Pharmacology of tacrine: Contribution of cholinergic and noncholinergic actions

N E U R O B I O L O G Y OF AGING, V O L U M E 11, 1990 ABSTRACTS OF SECOND I N T E R N A T I O N A L C O N F E R E N C E ON A L Z H E I M E R ' S DISEASE A N I M A L MODELS

we have described.' The right subclavian and its vertebral artery branch were left open to perfuse I~'ain. At specific time periods for 90 days, rat cortexhippocampus areas were examined in rive with =P- magnetic resonance spectroscopy and CBF recordings. PCr/IY-ATP spectra was reduced after several weeks and cortical CBF fell from 132 ml to 74 ml. After 15, 30, 60 and 90 days of Ixaln hypoperfusion, rats were killed and counts of CA1-CA4 neuron density were made. Group AM showed greater damage to hippocampal neurons than its younger MA counterpart. Energy metabolite reduction showed a variability consistent with the structural pathology. CBF remained relatively similar between younger and aged groups throughout the observation period. These results suggest that hippocampal neuron vulnerability to chronic brain ischemia is greater in aged than in mature young rats. I de la T0rre, J.C., F0rtin, T.: S0c Neur0 Abstr

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314 PHARMACOLOGY OF TACRINE: CONTRIBUTION OF CHOLINERGIC AND NONCHOLINERGIC ACTIONS. R.E. Davis, LL. Coughenour, D. Dudley, T.A. Pugsley, R.D. Schwarz, W.H. Moos. Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., Ann Arbor, MI, 48105, USA. Alzheimer's disease is characterized by significant neuronal pathology in discrete brain regions. Loss of forebrain cholinergic neurons accompanied by decreased neocortical choline acetyltransferase activity is consistently seen in brains of demented subjects and may be responsible in part for the cognitive dysfunction associated with this disease. One approach to the treatment of the cognitive symptoms of Alzheimer's disease is to increase the synaptic availability of acetylcholine (ACh) by preventing its breakdown through inhibition of acetylcholinasterase (ACHE). Tacrine (1,2,3,4tetrahydro-9-aminoacridine) was selected for clinical study on the basis of its ability to inhibit ACHE. However, several recent studies have suggested that Tacrine may have noncholinergic activities. The purpose of the present series of studies was to determine the relative contributions of cholinergic and noncholinergic actions to the cognition enhancing properties of Tacrine. Tacrine improves the ability of cognitively impaired mice and basal forebrain lesioned rats to find a hidden platform in a water-maze. Tacrine also modestly improved delayed match-to-sample performance of aged-rhesus monkeys. The mechanisms mediating these positive effects on cognition are not known. But, Tacrine is an acetylcholinesterase inhibitor (IC~=220 nM) which decreases the presynaptic release of ACh (0.3-3 ~M), inhibits high affinity choline uptake and increases the turnover of calecholamines through its ability to increase the synaptic availability of ACE Like other cholinomimetics in rats, Tacrine increases gastrointestinal motility, reduces scopolamine-induced and spontaneous swimming activity, decreases body temperature, and decreases the amount of high-voltage, low-frequency activity in the cortical EEG. This latter effect is also seen in rhesus monkeys. Unlike other cholinomimeties, however, tacrine does not increase local cortical blood flow. Tacrine also binds with ,uM affinity to numerous noncholinergic receptor and channel sites. At muscarinic receptors it exhibits a binding profile similar to weak muscarinic antagonists. Consistent with this at high concentrations Tacrine reverses arecoline-induced decreases in ACh release and carbachol-elicited increases in PI turnover. Tacrine also is an extremely potent inhibitor of butyrylcholinesterase activity (IC~=60 nM). Tacrine has been suggested to interact with aminopyridine-sensitive K+ channels. But, unlike simple aminopyridines Tacrine does not increase nonstimulated release of ACh. In addition, simple aminopyridines do not improve performance of mice in a water-maze task in a manner similar to Tacrine. Based on these data, Tacrine is a cholinomimetic agent at reasonable concentrations and cognitively active doses. Tacrine does exhibit noncholinergic activities 'in vitro' at high concentrations (>1 /~M) but these occur at concentrations higher than those necessary to inhibit AChE and do not appear to contribute significantly to its acute action 'in vivo' or its ability to improve cognitive function in animals and perhaps man.

