Differential inhibition of acetylcholinesterase molecular forms in normal and Alzheimer disease brain

Brain Research, 589 (1992) 307-312 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

307

BRES 18039

Differential inhibition of acetylcholinesterase molecular forms in normal and Alzheimer disease brain N o b u o O g a n e a, E z i o G i a c o b i n i a a n d R o b e r t S t r u b l e b Department of a Pharmacology and b Pathology and Psychiatry / Alzheimer Center, Southern Illinois University School of Medicine, Springfield, IL 62794-9230 (USA) (Accepted 7 April 1992)

Key words: Acetylcholinesterase; Molecular form; Acetylcholinesterase inhibitor; Heptyl-physostigmine; Physostigmine; Edrophonium; Alzheimer disease; Human brain

Molecular forms of acetylcholinesterase were studied in three brain regions from Alzheimer disease patients and non-demented, age-matched controls. In Alzheimer disease patients, the membrane-bound G 4 form was decreased in frontal ( - 71%) and parietal cortex (-45%) and in the caudate-putamen ( - 4 7 % ) from control levels. We also found a decrease of aqueous-soluble acetylcholinesterase molecular forms in the caudate-putamen region. The effect of three clinically significant acetylcholinesterase inhibitors, heptyl-physostigmine, physostigmine and edrophonium, on aqueous-soluble acetylcholinesterase molecular forms of the caudate-putamen was investigated. Heptyl-physostigmine, a physostigmine analogue, showed/preferential inhibition for the G t form. On the contrary, edrophonium inhibited the G 4 form more potently than the G 1 form. Physostigmine inhibited both forms with similar-potency. The clinical implications of selective acetylcholinesterase inhibitors are discussed.

INTRODUCTION Acetylcholinesterase (ACHE), as well as choline acetyltransferase (CHAT), is decreased in post-mortem brain tissue from Alzheimer disease (AD) patients compared to age-matched controls re,t3'2°. This has lead to clinical trials of anticholinesterases in attempts to increase cholinergic-function 3,27. However, the presence of multiple molecular forms of AChE makes it difficult to interpret the effect of these drugs on brain. Acetylcholinesterase can be separated into multiple molecular forms by means of their sedimentation coefficients s'24'29. Multiple molecular forms of AChE can be distinguished based on their shapes, collagen-tailed asymmetric forms (A n) and globular forms (G,). Globular forms can be subdivided by their solubility characteristics into two main classes: detergent-soluble forms (G~) and aqueous-soluble or low salt-soluble forms (G A) in the absence of detergent. Asymmetric forms are all hydrophilic and homologously high saltsoluble s'24. In mammalian brain, the bulk of AChE is

the G 4 (10 s) and Gt (4 s) forms with a minor amount of G 2 (6 s) and a trace (< 2%) of an AI2 form (16 s) 6'm. The proportion of these molecular forms varies depending upon the area of brain 2. Selective loss of the membrane-bound G 4 form has been reported in AD, suggesting a presynaptic localization of this form Lt6'ss. However, little is known of the aqueous-soluble AChE molecular forms in AD brain. Aqueous-soluble or secretory AChE molecular forms may have additional functions, such as the protease activity,34 peptidase activity for neuropeptides like substance P and enkephalins, ~L~2 and neurotransmitter modulatory functions in substantia nigra t9. In a previous study in the rat, we compared the relative inhibitory potency of a series of clinically significant AChE inhibitors on the G~ and G 4 forms 26. We found that heptyl-physostigmine (HEP), a novel carbamate derivative of physostigmine (PHY), ~5 showed preferential inhibition of the G t form, suggesting a possible therapeutical application of a G~ selective inhibitor in AD.

Correspondence: N. Ogane, Research Institute of Life Science, Snow Brand Milk Products Co. Ltd., Tochigi 329-05, Japan. Fax: (81) (285) 53-1314.

308 forms. Our data suggests differential selectivity of the three AChE inhibitors on AChE molecular forms.

