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Phospholipase C isozymes in the human brain and their changes in Alzheimer's disease
Pergamon
PII:
Neuroscience Vol. 82, No. 4, pp. 999–1007, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00342-4
PHOSPHOLIPASE C ISOZYMES IN THE HUMAN BRAIN AND THEIR CHANGES IN ALZHEIMER’S DISEASE S. SHIMOHAMA,*¶ Y. SASAKI,† S. FUJIMOTO,† S. KAMIYA,‡ T. TANIGUCHI,‡ T. TAKENAWA§ and J. KIMURA* *Department of Neurology, Faculty of Medicine, Kyoto University, Sakyoku, Kyoto 606, Japan Departments of †Environmental Biochemistry and ‡Neurobiology, Kyoto Pharmaceutical University, Yamashinaku, Kyoto 607, Japan §Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minatoku, Tokyo 108, Japan Abstract––Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-ä1, accumulates aberrantly in the brains of patients with Alzheimer’s disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer’s disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-ã1, exhibiting an isoelectric point value of 5.2, and phospholipase C-ä1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimer’s disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-ã1 and phospholipase C-ä1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-ã1 activity was significantly decreased and the phospholipase C-ä1 activity was significantly increased in Alzheimer’s disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimer’s disease. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: Alzheimer’s disease, phospholipase C, phospholipase C-ã1, phospholipase C-ä1, chromatofocusing, gel filtration chromatography.
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the progressive deterioration of cognitive function and memory. The characteristic lesions in AD brains include senile plaques (SPs), neurofibrillary tangles (NFTs) and neuronal loss. An important question is the nature of the key signaling that is responsible for the formation of these pathological hallmarks, as well as the neuronal dysfunction associated with this disease.4,9 There is increasing evidence that phosphoinositidespecific phospholipase C (PLC) is a key molecule in signal transduction. PLC catalyses the three phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol ¶To whom correspondence should be addressed. Abbreviations: AD, Alzheimer’s disease; BSA, bovine serum albumin; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; HEPES, N-2-hydroxyethylpiperazineN*-2-ethanesulfonic acid; NFT, neurofibrillary tangle; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; pI, isoelectric point; PLC, phospholipase C; SDS, sodium dodecyl sulfate; SP, senile plaque.
4,5-biphosphate, to generate diacylglycerol and three inositol phosphates, among which diacylglycerol and inositol 1,4,5-trisphosphate serve as intracellular messengers for protein kinase C activation and intracellular Ca2+ mobilization.1,12,14,16 To date, at least 10 PLC isozymes have been identified by protein chemistry or cDNA cloning methods.15 We have previously demonstrated that a PLC isozyme, PLC-ä1, accumulates abnormally in NFTs, the neurites surrounding SP cores, and neuropil threads in AD brains.19,21,22 We have also shown that this abnormal accumulation of PLC-ä1 in AD brains is due to an increase of the protein level of this enzyme, and that its specific activity is decreased in AD brains compared with controls,23 although total PLC activity is not changed in control and AD brains.20 However, as the protein character of PLC-ä1 as well as other PLC isozymes has not been well investigated so far, in the present study we examined the property of PLC isozymes in human control and AD brains by using the methods of chromatofocusing and gel filtration chromatography.
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Materials -á-Phosphatidyl(myo-inositol 2-3H(N)) (1.0 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, U.S.A.). -á-Phosphatidylinositol (Soybean, ammonium salt), phenylmethylsulfonyl fluoride and diisopropylfluorophosphate were obtained from Sigma Chemical Co. Sephadex G-25, Superdex 200 pg HiLoad and Mono P HR columns were obtained from Pharmacia LKB Biotechnology. Standard proteins used for calibration of the Sephadex G-25 and Superdex 200 pg columns were obtained from Boehringer Mannheim GmbH. All other chemicals were of reagent grade and were obtained commercially. Brain samples Brain tissues were obtained at autopsy from seven patients diagnosed clinically and histopathologically as having AD (mean age: 79 years; mean post mortem delay: 6 h), and from seven age-matched controls (mean age: 78 years; mean post mortem delay: 8 h) with no clinical or morphological evidence of brain pathology. All the AD patients were clinically severely demented, while those in control groups were not demented. Among the AD patients, the final cause of death was listed as cardiac insufficiency in three and a terminal respiratory condition in four. Among the control cases, four died from cardiac insufficiency and three from a terminal respiratory condition. Ante-post mortem hypoxic conditions exhibited essentially no significant difference between AD and control cases. Immediately after autopsy, the brains were divided sagittally into halves, with one half being used in the biochemical assays and the other half being examined histologically. The half to be used for the biochemical studies was stored at "80)C until thawing for homogenization and the biochemical studies. Temporal cortices were used for the present biochemical study. The neuropathological assessment of AD was made in accordance with the criteria of the Consortium to Establish a Registry for AD (CERAD).13 Tissue blocks were dissected and cut into 30-µm wedge microtome sections. Adjacent sections from the temporal cortices and the hippocampus of all brains were postfixed with 10% formaldehyde, and screened to provide a histological diagnosis. Control brains exhibited negligible microscopic neuropathology (0–2 SPs per low-power field). All the AD cases exhibited numerous SPs and NFTs throughout the neocortex. The locus coeruleus and nucleus basalis of Meynert showed severe atrophy, with NFTs in the remaining neurons. The substantia nigra was relatively well preserved and showed no Lewy bodies. Wistar rats were purchased from Japan SLC, Inc. The brains of seven-week-old Wistar rats were taken from animals. The animals were treated in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals. Preparation of brain extracts Brain tissue samples (1 g wet weight) were homogenized with a Teflon–glass homogenizer in 4 volumes of 10 mM HEPES buffer (pH 7.0) containing 0.32 M sucrose, 0.05% NaN3, 100 µM orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM diisopropylfluorophosphate, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, 5 µg/ml leupeptin, 5 mM benzamidine, 5 mM 2-mercaptoethanol and 3 mM EGTA. The homogenate was centrifuged at 105,000#g for 60 min and the supernatant thus obtained was used as the brain extract for further experiments. Chromatofocusing of brain extracts Brain extract was passed through a Sephadex G-25 column equilibrated with 25 mM bis-Tris–iminodiacetic acid buffer (pH 7.1). The active fractions were pooled and
the enzyme solution was applied to a Mono P HR column (0.5#20 cm2) equilibrated with the above buffer and eluted with 1:10 diluted Polybuffer 74 (pH 4.0). One-milliliter fractions were collected, analysed for PLC activity and pH, and used for immunochemical detection. Gel filtration of brain extracts Brain extract was loaded on to a Superdex 200 pg column (1.6#60 cm2) equilibrated and eluted with 25 mM HEPES buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1 M NaCl. Two-milliliter fractions were collected, assayed for PLC activity and used for immunochemical detection. Apparent molecular weights of PLC activity fractions were estimated by using an elution profile of standard proteins [ferritin, molecular weight: 450,000; catalase, molecular weight: 240,000; sweet potato acid phosphatase,24 molecular weight: 110,000; bovine serum albumin (BSA), molecular weight: 68,000] on the same Superdex 200 pg column used in gel filtration chromatography of brain extracts after washing the column thoroughly with the elution buffer. Enzyme assay The PLC activity was measured according to the method described previously.20 In brief, the reaction mixtures contained 280 µM phosphatidylinositol, 30,000 d.p.m. of -áphosphatidyl-(myo-inositol 2-3H(N)), 1 mg/ml sodium deoxycholate, 1.5 mM CaCl2, 50 mM HEPES (pH 7.0), and brain extract (10 µg protein) or eluted sample from the column (20 µl). After incubation for 60 min at 37)C, the reaction was stopped with 1 ml of chloroform/methanol/ concentrated HCl (50:50:0.3), followed by the addition of 0.3 ml of 1 N HCl containing 5 mM EGTA. After centrifugation for 10 min