Cerebral microvessels in Alzheimer's have reduced protein kinase C activity

Neurobiologyof Aging.Vol. 16, No. 4, pp. 563-569, 1995 Copyright © 1995ElsevierScienceLtd. Printed in the USA. All rights reserved 0197-4580/95 $9.50 + .00

Pergamon 0197-4580(95)00048-8

Cerebral Microvessels in Alzheimer's Have Reduced Protein Kinase C Activity P A U L A G R A M M A S , *l P E T E M O O R E , * T O Y A B O T C H L E T , * O L I V I A H A N S O N - P A I N T O N , * ' ~ D E N I S E R. COOPER,:I: M E L V Y N J. B A L L § A N D A L E X R O H E R #

*Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190 tDepartment of Chemistry, University of Central Oklahoma, Edmond, OK 73034 ~Department of Medicine, Veterans Hospital, Tampa, FL 33612 §Departments of Pathology & Neurology, Oregon Health Sciences University, Portland, OR 97201 #Department of Anatomy & Cell Biology, Wayne State University, Detroit, MI 48201 Received 24 M a y 1994; Revised 30 N o v e m b e r 1994; Accepted 15 D e c e m b e r 1994 GRAMMAS, P., P. MOORE, T. BOTCHLET, O. HANSON-PAINTON, D. R. COOPER, M. J. BALL AND A. ROHER. Cerebralmicrovesselsin Alzheirner's have reducedprotein kinase C activity. NEUROBIOL AGING 16(4) 563-569, 1995.Protein kinase C (PKC) is an important intracellular signalling enzyme. Numerous studies have suggested that alterations in this enzyme occur in aging and dementia. The objective of this study was to examine PKC in the cerebral microcirculation in aging and Alzheimer':~disease. PKC activity, amount, and isoform distribution were analyzed in microvesselsfrom adult and aged rodents as well as from Alzheimer patients and nondemented elderly controls. PKC activity was lower in Alzheimer vessels than in vessels from control brains, despite the presence of similar levels of PKC enzyme. In contrast, both activity and enzyme levelsin young and aged rats were comparable. The/3-isoform was present in both rat and human microvesselsand there were no age- or disea:~e-relatedalterations. The loss in activity in cerebromicrovascular PKC in Alzheimer's suggest that perturbations in phosphorylation signalling cascades may exist at the Alzheimer blood-brain barrier. Aging

Alzheimer's

Microvessels

Blood-brain barrier

PROTEIN kinase C (PKC) was initially identified as a protein kinase activated in vitro by partial proteolysis (32) and later shown to be reversibly actiwtted by calcium and phospholipids, especially phosphatidylserine (40). Phosphorylation of serine and threonine residues by PKC is an important mechanism of receptor-mediated signal transduction. Defects in postreceptor signalling pathways, including PKC, have been observed in aging and Alzheimer's disease (AD) (8,15,18,36,37,43,46). Fibroblasts from AD patients have both reduced PKC immunoreactivity and altered protein phosphorylation (3). In brain, PKC was shown to decrease 50% in the frontal cortex in AD in both amount, evaluated hy phorbol binding, and in enzyme activity, assessed by in vitro phosphorylation of histone (8). In addition, decreased phosphorylation of PKC substrates and diminished PKC activity have been demonstrated in several brain regions in AD (8,43) and changes in isoform distribution in AD appeared to be related to AD lesions. Using antibodies to the major calcium and phospholipid dependent isoforms, Masliah et al. (36) demonstrated that antibodies to/3I-PKC stained dystrophic plaque neurites and antibodies to c¢-PKC faintly stained entire plaques and surrounding glial cells, while in contrast,/3II-PKC antibodies reacted only to the amyloid-

Signaltransduction

Protein kinase C

containing portions of plaques and there was no staining for /3-PKC in any type of plaque. Because in rat brain there is a significant decrease in PKC activity with age (17), it is possible that AD can augment or accelerate the age-related reduction of PKC in brain. Although most studies have identified a decrease in PKC level or activity, changes in this enzyme in AD are not generalized in CNS because no alterations in PKC are discernible in the cerebellum. Furthermore, changes in PKC responsiveness in AD are not uniform and are variable in different areas of the brain (43). An important cell type in which PKC has not been examined in aging and AD is the cerebral endothelium, i.e., the blood-brain barrier. The notion that the cerebral microcirculation would be an important target in AD is supported by data showing selective structural and functional changes of the blood-brain barrier in AD (21,22,29,50). Furthermore, the immunocytochemical and electron microscopic demonstration that the amyloid of senile plaques is often observed in close proximity to affected microvessels (39,41,51,52) suggests a role for the vasculature in amyloid deposition and plaque formation in AD. The objective of this study was to examine PKC in cerebral microvessels, the constituent cell of the blood-brain barrier in