resorcinol reagents for sialic acid. Total ganglioside contents in the h e m i s p h e r e ipsilateral to the lesion site w e r e d e c r e a s e d by 10 to 20 p e r c e n t w h e n c o m p a r e d with the c o n t r a l a t e r a l region, in a g r e e m e n t with our previous findings. Differences in the i n c o r p o r a t i o n o f l a b e l l e d sugar into the gangliosides were even m o r e p r o n o u n c e d , with a n average of 50 percent reduction in the lesioned brain. F u t h e r m o r e , r a d i o l a b e l l e d gangliosides accounted for most of the radioactivity found in the total C M extracts of the control hemisphere. Analysis of histological sections showed the lesions to be fairly well confined to the n b m and i m m e d i a t e surrounding areas, with little evidence of n e u r o n a l destructions in the projected cortical regions, on which most of the chemical analyses w e r e performed. Thus, the consistent reductions in both total gangliosides and in the i n c o r p o r a t i o n of l a b e l l e d precursor sugar into these neuroconstituents, cannot be a t t r i b u t e d to n e u r o n a l loss. Rather, deafferentiation of the cortex resulting from the loss of cholinergic input from the nbm, a p p e a r s to be a contributing factor. It is p r o b a b l e that similar m e c h a n i s m s also operate in h u m a n AD. Since the gangliosides are i m p o r t a n t n e u r o n a l m e m b r a n e constituents with d e m o n s t r a b l e trophic properties, d e r a n g e m e n t s in their m e t a b o l i s m may be of significance in the pathophysiology of AD.

316 C O M P L E T E H O M O L O G Y OF THE A M Y L O I D A - P R O T E I N P R E C U R S O R IN M O N K E Y A N D HUMAN: S U P P O R T F O R A P R I M A T E M O D E L OF BAMYLOIDOSIS. *MB. P o d l i s n y 1,2, D. T o l a n 2, DJ. S e l k o e I . i H a r v a r d M e d i c a l Sch. / B r i g h a m & W o m e n 's Hosp., and 2Boston Univ., Boston, M A 02115 USA. Progressive cerebral deposition of the a m y l o i d h-protein (ASP) O c c u r s in A l z h e i m e r ' s d i s e a s e (AD) and d u r i n g n o r m a l a g i n g of c e r t a i n m a m m a l s (e °g., human; monkey; dog) but not o t h e r s (e .g. m o u s e ; rat) . We c l o n e d a n d s e q u e n c e d a f u l l - l e n g t h c D N A e n c o d i n g the Bprotein precursor (APP) of c y n o m o l o g u s monkey. The predicted amino acid sequence of t h e 695-residue protein is 100% h o m o l o g o u s to t h a t of h u m a n . The polymerase chain reaction (PCR) was u s e d to c l o n e the K u n i t z p r o t e a s e i n h i b i t o r d o m a i n in monkey, r e v e a l i n g only a single amino acid substitution in the 7 5 1 r e s i d u e f o r m of APP a n d four a m i n o a c i d s u b s t i t u t i o n s in A P P 7 7 0 . I m m u n o b l o t t i n g w i t h a p a n e l of a n t i b o d i e s to various APP domains showed highly similar APP polypeptides in h u m a n a n d m o n k e y b r a i n a n d n o n - n e u r a l tissues, in c o n t r a s t to t h o s e of m o u s e a n d rat. The l a t t e r d i f f e r e n c e s reflect t r a n s c r i p t i o n a l and p o s s i b l y p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n s w h i c h m a y d e c r e a s e the amyloidogenic potential of r o d e n t APP. Immunocytochemistry of aged cynomologus brain revealed ABP d e p o s i t e d in b l o o d v e s s e l s a n d m u l t i p l e p l a q u e s in the absence of neurofibrillary tangles. Our findings demonstrate t h a t the c y n o m o l o g u s monkey and perhaps o t h e r p r i m a t e s p r o v i d e an e x c e l l e n t a n i m a l m o d e l for examining early transcriptional and post-translational p r o c e s s i n g of APP that p r e c e d e s A~P d e p o s i t i o n d u r i n g a g i n g a n d in AD. S i n c e the a g i n g d o g is a l s o p r o n e to p r o g r e s s i v e cerebrovascular and c e r e b r a l d e p o s i t i o n of A~P a n d is m o r e a c c e s s i b l e for research, we are c u r r e n t l y s t u d y i n g the homology of the APP g e n e in dog and h u m a n u s i n g d i r e c t s e q u e n c i n g f r o m P C R of g e n o m i c DNA. We are also u s i n g t h i s t e c h n i q u e to test for s e q u e n c e h e t e r o g e n e i t y in the A P P gene of FAD p a t i e n t s . The results of these e x p e r i m e n t s will be p r e s e n t e d .