at 3000#g, a 700-µl aliquot of the supernatant was removed for liquid scintillation counting. Protein concentration was determined by the method of Bradford with BSA as the standard.2 Antibodies A specific antibody against PLC-ä1 was prepared using PLC-ä1 protein produced by an E. coli expression system.8 The characterization and specificity of the antibody against PLC-ä1 has been fully described previously.19 This antibody recognizes human PLC-ä1 as an 85,000 mol. wt protein on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), as reported previously.19 The specific antibodies against PLC-â1, -â2, -â3, -â4, -ã1, -ã2 and -ä2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Immunochemical detection The eluted samples from chromatofocusing and gel filtration chromatography in Laemmli sample buffer were subjected to electrophoresis on 4–20% SDS–PAGE or on 4–20% native PAGE, blotted on to Immobilon (Millipore), blocked with phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h, and incubated with the anti-PLC-â1 (1:500), -â2 (1:500), -â3 (1:500), -â4 (1:500), -ã1 (1:500), -ã2 (1:500), -ä1 (1:5000) and -ä2 (1:500) antibodies in PBS containing 3% BSA for 18 h at 4)C. Blots were then washed with PBS/0.1% Tween 20, incubated with horseradish peroxidase-linked antibody against rabbit immunoglobulin (diluted 1:1000). Subsequently, membrane-bound horseradish peroxidase-labeled antibodies were detected by the enhanced chemiluminescence detection system (ECL kit, Amersham). The protein bands that cross-reacted with antibodies could be detected in X-ray films (X-Omat JB-1, Kodak) 5–60 s after the exposure. The integrated optical density of the 85,000 mol. wt protein band recognized by the anti-PLC-ä1 antibody and that of the 145,000 mol. wt protein band recognized by the anti-PLC-ã1 antibody were
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Fig. 1. Separation of rat brain PLC by chromatofocusing. An extract of rat brain cytosol was passed through a Sephadex G-25 column equilibrated with 25 mM bis-Tris–iminodiacetic acid buffer (pH 7.1). The active fractions were pooled and the enzyme solution was applied to a Mono P HR column (0.5#20 cm2) equilibrated with the above buffer and eluted with 1:10 diluted Polybuffer 74 (pH 3.8). One-milliliter fractions were collected, analysed for PLC activity and pH (A), and used for immunochemical detection (B, C). The assay for PLC activity and the method for immunochemical detection are described in Experimental Procedures. B and C indicate the distribution of relative PLC-ã1 and PLC-ä1 immunoreactivity, respectively.
measured by a scanning densitometer (Arcus II, Agfa) and were taken to indicate the relative quantities of PLC-ä1 and PLC-ã1, respectively. RESULTS
Expression of phospholipase C isozymes in rat brain extracts To clarify which PLC isozymes are expressed in rat brain extracts, immunochemical analyses by the antiPLC-â1, -â2, -â3, -â4, -ã1, -ã2, -ä1 and -ä2 antibodies were applied to brain extracts. Only PLC-â1, -ã1 and -ä1 isozymes were detected in rat brain extracts (data not shown); therefore, the following immunochemical analyses were performed using the anti-PLC-â1, -ã1 and -ä1 antibodies. Chromatofocusing of brain extracts Figure 1 shows a typical chromatofocusing profile of rat brain PLC activity on a Mono P HR column. Two major peaks of PLC activity were obtained, and they exhibited isoelectric point (pI) values of 4.3 and 4.0, respectively. Immunochemical detection by the anti-PLC-ã1 and anti-PLC-ä1 antibodies revealed that the first form of PLC activity mainly corresponds to PLC-ã1, and the second form of PLC
activity mainly corresponds to PLC-ä1 (Fig. 1). To examine the post mortem stability of PLC, the rat brains were left for 0, 7 and 24 h at room temperature. The chromatofocusing profile of PLC activity was not changed even after the brains had been left for 24 h at room temperature (data not shown). Figure 2 shows a typical chromatofocusing profile of control human brain PLC activity. Two major peaks of PLC activity were obtained, and they exhibited pI values of about 5.2 and 4.6, respectively, in control human brains. Immunochemical detection by the anti-PLC-ã1 and anti-PLC-ä1 antibodies revealed that the first large form of PLC activity corresponds to both PLC-ã1 and PLC-ä1, and the second small form of PLC activity corresponds mainly to PLC-ä1. (Fig. 2). Figure 3 shows a typical chromatofocusing profile of AD brain PLC activity. Two major peaks of PLC activity were also obtained, and they exhibited pI values of about 5.2 and 4.6, respectively, in AD brains. Immunochemical detection by the antiPLC-ã1 and anti-PLC-ä1 antibodies revealed that the first large form of PLC activity corresponds to both PLC-ã1 and PLC-ä1, and the second small form of PLC activity corresponds mainly to PLC-ä1. (Fig. 3). It was difficult to recognize PLC-â1 by immunochemical detection on the chromatofocusing profiles
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Fig. 2. Separation of control human brain PLC by chromatofocusing. Samples were analysed as in Fig. 1. Symbols used are also as in Fig. 1.
of rat, control human and AD brain extracts. The chromatofocusing profile of human brain PLC activity on a Mono P HR column indicates that PLC-ã1 and PLC-ä1 are overlapped at pI values between 5.2 and 4.6, suggesting that these two PLC isozymes cannot be separated by this method (Figs 2, 3). Gel filtration chromatography of brain extracts Figure 4 shows a typical elution pattern of rat brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in rat brains. The first form was detected at the elution position having a molecular weight of about 220,000. The second form was detected at the elution position having a molecular weight of about 140,000. The first form of PLC activity was higher than the second form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in the rat brain (Fig. 4). PLC-â1 was detected as a very minor component before the elution position of PLC-ã1 (data not shown). Figure 5 shows a typical elution pattern of control human brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in control brains. The first form was detected at the elution position having a molecular weight of about
240,000. The second form was detected at the elution position having a molecular weight of about 140,000. The second form of PLC activity was higher than the first form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in the control brains (Fig. 5). PLC-â1 was detected as a very minor component before the elution position of PLC-ã1 (data not shown). Figure 6 shows a typical elution pattern of AD brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in AD brains. The first form was detected at the elution position having a molecular weight of about 240,000. The second form was detected at the elution position having a molecular weight of about 140,000. The second form of PLC activity was higher than the first form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in AD brains. It was difficult to recognize PLC-â1 by immunochemical detection. PLC-ã1 and PLC-ä1 were not detected at elution positions having molecular weights of 145,000 and 85,000, respectively, in rat, control human or AD brains (Figs 4–6). The elution profiles of control and AD brain PLC on Superdex 200 pg column gel
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Fig. 3. Separation of AD brain PLC by chromatofocusing. Samples were analysed as in Fig. 1. Symbols used are also as in Fig. 1.
filtration chromatography indicate that PLC-ã1 and PLC-ä1 isozymes can be separated by this method (Figs 5, 6); thus, the enzyme activities corresponding to PLC-ã1 and PLC-ä1, respectively, were compared between control and AD cases. The PLC-ã1 activity in AD brains was significantly lower than that in the control brains, whereas the PLC-ä1 activity was significantly higher than that in the control brains (Table 1). DISCUSSION
Although it is well recognized that PLC is a key molecule in signal transduction, little is known about the character of PLC isozymes in the brain. The present study intended to examine the properties of the brain PLC by chromatofocusing and gel filtration chromatography, and to investigate its changes in AD. Firstly, we examined which isozymes are mainly expressed in the cytosolic fraction of the rat brain. Immunochemical detection by anti-PLC-â1, -â2, -â3, -â4, -ã1, -ã2, -ä1 and -ä2 antibodies revealed that PLC-â1, -ã1 and -ä1 isozymes are recognized in the cytosolic fraction of the rat brain. As the specific antibodies against PLC-ä3 and -ä4 isozymes could not be obtained, expressions of PLC-ä3 and -ä4 isozymes in the rat brain were not clarified in the present study. However, Lee and Rhee10 very recently reported that PLC-ä4 is expressed at low concentrations in a limited number of rat tissues.
