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Keywords:

  • amyloid precursor protein;
  • apolipoprotein E;
  • endocytosis;
  • low-density lipoprotein receptor-related protein;
  • memory consolidation;
  • synaptic plasticity

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Despite advances in our understanding of the basic biology of amyloid precursor protein (APP), the normal physiological function(s) of APP in learning and memory remains unclear. Here we show increased APP degradation in the hippocampus to be associated with the consolidation of a passive avoidance response. Neurone-specific APP695 expression became transiently reduced 2–4 h post-training through association with endosomal adaptin proteins and enhanced internalization. By contrast, internalization of glial-associated APP containing a Kunitz protease inhibitor-like domain (APP-KPI) was dependent on the low-density lipoprotein receptor-related protein (LRP). In addition, LRP expression and association with apolipoprotein E increased in the 2–4 h post-training period. The LRP antagonist receptor-associated protein prevented the APP-KPI internalization and LRP–apolipoprotein E association and this resulted in amnesia. Degradation of APP695 and APP-KPI did not appear to be related to α-secretase activity, as no learning-associated increase of secreted APP was observed in the CSF. Moreover, as internalization of APP isoforms was observed only in dentate gyrus, it probably relates to the learning-associated restructuring of the perforant path terminals. Memory-associated APP processing in both neuronal and glial compartments points to a role for glial unsheathing of synaptic connections, an event required for the synaptic restructuring that accompanies memory consolidation. These observations may have a direct relevance to understanding the pathophysiology of Alzheimer's disease as β/γ-secretase-derived β-amyloid is formed following internalization of cell surface APP into the endosomal compartment.

Abbreviations used
AD

Alzheimer's disease

AP

adaptin protein

ApoE

apolipoprotein E

APP

amyloid precursor protein

APP-KPI

amyloid precursor protein containing a Kunitz protease inhibitor-like domain

GST

glutathione S-transferase

LRP

low-density lipoprotein receptor-related protein

RAP

receptor-associated protein

sAPP

soluble amyloid precursor protein

Alzheimer's disease (AD) is a neurodegenerative condition where the associated neuronal cell loss has been attributed to the toxicity of β-amyloid, a proteolytic derivative of amyloid precursor protein (APP) (Masters et al. 1985; Gomez-Isla et al. 1996; Kim et al. 2003). Despite advances in our understanding of the basic biology of APP, the normal physiological function(s) of APP and its processing remains unclear. APP exists mainly as three spliced variants of which APP695 is principally expressed by neurones and the Kunitz protease inhibitor-like domain-containing isoforms, APP751 and APP770, are expressed by glial cells (Kang and Muller-Hill 1990; LeBlanc et al. 1991, 1996). Cell surface expression of APP is regulated by enzymatic cleavage to yield soluble APP (sAPP) (Parvathy et al. 1999) or, alternatively, by internalization and degradation within the endosomal pathway (Haass et al. 1992; Koo and Squazzo 1994; Perez et al. 1999).

Several proteins that regulate APP processing have been implicated in the pathophysiology of AD. For example, degradation of APP follows clathrin-coated pit-mediated internalization, a process regulated by adaptin proteins (APs) AP1, AP2 and AP180 (Marsh and McMahon 1999). These APs are down-regulated in AD (Yao et al. 1999, 2000) and associated with abnormalities in the endosomal pathway early in the disease process (Cataldo et al. 2000). APP internalization is also facilitated by low-density lipoprotein receptor-related protein (LRP) (Ulery et al. 2000; Rebeck et al. 2001). Apolipoprotein E (ApoE), a protein internalized by LRP (Beisiegel et al. 1989), also modulates APP processing (Vincent and Smith 2001; Dodart et al. 2002). ApoE alleles and LRP polymorphisms pre-dispose individuals to developing AD, further underscoring their importance in the appropriate regulation of APP expression (Kang et al. 1997; Strimmatter et al. 1993).

