Brain Research, 579 (1992) 347-349 t~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
347
BRES 25165
Phosphatidylinositol-specific phospholipase C activity in the postmortem human brain: no alteration in Alzheimer's disease Shun S h i m o h a m a a, Sadaki Fujimoto b, Takashi Taniguchi c and Jun Kimura a aDepartment of Neurology, Faculty of Medicine, Kyoto University, Kyoto (Japan) and Departments of bBiochemistry and CNeurobiology, Kyoto Pharmaceutical University, Kyoto (Japan) (Accepted 29 January 1992)
Key words: Phospholipase C; Phosphatidylin0sitol; Activity; Human brain; Alzheimer's disease; Postmortem stability
The activity of phospholipase C (PLC) which hydrolyzes exogenous phosphatidylinositol (PI), was investigated in samples prepared from postmortem normal human brains and Alzheimer disease brains. The enzyme activity did not change signifcantly after rat brains were left for 24 h at room temperature. The PI-specific PLC activity in the Alzheimer cytosolic and particulate fractions was not significantly different from that in the control fractions. The PI-specific PLC activity as a function of the free Ca2+ concentration was also similar between control and Alzheimer brains. These results suggest that the PI-specific PLC activity is not altered in Alzheimer's disease. Evidence is mounting that inositol phospholipid-specific phospholipase C (PLC) is a key molecule in signal transduction. PLC catalyzes the three phosphoinositides, phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2), to generate diacylglycerol and three inositol phosphates, among which diacylglycerol and inositol 1,4,5-trisphosphate serve as intracellular messengers for protein kinase C (PKC) activation and intracellular Ca 2+ mobilization 1,3,6,9-11. We have already reported the alterations of muscarinic cholinergic, a-adrenergic, and glutamatergic receptors that trigger the activation of PLC 8'14'15, and have demonstrated the involvement of PKC in Alzheimer's disease (AD) pathology 7'16. These findings have suggested a crucial role for PLC in the aberrant signalling that occurs in AD brains. To examine the involvement of PLC in the pathogenesis of AD, we investigated here the PI-specific PLC activity in the postmortem normal human brain and that in the Alzheimer brain. L-a-myo-Inositol-2-3H(N) (20 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Phosphatidylinositol (Soybean, ammonium salt), phenylmethylsulfonyl fluoride and diisopropylfluorophosphate were obtained from Sigma Chemical Co. All other chemicals were of reagent grade or high purity and were obtained from the usual laboratory suppliers. Brain tissues were obtained at autopsy from 10 patients diagnosed clinically and histopathologically as hav-
ing AD (mean age: 79 years; mean postmortem delay: 9 h), and from 10 age-matched controls (mean age: 83 years; mean postmortem delay: 8 h) with no clinical or morphological evidence of brain pathology. The causes of death were similar in both groups and were usually linked to cardiac insufficiency or to a terminal respiratory condition. Immediately after autopsy, the brains were divided sagitally into halves; one half was used in the biochemical assays and the other half was examined histologically. The diagnosis of AD was made in accordance with the National Institute on Aging criteria 5. Tissue blocks were dissected out and cut into 30-pm wedge microtome sections. Sections from the frontal cortex and the hippocampus of all brains were postfixed with 10% formaldehyde solution, and screened for a histological diagnosis and the detection of senile plaques using modified Bielschowsky staining. Control brains exhibited negligible microscopic neuropathology (0-2 senile plaques per low-power field). AD was diagnosed on the basis of the abundant presence of senile plaques and neurofibrillary tangles in the neocortex and hippocampus. Brain extracts were prepared according to a modification of the method of Ryu et a1.12. In brief, brain tissues were homogenized in a glass homogenizer with a buffer containing (in mM) Tris-HCl 10 (pH 7.2), EGTA, 4, phenylmethylsulfonyl fluoride 1, diisopropylfluorophosphate 0.5, and dithiothreitol 0.1. The homogenate was centrifuged for 1 h at 105,000 x g and the superna-
Correspondence: S. Shimohama, Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoinkawaharacho, Sakyo-ku, Kyoto 606, Japan.