Brain was one of the rat tissues relatively rich in PLC-ä4, but the enzyme concentration of 7.2 ng per milligram of crude extract protein is significantly lower than those of PLC-â1 (70 ng/mg), PLC-ã1 (140 ng/mg) and PLC-ä1 (180 ng/mg). Moreover, PLC-ä4 appeared mostly in the nuclear fraction, while PLC-ã1 and -ä1 were in the cytosol, and PLC-â1 was mainly detected in the nucleus.11 These reports and our present results suggest that PLC-ã1 and -ä1 are mainly present in the cytosolic fraction of the brain. The chromatofocusing profile of PLC activity indicates a species difference between rats and humans; although two major peaks of PLC activity were obtained, they exhibited pI values of 4.3 and 4.0, and 5.2 and 4.6, respectively, in rat and human brains. Immunochemical detection by the antiPLC-ã1 and anti-PLC-ä1 antibodies revealed that the first form corresponds mainly to PLC-ã1, and the second form corresponds mainly to PLC-ä1 in the cytosolic fraction of the rat brain, whereas the first form corresponds to both PLC-ã1 and PLC-ä1, and the second form corresponds mainly to PLC-ä1 in the cytosolic fraction of the human brain, suggesting that these two PLC isozymes in the cytosolic fraction of the human brains cannot be separated by this method. Interestingly, the human brain had two types of PLC-ä1, exhibiting pI values of 5.2 and 4.6, in contrast to rat brain, which had only one, exhibiting a pI value of 4.0. As anti-PLC-ä1 antibody
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Fig. 4. Separation of rat brain PLC by Superdex 200 pg gel filtration chromatography. An extract of rat brain cytosols was applied to a Superdex 200 pg column (1.6#60 cm2) equilibrated and eluted with 25 mM HEPES buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1 M NaCl. Onemilliliter fractions were collected, assayed for PLC activity (A) and used for immunochemical detection (B, C). The assay for PLC activity and the method for immunochemical detection are described in Experimental Procedures. Arrows at the top of the figure show the elution position of standard proteins: (1) ferritin (molecular weight: 450,000); (2) catalase (molecular weight: 240,000); (3) sweet potato acid phosphatase24 (molecular weight: 110,000); (4) BSA (molecular weight: 68,000). B and C indicate the distribution of relative PLC-ã1 and PLC-ä1 immunoreactivity, respectively.
specifically recognizes PLC-ä119 and rat brain had only one type of PLC-ä1, PLC-ä1 in the human brain might be markedly post-translationally modified, or another unknown PLC-ä1 isozyme might be present in the cytosolic fraction of the human brain. PLC-ä4 in the brain exhibited an apparent molecular mass of 90,000 on SDS–PAGE,10 different from PLC-ä1, which exhibited an apparent molecular mass of 85,000 in the brain.19 The chromatofocusing profile of AD brain PLC activity is essentially the same as that of controls, with approximately the same pI values. The elution pattern of the PLC activity on Superdex 200 pg column gel filtration chromatography showed two major peaks of PLC activity in the cytosolic fraction of both rat and human brains. The first major peak corresponds mainly to PLC-ã1, and the second to PLC-ä1 in the cytosolic fraction of both rat and human brains, although the first peak of PLC activity was higher than the second peak in the rat brain, whereas the second peak is higher than the first peak in the human control brain. These results suggest that the dominant cytosolic PLC isozymes which can hydrolyse phosphatidylinositol are differ-
Fig. 5. Separation of control human brain PLC by Superdex 200 pg gel filtration chromatography. Samples were analysed as in Fig. 4. Symbols used are also as in Fig. 4.
Fig. 6. Separation of AD brain PLC by Superdex 200 pg gel filtration chromatography. Samples were analysed as in Fig. 4. Symbols used are also as in Fig. 4.
ent between rat and human brains. In addition, PLC-ã1 and PLC-ä1 were detected at elution positions having molecular weights of about 240,000 and 140,000, respectively, in human brain. The molecular weights of these two PLC isozymes are different from those obtained on SDS–PAGE; PLC-ã1 was detected as a protein with a molecular weight of 145,000, and PLC-ä1 was detected as a protein with a molecular weight of 85,000 on SDS–PAGE.19 Ryu et al.17 have analysed purified enzymes of bovine PLC-ã1 and
PLC isozymes in control and Alzheimer brain Table 1. The phospholipase C-ã1 and phospholipase C-ä1 activities in control and Alzheimer’s disease brain samples
PLC-ã1 activity (d.p.m.) PLC-ä1 activity (d.p.m.)
Controls (n=7)
Alzheimer (n=7)
2521&127 4503&136
1138&132* 5552&135*
The total PLC activity of the fractions which are stained with anti-PLC-ã1 and anti-PLC-ä1 antibodies in Superdex 200 pg gel filtration was designated as the PLC-ã1 activity and PLC-ä1 activity, respectively. Results show mean values&S.E.M. The PLC-ã1 activity was significantly decreased in the AD group (n=7) as compared with the control group (n=7): *P<0.001 by Dunnett’s two-tailed test. The PLC-ä1 activity was significantly increased in the AD group (n=7) as compared with the control group (n=7): *P<0.001 by Dunnett’s two-tailed test.