Appropriate expression of APP also appears to be necessary for the neuroplastic events that accompany learning and memory in animal models. Perturbation of APP function by intraventricular administration of antibodies or antisense oligonucleotides results in profound amnesia for avoidance conditioning paradigms (Doyle et al. 1990; Huber et al. 1993; Mileusnic et al. 2000). Similarly, mice expressing mutant APP exhibit age-dependent onset of spatial learning deficits (Westerman et al. 2002). Evidence of a role for LRP is less compelling; however, its functional disruption produces significant deficits in long-term potentiation, a cellular model of learning and memory (Zhou et al. 2000). Despite these findings, a direct relationship between APP processing and the cellular events that accompany learning and memory within the medial temporal lobe, and the hippocampus in particular, has yet to be demonstrated. Given its synaptic association and proposed function as a cell adhesion molecule (Koo et al. 1990; Coulson et al. 1997; Lyckman et al. 1998), we determined whether, as has been observed for other cell adhesion molecules (Foley et al. 2000), change in APP processing is integral to the neuroplastic events that underpin hippocampal-dependent memory consolidation.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

The anaesthetic agents ketamine hydrochloride and medetomidine hydrochloride were purchased from Pharmacia Animal Health (Limerick, Ireland) and Pfizer (Cork, Ireland), respectively. Proteins for the interventive study, rat receptor-associated protein (RAP) and Escherichia coli glutathione S-transferase (GST), were purchased from Maine Biotechnology Services (Portland, ME, USA) and Sigma (St Louis, MO, USA), respectively. Monoclonal antibodies to a synthetic actin C-terminal peptide and bovine AP1/2 and AP180 were obtained from Sigma. Monoclonal anti-human ApoE was purchased from BD Transduction Laboratories (Oxford, UK). Monoclonal antibodies to recombinant human APP (clone 22C11) and APP containing a Kunitz protease inhibitor-like domain (APP-KPI) were obtained from Chemicon (Temecula, CA, USA) and Upstate Biotechnology (Lake Placid, NY, USA), respectively. Monoclonal antibodies to human LRP were purchased from American Diagnostica (Stamford, CT, USA). All secondary antibodies and routine laboratory chemicals were from Sigma. Polyclonal rabbit anti-rat albumin conjugated to peroxidase was purchased from Autogen Bioclear (Calne, UK).

Animal maintenance and behavioural assessment

Post-natal day 80 male Wistar rats (300–350 g) were obtained from the Biomedical Facility at University College Dublin. The animals were introduced into the experimental room 5 days prior to commencement of passive avoidance training. Animals were housed singly and left to habituate for days 1–3. On days 4–5 the animals were handled, their weights monitored and spontaneous behaviour assessed in an open field apparatus (620 mm long, 620 mm wide, 150 mm high). The floor of the open field was ruled into a series of squares (77 × 77 mm), the animal was placed in the centre and the number of lines crossed in a 5-min period counted. Other behaviours assessed included rearing, grooming, piloerection, defaecation and posture. All observations were carried out in a quiet room under low-level red illumination between 08:00 and 12:00 h to minimize circadian influence. On the day of training and immediately preceding recall these spontaneous behaviours were reassessed.

Passive avoidance conditioning

Animals were trained in a single-trial, step-through, light–dark passive avoidance paradigm, as described previously (Fox et al. 1995). The training apparatus was divided into two chambers, the smaller illuminated chamber being separated from the larger dark chamber by a shutter that contained a small entrance. The floor of the training apparatus consisted of a grid of 16 stainless steel bars through which a remotely controlled, scrambled shock (0.75 mA every 0.5 ms) of 5 s duration could be delivered when the animal entered the dark chamber. Passive control animals received no foot shock but were allowed to explore the apparatus for a time similar to that of the trained animals. All animals, except those taken at 0 h post-training, were tested for recall of this inhibitory stimulus immediately prior to killing by placing them into the light chamber and recording their latency to enter the dark chamber. A maximum criterion time of 600 s was used; however, any animal that re-entered the dark compartment in under 300 s was deemed not to have acquired the task and therefore was excluded from subsequent experimental procedures. Passive animals were also returned to the passive avoidance apparatus and allowed to freely explore both compartments for a period of time matched to their trained counterparts. Animals were killed either immediately following training (0 h) or at recall (2, 4, 6 and 12 h post-training). A minimum of three animals were trained at each time point.