348 120 -
TABLE II
1 O0
Phosphatidylinositol-specific phospholipase C activity in control and Alzheimer brains
The means + S.D. are shown. 80
Phospholipase C activity - PI hydrolysis (nmol/min/mg protein) --
60
=1
40
Controls (n = 10) Alzheimer (n = 10)
Cytosolic fraction
Particulate fraction
31.5 + 8.5 28.6 + 4.0
9.0 + 1.2 10.1 + 1.6
20
Cael2 (raM) Fig. 1. Effects of changes in the free Ca 2+ concentration on the phosphatidylinositol-specific PLC activity in control and AD brains. Free Caz+ concentrations were varied using CaClz/EGTA buffers. Data show the mean of three independent experiments using control (r-q) and AD (0) brains.
tant thus o b t a i n e d was designated as the cytosolic fraction. The pellet was resuspended in the extraction buffer, rehomogenized, and then centrifuged for 1 h at 105,000 x g. Then the precipitate was collected, dissolved in the same buffer, and designated as the particulate fraction. The PI-specific PLC activity was measured according to the m e t h o d of H o f m a n n and Majerus 2. Reaction mixtures contained 280/~M phosphatidylinositol, 30,000 d p m of L - a - ( m y o - i n o s i t o l - 2 - 3 H ( N ) ) , 1 mg/ml sodium deoxycholate, 1 m M CaC1 a, 50 m M H E P E S (pH 7.0), and approximate amounts of brain extracts. A f t e r incubation for 15 min at 37°C, the reaction was stopped with 1 ml of chloroform/methanol/concentrated HC1 (50:50:0.3), followed by 0.3 ml of 1 N HC1 containing 5 m M E G T A . A f t e r centrifugation for 10 min at 3,000 × g, a 700-kd aliquot of the supernatant was r e m o v e d for liquid scintillation counting. Protein content was measured using bicinchoninic acid TM. To examine the p o s t m o r t e m stabil-
TABLE I Postmortem stability o f phosphatidylinositol-specific phospholipase C activity
Whole rat brains were left standing for 0, 7 and 24 h at room temperature (22°C). The means + S.D. of three experiments are shown. Phospholipase C activity -- PI hydrolysis (nrnol/min/mg protein) --
Time from death 0h 7h 24 h
Cytosolic fraction
Particulate fraction
30.0 + 0.8 35.5 + 0.7 35.9 _+_+0.4
9.2 _+ 0.3 7.9 _+ 0.4 8.1 _+ 0.4
ity of PI-specific PLC, rat brains were left for 0, 7 and 24 h at r o o m temperature. Free Ca 2+ concentrations in the range of 0 - 5 . 0 m M were achieved using CaC12/ E G T A buffers as described previously 15. The PI-specific PLC activity increased in a linear fashion along with increase in the concentration of human brain extract over the range of 1-4 mg protein/ml (data not shown). The activity was also linear with respect to time, at least up to 30 min (data not shown). The PIspecific PLC activity of the human brain extracts was o p t i m u m around neutral p H (data not shown), and at a Ca 2+ concentration of 1.2 m M (Fig. 1). The enzyme activity did not change significantly in rat brains after they were left for 24 h at r o o m t e m p e r a t u r e (Table I). The activity was about three times higher in the cytosolic fraction than in the particulate fraction (Table II). The PI-specific PLC activity in the A l z h e i m e r brain cytosolic and particulate fractions was not significantly different from that in the control fractions (Table II). The PI-specific PLC activity as a function of the free Ca 2÷ concentration also showed no significant difference between control and A l z h e i m e r brains (Fig. 1). Comparison of the hydrolysis of phosphatidylinositol in samples p r e p a r e d from p o s t m o r t e m human brain and fresh rat brains revealed an enzyme activity with essentially similar properties (Table I and II). The activity was higher in the cytosolic fraction than in the particulate fraction, a finding consistent with the p r e d o m i n a n t localization of PLC in the cytosol ~7. H u m a n brain PI hydrolysis