PLC-ä1 on non-denaturing polyacrylamide gradient gels and demonstrated that PLC-ã1 is predominantly monomeric and, to a smaller extent, dimeric. In contrast, two broad bands were observed for PLCä1. The estimated molecular weight values were 150,000–170,000 for the intense band and about 250,000 for the faint band, suggesting that PLC-ä1 exists mainly in a dimeric form.17 Our present results suggest that PLC-ä1 is present as a dimer, or some protein is bound to this enzyme. As for PLC-ã1, some protein might be bound to this enzyme in the natural form. These findings differ slightly from those reported by Ryu et al.17 Therefore, further study will be necessary to clarify this point. The elution pattern of AD brain PLC activity was almost the same as that of controls, with two major peaks of PLC activity: one at the elution position having a molecular weight of about 240,000, which is immunochemically identified with PLC-ã1, and the other at the elution position having a molecular weight of about 140,000, which is immunochemically identified with PLC-ä1. PLC-ã1 and PLC-ä1 were not detected at elution positions having molecular weights of 145,000 and 85,000, respectively, in AD brains, suggesting that no remarkable structural changes of either PLC-ã1 or PLC-ä1 occur in AD brains. As gel filtration chromatography can differentiate PLC-ä1 from PLC-ã1 in control and AD brains, in contrast to chromatofocusing, the enzyme activities corresponding to PLC-ã1 and PLC-ä1, respectively, could be compared between control and AD samples. The PLC-ã1 activity in the cytosolic fraction of AD brains was significantly lower than that in the control brains, whereas the PLC-ä1 activity in the cytosolic fraction of AD brains was significantly higher than that in the control brains. Total PLC activity was not changed in the cytosolic fraction of control and AD brains, which was consistent with the previous study.20 Thus, these results suggest that although total PLC activity was not
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changed in AD brains, PLC-ã1 and -ä1 isozymes are differentially involved in AD. The previous studies indicated that PLC-ä1 was abnormally accumulated in NFTs, the neurites surrounding SP cores, and neuropil threads, as well as in the cytosol of neurons, in AD brains when compared with control brains.19,22 The cytosolic PLC-ä1 activity was not significantly increased, although its protein level was raised more than two-fold in AD brains, suggesting that the specific activity of PLC-ä1 is decreased in AD brains.23 Several mechanisms, such as an increase of inhibitors, a decrease of activators, or post-translational modifications such as phosphorylation, glycosylation or oxidation, were considered to explain the inactivation of PLC-ä1 activity.23 The present gel filtration chromatography and chromatofocusing analysis demonstrated that the PLC-ä1 present in the AD brains has essentially the same molecular weight and pI values as that in the control brains, suggesting that marked post-translational modifications of the PLC-ä1 protein do not occur in AD. As the gel filtration chromatography indicated that the PLC-ä1 activity in the AD brains was higher than that in the control brains, inhibitors of PLC-ä1 or some PLC-ä1 binding proteins which reduce the catalytic activity of PLC-ä1 might be removed by gel filtration chromatography, although further work will be necessary to identify the inhibitors or binding proteins for PLCä1. Recent studies on crystal structure of a mammalian PLC-ä1 molecule might be useful to clarify the regulatory mechanism of PLC-ä1 activity.5,6 The present study first demonstrated that the cytosolic PLC-ã1 activity in AD brains is significantly decreased compared with that in control brains. The gel filtration chromatography and chromatofocusing analysis showed that the PLC-ã1 present in the AD brains has essentially the same molecular weight and pI values as that in the control brains, suggesting that specific post-translational modifications of the PLC-ã1 protein do not occur in AD brains. It is now recognized that PLC-ã1 is activated by the phosphorylation of its tyrosine residue induced by tyrosine kinase,15 and thus inactivation of tyrosine kinase might occur in AD, although protein tyrosine kinase activity in AD brains has not yet been studied in great detail.7 Indeed, PLC-ã1 activity has not been measured previously in AD brain, and several studies have reported tyrosine kinase activities in AD brain. The zinc- and magnesium-stimulated protein kinase activities, distinct from the activity of p60c-src, were decreased in AD hippocampus.25 The specific activity of protein tyrosine kinases in the particulate fractions decreased significantly in AD frontal cortex, although cytosolic protein tyrosine kinase activity in AD tissue was not significantly different from that in control tissue.18 Although the mechanism that the cytosolic PLC-ã1 activity is significantly decreased in AD brains should be elucidated in the future, several
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studies suggest that activation of PLC-ã1 is important for the regulation of cytoskeletal systems and maintenance of cell function. Recent data suggest that profilin, actin monomer sequester, is activated by the coordinated action of receptor tyrosine kinases and PLC-ã1 to stimulate the stabilization of actin filaments.3,26 Therefore, down-regulation of PLC-ã1 might be related to the neuronal dysfunction associated with AD. CONCLUSIONS
The PLC isozymes in human control and AD brains were examined using the methods of chromatofocusing and gel filtration chromatography. The gel filtration chromatography revealed that the PLC-ã1 activity was significantly decreased and the
PLC-ä1 activity was significantly increased in AD brains compared with controls. These alterations of phospholipid-specific signal transduction might be associated with key features of AD such as neuronal dysfunction and neuronal death.
Acknowledgements—This work was supported by Grantsin-Aid for Scientific Research (A and B), for Scientific Research on Priority Areas, and for Exploratory Research from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Ministry of Welfare of Japan, the Smoking Research Foundation, the Mitsui Life Social Welfare Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders and the Takeda Medical Research Foundation. We also thank Drs George Perry and Peter J. Whitehouse at Case Western Reserve University (Cleveland, OH, U.S.A.) for providing some autopsy samples used in the present study.
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Berridge M. J. and Irvine R. F. (1984) Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature 312, 315–321. Bradford M. M. (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analyt. Biochem. 72, 248–254. Chan A. C., Irving B. A., Fraser J. D. and Weiss A. (1991) The æ chain is associated with a tyrosine kinase and upon T-cell antigen receptor stimulation associates with ZAP-70, a 70-kDa tyrosine phosphoprotein. Proc. natn. Acad. Sci. U.S.A. 359, 66–70. Coleman P. D. and Flood D. G. (1987) Neuron numbers and dendritic extent in normal aging and Alzheimer’s disease. Neurobiol. Aging 8, 521–545. Essen L.-O., Perisic O., Cheung R., Katan M. and Williams R. L. (1996) Crystal structure of a mammalian phosphoinositide-specific phospholipase C-ä. Nature 380, 595–602. Ferguson K. M., Lemmon M. A., Schlessinger J. and Sigler P. B. (1995) Structure of the high affinity complex of inositol trisphosphate with a phospholipase C pleckstrin homology domain. Cell 83, 1037–1046. Fulop T. Jr and Seres I. (1994) Age-related changes in signal transduction. Implications for neuronal transmission and potential for drug intervention. Drugs Aging 5, 366–390. Homma Y., Emori Y., Shibasaki F., Suzuki K. and Takenawa T. (1990) Isolation and characterization of a ã-type phosphoinositide-specific phospholipase C (PLC-ã2). Biochem. J. 269, 13–18. Katzman R. (1986) Alzheimer’s disease. New Engl. J. Med. 314, 964–973. Lee S. B. and Rhee S. G. (1996) Molecular cloning, splice variants, expression, and purification of phospholipase C-ä4. J. biol. Chem. 271, 25–31. Liu N., Fukami K., Yu H. and Takenawa T. (1996) A new phospholipase C ä4 is induced at S-phase of the cell cycle and appears in the nucleus. J. biol. Chem. 271, 355–360. Majerus P. W., Connolly T. M., Deckmyn H., Ross T. S., Bross T. E., Bansal S. and Wilson D. B. (1986) The metabolism of phosphoinositide-derived messenger molecules. Science 234, 1519–1526. Mirra S. S., Heyman A., Mckeel D., Sumi S. M., Crain B. J., Brownlee L. M., Vogel F. S., Hughes J. P., van Belle G. and Berg L. (1991) The consortium to establish a registry for Alzheimer’s disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology 41, 479–486. Nishizuka Y. (1988) The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature 334, 661–665. Noh D. Y., Shin S. H. and Rhee S. G. (1995) Phosphoinositide-specific phospholipase C and mitogenic signaling. Biochim. biophys. Acta 1242, 99–114. Rhee S. G., Suh P. G., Ryu S. H. and Lee S. Y. (1989) Studies of inositol phospholipid-specific phospholipase C. Science 244, 546–550. Ryu S. H., Cho K. S., Lee K. Y., Suh P. G. and Rhee S. G. (1987) Purification and characterization of two immunologically distinct phosphoinositide-specific phospholipase C from bovine brain. J. biol. Chem. 262, 12,511–12,518. Shapiro I. P., Masliah E. and Saitoh T. (1991) Altered protein tyrosine phosphorylation in Alzheimer’s disease. J. Neurochem. 56, 1154–1162. Shimohama S., Homma S., Suenaga T., Fujimoto S., Taniguchi T., Araki W., Yamaoka Y., Takenawa T. and Kimura J. (1991) Aberrant accumulation of phospholipase C-delta in Alzheimer brains. Am. J. Path. 139, 737–742. Shimohama S., Fujimoto S., Taniguchi T. and Kimura J. (1992) Phosphatidylinositol-specific phospholipase C activity in the postmortem human brain: no alteration in Alzheimer’s disease. Brain Res. 579, 347–349. Shimohama S., Perry G., Richey P., Takenawa T., Whitehouse P. J., Miyoshi K., Suenaga T., Matsumoto S., Nishimura M. and Kimura J. (1993) Abnormal accumulation of phospholipase C-ä in filamentous inclusions of human neurodegenerative disease. Neurosci. Lett. 162, 183–186. Shimohama S., Perry G., Richey P., Praprotnik D., Takenawa T., Fukami K., Whitehouse P. J. and Kimura J. (1995) Characterization of the association of phospholipase C-ä with Alzheimer neurofibrillary tangles. Brain Res. 669, 217–224.
