A secreted form of the β-amyloid precursor protein (sAPP695) improves spatial recognition memory in OF1 mice

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Abstract

The β-amyloid precursor protein (APP) plays a central role in Alzheimer’s disease (AD) and appears to be a multifunctional protein. Secreted forms of APP (sAPP) have memory-enhancing effects in certain behavioral paradigms. To investigate sAPP’s role in spatial memory processes, we adapted a spatial recognition task and evaluated (1) the performance of OF1 mice after massed training (single 15-min acquisition session) and distributed training (three 5-min acquisition sessions), (2) the decline of spatial recognition performance by introducing different delays (5 min, 1, 3, and 24 h) between the acquisition and retention phases, and (3) the effects of sAPP695 on spatial recognition memory. In the present study, mice selectively reacted to a change in the spatial configuration of five objects. Indeed, 3 min post-acquisition, mice performed similarly in the massed and distributed versions of the task, by re-exploring the two displaced objects only, whereas mice exposed to the same spatial configuration did not. Additionally, all mice did react to a novel object in a subsequent object recognition phase. Mice detected object displacements 5 min, 1 h, or 3 h post-acquisition, but no more at a 24 h-delay. Finally, mice treated with sAPP695 intracerebroventricularly at a dose of 0.5 pg/4 μL/mouse, 20-min pre-acquisition or 5-min post-acquisition, still reacted to a spatial change in objects position 24 h post-acquisition, in marked contrast to NaCl-treated mice. Our data demonstrate that sAPP695 significantly improves a form of spatial memory, and confirms the hypothesis of an action of this protein on early memory processes.

Introduction

Researches on Alzheimer’s disease (AD),1 the most common cause of senile dementia, strongly focus on the disruption of amyloid precursor protein (APP) metabolism. The APP gene maps to chromosome 21 in humans, and alternative splicing of APP’s mRNA leads to the production of different isoforms of the protein (for review: Rossner, Ueberham, Schliebs, Perez-Polo, & Bigl, 1998). These isoforms are named according to their length in amino acids (from 365 to 770), and the three major forms are APP770, APP751, and APP695. The first two isoforms contain a Kunitz-like protease inhibitor (KPI) domain and are expressed in most peripheral tissues and mainly glial cells in the brain. The third isoform, APP695, lacks the KPI domain and is more selectively expressed in neurons (Rohan de Silva et al., 1997). All these APPs are membrane-anchored glycoproteins with a long extracellular N-terminal domain, the amyloid β-protein (Aβ) sequence and a short C-terminal intracellular domain. Proteolytic processing of APP by β- and γ-secretases leads to the production of Aβ: its accumulation in extracellular neuritic plaques represents a main feature of AD (Selkoe, 1994). This amyloidogenic pathway also leads to the formation of a truncated secreted form of APP called sAPPβ. In contrast, normal processing of APP mainly involves α-secretase activity that cleaves the protein within the sequence of Aβ. This non-amyloidogenic pathway leads to the secretion of sAPPα, a soluble form containing only a fragment of the Aβ-sequence at the C-terminal portion (Dodart, Mathis, & Ungerer, 2000).

Physiological properties of APP and its derivatives remain poorly understood. APP is a multifunctional glycoprotein with neurotrophic and neuroprotective activities (Isacson, Seo, Lin, Albeck, & Granholm, 2002; Qiu, Ferreira, Miller, Koo, & Selkoe, 1995). It can act as a cell adhesion molecule (Storey, Beyreuther, & Masters, 1996), promote neurite outgrowth or serve as a receptor coupled to a G protein (Okamoto, Takeda, Murayama, Ogata, & Nishimoto, 1995). Variations in expression of the different isoforms during development suggest their differential implication in nervous system ontogenesis, with a role for APP751 in promoting cellular growth and synaptogenesis, and a role for APP695 in establishing mature connectional patterns between neurons (Apelt, Schliebs, Beck, Rossner, & Bigl, 1997). Secreted forms also have neuroprotective and neurotrophic effects (Barger & Mattson, 1996; Mattson et al., 1997), and can activate protein kinase C and phospholipase C pathways (Ishiguro, Ohsawa, Takamura, Morimoto, & Kohsaka, 1998).

Several studies suggest that APP and its derivatives could play a role in structural and neurochemical changes underlying memory processes (for review: Dodart et al., 2000). Anti-APP antibodies, administered intracerebroventricularly (i.c.v.), induce memory deficits in rats and chicks in passive and active avoidance tasks (Doyle et al., 1990; Huber, Martin, Loffler, & Moreau, 1993; Mileusnic, Lancashire, Johnston, & Rose, 2000). Moreover, secreted forms themselves may participate to neuronal plasticity by promoting neurite extension (Qiu et al., 1995) and modulating long-term potentiation or depression in hippocampal slices (Ishida, Furukawa, Keller, & Mattson, 1997). Furthermore, spatial memory deficits in aged rats are correlated with low levels of sAPP in cerebrospinal fluid (Anderson et al., 1999). Finally, i.c.v. administration of sAPP in mice (1) improves memory performance when injected 20 min before the acquisition phase of a go-no go and an object recognition tasks and (2) blocks scopolamine-induced deficits in a go-no go visual discrimination task, an object recognition task and a lever-press task (Meziane et al., 1998).