was found to have an optimal p H between 7.0 and 7.5 in the presence of 1 m M deoxycholate, a finding which is in agreement with a r e p o r t on purified particulate bovine brain P L C 4. The PI-specific PLC activity did not change significantly after fresh rat brains were left for 24 h at r o o m t e m p e r a t u r e , suggesting that postmortem delay would have little effect on the human PI-specific P L C activity. The PI-specific PLC activity of A l z h e i m e r brains has not been examined previously. The present study revealed that the PI-specific PLC activity in A l z h e i m e r cy-
349 tosolic and particulate fractions was not significantly different from that in control brains, and that the PIspecific P L C activity as a function of free Ca 2÷ was also similar in both groups of brains. These results indicate that PI-specific P L C activity is not significantly altered in the A l z h e i m e r brain. H o w e v e r , the present results do not rule out the involvement of PLC, which hydrolyzes other phosphoinositides like PIP and PIP 2 in Alzheim e r ' s disease. I n d e e d , PIP2-specific P L C generates diacylglycerol and inositol 1,4,5-trisphosphate which serve as intracellular messengers for P K C activation and intracellular Ca 2÷ mobilization. Several P L C isozymes have been d e m o n s t r a t e d by protein purification and c D N A cloning 11. We have recently used four antibodies against
different P L C isozymes to immunostain in A l z h e i m e r brain tissues, and found that the isozyme PLC-6 was abnormally accumulated in neurofibrillary tangles, the neurites surrounding senile plaque cores and neuropil threads 17. This finding strongly suggested a crucial involvement of P L C in A D pathology. Thus, further studies are n e e d e d to define the exact pathophysiological role of P L C in A D .
1 Berridge, M.J. and Irvine, R.E, Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature, 312 (1984) 315-321. 2 Hofrnann, S.L. and Majerus, P.W., Identification and properties of two distinct phosphatidylinositol-specific phospholipase C enzymes from sheep seminal vesicular glands, J. Biol. Chem., 257 (1982) 6461-6469. 3 Homma, Y., Emori, Y., Shibasaki, E, Suzuki, K. and Takenawa, T., Isolation and characterization of a ~,-type phosphoinositide-specific phospholipase C (PLC-),2), Biochem. J., 269 (1990) 13-18. 4 Katan, M. and Parker, P.J., Purification of phosphoinositidespecific phospholipase C from a particulate fraction of bovine brain, Eur. J. Biochem., 168 (1987) 413-418. 5 Khachaturian, Z.S., Diagnosis of Alzheimer's disease, Arch. Neurol., 42 (1985) 1097-1105. 6 Majerus, P.W., Conolly, T.M., Deckmyn, H., Ross, T.S., Bross, T.E., Ishii, H., Bansal, V.S. and Wilson, D.W., The metabolism of phosphoinositide-derived messenger molecules, Science, 234 (1986) 1519-1526. 7 Masliah, E., Cole, G., Shimohama, S., Hansen, L., DeTeresa, R., Terry, D.T. and Saitoh, T., Differential involvement of protein kinase C isozymes in Alzheimer's disease, J. Neurosci., 10 (1990) 2113-2124. 8 Ninomiya, H., Taniguchi, T., Fujiwara, M., Shimohama, S. and Kameyama, M., [3H]N-[1-(2-Thienyl)cyclohexyl}-3,4-piperidine ([3H]TCP) binding in human frontal cortex: decreases in .~lzheimer-type dementia, J. Neurochem., 54 (1990) 526--532 9 Nishizuka, Y., Studies and perspectives of protein kinase C, Science, 233 (1986) 1365-1370. 10 Nishizuka, Y., The molecular heterogeneity of protein kinase C