Requests for reprints shoultdbe addressed to Paula Grammas, Ph.D., University of Oklahoma Health SciencesCenter, Department of Pathology, P.O. BOX 26901, Oklahoma City, OK 73190. 563

564

GRAMMAS ET AL.

aging and AD. Enzyme activity, level, and isoforms were compared in microvessels isolated from adult and aged rats as well as in vessels from AD patients and age-matched controls.

described (23). Protein concentrations were determined by the method of Bradford (2) using BSA as a standard.

PKC Activity Assay METHOD

Brains were studied from 10 patients who died as a result of AD (mean age 79.6 + 3.7 years) and 10 elderly controls (mean age 69.5 _+ 9.5 years) whose neuropathology and clinical history showed no neurological or psychiatric disease. The clinical diagnosis of primary degenerative dementia of Alzheimer type was confirmed by neuropathological examination and accepted quantitative criteria. Each case met with the diagnostic criteria recommended by the NIH Neuropathology Panel (31) and by the Consortium to Establish a Registry for AD (1,38). The postmortem time for control samples (5.5 _+2.2 h) was not significantly different from the times for AD samples (6.4 + 1.6 h).

Microvessel Isolation A microvessel preparation consisting primarily of capillary segments was isolated from the cerebral cortices of 18 male adult (1 to 3 months) and aged (18 to 24 months) Fischer 344 rats as previously described (10). Briefly, the cerebral cortex was minced in cold Ca 2+ and Mg 2+ free Hank's balanced salt solution (HBSS) and homogenized using 20 up and down strokes in a glass homogenizer fitted with a serrated Teflon pestle. The homogenate was centrifuged at 2,000 g for 15 min, the pellet resuspended in 15% dextran containing 5°70 fetal calf serum (FCS) and centrifuged at 3,000 g for 20 min. The supernatant was discarded and the pellet was resuspended in HBSS and filtered through a 150 t~m nylon mesh. The filtrate was applied to glass bead columns, washed, and recovered in HBSS. From 15 g of cortex the rat isolation yielded 10-12 mg microvessel protein. The procedure for the isolation of cerebral microvessels from human brain utilized pooled temporal, parietal, and frontal cortices and is a modification of the procedure for the rat isolation using filtration through a 210/~m sieve and a final microvessel collection on a 53 izm sieve (21). This procedure yields approximately 6 to 10 mg microvessel protein from 15 g of human cortex. A separate microvessel preparation was isolated from each human brain. The purity of the microvessel preparations was routinely monitored by phase-contrast microscopy. Both rat and human microvessel pellets were resuspended in Dulbecco's modified Eagle's medium containing 20% dimethylsulfoxide and supplemented with 10°70 FCS and then stored in liquid nitrogen until use. We have previously observed that PKC activity levels were similar between freshly prepared microvessel fractions and those from previously frozen vessels (23). Frozen microvessels were thawed rapidly at 37°C, centrifuged at 6000 g for 10 min in a rotor precooled to 4°C, then resuspended in HBSS. The centrifugation and wash in HBSS was repeated twice more. Protein concentrations were determined according to the method of Lowry et al. (34) using bovine serum albumin (BSA) as a standard.

Preparation and Fractionation of Tissue Homogenates Cytosolic and particulate fractions were prepared from microvessel homogenates by a modification (23) of the method of Kikkawa et al. (32). Microvessels were resuspended in 1.0 ml of ice cold homogenization buffer (20 mM Tris-HC1, pH 7.5, 2.0 mM EDTA, 5.0 mM EGTA, 10.0 mM B-mercaptoethanol, and 100/~g/ml leupeptin), homogenized on a Polytron at 12,000 rpm for 1 min at 4°C, and then centrifuged at 100,000 g for 40 rain at 4°C. If not used immediately, the samples were frozen at - 7 0 ° C until use. Brain cytosol was prepared as previously

