E L S E V I E R Brain Research 731 (1996) 249-253

BRAIN RESEARCH

Shor t c o m m u n i c a t i o n

Reversion of [325_35-amyloid peptide-induced amnesia by NMDA receptor-associated glycine site agonists

Tangui Maurice a, *, Brian P. Lockhart a,l, Tsung-Ping Su b, Alain Privat a

a INSERM U. 336, Ddveloppement, Plasticit~ et Vieillissement du Syst~me Nerveux, Ecole Nationale Supdrieure de Chimie, 8, Rue de l'Ecole Normale, 34053 Montpellier, France

b NIDA, Intramural Research Program, NIH, P.O. Box 5180, Baltimore, MD 21224, USA

Accepted 11 June 1996

Abstract

The effects of D-cycloserine (DCS), a N-methyl-D-aspartate receptor-associated glycine site agonist, and milacemide (MIL), a glycine prodrug, were examined on learning impairments induced by administration of 13z5_35-amyloid peptide (3 nmol i.c.v.). Mice were examined for spontaneous alternation and step-down passive avoidance. 7 and 14 days after 1325_35, respectively. The 1325_35-induced deficits were reversed by DCS, 1-30 mg/kg i.p., or MIL, 3-100 mg/kg i.p., each drug being ineffective on control mice behaviours. These observations strengthen the therapeutic potential of glycine site agonists against the memory impairments induced by [3-amyloid peptides.

Keywords: I>Cycloserine; Milacemide; NMDA/glycine site; [3-Amyloid; Amnesia

Deposition of amyloid plaques, mainly constituted of [31_40- and [3~_42-amyloid proteins, and intraneuronal neu- rofibrillary tangles formation are the most stereotypic neu- ropathological features of Alzheimer's disease. Their ex- tent correlates with the progressive cognitive deficits and memory impairment [22]. The mechanism of the neurotox- icity induced by [3-amyloid proteins may involve their ability to self-aggregate and form Ca2+-permeable chan- nels in membranes, resulting in excessive Ca 2÷ influx and induction of neurotoxic cascades [1,12]. We previously reported that central administration of [325_35 peptide, in its aggregated form, into mouse brain induces Congo-red stained amyloid-like deposits and neurodegeneration in the hippocampus and cortex, and impairment of the sponta- neous alternation, a behaviour related to working memory, and delayed amnesia in a passive avoidance task and in place learning in a water-maze [13]. The 1325_35-induced amnesia could be reversed by the acetylcholinesterase inhibitor tacrine or by the cholinergic agonist nicotine, indicating that memory dysfunctions involved deregulation of the cholinergic systems.

* Corresponding author. Fax: (33) 67540610; e-mail: maurice@cit.en- scm.fr

Present address: Institut de Recherche SERVIER, 125, Chemin de RoMe, 78290 Croissy-sur-Seine, France.

The N-methyl-D-aspartate (NMDA) receptor activation represents a key process in learning. Pharmacological an- tagonism of the NMDA receptor blocks induction of long- term potentiation in hippocampal CA 1 pyramidal neurons. a form of synaptic plasticity that may be involved in the encoding of memory, and impair learning of several tasks in rodents [16]. Agonists at the glycine modulatory site have been shown to facilitate the induction of long-term potentiation [ 10]. Two compounds appear of interest, which readily cross the blood-brain barrier: D-cycloserine (DCS). a partial glycine site agonist, and milacemide (MIL), a glycine prodrug [4,7,9,17,19]. The drugs facilitate learning in several tasks, by potentiating the NMDA activity. We thus examined the ability of DCS and MII. in attenuating the learning impairments induced by central administration of [325_35-amyloid peptide, by using the spontaneous alter- nation and passive avoidance tests in mice.

