fibroblasts specifically; staining was absorbed by a previous incubation of the antibody with purified CK-II