PLC isozymes in control and Alzheimer brain 23.
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Shimohama S., Fujimoto S., Matsushima H., Takenawa T., Taniguchi T., Perry G., Whitehouse P. J. and Kimura J. (1995) Alteration of phospholipase C-ä protein level and specific activity in Alzheimer’s disease. J. Neurochem. 64, 2629–2634. 24. Sugiura Y., Kawabe H., Tanaka H., Fujimoto S. and Ohara A. (1981) Purification, enzymatic properties, and active site environment of a novel manganese (III)-containing acid phosphatase. J. biol. Chem. 256, 10,664–10,670. 25. Vener A. V., Aksenova M. V. and Burbaeva G. Sh. (1993) Drastic reduction of the zinc- and magnesium-stimulated protein tyrosine kinase activities in Alzheimer’s disease hippocampus. Fedn Eur. biochem. Socs Lett. 328, 6–8. 26. Wange R. L., Kong A.-N. T. and Samelson L. E. (1992) A tyrosine-phosphorylated 70-kDa protein binds a photoaffinity analogue of ATP and associates with the æ chain and CD3 components of the activated T cell antigen receptor. J. biol. Chem. 267, 11,685–11,688. (Accepted 8 July 1997)
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Neuroscience Vol. 82, No. 4, pp. 999–1007, 1998 Copyright ? 1997 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/98 $19.00+0.00 S0306-4522(97)00342-4
PHOSPHOLIPASE C ISOZYMES IN THE HUMAN BRAIN AND THEIR CHANGES IN ALZHEIMER’S DISEASE S. SHIMOHAMA,*¶ Y. SASAKI,† S. FUJIMOTO,† S. KAMIYA,‡ T. TANIGUCHI,‡ T. TAKENAWA§ and J. KIMURA* *Department of Neurology, Faculty of Medicine, Kyoto University, Sakyoku, Kyoto 606, Japan Departments of †Environmental Biochemistry and ‡Neurobiology, Kyoto Pharmaceutical University, Yamashinaku, Kyoto 607, Japan §Department of Biochemistry, Institute of Medical Science, University of Tokyo, Minatoku, Tokyo 108, Japan Abstract––Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-ä1, accumulates aberrantly in the brains of patients with Alzheimer’s disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimer’s disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-ã1, exhibiting an isoelectric point value of 5.2, and phospholipase C-ä1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimer’s disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-ã1 and phospholipase C-ä1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-ã1 activity was significantly decreased and the phospholipase C-ä1 activity was significantly increased in Alzheimer’s disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimer’s disease. ? 1997 IBRO. Published by Elsevier Science Ltd. Key words: Alzheimer’s disease, phospholipase C, phospholipase C-ã1, phospholipase C-ä1, chromatofocusing, gel filtration chromatography.
Alzheimer’s disease (AD) is a neurodegenerative disease characterized by the progressive deterioration of cognitive function and memory. The characteristic lesions in AD brains include senile plaques (SPs), neurofibrillary tangles (NFTs) and neuronal loss. An important question is the nature of the key signaling that is responsible for the formation of these pathological hallmarks, as well as the neuronal dysfunction associated with this disease.4,9 There is increasing evidence that phosphoinositidespecific phospholipase C (PLC) is a key molecule in signal transduction. PLC catalyses the three phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol ¶To whom correspondence should be addressed. Abbreviations: AD, Alzheimer’s disease; BSA, bovine serum albumin; EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; HEPES, N-2-hydroxyethylpiperazineN*-2-ethanesulfonic acid; NFT, neurofibrillary tangle; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; pI, isoelectric point; PLC, phospholipase C; SDS, sodium dodecyl sulfate; SP, senile plaque.
4,5-biphosphate, to generate diacylglycerol and three inositol phosphates, among which diacylglycerol and inositol 1,4,5-trisphosphate serve as intracellular messengers for protein kinase C activation and intracellular Ca2+ mobilization.1,12,14,16 To date, at least 10 PLC isozymes have been identified by protein chemistry or cDNA cloning methods.15 We have previously demonstrated that a PLC isozyme, PLC-ä1, accumulates abnormally in NFTs, the neurites surrounding SP cores, and neuropil threads in AD brains.19,21,22 We have also shown that this abnormal accumulation of PLC-ä1 in AD brains is due to an increase of the protein level of this enzyme, and that its specific activity is decreased in AD brains compared with controls,23 although total PLC activity is not changed in control and AD brains.20 However, as the protein character of PLC-ä1 as well as other PLC isozymes has not been well investigated so far, in the present study we examined the property of PLC isozymes in human control and AD brains by using the methods of chromatofocusing and gel filtration chromatography.