Intracerebroventricular cannula placement and low-density lipoprotein receptor-related protein antagonist administration

Animals were anaesthetized by intraperitoneal administration of a solution (1 mL/kg) containing 4 parts ketamine hydrochloride (100 mg/mL) and 3 parts medetomidine hydrochloride (20 mg/mL) mixture. Depth of anaesthesia was assessed by monitoring respiration rate and palpebral and pedal withdrawal reflexes. Once the animal was anaesthetized, the skull was exposed and trepanned (1 mm in diameter) and a guide cannula was stereotactically implanted (1 mm deep) at a position 1 mm posterior and 1.6 mm lateral to Bregma. Two flat-bottomed stainless steel screws were placed laterally to the cannula and the entire complex was secured with dental acrylic. When the acrylic had polymerized, a dummy cannula, cut to the same dimensions as the guide cannula, was screwed into place to maintain the patency of the guide cannula and minimize infection. Following closure of the incision with interrupted silk sutures, the animal was placed in a heated cage (35°C) and monitored carefully for complete recovery. No further procedures were allowed for a minimum of 1 week post-surgery. During this period body weight was monitored.

All agents were delivered by inserting an internal cannula (28 gauge, 16 mm long) through the guide cannula and into the right lateral ventricle. Each treatment was delivered to the unrestrained, conscious rat in a 5 µL volume at a rate of 1 µL/min of each treatment by a micropipette connected to the internal cannula. Aliquots of the LRP antagonist RAP (0.32 µg), produced as a GST fusion protein, or an equivalent amount of GST alone (0.128 µg) were injected at 3 h post-training. Following administration the internal cannula was left in position for 1 min and then withdrawn slowly. The dummy cannula was replaced and the animal returned to its home cage. The effect of RAP-GST and the GST control on memory formation was assessed on task recall at 48 h post-training.

In a separate series of experiments the effect of RAP-GST (0.320 µg) and GST (0.128 µg) on APP and LRP expression was determined in tissue obtained from animals killed 1 h after intracerebroventricular administration.

Tissue collection and preparation

Animals trained in the avoidance task were killed by cervical dislocation at increasing post-training times. Untrained naive and passive animals served as controls. The dentate gyrus, combined CA regions and entorhinal cortex were separately dissected, immediately snap frozen in liquid nitrogen and stored at −80°C. Prior to electrophoresis and immunoblotting, each brain region was homogenized in ice-cold 0.32 m sucrose and protein concentrations were determined according to the method of Lowry et al. (1951). Samples of equal protein concentration were then boiled for 10 min in a reducing sample buffer of 70 mm Tris-HCl, pH 6.8, 33 mm NaCl, 1 mm EDTA, 2% (w/v) sodium dodecyl sulphate, 0.01% (w/v) bromophenol blue, 10% glycerol and 3% (v/v) dithiothreitol reducing agent. Samples used for LRP immunoblotting procedures were prepared in a non-reducing sample buffer of 0.15 mm Tris-HCl, pH 6.8, containing 10% (v/v) glycerol and 0.15% (w/v) bromophenol blue.

CSF collection and preparation

At increasing post-training times, animals were anaesthetized as for intracerebroventricular surgery, placed in a stereotaxic frame and the atlanto-occipital membrane at the back of the head was exposed by dissection. An aliquot of 100–125 µL CSF was collected from each animal by puncturing the membrane with a 30-gauge hypodermic needle attached to a 1-mL syringe and immediately snap frozen in liquid nitrogen. The animal was then killed by cervical dislocation. Given the small sample size, CSF protein concentrations were determined according to the methods of Bradford (1976) and samples of equal protein concentration were prepared for electrophoresis and immunoblotting procedures as described below.