and its implications for cellular regulation, Nature, 334 (1988) 661-665. 11 Rhee, S.G., Suh, P.G., Ryu, S.H. and Lee, S.Y., Studies of inositol phospholipid-specific phospholipase C, Science, 244 (1989) 546-550. 12 Ryu, S.H., Cho, K.S., Lee, K.Y., Suh, P.-G. and Rhee, S.G., Two forms of phosphatidylinositol-specific phospholipase C from bovine brain, Biochem. Biophys. Res. Commun., 26 (1986) 137-144. 13 Schatzmann, H.J., Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells, J. Physiol., 235 (1973) 551-569. 14 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia, J. Neurochem., 46 (1986) 288-293. 15 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Biochemical characterization of a-adrenergic receptors in human brain and changes in Alzheimer-type dementia, J. Neurochem., 47 (1986) 1294-1301. 16 Shimohama, S., Ninomiya, H., Saitoh, T., Terry, R.D., Fukunaga, R., Taniguchi, T., Fujiwara, M., Kimura, J. and Kameyama, M., Changes in signal transduction in Alzheimer's disease, J. Neural Transm., 30 (Suppl.) (1990) 69-78. 17 Shimohama, S., Homma, Y., Suenaga, T., Fujimoto, S., Taniguchi, T., Araki, W., Yamaoka, Y., Takenawa, T. and Kimura, J., Aberrant accumulation of phospholipase C-6 in Alzheimer brains, Am. J. Pathol., 139 (1991) 737-742. 18 Smith, P.K., Krohn, R.I., Hermason, G.T., Mallia, A.K., Gartner, EH., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenl, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150 (1985) 76-85.
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (02240215, 03224105 and 03263213) from the Ministry of Education, Science and Culture, Japan and by grants from the Sasagawa Research Foundation, the Japan Brain Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
347
BRES 25165
Phosphatidylinositol-specific phospholipase C activity in the postmortem human brain: no alteration in Alzheimer's disease Shun S h i m o h a m a a, Sadaki Fujimoto b, Takashi Taniguchi c and Jun Kimura a aDepartment of Neurology, Faculty of Medicine, Kyoto University, Kyoto (Japan) and Departments of bBiochemistry and CNeurobiology, Kyoto Pharmaceutical University, Kyoto (Japan) (Accepted 29 January 1992)
Key words: Phospholipase C; Phosphatidylin0sitol; Activity; Human brain; Alzheimer's disease; Postmortem stability
The activity of phospholipase C (PLC) which hydrolyzes exogenous phosphatidylinositol (PI), was investigated in samples prepared from postmortem normal human brains and Alzheimer disease brains. The enzyme activity did not change signifcantly after rat brains were left for 24 h at room temperature. The PI-specific PLC activity in the Alzheimer cytosolic and particulate fractions was not significantly different from that in the control fractions. The PI-specific PLC activity as a function of the free Ca2+ concentration was also similar between control and Alzheimer brains. These results suggest that the PI-specific PLC activity is not altered in Alzheimer's disease. Evidence is mounting that inositol phospholipid-specific phospholipase C (PLC) is a key molecule in signal transduction. PLC catalyzes the three phosphoinositides, phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2), to generate diacylglycerol and three inositol phosphates, among which diacylglycerol and inositol 1,4,5-trisphosphate serve as intracellular messengers for protein kinase C (PKC) activation and intracellular Ca 2+ mobilization 1,3,6,9-11. We have already reported the alterations of muscarinic cholinergic, a-adrenergic, and glutamatergic receptors that trigger the activation of PLC 8'14'15, and have demonstrated the involvement of PKC in Alzheimer's disease (AD) pathology 7'16. These findings have suggested a crucial role for PLC in the aberrant signalling that occurs in AD brains. To examine the involvement of PLC in the pathogenesis of AD, we investigated here the PI-specific PLC activity in the postmortem normal human brain and that in the Alzheimer brain. L-a-myo-Inositol-2-3H(N) (20 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO). Phosphatidylinositol (Soybean, ammonium salt), phenylmethylsulfonyl fluoride and diisopropylfluorophosphate were obtained from Sigma Chemical Co. All other chemicals were of reagent grade or high purity and were obtained from the usual laboratory suppliers. Brain tissues were obtained at autopsy from 10 patients diagnosed clinically and histopathologically as hav-