Our previous studies of PKC in cerebral microvessels demonstrated that the majority of enzyme activity was recoverable in the cytosolic fraction (23). Therefore, in the present study all PKC activity measurements, except translocation experiments, were performed on the cytosolic fractions. The cytosolic fraction was enriched on Q-Sepharose and PKC activity was assayed as previously described (23) in a total volume of 130 IA containing 100 #1Q-Sepharose eluate (15 to 27/zg protein), in 20.0 mM TrisHC1, pH 7.5, containing 26/zg of histone H1 type IIIs, 1.0 mM magnesium acetate, 1.3 mM ATP, 3 to 4 x 106 cpm ('y-32p)ATP, 0.5 mM free CaC12, and in the presence or absence of 13 #g of phosphatidylserine and 2.6 #g of 1,2-dioleoyl-sn-glycerol dispersed by sonication in 20 mM Tris-HCl, pH 7.5. Reactions were initiated by addition of 10 izl of 2.6 mg/ml histone H1 and 3 to 4 × 106 cpm (3,-32p)ATP, incubated with shaking for 7 min at 30°C, and terminated by addition of 75/A of glacial acetic acid. The samples were then applied to 1" x 1" Whatman P81 phosphocellulose filters, washed in 33% glacial acetic acid, air dried, and counted. Samples were assayed in duplicate.

Translocation by Phorbol Ester Frozen microvessels were rapidly thawed at 37°C and placed on ice or at 4°C for the remainder of the manipulations. The microvessels were transferred to homogenization buffer containing protease inhibitor as defined above by washing two times by centrifugation at 7000 g for 10 min and resuspension in 1.0 ml. The final pellet was resuspended in 1.25 ml and aliquots were taken for protein determination and for treatment with phorbol ester. Phorbol 12-myristate (PMA) was added to 1 aliquot to a final concentration of 100 nM, followed by incubation at 37°C for 10 min. The control aliquot without P M A was incubated in parallel. Samples were then diluted with 1.0 ml homogenization buffer, homogenized, and centrifuged as above. Each sample was treated with 1070Triton X-100 and the PKC activity assayed.

Phorbol Ester Binding Assay The phorbol binding assay was performed according to published methods (23,44). The unfractionated microvessels were resuspended in assay buffer (50 mM Tris-HC1, pH 7.5, 2.5 mM KC1, 8 mM MgCI2, 0.25 M sucrose, 0.5 mM CaCI2, 0.02 mg/ 1.0 ml phosphatidylserine and 0.5 mg/ml BSA) and suspensions containing 100/zg of protein in a total volume of 0.10 ml and incubated for 30 min at 24°C. The incubation was terminated by the addition of cold assay buffer and the contents filtered using Whatman G F / C filters under vacuum. Each binding determination was performed in triplicate and triplicate samples containing 10 #M phorbol dibutyrate (phorbol ester) were included to determine nonspecific binding.

Immunoblotting c~-,/3-, and 3'-PKC isoform expression was determined by immunoblot analysis as previously described (13,23). Polyclonal antipeptide antibodies to a- and 3'- PKC were purchased from GIBCO BRL. Anti/3-PKC was provided by B. Roth (42). Both particulate and cytosolic fractions (5 to 10 ~g) were resolved on 9% denaturing polyacrylamide gels (33) and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding sites were blocked by incubation for 1 h in TBS (20 mM Tris,

BLOOD-BRAIN BARRIER PKC IN ALZHEIMER'S pH 7.5,500 mM NaC1) containing 3% gelatin. Membranes were washed with TBS containing 0.05°7o Tween-20, incubated for 12 to 18 h with the appropriate PKC antibodies at dilutions of l: 1000 to 1:2000 in TBS con~Laining1°70gelatin, then incubated for 1 to 2 h with goat anti-rabbit 3,-globulin coupled to alkaline phosphatase and developed using nitro blue tetrazolium for detection. The specificity of the 80--90 kDa immunoreactive bands of PKC was verified by comparison to the migration of microvessel immunoreactive bands to that of purified whole rat brain PKC standards. In addition, the specificity was confirmed by the specific loss of immunoreactive bands in the presence of the immunogenic synthetic peptide and by detailed comparison of the cross reaction of the antibodies in numerous rat tissues (l 3).