315 _IN VIVO INCORPORATION OF (C-14) NACETYLMANNOSAMINE I N T O B R A I N G A N G L I O S I D E S IN H Y P O C H O L I N E R G I C RATS. *N.M.K. N G Y I N G KIN A N D D. CHUNG. Dept. of Psychiatry , McGill University and D o u g l a s H o s p i t a l R e s e a r c h Centre, Verdun, Q u e b e c , Canada. H 4 H 1 R 3 . R a t s with quinolinic acid-induced lesions of the f o r e b r a i n nucleus basalis magnocellularis (nbm) exhibit central cholinergic hypofunctions and are p u t a t i v e a n i m a l m o d e l s for A l z h e i m e r ' s disease (AD). R e c e n t l y , we r e p o r t e d that in both A D and the l e s i o n e d rats, b r a i n gangliosides were significantly r e d u c e d (Neurosci. Abs., 1989, 15. 344). To investigate the possibility of a l t e r e d biosynthesis of gangliosides, we studied the m e t a b o l i c fate of (C 14) NA c e t y l m a n n o s a n i n e , a precursor of sialic acid,in the brains of rats with u n i l a t e r a l lesions. T h e l a b e l l e d sugar was injected i n t r a c e r e b r a l l y a r o u n d the skull ( n = 5 ) . 24 hours later, the rats w e r e sacrificed and the b r a i n cortical tissues subjected to chloroform: m e t h a n o l ( C M ) extractions and Folch partitioning. Gangliosides w e r e isolated from the non-dialysable fractions of the u p p e r p h a s e and quantified using the

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REVERSAL OF LESION-INDUCED MEMORY DEFICITS AND ENHANCEMENT OF A CHOLINERGIC MARKER BY CHROMAFFIN CELL GRAFTS TO RAT CEREBRAL CORTEX. *S.A. Welner, Z. Koty, P. Boksa. McGill University, Dept. Psychiatry, Douglas Hospital Research Centre, Montreal, Quebec, H4H 1R3 Canada. Lesions of the nucleus basalis magnocellularis (NBM) in the rat produce deficits in spatial memory, usually suggested to be due to a loss in cholinergic input to the cerebral cortex. In the present study, adrenal chromaffin cells, which are normally catecholaminergic, but can express a cholinergic phenotype under certain conditions, were isolated from donor adult rats and transplanted to the cerebral cortex of bilaterally NBM-lesioned rats. Effects of grafts on lesion-induced memory impairments were tested in a T-maze alternation task. Chromaffin cell grafts to lesioned animals completely reversed the spatial memory deficit seen in lesioned alone animals such that Tmaze performance of the grafted group was at the level of unlesioned-