ing AD (mean age: 79 years; mean postmortem delay: 9 h), and from 10 age-matched controls (mean age: 83 years; mean postmortem delay: 8 h) with no clinical or morphological evidence of brain pathology. The causes of death were similar in both groups and were usually linked to cardiac insufficiency or to a terminal respiratory condition. Immediately after autopsy, the brains were divided sagitally into halves; one half was used in the biochemical assays and the other half was examined histologically. The diagnosis of AD was made in accordance with the National Institute on Aging criteria 5. Tissue blocks were dissected out and cut into 30-pm wedge microtome sections. Sections from the frontal cortex and the hippocampus of all brains were postfixed with 10% formaldehyde solution, and screened for a histological diagnosis and the detection of senile plaques using modified Bielschowsky staining. Control brains exhibited negligible microscopic neuropathology (0-2 senile plaques per low-power field). AD was diagnosed on the basis of the abundant presence of senile plaques and neurofibrillary tangles in the neocortex and hippocampus. Brain extracts were prepared according to a modification of the method of Ryu et a1.12. In brief, brain tissues were homogenized in a glass homogenizer with a buffer containing (in mM) Tris-HCl 10 (pH 7.2), EGTA, 4, phenylmethylsulfonyl fluoride 1, diisopropylfluorophosphate 0.5, and dithiothreitol 0.1. The homogenate was centrifuged for 1 h at 105,000 x g and the superna-
Correspondence: S. Shimohama, Department of Neurology, Faculty of Medicine, Kyoto University, 54 Shogoinkawaharacho, Sakyo-ku, Kyoto 606, Japan.
348 120 -
TABLE II
1 O0
Phosphatidylinositol-specific phospholipase C activity in control and Alzheimer brains
The means + S.D. are shown. 80
Phospholipase C activity - PI hydrolysis (nmol/min/mg protein) --
60
=1
40
Controls (n = 10) Alzheimer (n = 10)
Cytosolic fraction
Particulate fraction
31.5 + 8.5 28.6 + 4.0
9.0 + 1.2 10.1 + 1.6
20
Cael2 (raM) Fig. 1. Effects of changes in the free Ca 2+ concentration on the phosphatidylinositol-specific PLC activity in control and AD brains. Free Caz+ concentrations were varied using CaClz/EGTA buffers. Data show the mean of three independent experiments using control (r-q) and AD (0) brains.
tant thus o b t a i n e d was designated as the cytosolic fraction. The pellet was resuspended in the extraction buffer, rehomogenized, and then centrifuged for 1 h at 105,000 x g. Then the precipitate was collected, dissolved in the same buffer, and designated as the particulate fraction. The PI-specific PLC activity was measured according to the m e t h o d of H o f m a n n and Majerus 2. Reaction mixtures contained 280/~M phosphatidylinositol, 30,000 d p m of L - a - ( m y o - i n o s i t o l - 2 - 3 H ( N ) ) , 1 mg/ml sodium deoxycholate, 1 m M CaC1 a, 50 m M H E P E S (pH 7.0), and approximate amounts of brain extracts. A f t e r incubation for 15 min at 37°C, the reaction was stopped with 1 ml of chloroform/methanol/concentrated HC1 (50:50:0.3), followed by 0.3 ml of 1 N HC1 containing 5 m M E G T A . A f t e r centrifugation for 10 min at 3,000 × g, a 700-kd aliquot of the supernatant was r e m o v e d for liquid scintillation counting. Protein content was measured using bicinchoninic acid TM. To examine the p o s t m o r t e m stabil-
TABLE I Postmortem stability o f phosphatidylinositol-specific phospholipase C activity