Statistical A nalysis A comparison of PKC activity between microvessels from aged and adult rats and from control and Alzheimer subjects was performed using the hypothesis test for difference between two group means. Statistical significance was determined at p < 0.05.

565 for this difference, PKC was measured in rat brain cytosol over an 8 hour postmortem interval. There was a rapid decrease of 34°7o at 1 h postmortem, but between 2 and 8 h there was little additional decline (Fig. 2).

PKC Quantitation by Phorbol Binding Specific binding of phorbol ester to rat and human brain microvessels was saturable (Fig. 3). A composite saturation binding isotherm for each group is shown in Fig. 3. Scatchard analysis of these composite data indicated a KD of 122 nM for aged and 84 nM for adult-derived microvessels and/3max of 31.9 pmol/mg for aged and 30.2 pmol/mg for adult-derived microvessels. Scatchard analysis of the data from the four individual experiments (K D 81.9 _+ 20,/3max 25.6 _+7 for adult; K D 101 + 27;/3max 25.3 _+ g for aged) were similar, confirming that the parameters derived from the composite data were valid. Phorbol ester binding to human microvessel preparations was lower than that to rat vessels. The composite data for human microvessels are shown in Fig. 3. Scatchard analysis of these data indicated a K D of 81.9 nM for elderly controls and 38 nM for AD-derived microvessels and a/3max of 48 pmol/mg for con-

1;'ESULTS Q-Sepharose Purified PKC Activity in Rat and Human Microvessels: PKC activity measured in isolated microvessels from AD patients was significantly (p < 0.05) lower when compared to enzyme activity in microvessels from control brains (Table 1). In contrast, PKC activity in microvessels from aged and adult rodents was not significantly different (Table 1). To confirm that the measured kinase activity was due to PKC, phosphorylation was measured in the presence of PKC inhibitors (24,47,48). Staurosporine (7 #M) inhibited PKC activity in both rat and human microvessels (Fig. 1). In addition, bisindolylmaleimide GF109203X, (1 #M) a more specific PKC inhibitor (48), completely blocked kinase activity in both rat and human microvessels (data not shown). Interestingly, although PMA caused a translocation of PKC activity from cytosol to particulate fraction in rat microvessels (30(70 translocation), no phorbol ester effect was demonstrable in human tissue from either AD or control-derived microvessels. The level of PKC activity in human autopsy microvessel preparations was considerably lower than activity in rat microvessels. To determine if postmortem autolysis was responsible

A 120

110 100 9o

"~ ~ ~ ~ ~-

a0 r0 so 50 40 3o 20

lO 0

B

adult

aged

human control

Alzheimer'a disease

120 110 100 90

TABLE 1 PKC ACTIVITYIN CEREBRALMICROVESSELCYTOSOL MicrovesselSource

Activity (pmol/min/mg)

Rat Adult Aged

232.7 ± 16 208.0 ± 20

Human Alzheimer Control

-~

80

~

70

15

6O

50

40 30

20 10 0

10.5 +_2.5* 19.5 ± 4.0

Samples from aged and adult rats and Alzheimer and human controis were utilized. Cytosolic PKC activity of Q-Sepharose purified microvesselswas determined by measuring phosphorylation of histone (200/zg/ml) by 15-27 #g protein in a 130 #1 total volume for 7 min. at 30°C. Data are means ± SEM of 6 separate experimentsfor human samples and 6 for rodent. *Significantly different than human control.

FIG. 1. PKC activity in human and rat microvesselsin the absence (11) and presence ([D)of staurosporine (7/~M). PKC activity in the absence of staurosporine is defined as 100o70. Cytosolic PKC activity of Q-Sepharose purified microvesselsfrom adult and aged rats (A) and AD and human controls (B) was determined by measuring phosphorylation of histone (200 tzg/ml) by 15-27/zg protein in a 130/xltotal volume for 7 min at 30°C. Data are mean + SEM of three separate experimentsperformed in triplicate.

G R A M M A S ET AL.