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Materials -á-Phosphatidyl(myo-inositol 2-3H(N)) (1.0 Ci/mmol) was purchased from American Radiolabeled Chemicals Inc. (St Louis, MO, U.S.A.). -á-Phosphatidylinositol (Soybean, ammonium salt), phenylmethylsulfonyl fluoride and diisopropylfluorophosphate were obtained from Sigma Chemical Co. Sephadex G-25, Superdex 200 pg HiLoad and Mono P HR columns were obtained from Pharmacia LKB Biotechnology. Standard proteins used for calibration of the Sephadex G-25 and Superdex 200 pg columns were obtained from Boehringer Mannheim GmbH. All other chemicals were of reagent grade and were obtained commercially. Brain samples Brain tissues were obtained at autopsy from seven patients diagnosed clinically and histopathologically as having AD (mean age: 79 years; mean post mortem delay: 6 h), and from seven age-matched controls (mean age: 78 years; mean post mortem delay: 8 h) with no clinical or morphological evidence of brain pathology. All the AD patients were clinically severely demented, while those in control groups were not demented. Among the AD patients, the final cause of death was listed as cardiac insufficiency in three and a terminal respiratory condition in four. Among the control cases, four died from cardiac insufficiency and three from a terminal respiratory condition. Ante-post mortem hypoxic conditions exhibited essentially no significant difference between AD and control cases. Immediately after autopsy, the brains were divided sagittally into halves, with one half being used in the biochemical assays and the other half being examined histologically. The half to be used for the biochemical studies was stored at "80)C until thawing for homogenization and the biochemical studies. Temporal cortices were used for the present biochemical study. The neuropathological assessment of AD was made in accordance with the criteria of the Consortium to Establish a Registry for AD (CERAD).13 Tissue blocks were dissected and cut into 30-µm wedge microtome sections. Adjacent sections from the temporal cortices and the hippocampus of all brains were postfixed with 10% formaldehyde, and screened to provide a histological diagnosis. Control brains exhibited negligible microscopic neuropathology (0–2 SPs per low-power field). All the AD cases exhibited numerous SPs and NFTs throughout the neocortex. The locus coeruleus and nucleus basalis of Meynert showed severe atrophy, with NFTs in the remaining neurons. The substantia nigra was relatively well preserved and showed no Lewy bodies. Wistar rats were purchased from Japan SLC, Inc. The brains of seven-week-old Wistar rats were taken from animals. The animals were treated in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals. Preparation of brain extracts Brain tissue samples (1 g wet weight) were homogenized with a Teflon–glass homogenizer in 4 volumes of 10 mM HEPES buffer (pH 7.0) containing 0.32 M sucrose, 0.05% NaN3, 100 µM orthovanadate, 0.1 mM phenylmethylsulfonyl fluoride, 0.5 mM diisopropylfluorophosphate, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, 5 µg/ml leupeptin, 5 mM benzamidine, 5 mM 2-mercaptoethanol and 3 mM EGTA. The homogenate was centrifuged at 105,000#g for 60 min and the supernatant thus obtained was used as the brain extract for further experiments. Chromatofocusing of brain extracts Brain extract was passed through a Sephadex G-25 column equilibrated with 25 mM bis-Tris–iminodiacetic acid buffer (pH 7.1). The active fractions were pooled and
the enzyme solution was applied to a Mono P HR column (0.5#20 cm2) equilibrated with the above buffer and eluted with 1:10 diluted Polybuffer 74 (pH 4.0). One-milliliter fractions were collected, analysed for PLC activity and pH, and used for immunochemical detection. Gel filtration of brain extracts Brain extract was loaded on to a Superdex 200 pg column (1.6#60 cm2) equilibrated and eluted with 25 mM HEPES buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1 M NaCl. Two-milliliter fractions were collected, assayed for PLC activity and used for immunochemical detection. Apparent molecular weights of PLC activity fractions were estimated by using an elution profile of standard proteins [ferritin, molecular weight: 450,000; catalase, molecular weight: 240,000; sweet potato acid phosphatase,24 molecular weight: 110,000; bovine serum albumin (BSA), molecular weight: 68,000] on the same Superdex 200 pg column used in gel filtration chromatography of brain extracts after washing the column thoroughly with the elution buffer. Enzyme assay The PLC activity was measured according to the method described previously.20 In brief, the reaction mixtures contained 280 µM phosphatidylinositol, 30,000 d.p.m. of -áphosphatidyl-(myo-inositol 2-3H(N)), 1 mg/ml sodium deoxycholate, 1.5 mM CaCl2, 50 mM HEPES (pH 7.0), and brain extract (10 µg protein) or eluted sample from the column (20 µl). After incubation for 60 min at 37)C, the reaction was stopped with 1 ml of chloroform/methanol/ concentrated HCl (50:50:0.3), followed by the addition of 0.3 ml of 1 N HCl containing 5 mM EGTA. After centrifugation for 10 min at 3000#g, a 700-µl aliquot of the supernatant was removed for liquid scintillation counting. Protein concentration was determined by the method of Bradford with BSA as the standard.2 Antibodies A specific antibody against PLC-ä1 was prepared using PLC-ä1 protein produced by an E. coli expression system.8 The characterization and specificity of the antibody against PLC-ä1 has been fully described previously.19 This antibody recognizes human PLC-ä1 as an 85,000 mol. wt protein on sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE), as reported previously.19 The specific antibodies against PLC-â1, -â2, -â3, -â4, -ã1, -ã2 and -ä2 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, U.S.A.). Immunochemical detection The eluted samples from chromatofocusing and gel filtration chromatography in Laemmli sample buffer were subjected to electrophoresis on 4–20% SDS–PAGE or on 4–20% native PAGE, blotted on to Immobilon (Millipore), blocked with phosphate-buffered saline (PBS) containing 0.1% Tween 20 for 1 h, and incubated with the anti-PLC-â1 (1:500), -â2 (1:500), -â3 (1:500), -â4 (1:500), -ã1 (1:500), -ã2 (1:500), -ä1 (1:5000) and -ä2 (1:500) antibodies in PBS containing 3% BSA for 18 h at 4)C. Blots were then washed with PBS/0.1% Tween 20, incubated with horseradish peroxidase-linked antibody against rabbit immunoglobulin (diluted 1:1000). Subsequently, membrane-bound horseradish peroxidase-labeled antibodies were detected by the enhanced chemiluminescence detection system (ECL kit, Amersham). The protein bands that cross-reacted with antibodies could be detected in X-ray films (X-Omat JB-1, Kodak) 5–60 s after the exposure. The integrated optical density of the 85,000 mol. wt protein band recognized by the anti-PLC-ä1 antibody and that of the 145,000 mol. wt protein band recognized by the anti-PLC-ã1 antibody were
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Fig. 1. Separation of rat brain PLC by chromatofocusing. An extract of rat brain cytosol was passed through a Sephadex G-25 column equilibrated with 25 mM bis-Tris–iminodiacetic acid buffer (pH 7.1). The active fractions were pooled and the enzyme solution was applied to a Mono P HR column (0.5#20 cm2) equilibrated with the above buffer and eluted with 1:10 diluted Polybuffer 74 (pH 3.8). One-milliliter fractions were collected, analysed for PLC activity and pH (A), and used for immunochemical detection (B, C). The assay for PLC activity and the method for immunochemical detection are described in Experimental Procedures. B and C indicate the distribution of relative PLC-ã1 and PLC-ä1 immunoreactivity, respectively.
measured by a scanning densitometer (Arcus II, Agfa) and were taken to indicate the relative quantities of PLC-ä1 and PLC-ã1, respectively. RESULTS
Expression of phospholipase C isozymes in rat brain extracts To clarify which PLC isozymes are expressed in rat brain extracts, immunochemical analyses by the antiPLC-â1, -â2, -â3, -â4, -ã1, -ã2, -ä1 and -ä2 antibodies were applied to brain extracts. Only PLC-â1, -ã1 and -ä1 isozymes were detected in rat brain extracts (data not shown); therefore, the following immunochemical analyses were performed using the anti-PLC-â1, -ã1 and -ä1 antibodies. Chromatofocusing of brain extracts Figure 1 shows a typical chromatofocusing profile of rat brain PLC activity on a Mono P HR column. Two major peaks of PLC activity were obtained, and they exhibited isoelectric point (pI) values of 4.3 and 4.0, respectively. Immunochemical detection by the anti-PLC-ã1 and anti-PLC-ä1 antibodies revealed that the first form of PLC activity mainly corresponds to PLC-ã1, and the second form of PLC
activity mainly corresponds to PLC-ä1 (Fig. 1). To examine the post mortem stability of PLC, the rat brains were left for 0, 7 and 24 h at room temperature. The chromatofocusing profile of PLC activity was not changed even after the brains had been left for 24 h at room temperature (data not shown). Figure 2 shows a typical chromatofocusing profile of control human brain PLC activity. Two major peaks of PLC activity were obtained, and they exhibited pI values of about 5.2 and 4.6, respectively, in control human brains. Immunochemical detection by the anti-PLC-ã1 and anti-PLC-ä1 antibodies revealed that the first large form of PLC activity corresponds to both PLC-ã1 and PLC-ä1, and the second small form of PLC activity corresponds mainly to PLC-ä1. (Fig. 2). Figure 3 shows a typical chromatofocusing profile of AD brain PLC activity. Two major peaks of PLC activity were also obtained, and they exhibited pI values of about 5.2 and 4.6, respectively, in AD brains. Immunochemical detection by the antiPLC-ã1 and anti-PLC-ä1 antibodies revealed that the first large form of PLC activity corresponds to both PLC-ã1 and PLC-ä1, and the second small form of PLC activity corresponds mainly to PLC-ä1. (Fig. 3). It was difficult to recognize PLC-â1 by immunochemical detection on the chromatofocusing profiles
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Fig. 2. Separation of control human brain PLC by chromatofocusing. Samples were analysed as in Fig. 1. Symbols used are also as in Fig. 1.