Whole rat brains were left standing for 0, 7 and 24 h at room temperature (22°C). The means + S.D. of three experiments are shown. Phospholipase C activity -- PI hydrolysis (nrnol/min/mg protein) --
Time from death 0h 7h 24 h
Cytosolic fraction
Particulate fraction
30.0 + 0.8 35.5 + 0.7 35.9 _+_+0.4
9.2 _+ 0.3 7.9 _+ 0.4 8.1 _+ 0.4
ity of PI-specific PLC, rat brains were left for 0, 7 and 24 h at r o o m temperature. Free Ca 2+ concentrations in the range of 0 - 5 . 0 m M were achieved using CaC12/ E G T A buffers as described previously 15. The PI-specific PLC activity increased in a linear fashion along with increase in the concentration of human brain extract over the range of 1-4 mg protein/ml (data not shown). The activity was also linear with respect to time, at least up to 30 min (data not shown). The PIspecific PLC activity of the human brain extracts was o p t i m u m around neutral p H (data not shown), and at a Ca 2+ concentration of 1.2 m M (Fig. 1). The enzyme activity did not change significantly in rat brains after they were left for 24 h at r o o m t e m p e r a t u r e (Table I). The activity was about three times higher in the cytosolic fraction than in the particulate fraction (Table II). The PI-specific PLC activity in the A l z h e i m e r brain cytosolic and particulate fractions was not significantly different from that in the control fractions (Table II). The PI-specific PLC activity as a function of the free Ca 2÷ concentration also showed no significant difference between control and A l z h e i m e r brains (Fig. 1). Comparison of the hydrolysis of phosphatidylinositol in samples p r e p a r e d from p o s t m o r t e m human brain and fresh rat brains revealed an enzyme activity with essentially similar properties (Table I and II). The activity was higher in the cytosolic fraction than in the particulate fraction, a finding consistent with the p r e d o m i n a n t localization of PLC in the cytosol ~7. H u m a n brain PI hydrolysis was found to have an optimal p H between 7.0 and 7.5 in the presence of 1 m M deoxycholate, a finding which is in agreement with a r e p o r t on purified particulate bovine brain P L C 4. The PI-specific PLC activity did not change significantly after fresh rat brains were left for 24 h at r o o m t e m p e r a t u r e , suggesting that postmortem delay would have little effect on the human PI-specific P L C activity. The PI-specific PLC activity of A l z h e i m e r brains has not been examined previously. The present study revealed that the PI-specific PLC activity in A l z h e i m e r cy-
349 tosolic and particulate fractions was not significantly different from that in control brains, and that the PIspecific P L C activity as a function of free Ca 2÷ was also similar in both groups of brains. These results indicate that PI-specific P L C activity is not significantly altered in the A l z h e i m e r brain. H o w e v e r , the present results do not rule out the involvement of PLC, which hydrolyzes other phosphoinositides like PIP and PIP 2 in Alzheim e r ' s disease. I n d e e d , PIP2-specific P L C generates diacylglycerol and inositol 1,4,5-trisphosphate which serve as intracellular messengers for P K C activation and intracellular Ca 2÷ mobilization. Several P L C isozymes have been d e m o n s t r a t e d by protein purification and c D N A cloning 11. We have recently used four antibodies against
different P L C isozymes to immunostain in A l z h e i m e r brain tissues, and found that the isozyme PLC-6 was abnormally accumulated in neurofibrillary tangles, the neurites surrounding senile plaque cores and neuropil threads 17. This finding strongly suggested a crucial involvement of P L C in A D pathology. Thus, further studies are n e e d e d to define the exact pathophysiological role of P L C in A D .