Male Swiss mice (Iffa-Credo, L'Arbresle, France), aged 4-5 weeks and weighing 30 35 g, were housed in plastic cages in a regulated environment (23 _+ I°C, 50% humid- ity, 12-h light/dark cycle), with free access to laboratory food and water, except during behavioral observations. Experiments were carried out between 10:00 and 18:00 in an air-regulated and soundproof laboratory, in which mice were habituated 30 min before each experiment. Mice were examined either for spontaneous alternation be-

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250 T. Maurice et al. / Brain Research 731 (1996) 249-253

haviour in a Y-maze 7 days after ~25-35 peptide adminis- tration, or for passive avoidance, 14 days after [3~5_35. Spontaneous alternation behaviour in a Y-maze allows the measurement of the spatial working memory perfor- mances, as detailed in [13]. In brief, mice freely explored a Y-shaped maze during an 8-rain session. The series of arm entries, including possible returns into the same arm, was recorded using an Apple IIe computer. An alternation was defined as entries into all three arms on consecutive occa- sions. The percentage of alternation and the total number of arm entries were determined. The step-down type of passive avoidance was used to examine the long-term memory, as detailed in [13]. In brief, training was carried out in two similar sessions at a 90-rain time interval. Each mouse was placed on a wooden platform, set at the centre of a clear plexiglas cage equipped with a grid floor. When it stepped down and placed its four paws on the grid floor, electric shocks (1 Hz, 500 ms, 45 V DC) were delivered for 15 s. Step-down latency (SDL), and the numbers of flinching reactions and vocalizations were measured. The retention was carried out 24 h after training similarly, but no shock was applied. Two parametric measures for mnesic capacities were measured: (1) the SDL, recorded up to 300 s; and (2) an avoidance criterion, defined as correct if the SDL during retention was higher than 3-fold the SDL during the second training session and at least higher than 1 rain. Using this procedure, SDL is usually considered as a qualitative index for mnesic capacities, whereas the percentage of animals to criterion is considered as a quan-

titative index. D-4-Amino-3-isoxazolidone (D-cycloserine, DCS) was obtained from R.B.I. (Natick, MA) and 2-N- pentylaminoacetamide (milacemide, MIL) was donated by Dr. Robert Hefting (Searle, Skokie, IL). Vehicle was 0.9% saline solution and injection volume was 100 Ixl/20 g body weight. The [325_35-amyloid and scrambled ~5-35- amyloid peptides (Neosystems Labs., Strasbourg, France) was dissolved in sterile distilled water (vehicle), aggre- gated by incubation at 37°C for 4 days, at 1 mg /ml [11,13], and administered i.c.v, as aggregates [13]. Results are expressed as mean + S.E.M, except for SDL, which are expressed in terms of medians and interquartile ranges. Data were analyzed by the non-parametric Kruskal-Wallis ANOVA (KW value), followed by Dunn's multiple com- parisons test. Statistical levels of significance were: NS, not significant, P < 0.05, P < 0.01.

As shown in Fig. 1, neither DCS nor MIL, administered i.p. 30 min before the test, affected the spontaneous alter- nation behaviour in animals, administered with distilled water i.c.v. 7 days before (KW = 4.91, NS). Mice treated with scrambled [3~5_35 peptide, 3 nmol i.c.v. 7 days before, did also not exhibited deficits in spontaneous alternation, whereas animals treated with [3~5_55 peptide showed a decrease in spontaneous alternation (P < 0.05; Fig. 1). No difference was observed in the number of arms entered during the 8-rain session ( 3 0 + 2 for the scrambled [3~5_~5-treated group, 32 __+ 2 for the [3~5_35-treated group and 32 + 3 for the control group, NS), indicating that none of the treatments affected the motility of the animals. The

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Fig. 1. Dose-response effect of D-cycloserine (DCS) and milacemide (MIL) on the spontaneous alternation behaviour in [325_35-treated mice. The ~3z5_35, or scrambled [325_35 peptide, or vehicle was administered i.c.v. 7 days before the test. DCS, MIL or saline (Sal) was administered 30 min before the test. The total numbers of arm entries did not significantly vary among groups, and were in the 28-33 range. The number of mice used is shown below each

column. * P < 0.05, * * P < 0.01 vs. (Veh + Sal)-treated group; + P < 0.05, ++ P < 0.01 vs. ([325-35 + Sal)-treated group (Dunn's test).