of rat, control human and AD brain extracts. The chromatofocusing profile of human brain PLC activity on a Mono P HR column indicates that PLC-ã1 and PLC-ä1 are overlapped at pI values between 5.2 and 4.6, suggesting that these two PLC isozymes cannot be separated by this method (Figs 2, 3). Gel filtration chromatography of brain extracts Figure 4 shows a typical elution pattern of rat brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in rat brains. The first form was detected at the elution position having a molecular weight of about 220,000. The second form was detected at the elution position having a molecular weight of about 140,000. The first form of PLC activity was higher than the second form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in the rat brain (Fig. 4). PLC-â1 was detected as a very minor component before the elution position of PLC-ã1 (data not shown). Figure 5 shows a typical elution pattern of control human brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in control brains. The first form was detected at the elution position having a molecular weight of about
240,000. The second form was detected at the elution position having a molecular weight of about 140,000. The second form of PLC activity was higher than the first form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in the control brains (Fig. 5). PLC-â1 was detected as a very minor component before the elution position of PLC-ã1 (data not shown). Figure 6 shows a typical elution pattern of AD brain PLC on a Superdex 200 pg column. Two major peaks of PLC activity were obtained in AD brains. The first form was detected at the elution position having a molecular weight of about 240,000. The second form was detected at the elution position having a molecular weight of about 140,000. The second form of PLC activity was higher than the first form. Immunochemical detection by specific antibodies against PLC isozymes revealed that the first major peak of PLC activity corresponds mainly to PLC-ã1, and the second major peak corresponds mainly to PLC-ä1 in AD brains. It was difficult to recognize PLC-â1 by immunochemical detection. PLC-ã1 and PLC-ä1 were not detected at elution positions having molecular weights of 145,000 and 85,000, respectively, in rat, control human or AD brains (Figs 4–6). The elution profiles of control and AD brain PLC on Superdex 200 pg column gel
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Fig. 3. Separation of AD brain PLC by chromatofocusing. Samples were analysed as in Fig. 1. Symbols used are also as in Fig. 1.
filtration chromatography indicate that PLC-ã1 and PLC-ä1 isozymes can be separated by this method (Figs 5, 6); thus, the enzyme activities corresponding to PLC-ã1 and PLC-ä1, respectively, were compared between control and AD cases. The PLC-ã1 activity in AD brains was significantly lower than that in the control brains, whereas the PLC-ä1 activity was significantly higher than that in the control brains (Table 1). DISCUSSION
Although it is well recognized that PLC is a key molecule in signal transduction, little is known about the character of PLC isozymes in the brain. The present study intended to examine the properties of the brain PLC by chromatofocusing and gel filtration chromatography, and to investigate its changes in AD. Firstly, we examined which isozymes are mainly expressed in the cytosolic fraction of the rat brain. Immunochemical detection by anti-PLC-â1, -â2, -â3, -â4, -ã1, -ã2, -ä1 and -ä2 antibodies revealed that PLC-â1, -ã1 and -ä1 isozymes are recognized in the cytosolic fraction of the rat brain. As the specific antibodies against PLC-ä3 and -ä4 isozymes could not be obtained, expressions of PLC-ä3 and -ä4 isozymes in the rat brain were not clarified in the present study. However, Lee and Rhee10 very recently reported that PLC-ä4 is expressed at low concentrations in a limited number of rat tissues.
Brain was one of the rat tissues relatively rich in PLC-ä4, but the enzyme concentration of 7.2 ng per milligram of crude extract protein is significantly lower than those of PLC-â1 (70 ng/mg), PLC-ã1 (140 ng/mg) and PLC-ä1 (180 ng/mg). Moreover, PLC-ä4 appeared mostly in the nuclear fraction, while PLC-ã1 and -ä1 were in the cytosol, and PLC-â1 was mainly detected in the nucleus.11 These reports and our present results suggest that PLC-ã1 and -ä1 are mainly present in the cytosolic fraction of the brain. The chromatofocusing profile of PLC activity indicates a species difference between rats and humans; although two major peaks of PLC activity were obtained, they exhibited pI values of 4.3 and 4.0, and 5.2 and 4.6, respectively, in rat and human brains. Immunochemical detection by the antiPLC-ã1 and anti-PLC-ä1 antibodies revealed that the first form corresponds mainly to PLC-ã1, and the second form corresponds mainly to PLC-ä1 in the cytosolic fraction of the rat brain, whereas the first form corresponds to both PLC-ã1 and PLC-ä1, and the second form corresponds mainly to PLC-ä1 in the cytosolic fraction of the human brain, suggesting that these two PLC isozymes in the cytosolic fraction of the human brains cannot be separated by this method. Interestingly, the human brain had two types of PLC-ä1, exhibiting pI values of 5.2 and 4.6, in contrast to rat brain, which had only one, exhibiting a pI value of 4.0. As anti-PLC-ä1 antibody
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Fig. 4. Separation of rat brain PLC by Superdex 200 pg gel filtration chromatography. An extract of rat brain cytosols was applied to a Superdex 200 pg column (1.6#60 cm2) equilibrated and eluted with 25 mM HEPES buffer (pH 7.0) containing 1 mM dithiothreitol and 0.1 M NaCl. Onemilliliter fractions were collected, assayed for PLC activity (A) and used for immunochemical detection (B, C). The assay for PLC activity and the method for immunochemical detection are described in Experimental Procedures. Arrows at the top of the figure show the elution position of standard proteins: (1) ferritin (molecular weight: 450,000); (2) catalase (molecular weight: 240,000); (3) sweet potato acid phosphatase24 (molecular weight: 110,000); (4) BSA (molecular weight: 68,000). B and C indicate the distribution of relative PLC-ã1 and PLC-ä1 immunoreactivity, respectively.
specifically recognizes PLC-ä119 and rat brain had only one type of PLC-ä1, PLC-ä1 in the human brain might be markedly post-translationally modified, or another unknown PLC-ä1 isozyme might be present in the cytosolic fraction of the human brain. PLC-ä4 in the brain exhibited an apparent molecular mass of 90,000 on SDS–PAGE,10 different from PLC-ä1, which exhibited an apparent molecular mass of 85,000 in the brain.19 The chromatofocusing profile of AD brain PLC activity is essentially the same as that of controls, with approximately the same pI values. The elution pattern of the PLC activity on Superdex 200 pg column gel filtration chromatography showed two major peaks of PLC activity in the cytosolic fraction of both rat and human brains. The first major peak corresponds mainly to PLC-ã1, and the second to PLC-ä1 in the cytosolic fraction of both rat and human brains, although the first peak of PLC activity was higher than the second peak in the rat brain, whereas the second peak is higher than the first peak in the human control brain. These results suggest that the dominant cytosolic PLC isozymes which can hydrolyse phosphatidylinositol are differ-
Fig. 5. Separation of control human brain PLC by Superdex 200 pg gel filtration chromatography. Samples were analysed as in Fig. 4. Symbols used are also as in Fig. 4.