In the present study, we examined the distribution of the membrane-bound and aqueous-soluble AChE molecular forms in the brains of AD patients and age-matched controls. Furthermore, we studied the effects of three AChE inhibitors of clinical interest and with differential binding properties for ACHE, i.e. HEP, PHY and edrophonium (EDR), on the G4 and G~

MATERIALS AND METHODS Brain tissue was obtained from the Brain Bank of Southern Illinois University Center for Alzheimer Disease. Following autopsy within 24 h after death, tissue was rapidly frozen and stored at -90°C. All specimens were subjected to neuropathological examina-

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309 tion. For control brains, the diagnosis of a neurological disease was ruled out. All AD cases met or exceeded the criteria for the neuropathological diagnosis of AD -'3. Age and sex (mean+S.D., m e n / w o m e n ) of subjects were 78.0+8.2, 2/1 for control and 78.3± 5.7, 3 / 0 for AD. Three brain areas, frontal cortex (Brodmann area 8), parietal cortex (Brodmann area 7) and rostral caudate-putamen were selected. The brain tissue was homogenized with a Teflon-glass Potter homogenizer (3 sets x 10 strokes) and re-homogenized with a Polytron (15 s x 2 cycles) in five volumes Krebs Ringer solution (120 mM NaCI, 4.6 mM KCI, 2.4 mM CaCI2, 1.2 mM KH2PO 4, 1.2 mM MgSO4, 2.5 mM NaHCO 3, 9.9 mM glucose, pH 7.0) in the presence of I m g / m i bacitracin. The homogenate was centrifuged (200,000 x g, 30 min, 4°C) in a Beckman SW 60 Ti rotor. Its supernatant represents the "aqueous extract". The pellet was resuspended and homogenized in five volumes Kr~bs Ringer solution in the presence of 1 m g / m l bacitracin and 1% Triton X-100. The homogenate was centrifuged (200,000x g, 30 min, 4°C). Its supernatant represents the "detergent extract". Aliquots (100-200/zi) of each extract were layered onto 5-25% (w/v) sucrose gradients in saline buffer (1 M NaCi, 50 mM MgCI2, 10 mM Tris-HCI, pH 7.0) in the presence or absence of 1% Triton X-100. Centrifugation was performed at 160,000x g for 21 h, 4°C in a Beckman SW 60 Ti rotor. Catalase from beef liver (11.3 s) and bovine serum albumin (4.3 s) were used as internal sedimentation markers. Human erythrocyte AChE (Sigma, Type XIII) was used in order to confirm the sedimentation coefficient of the dimeric globular form (G,). Acetylcholinesterase activity was assayed according to a modification of the method of Johnson and Russell": in the presence of 100 /zM tetramonoisopropyl pyrophosphortetramide to inhibit butyrylcholinesterase activity "~t. Aliquots of fractions from the sucrose gradient centrifugates were preincubated for 30 min at 37°C with HEP, PHY and EDR, in 50 mM Tris-HCI buffer (pH 7.4). Assays were carried out for 30 min with ! mM ['~H]acetylcholine (0.46/zCi/ml) as substrate. The ICs0 values were obtained from pseudo Hill plots as values of zero interception. One-way ANOVA was used for all statistical evaluations. Heptyl-physostigmine was provided by Mediolanum Farmaceutici

S.r.i. (Milano, Italy). Acetylcholine iodide ([acetyl-aH]-, spec. act, 90 mCi/mmol) was from New England Nuclear (Boston, MA). All other chemicals were analytical grade and were obtained from com. mercial sources.

RESULTS

Separation of AChE molecular forms in control and AD brains The activity and proportion of AChE molecular forms varied depending on the region of brain and on the type of extract (Fig. 1). In detergent extracts of control: brain, total AChE activity of caudate-putamen was over 50-fold higher than in the parietal cortex (Fig. 1C vs 1B). The G 4 / G ~ ratio in a detergent extract of the caudate-putamen was much higher than in an aqueous extract (compare Fig. 1C vs 1F). Values of AChE activity in each molecular form in AD and age-matched control brains are reported in Table I. The tetrameric G 4 form from detergent extracts of AD brain was significantly decreased in frontal cortex(-71%), parietal cortex ( - 4 5 % ) and caudateputamen ( - 4 7 % ) compared to control brain (P < 0.05).