T. Maurice et al. / Brain Research 731 (1996) 249-253 251

decrease in alternation observed after 1325 35 thus directly reflected the altered working memory. Systemic i.p. ad- ministrations of DCS or MIL, 30 min before the test, induced a significant reversion of the decrease in alterna- tion (KW = 37.43, P < 0.01). The dose- response curves were bell-shaped for both compounds, significant effects being observed at 10 m g / k g for DCS and 30 m g / k g for MIL. Neither the DCS, nor the MIL treatment affected the

total numbers of arm entries among groups (data not shown). In the passive avoidance training sessions, no significant differences in the numbers of vocalizations or flinchings were observed among groups, indicating that none of the treatments affected the sensitivity to the shocks (data not shown). As shown in Fig. 2, neither DCS nor

M1L affected the passive avoidance behaviour, as com- pared to control animals, in terms of SDL (KW = 8.62, NS, Fig. 2A), and in terms of the number of animals to criterion (KW = 2.95, NS, Fig. 2B). Both the SDL and the number of animals to criterion were significantly de-

creased with the #25-35 treatment, but not with the scram- bled peptide (Fig. 2A,B). The groups treated with DCS or MIL showed dose-dependent reversions of the ~25_35-in- duced decreases in SDL (KW = 32.21, P < 0.01, Fig. 2A), and in the number of animals to criterion (KW = 28.20, P < 0.01, Fig. 2B). Significant effects were observed at 10 m g / k g for DCS and 10-30 m g / k g for MIL. The dose - r e - sponse curves appeared bell-shaped for both compounds.

These results showed that acute treatment with DCS or

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Fig. 2. Dose-response effect of D-cycloserine (DCS) and milacemide (MIL) on the 1325_35-induced delayed amnesia in the step-down type passive avoidance test: (A) step-down latencies (SDL), (B) percentages of animals to criterion. Peptides or vehicle were administered 14 days before training, and drugs were administered 30 min before training. (A) shows the median and interquartile range of number of mice indicated below each bar in (B). * P < 0.05, * * P < 0.01 vs. (Veh + Sal)-treated group; + P < 0.05, ++ P < 0.01 vs. (~325_35 + Sal)-treated group (Dunn's test).

252 T. Maurice et al. / Brain Research 731 (1996)249-253

MIL reversed the memory deficits in mice treated with the aggregated [3-amyloid peptide fragment [325-35. Effects were observed on spontaneous alternation behaviour, a behaviour relevant to working memory, and on retention in the step-down passive avoidance task. indicating a facilita- tion of long-term memory capacities [13]. We previously reported that central administration of [325-35 amyloid peptide induces in mice Congo red-stained amyloid-like deposits, neuronal loss. evidenced on Cresyl violet-stained brain slices, and delayed amnesia [13]. Preliminary in vitro incubation of the 1325_35 peptide, 4 days at 37°C, allows the formation of two types of insoluble precipitates, bire- fringent fibril-like structures and amorphous aggregates. It has been demonstrated that exposure of hippocampal cul- tures to this aggregated form of the [325-35 peptide markedly facilitates the apoptotic-mediated neuronal toxic- ity [11]. In vivo, a facilitation of the delayed amnesia was observed with the aggregated peptide [13]. Furthermore, acute administration of the cholinomimetics tacrine and nicotine, before either the Y-maze test session or training sessions in the passive avoidance test or in place learning in a water-maze, allowed significant reversions of the 1325_35-induced delayed amnesia, indicating that dysfunc- tion of the cholinergic systems was implicated in its appearance. The results presented in this study confirmed the validity of the model by showing the deficits can be attributed specifically to [325- 35, since the scrambled ~ 25- 35 peptide is inactive.

The glycine site agonist DCS has been reported to facilitate passive avoidance and place learning in the T- maze in young adult rats. at 0.3-10 mg/kg i.p. [15]. DCS also facilitated learning in 2-month-old mice subjected to weak training in the T-maze, and in senescence-accelerated mice (SAM/P8), a mutant inbred strain which shows memory impairments of early onset and rapidly increasing with age [6]. Similarly, the glycine prodrug MIL has also been reported to enhance learning performances in a pas- sive avoidance task in young adult rats. in spatial learning in the Morris-type water-maze task using 6-month-old mice, and to reverse amnesia induced by drugs acting through different neurotransmitter systems (scopolamine, AP-7, diazepam) in a one-trial alternation behaviour in a Y-maze [5,7]. Furthermore, Quartermain et al. [19] re- ported that MIL alleviates spontaneous forgetting, in paral- lel to its memory enhancing effect in mice. All these effects have been directly related to the increased glyciner- gic neurotransmission on the NMDA receptor complex, resulting in the improvement of the NMDA-dependent learning and memory processes.