Fig. 6. Separation of AD brain PLC by Superdex 200 pg gel filtration chromatography. Samples were analysed as in Fig. 4. Symbols used are also as in Fig. 4.
ent between rat and human brains. In addition, PLC-ã1 and PLC-ä1 were detected at elution positions having molecular weights of about 240,000 and 140,000, respectively, in human brain. The molecular weights of these two PLC isozymes are different from those obtained on SDS–PAGE; PLC-ã1 was detected as a protein with a molecular weight of 145,000, and PLC-ä1 was detected as a protein with a molecular weight of 85,000 on SDS–PAGE.19 Ryu et al.17 have analysed purified enzymes of bovine PLC-ã1 and
PLC isozymes in control and Alzheimer brain Table 1. The phospholipase C-ã1 and phospholipase C-ä1 activities in control and Alzheimer’s disease brain samples
PLC-ã1 activity (d.p.m.) PLC-ä1 activity (d.p.m.)
Controls (n=7)
Alzheimer (n=7)
2521&127 4503&136
1138&132* 5552&135*
The total PLC activity of the fractions which are stained with anti-PLC-ã1 and anti-PLC-ä1 antibodies in Superdex 200 pg gel filtration was designated as the PLC-ã1 activity and PLC-ä1 activity, respectively. Results show mean values&S.E.M. The PLC-ã1 activity was significantly decreased in the AD group (n=7) as compared with the control group (n=7): *P<0.001 by Dunnett’s two-tailed test. The PLC-ä1 activity was significantly increased in the AD group (n=7) as compared with the control group (n=7): *P<0.001 by Dunnett’s two-tailed test.
PLC-ä1 on non-denaturing polyacrylamide gradient gels and demonstrated that PLC-ã1 is predominantly monomeric and, to a smaller extent, dimeric. In contrast, two broad bands were observed for PLCä1. The estimated molecular weight values were 150,000–170,000 for the intense band and about 250,000 for the faint band, suggesting that PLC-ä1 exists mainly in a dimeric form.17 Our present results suggest that PLC-ä1 is present as a dimer, or some protein is bound to this enzyme. As for PLC-ã1, some protein might be bound to this enzyme in the natural form. These findings differ slightly from those reported by Ryu et al.17 Therefore, further study will be necessary to clarify this point. The elution pattern of AD brain PLC activity was almost the same as that of controls, with two major peaks of PLC activity: one at the elution position having a molecular weight of about 240,000, which is immunochemically identified with PLC-ã1, and the other at the elution position having a molecular weight of about 140,000, which is immunochemically identified with PLC-ä1. PLC-ã1 and PLC-ä1 were not detected at elution positions having molecular weights of 145,000 and 85,000, respectively, in AD brains, suggesting that no remarkable structural changes of either PLC-ã1 or PLC-ä1 occur in AD brains. As gel filtration chromatography can differentiate PLC-ä1 from PLC-ã1 in control and AD brains, in contrast to chromatofocusing, the enzyme activities corresponding to PLC-ã1 and PLC-ä1, respectively, could be compared between control and AD samples. The PLC-ã1 activity in the cytosolic fraction of AD brains was significantly lower than that in the control brains, whereas the PLC-ä1 activity in the cytosolic fraction of AD brains was significantly higher than that in the control brains. Total PLC activity was not changed in the cytosolic fraction of control and AD brains, which was consistent with the previous study.20 Thus, these results suggest that although total PLC activity was not
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changed in AD brains, PLC-ã1 and -ä1 isozymes are differentially involved in AD. The previous studies indicated that PLC-ä1 was abnormally accumulated in NFTs, the neurites surrounding SP cores, and neuropil threads, as well as in the cytosol of neurons, in AD brains when compared with control brains.19,22 The cytosolic PLC-ä1 activity was not significantly increased, although its protein level was raised more than two-fold in AD brains, suggesting that the specific activity of PLC-ä1 is decreased in AD brains.23 Several mechanisms, such as an increase of inhibitors, a decrease of activators, or post-translational modifications such as phosphorylation, glycosylation or oxidation, were considered to explain the inactivation of PLC-ä1 activity.23 The present gel filtration chromatography and chromatofocusing analysis demonstrated that the PLC-ä1 present in the AD brains has essentially the same molecular weight and pI values as that in the control brains, suggesting that marked post-translational modifications of the PLC-ä1 protein do not occur in AD. As the gel filtration chromatography indicated that the PLC-ä1 activity in the AD brains was higher than that in the control brains, inhibitors of PLC-ä1 or some PLC-ä1 binding proteins which reduce the catalytic activity of PLC-ä1 might be removed by gel filtration chromatography, although further work will be necessary to identify the inhibitors or binding proteins for PLCä1. Recent studies on crystal structure of a mammalian PLC-ä1 molecule might be useful to clarify the regulatory mechanism of PLC-ä1 activity.5,6 The present study first demonstrated that the cytosolic PLC-ã1 activity in AD brains is significantly decreased compared with that in control brains. The gel filtration chromatography and chromatofocusing analysis showed that the PLC-ã1 present in the AD brains has essentially the same molecular weight and pI values as that in the control brains, suggesting that specific post-translational modifications of the PLC-ã1 protein do not occur in AD brains. It is now recognized that PLC-ã1 is activated by the phosphorylation of its tyrosine residue induced by tyrosine kinase,15 and thus inactivation of tyrosine kinase might occur in AD, although protein tyrosine kinase activity in AD brains has not yet been studied in great detail.7 Indeed, PLC-ã1 activity has not been measured previously in AD brain, and several studies have reported tyrosine kinase activities in AD brain. The zinc- and magnesium-stimulated protein kinase activities, distinct from the activity of p60c-src, were decreased in AD hippocampus.25 The specific activity of protein tyrosine kinases in the particulate fractions decreased significantly in AD frontal cortex, although cytosolic protein tyrosine kinase activity in AD tissue was not significantly different from that in control tissue.18 Although the mechanism that the cytosolic PLC-ã1 activity is significantly decreased in AD brains should be elucidated in the future, several
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studies suggest that activation of PLC-ã1 is important for the regulation of cytoskeletal systems and maintenance of cell function. Recent data suggest that profilin, actin monomer sequester, is activated by the coordinated action of receptor tyrosine kinases and PLC-ã1 to stimulate the stabilization of actin filaments.3,26 Therefore, down-regulation of PLC-ã1 might be related to the neuronal dysfunction associated with AD. CONCLUSIONS
The PLC isozymes in human control and AD brains were examined using the methods of chromatofocusing and gel filtration chromatography. The gel filtration chromatography revealed that the PLC-ã1 activity was significantly decreased and the
PLC-ä1 activity was significantly increased in AD brains compared with controls. These alterations of phospholipid-specific signal transduction might be associated with key features of AD such as neuronal dysfunction and neuronal death.
Acknowledgements—This work was supported by Grantsin-Aid for Scientific Research (A and B), for Scientific Research on Priority Areas, and for Exploratory Research from the Ministry of Education, Science, Sports and Culture, Japan, grants from the Ministry of Welfare of Japan, the Smoking Research Foundation, the Mitsui Life Social Welfare Foundation, the Yamanouchi Foundation for Research on Metabolic Disorders and the Takeda Medical Research Foundation. We also thank Drs George Perry and Peter J. Whitehouse at Case Western Reserve University (Cleveland, OH, U.S.A.) for providing some autopsy samples used in the present study.
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