Adaptin and apolipoprotein E immunoprecipitation

Tissue samples containing 250 µg of protein, in a final volume of 500 µL, were prepared in an immunoprecipitation buffer comprised of 10 mm Tris-HCl, pH 7.4, containing 1% (v/v) Triton X-100, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 0.2 mm phenylmethylsulphonyl fluoride and 0.5% (v/v) NP-40. The samples were incubated overnight at 4°C with 1–2 µL of anti-adaptin antibody. The same buffer was used in the anti-ApoE immunoprecipitations but without the addition of EGTA. Subsequently, the anti-adaptin immunocomplex was captured with protein G sepharose beads and the anti-ApoE complex with anti-mouse IgG agarose by rotation for 2 h at 4°C. The beads were washed three times in immunoprecipitation buffer, resuspended in the electrophoresis reducing sample buffer, the immunocomplex released by boiling and the beads removed by centrifugation.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and immunoblotting

Protein samples containing equal amounts of protein were separated on polyacrylamide minigels and electrophoretically transferred to nitrocellulose membranes. Equal protein loading was confirmed by ponceau S staining of the membrane (not shown). The nitrocellulose was blocked using 10 mm Tris-HCl, pH 7.4, containing 150 mm NaCl, 0.05% (v/v) Tween-20 (TBS-T) and 5% (w/v) non-fat milk powder for 1 h at room temperature (22°C). Monoclonal antibodies to actin, AP1/AP2, AP180, APP (clone 22C11), APP-KPI and LRP were diluted in blocking buffer and incubated overnight at 4°C. The membranes were then incubated for 1 h at room temperature with the appropriate secondary horseradish peroxidase-linked antibodies diluted in blocking buffer. Following membrane washing with TBS-T buffer, the immunocomplexes were visualized using a chemiluminescence peroxidase substrate and exposing the membranes to X-ray film (Kodak, Dublin, Ireland). Equal protein loading was verified using anti-actin or, in the case of CSF samples, polyclonal anti-rat albumin directly conjugated to peroxidase.

The X-ray films were scanned using Adobe Photoshop 7.0 and quantitative densitometry of the electrophoretic bands was performed using NIH Image software (version 1.62c). Protein expression levels were quantified as percent of that observed in naive animals.

Statistical analysis

Group comparisons of modulations in protein expression were determined by anova followed by a Bonferroni post-hoc analysis. Single comparisons were made using Student's t-test. The effect of RAP-GST and GST on passive avoidance learning was analysed using the Mann–Whitney U-test for non-parametric data. In all cases, p-values less than 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Amyloid precursor protein expression becomes transiently decreased in the rat hippocampal dentate gyrus during memory consolidation

Total APP expression was evaluated in the hippocampal dentate gyrus of adult Wistar rats, a brain structure required in consolidation of the passive avoidance paradigm (Murphy and Regan 1999). Monoclonal antibodies specific for total APP (22C11; Weidemann et al. 1989) and APP isoforms containing the Kunitz protease inhibitor domain (anti-APP-KPI; Refolo et al. 1989) were employed for immunoblot analysis of dentate gyrus APP expression, as these antibodies have limited cross-reactivity with amyloid precursor-like proteins 1 and 2, respectively (Kim et al. 1995; Pangalos et al. 1995). Antibody 22C11 detected diffuse bands of between 97 and 116 kDa and these corresponded to the molecular weights expected for the glycosylated variants of both immature and mature forms of APP695 (Weidemann et al. 1989; Buxbaum et al. 1998) (Fig. 1a). Although the 22C11 monoclonal antibody cross-reacts with all isoforms of APP, the APP695 variant was predominately detected under the conditions employed in this study, probably due to this transcript having 30-fold higher expression than that of APP-KPI in the adult rat brain (LeBlanc et al. 1991). Moreover, the molecular weight of the APP isoform detected by antibody 22C11 is much less than the 140–150 kDa expected for APP-KPI (Weidemann et al. 1989; Sisodia et al. 1993).

image

Figure 1. Transient decrease in amyloid precursor protein (APP) expression in the rat dentate gyrus following passive avoidance training. (a and b) Representative immunoblots of dentate gyrus APP695 (97–116 kDa) and APP containing a Kunitz protease inhibitor-like domain (APP-KPI) (140 kDa) expression, respectively, in naive (N) and trained animals killed at increasing post-training times. Also shown are 2 h passive control animals (P) immunoblotted for APP695. Equal protein loading is illustrated by unchanged actin immunoreactivity. (c) Quantitative densitometric analysis of dentate gyrus APP695 (N, n = 15; 0 h, n = 3; 2 h, n = 10; 2 h passive, n = 3; 4 h, n = 9; 6 h, n = 8; 12 h, n = 9; ▪) and APP-KPI (N, n = 6; 0 h, n = 3; 2 h, n = 3; 4 h, n = 3; 6 h, n = 3; 12 h, n = 3; □) expression at increasing post-training times and 2 h passive (2P) control APP695 (n = 3; bsl00077). The quantitative data are expressed as percent of protein expression in the naive animal and are the mean ± SEM. Significant changes in protein expression were determined using a one-way anova followed by Bonferroni post-hoc analysis. *Values significantly different from naive animal (p < 0.05).