Increasing evidence indicates that [3-amyloid deposition is the primo movins event in the genesis of AD. preceding neurofibrillary tangles formation. Although the presented model fails to encompass the plethora of neuropathological mechanisms involved in the genesis of AD. it does how- ever implicate a crucial initial pathological event, namely the abnormal deposition of ~-amyloid proteins. Moreover,

the ability of glycinergic agonists to reverse the delayed amnesia induced by i.c.v, administration of [325_35 indi- cates that the glutamatergic system is evidently one of the primary neurotransmitter systems affected by the exagger- ated deposition of aggregated [3-amyloid proteins in the brain, and delineates a potentially important therapeutic target. The present evidences that glycinergic agonists markedly reversed [3~s_3s-induced delayed amnesia indi- cate that the activation of the NMDA receptor may be altered by [3-amyloid deposition in the brain. The NMDA receptor-mediated neurotransmission has been reported to be affected in AD [3,14]. In particular, a reduced stimula- tion of the NMDA receptor complex activation by glycine and glycine site agonists has been reported in patients with AD [2,23]. Thus, enhancing the NMDA-type of gluta- matergic neurotransmission through the glycine modula- tory site may alleviate the symptomatic cognitive impair- ments of AD patients.

Clinical studies in AD patients with such drugs have experienced limited success. A single study with DCS has been published, with negative results [20]. Twelve patients received daily oral doses of DCS ranging from 25 to 500 mg during two weeks, followed by two weeks of placebo. No significant effects on neuropsychological outcome measures were observed, leading the authors to conclude that short-term positive modulation of the glutamatergic neurotransmission is unlikely t o produce significant im- provement of cognitive function in patients with AD. Human subjects treated with MIL recalled better than controls in two separate word retrieval tasks, and in older subjects MIL improved the abilities generally impaired in AD [21]. However, chronic treatment with MIL in AD patients failed to produce any positive results [8]. A chronic treatment with MIL induces the inactivation of the enzyme involved in its conversion to glycine, and thus prevents any long-term cognitive enhancing effect. Furthermore, chronic administration of DCS, 3 mg/kg twice daily for 15 days, has also been reported to induce some desensitiza- tion to the memory-enhancing effects of DCS [18]. How- ever, it remains to be clearly established if such tolerance to the memory improving effects of glycinergic agonists could be a common problem involving an autoregulatory mechanism through the glycine site on the NMDA recep- tor-associated responses.

In conclusion, our results further support the concept that acute administration in mice of glycinergic agonists on the NMDA receptor complex is able to reverse the learn- ing and memory impairments induced by ~3-amyloid re- lated peptides. Therefore, such compounds still constitute a therapeutic approach of interest for AD patients, if some derivative could be obtained, devoid of the side-effects that impeded the chronic therapeutic use of these drugs. On the other hand, the evident complexity of the myriad of subse- quent mechanisms implicated in the pathological progres- sion of AD, such as Apolipoprotein E genotype, neuronal degeneration, inflammation and oxidative stress, must also

T. Maurice et al. / Brain Research 731 (1996)249-253 253

taken into consideration to evaluate the therapeutic impact of cognitive enhancers. It is evident that full clinical benefit from such drugs will only be achieved in combina- tion with therapeutic interventions aimed at reducing the mechanisms responsible for the progressive neuronal loss and amyloid deposition in AD.

Acknowledgements

Thanks are due to Dr. Robert L. Herting (Searle, Skokie, IL) for providing us milacemide, to Jean Bayle, for build- ing the apparatus used for behavioral testing, and to Didier Petite for incubation of [3-amyloid peptides.

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