1 Berridge, M.J. and Irvine, R.E, Inositol trisphosphate, a novel second messenger in cellular signal transduction, Nature, 312 (1984) 315-321. 2 Hofrnann, S.L. and Majerus, P.W., Identification and properties of two distinct phosphatidylinositol-specific phospholipase C enzymes from sheep seminal vesicular glands, J. Biol. Chem., 257 (1982) 6461-6469. 3 Homma, Y., Emori, Y., Shibasaki, E, Suzuki, K. and Takenawa, T., Isolation and characterization of a ~,-type phosphoinositide-specific phospholipase C (PLC-),2), Biochem. J., 269 (1990) 13-18. 4 Katan, M. and Parker, P.J., Purification of phosphoinositidespecific phospholipase C from a particulate fraction of bovine brain, Eur. J. Biochem., 168 (1987) 413-418. 5 Khachaturian, Z.S., Diagnosis of Alzheimer's disease, Arch. Neurol., 42 (1985) 1097-1105. 6 Majerus, P.W., Conolly, T.M., Deckmyn, H., Ross, T.S., Bross, T.E., Ishii, H., Bansal, V.S. and Wilson, D.W., The metabolism of phosphoinositide-derived messenger molecules, Science, 234 (1986) 1519-1526. 7 Masliah, E., Cole, G., Shimohama, S., Hansen, L., DeTeresa, R., Terry, D.T. and Saitoh, T., Differential involvement of protein kinase C isozymes in Alzheimer's disease, J. Neurosci., 10 (1990) 2113-2124. 8 Ninomiya, H., Taniguchi, T., Fujiwara, M., Shimohama, S. and Kameyama, M., [3H]N-[1-(2-Thienyl)cyclohexyl}-3,4-piperidine ([3H]TCP) binding in human frontal cortex: decreases in .~lzheimer-type dementia, J. Neurochem., 54 (1990) 526--532 9 Nishizuka, Y., Studies and perspectives of protein kinase C, Science, 233 (1986) 1365-1370. 10 Nishizuka, Y., The molecular heterogeneity of protein kinase C
and its implications for cellular regulation, Nature, 334 (1988) 661-665. 11 Rhee, S.G., Suh, P.G., Ryu, S.H. and Lee, S.Y., Studies of inositol phospholipid-specific phospholipase C, Science, 244 (1989) 546-550. 12 Ryu, S.H., Cho, K.S., Lee, K.Y., Suh, P.-G. and Rhee, S.G., Two forms of phosphatidylinositol-specific phospholipase C from bovine brain, Biochem. Biophys. Res. Commun., 26 (1986) 137-144. 13 Schatzmann, H.J., Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells, J. Physiol., 235 (1973) 551-569. 14 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Changes in nicotinic and muscarinic cholinergic receptors in Alzheimer-type dementia, J. Neurochem., 46 (1986) 288-293. 15 Shimohama, S., Taniguchi, T., Fujiwara, M. and Kameyama, M., Biochemical characterization of a-adrenergic receptors in human brain and changes in Alzheimer-type dementia, J. Neurochem., 47 (1986) 1294-1301. 16 Shimohama, S., Ninomiya, H., Saitoh, T., Terry, R.D., Fukunaga, R., Taniguchi, T., Fujiwara, M., Kimura, J. and Kameyama, M., Changes in signal transduction in Alzheimer's disease, J. Neural Transm., 30 (Suppl.) (1990) 69-78. 17 Shimohama, S., Homma, Y., Suenaga, T., Fujimoto, S., Taniguchi, T., Araki, W., Yamaoka, Y., Takenawa, T. and Kimura, J., Aberrant accumulation of phospholipase C-6 in Alzheimer brains, Am. J. Pathol., 139 (1991) 737-742. 18 Smith, P.K., Krohn, R.I., Hermason, G.T., Mallia, A.K., Gartner, EH., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J. and Klenl, D.C., Measurement of protein using bicinchoninic acid, Anal. Biochem., 150 (1985) 76-85.
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (02240215, 03224105 and 03263213) from the Ministry of Education, Science and Culture, Japan and by grants from the Sasagawa Research Foundation, the Japan Brain Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research.