TABLE

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Actirity of ace~.lchofinesterase mok,cular forms in hummz brain Molecular form Frontal cortex Detergent-soluble

Aqueous-soluble

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Aqueous-soluble

Caudate-putamen Detergent-soluble

Aqueous-soluble

Controls

AD

G2 Gl G4 G2 GI

41.9±4.0 5.3±.5 12.6±2.1 6.2±0.6 1.9±0.2 4.4±0.5

12.1±0.5"* 3.3±0.9 8.1±0.8 5.1±0.9 1.6±0.5 3.6±0.7

G4 G2 G, G4 G2 Gt

35.2+_3.5 4.4±0.7 12.0±!.1 9.0±2.1 3.8±l.6 6.3±i.4

19.3+_4.3" 6.1±0.8 9.6±0.9 8.2±1.2 2.6±0.4 4.4±0.8

G4 G2 G, G4

2027 ± 19 109+ 10 180+_29 384+_26 65+7 298+_27

! 083 _+336 * 66+_ 26 135+_62 210+_52 * 30+5 * 158+_35*

G4

G2 Gt

Values are mean + S.E.M. (nmol A C h / m i n / m l fraction, n = 3). Significantly different from controls, ** p < 0.01, * p < 0.05, One-way ANOVA. AD: AIzheimer disease.

A significant decrease (about 50%) in AD was observed in all three aqueous-soluble globular forms in the caudate-putamen.

Effects of AChE inhibitors on the tetrameric (;4 and monomeric G I forms Multiple molecular forms were separated from aqueous extracts of caudate-putamen by sucrose density gradient in the absence of Triton X-100 (Fig. 2). The sedimentation coefficients of all three globular

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310 ble to those of our previous report on rat brain 26. Pools of three fractions sedimented as G 4 and G~ peaks were used to assess the effects of the three AChE inhibitors. Fig. 3 shows the inhibition curves for the G 4 and G I forms from control brains. Heptyl-physostigmine inhibited the GI form more potently than the G4 form. On the contrary, EDR inhibited the G4 somewhat more than G~. Physostigmine inhibited the G 4 and Gm forms equally (Table If). The IC50 values of these AChE inhibitors for the molecular forms in control and AD Q brains are reported in "Table II. In cases of HEP and EDR, with the exception of PHY, there were significant differences in IC5o between G4 and G I forms. The IC5o found using material from AD brain was not significantly different in any case from that using from controls.

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forms were shifted to the left compared to the sedimentation pattern of the same sample in the presence of Triton X-100 (Fig. 1F). These results are comparaTABLE I!

ICsos of three acetylcholinesterase inhibitors for human brain acetyicholinesterase molecular forms Inhibitor

Molecular Controls form

heptylphysostigmine

G4 GI

2.144-0.59 2.214-0,83 0.51.+.0.08 0.334.0.03

[10 -s M] [10 -H M]

physostigmine

G4 Gt

3.66+0.48 3.124.0.51

3.974. 0.54 2.36+0.19

[10-s M] [10 -s M]

.:drophonium

G4

0.814-0.33 1.694-0.29

0.914-0.18 1.524-0.11

[10 -s M] [10 -5 M]

G~

AD

[unit]

Values are mean +S.E.M, (M). Number of experiments are 3-6. With the exception of physostigmine, there were significant differences of IC5, between G 4 and G I forms (p<0.05; One-way ANOVA). The ICso found using material from AD brain was not significantly different in any case from that using material from control.