566 -7OO

Subtypes of PKC Present in Microvessels In a previous study, we determined that in rat cerebral microvessels, calcium-independent PKC activity was not detected (23). Therefore, we compared the calcium-dependent isoform distribution in isolated microvessels from aged rats to that in adult as well as the distribution in A D and control patients. Immunoblot analysis, using isozyme specific antibodies for ~-,/3-, and 7-PKC showed staining in either rat or human microvessels to .y-PKC antibody and no reactivity to o~-PKC antibody in human microvessels and faint staining for c~-PKC in rat (data not shown). The immunoblot using anti-/3 PKC antibody (Fig. 4) demonstrated that the/3-isoform was present in both rat and human microvessels. In addition, there did not appear to be any quantitative difference in/3-PKC expression in rodent microvessels from adult and with aged rats or between human vessels from A D patients and elderly controls (Fig. 4).

600

500

300

E Q.

200

100

DISCUSSION

0

1

2

3

4

5

6

7

8

hours postmortem

FIG. 2. Rat brain cytosol PKC activity as a function postmortem time. Rats were euthanized and brain cytosol prepared at 1-8 h postmortem. PKC activity was assessed by incubation of 5 #g brain cytosol in a total volume of 130 #1 for 7 min at 30°C. Data mean +_ SEM of three separate experiments performed in triplicate.

trol and 6.5 p m o l / m g for AD-derived microvessels. A comparison o f three separate assays yielded a K o o f 114 _+ 21 nM and a/~max of 5.57 _+ 2.8 p m o l / m g for elderly control vessels and a K o of 80.8 _ 55 nM a n d / ~ m a x of 8.51 +_ 3.1 p m o l / m g for AD-derived microvessels.

Protein phosphorylation, a ubiquitous mechanism for the posttranslationa] control of protein function, is a likely target in AD. Abnormalities of phosphorylation cascades, including PKC, have been documented in agingand AD (Reviewedin 43). For example, Masliah et al. (36) demonstrated an increased staining of AD neurons with antibodies to ~II-PKC. However, total PKC activityhas been shown to be decreasedin AD brains relative to age-matchedcontrols (8,43) and phorbol ester binding to brain homogenate is apparently lowerin AD (8). In addition, phosphorylation of several PKC substrates, including GAP43, is reduced in AD (35,43). At least some PKC decreases may be age-related, since PKC activity and translocation in response to K+ depolarization and phorbol esters were decreased in cortical brain slicesof aged rats (17). In our study, the central finding is that PKC activity is significantlydecreasedin microvesselsfrom Alzheimer'scompared to controls but there is no change in enzyme level. Therefore,

C

30

P C

P

C

P C

P

"•25 m

112 kD 79.5 kD

?,

~,5 kD

11110

36 kD

0

40

80

120

160

200

240

280

320

[PDBu] nM FIG. 3. PKC quantitation by phorbol binding in microvessels from aged and young rats as well as AD and control brains. Specific binding of phorbol ester to unfractionated brain microvessels from adult (A) and aged (~1,)rats as well as from AD (0) and control patients ( I ) as a function of phorbol ester concentration. Samples of microvessels containing 100 #g protein were incubated for 30 rain at 24°C with varying concentrations of phorbol ester. Each point represents the mean of 34 separate experiments performed in triplicate.

A l z h e i m e r ' s Control Human

Aged

Adult Rat

FIG. 4. [mmunoblot analysisfor/3-PKC isoform. Cytosolic and particulate fractions (10-50 #g) of AD and human control derived-microvessels (left) and adult and aged rat microvessels (right) were prepared and subjected to SDS - polyacrylamide gel (9070) electrophoresis. After electrophoretic transfer to nitrocellulose, the blot was preincubated for 1 h in 20 mM Tris-HCl, pH 7.5, 500 mM NaCI (TBS) containing 3070 gelatin, washed with TBS containing 0.05°70 Tween 20, and incubated overnight in a 1:2000 dilution of the/3-PKC antibody in TBS containing 1070gelatin. Blots were developed following incubation with goat anti-rabbit IgG conjugated to alkaline phosphatase.