Among the main symptoms of AD are spatial disorientation, alterations of visuospatial memory with both spatially- and object-based recognition deficits, and attentional impairments (Buck, Black, Behrmann, Caldwell, & Bronskill, 1997; Dubois, Tounsi, Michon, & Deweer, 1997; Fujimori, Imamura, Yamashita, Hirono, & Mori, 1997; Kalman, Magloczky, & Janka, 1995; Keri, Antal, Kalman, Janka, & Benedek, 1999; Keri & Janka, 2001). The concept of spatial representations implies that both rodents and humans construct a cognitive representation of their environment through the organization of acquired spatial informations. Animals can also react to stimuli that are not immediately present, because spatial representations maintain relationships between such stimuli, and those effectively perceived. Animals are thus able to manage spatial recognition and identification: if not, exploratory behavior starts again and leads to the formation of a new spatial representation, or to update the former one (for review: Poucet, 1993). This renewal of exploratory behavior has previously been observed in hamsters after the change of spatial configuration of objects in an arena: these rodents explored displaced objects more than non-displaced ones (Poucet, Chapuis, Durup, & Thinus-Blanc, 1986). The object recognition task used with rats and mice is also based on the quantification of exploratory behavior after spatial change (when objects have been displaced, by shifting two familiar objects or moving an object to a new position) or after non-spatial change, i.e., when a new object has been added and substituted for a familiar one (Ennaceur & Delacour, 1988; Thinus-Blanc, Save, Rossi-Arnaud, Tozzi, & Ammassari-Teule, 1996).

Considering the fact that visuospatial memory disorder is one of the main symptoms of AD, and the strong evidence for a role of the secreted forms of APP in neuronal plasticity and memory formation, we hypothesized that these compounds might modulate spatial memory processes. Since direct effects of sAPP on spatial memory have not yet been tested, we evaluated the effects of (α)sAPP695 in a spatial recognition task in mice. In the first experiment, we validated a new protocol characterized by a reduced number of sessions, compared to that used in other laboratories (Adriani et al., 1998; Adriani, Sargolini, Coccurello, Oliverio, & Mele, 2000; Roullet, Sargolini, Oliverio, & Mele, 2001; Save, Poucet, Foreman, & Buhot, 1992; Thinus-Blanc et al., 1996). The aim was to limit handling and stress between sessions, and thus we grouped separate sessions in three “unified-sessions” corresponding to the three different phases of the task (acquisition, retention, and object recognition testing). In the second experiment, we established a retention memory performance curve by introducing different delays (5 min, 1 h, 3 h, and 24 h) between acquisition and retention sessions. Considering the promnestic effects of sAPP found by Meziane et al. (1998), sAPP695 was tested at the delay for which mice performance was low. Treatment (sAPP or NaCl) was injected either 20 min pre-training or 5 min post-training, two injection times for which promnestic effects of sAPP695 have been previously demonstrated in object recognition (“20 min pre-training”: Meziane et al., 1998; “5 min post-training”: unpublished data). The dose used in our study (0.5 pg/4 μl/mouse) was the lowest dose inducing promnestic effects in most behavioral tasks screened yet, including the object recognition task (Meziane et al., 1998).

Section snippets

Animals

One hundred and fifty-eight male “swiss” mice from the outbred OF1 strain (2–4 months old, Charles River Laboratories IFFA-CREDO, L’Arbresle, France) were maintained at 23 ± 1 °C under a 12–12 h light–dark cycle (light on at 07:00 h) with ad libitum access to standard chow and tap water. Upon arrival from the breeding center, mice were group-housed five per cage during at least one week before the beginning of the experiment. Mice were then housed individually 5 days (in experiments 1 and 2) or 7

Animal exclusions

Eight mice were excluded from statistical analyzes in experiment 3 according to the exclusion criteria described in Section 2. The number of mice changed to n=11 for each treated group (“NaCl, −20 min”; “NaCl, +5 min”; “sAPP, −20 min”; “sAPP +5 min”), and the “intact mice” group remained at n=12. Data collection for object exploration failed for one mouse during the second 5-min period of the retention phase, so for some analyzes including this period, group “sAPP, +5 min” had only n=10.

Locomotor activity (data not shown)

During the

Discussion

In the present study, we analyzed exploratory patterns of mice submitted to a spatial recognition memory task, and several factors susceptible to alter recognition performances (i.e., massed versus distributed sessions, change or no change in spatial configuration, delay between acquisition and retention phases).

In our first experiment, mice clearly reexplored displaced objects during the first 5 min of the retention phase of the task, whatever the protocol used. Thus, mice detected object

Acknowledgements

We are grateful to Dr. Steven M. Paul from Eli Lilly Research Laboratories (Indianapolis, USA) for initiating and encouraging our studies on sAPP, to Bernard Neusch for animal care, and to Mathias De Roo and Claire Leroy for their enthusiastic participation in testing the animals in experiment 2. The present work was supported by the CNRS and the Université Louis Pasteur (France), and also by a grant to A.B. from the Association Alsace Alzheimer (Mulhouse, France).

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