The present study confirms the previous findings that detergent-soluble, membrane-bound G4 form is selectively decreased in frontal and parietal cortex in AD brain ~'m6'3s. The decrease in membrane-bound G 4 form in AD brains is more pronounced in frontal ( - 71%) than in parietal cortex (-45%). The decrease "of ChAT activity is more pronounced in frontal ( - 47%) than in parietal cortex ( - 3 6 % ) , too (data not shown). These results suggest that cholinergic neurons in frontal cortex (Brodmann area 8) are more affected than those in parietal cortex (Brodmann area 7) in AD brain. In AD patients, a decrease of AChE activity in the caudate-putame~ was also reported ")'~. However, it was not clear which molecular form was identified. We found a decrease of all aqueous.soluble globular forms, as well as of the membrane-bound G4 form. The caudate-putamen region (striatum) includes large cholinergic interneurons with high ChAT and AChE activity 25, but also non-cholinergic, dopaminergic, GABAergic terminals and neurons ~7. Bolam et al. 5 suggested that AChE may be present in both somatostatin- and GABA-containing neurons of mice neostriatum. In the striatum, AChE is also released or secreted as a soluble form and might have a noncholinergic action 14'19. These data suggest that a loss of aqueous-soluble AChE in caudate-putamen may be, at least partly, associated with a non-cholinergic deficit in AD brain. Despite equivalents of catalytic activity per active site for all molecular forms, 37 some AChE inhibitors show selectivity for certain AChE molecular forms. Skau 32'33 demonstrated that ethopropazine shows a stronger inhibition for the G~ than for the G 4 form; while on the contrary, dithiothreitol shows preferential

311 inhibition for the G 4 form solubilized from rat diaphragm muscle. In our previous study on rat brain, we showed that HEP has G~ specificity, but its parent compound, PHY, does not separate the G4 from the Gm form 26. In this study, we confirmed these effects of HEP and PHY on brain AChE both in controls and AD patients. Moreover, we found a preferential G 4 inhibition by EDR under the same conditions. In contrast to PHY and HEP, EDR is believed to inhibit AChE by occupying the anionic site with no direct reaction with the esteratic site a~. Edrophonium is used for affinity chromatography as a ligand to separate the secreted AChE form (G A)2I. The greater preferency of EDR for G4 could be associated with an intrinsic and preferential affinity for this form. Consequently, a selective inhibition of AChE molecular forms may depend on intrinsic and selective affinities of the AChE inhibitor for hydrophobic binding sites. The presence of hydrophobic or anionic binding sites is well documented by many investigators also for aqueous-soluble AChE 7'2~'3°. The degree of hydrophobicity is different among different globular forms. The G4v form, for example, has at least three differential hydrophobic subdomains: a proximal domain containing a disulfate bridge, a proteinase K-sensitive intermediate subdomain, and a pronase-resistant subdomain which anchors to the membrane, phospholipid bilayer 3~'.The G A form is supposed to be composed of only a catalytic subunit. Sussman et ai a5 suggested that a variety of hydrophobic and anionic sites may be explained by the existence of an aromatic active site gorge. They also suggested that the aromatic site lines a deep and narrow gorge in ACHE, and that, therefore, there must be many differential ways and places for substrate, agonists, and inhibitors to bind to ACHE. These reports lead to the suggestion that the degree and type of hydrophobic binding site may not be equivalent between G4 and G~ forms. Differences in alIosteric effects or differential affinities for G4 and G~ forms could explain the inhibitor selectivities found by US.

Acetylcholinesterase inhibitors are currently used for clinical treatment of myasthenia gravis, glaucoma, and, in experimental clinical trials, of AD a'27. There is also evidence of a relation between AChE molecular form and an abnormal development of nervous system. The presence of a fast migrating AChE molecular form 4 (probably G 4 form) in amniotic fluid is used for prenatal diagnosis of neural tube defects ~. In AD, several brain areas show a selective loss of G 4 form re't6'38. Chracteristic changes on density and regional ditribution of binding with a G 4 selective inhibitor could be used for diagnostic applications. On

the other hand, a G~ selective inhibitor may have therapeutic applications in inhibiting the G~ form which are relatively unchanged in AD brain. Acknowledgements: This study was supported by National Institute of Aging Alzheimer Disease Center Core Grant P30AGO8014 to E.G. and Robert Becker; and by a grant from the Snow Brand Milk Products Co. Ltd. (Tochigi, Japan) to N.O. The authors thank Mrs. Elizabeth Williams for her technical assistance; and Mrs. Diana Smith for typing and editing the manuscript.

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