BLOOD-BRAIN BARRIER PKC IN ALZHEIMER'S

567

the demonstration of decreased enzyme activity measured on exogenous substrate with added cofactors argues for a specific alteration in the chemical structure or activation state of PKC. We believe it is unlikely to occur in the lipid binding site because phorbol ester binding was unaffected. Differences in processing of PKC by other kinases could play a role, since activation of PKC occurs via phospho'rylation by other endogenous protein kinases (14). In support of this notion, abnormalities of protein phosphorylation in AD have been identified for casein kinase II (43) and for protein tyrosine kinases (45). Alternatively, because endogenous diacylglycerol causes some conformational change in the catalytic site of PKC (30); PKC may not be maximally activated if diacylglycerol levels are lower in AD. Lipid abnormalities have been dernonstrated at the blood-brain barrier in aging (49) and in AD (19). In addition, the elevated ~-amyloid levels in AD vessels may reduce the activity, because amyloid proteins are known to inhibit PKC at high concentrations (6). Finally, expression of endogenous PKC inhibitors (16) may be different in AD. Despite lower PKC activity we observed no differences in PKC levels between AD and control-derived microvessels, as determined by phorbol ester binding. Lack of changes in PKC level, measured by autoradiography (25), and anti-PKC antibodies (7), has also been reported in the Alzheimer brain. In addition, we found no apparent changes in isoform distribution in microvessels from aged versus adult rats, or from AD and control brains. The decrease in PKC activity reported in our study could reflect the appearance of atypical PKC isoforms (6-, E-, ~-, ~-, and 0-PKC), because these may not phosphorylate histone as efficiently (9). In the experiments on rat brain PKC, there appeared to be an overall decline in PKC activity with postmortem time. This decrease was most pronounced in the first 2 h with little additional change between 3 and 8 h. Therefore, we believe it is unlikely that the postmortem interval, which averaged 6.4 h in AD and 5.5 h in control samples, would contribute to the difference in PKC activity observed between AD and control microvessels. The reasons for the inability of PKC to translocate in response to PMA in both human control and AD-derived microvessel preparations are unclear. It is possible that the membrane association of PKC could change during the microvessel isolation procedure. The lack of PMA sensitivity was not the result of the microvessel preparation itself, because PMA could stimulate translocation of F'KC in rat microvessels. Alteration of the lipid binding site is probably unlikely since [3H]-phorbol ester binding yielded consistent measurements in both rat and human microvessels. Finally, other authors have observed a decreased translocation resly~nse with age (17). Because both AD

and human control samples were from elderly individuals, it is possible that the ability of PKC to translocate in response to PMA was reduced in the microvessels of both groups. The cerebral microcirculation plays a key role in regulating permeability of polar molecules into and out of the brain. It is likely that PKC may be an important mediator of blood-brain barrier permeability because PKC has been shown in several cell types to modulate macromolecular ionic permeability by its effect on glucose transport, amino acid uptake, and ion channel activity (12,40). There is evidence that PKC regulates ion transport at the blood-brain barrier. Johshita et al. (28) recently demonstrated that diacylglycerol activation of PKC stimulated (Na ÷ + K+)-ATPase, an enzyme critical to brain ion and water permeability. Alterations of vascular PKC activity in AD may be important because PKC appears to play a central role in processing of ~-amyloid precursor protein (/~APP) into soluble NH2terminal APP derivatives and in inhibiting A/~ production (4,5,11). Caporaso et al. (5) demonstrated that phorbol esters increased secretion of APP695, 751, and 770 in PC12 cells and that this increase was potentiated by the protein phosphatase inhibitor okadaic acid. In endothelial cells, the ability of cytokines such as IL-I to upregulate message for/3APP is mediated through PKC (20). It has also been shown that an increase in PKC-mediated phosphorylation may induce proteolysis of /~APP695 into nonamyloidogenic forms (27). The neurologic deficits that characterize Alzheimer dementia are not confined to a single neurotransmitter system but involve numerous receptor families and subtypes. This observation suggests that abnormalities at postreceptor signalling pathways, where numerous receptor-mediated signals converge could be targets in AD. Our previous observation that cAMP levels are elevated in microvessels from AD brains (22) and the present observation that PKC activity is reduced in AD suggest that in cerebral endothelium the balance of protein phosphorylation mediated by cAMP. Dependent kinase and by PKC is disturbed. It has been suggested that "cross-talk" between distinct signalling systems such as cAMP-dependent k i n ~ e and PKC serves as an important control mechanism in response to extracellular signals (11,26). The balance between these two intracellular pathways may go awry in the Alzheimer bloodbrain barrier. ACKNOWLEDGEMENTS This work was supported by grants from NIH NS30457; Oklahoma Center for the Advancement of Scienceand Technology;and the Glenn Foundation (Paula Grammas) and NIH P30AG08017 (MelvynJ. Ball). We thank Melanie Beery for secretarial assistance.

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