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A significant temporal modulation in APP695 expression was evident following passive avoidance training (one-way anova; F6,50 = 3.69, p = 0.004). APP expression was the same in 0 h trained animals and controls indicating that handling and/or exposure to the avoidance paradigm had no effect on basal APP expression (Fig. 1a). Bonferroni post-hoc analysis revealed a significant decrease in APP695 expression at the 2 h (79.7 ± 3.6%; p < 0.01) and 4 h (84.6 ± 3.1%; p < 0.05) post-training times compared with naive control (98.82 ± 2.76%) (Fig. 1c). This down-regulation in APP expression was specific to the 2–4 h post-training period as it was unaltered immediately after training (0 h; 103.79 ± 4.25%), returned to control levels by 6 h (94.15 ± 6.31%) and thereafter remained unchanged up to the 12 h (91.89 ± 6.77%) post-training time. In addition, the 2 h post-training modulation of APP was learning specific as its expression was unchanged in naive and 2 h passive control animals (Figs 1a and c). Anti-APP-KPI detected a band of approximately 140 kDa (Fig. 1b). As with APP695, a temporal modulation in APP-KPI expression was evident following passive avoidance training (F5,15 = 3.69; p = 0.02). Post-hoc analysis of APP-KPI expression became significantly reduced at the 2 h (77.9 ± 4.9%; p < 0.05), 4 h (75.9 ± 10.4%; p < 0.05) and 6 h (72.7 ± 1.8%; p < 0.05) post-training times compared with naive control and thereafter returned to levels observed in the control animal by 12 h post-training (n = 3) (Figs 1b and c). The learning-associated decrease in APP expression was specific to the dentate gyrus as no such temporal modulation in APP processing occurred in the entorhinal cortex or CA1–CA3 regions of the hippocampal formation at any of the post-training times examined (Figs 2a–d). Three animals were assessed in each brain region at each time.

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Figure 2. Amyloid precursor protein (APP) expression does not transiently decrease in the rat entorhinal cortex and hippocampal CA region following passive avoidance training. (a and b) Representative immunoblots of APP695 and APP containing a Kunitz protease inhibitor-like domain (APP-KPI) in the entorhinal cortex and hippocampal CA region, respectively, in naive (N, n = 3) and trained animals (n = 3) killed at increasing post-training times. (c and d) Quantitative densitometric analysis of APP expression in the entorhinal cortex and hippocampal CA regions, respectively. ▪, APP695 expression; □, APP-KPI expression. The quantitative data are expressed as percent of protein expression in the naive animal and are the mean ± SEM. No significant change in the expression of APP695 or APP-KPI was found.

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To determine whether proteolytic processing and release of sAPP accounted for the learning-associated decrease in APP expression, we measured its expression in the rat CSF in the 2–6 h post-training period as this has previously been demonstrated to be effective in relating modulations in hippocampal APP expression to α-secretase activity (Lin et al. 1999). In rat CSF, antibody 22C11 detected a discrete APP band of approximately 97 kDa (Fig. 3a). Anti-APP-KPI recognized a band that was substantially larger (∼200 kDa) than the full-length APP-KPI observed in the dentate gyrus (Fig. 3b). The latter high molecular weight band probably represents an APP-KPI–substrate complex, as the soluble domain of APP-KPI is known to form sodium dodecyl sulphate-stable complexes with its substrates (Van Nostrand et al. 1990). A temporal modulation in CSF sAPP expression was evident following training (F4,14 = 11.31, p = 0.0003) with significant reductions occurring at the 2 h (49.9 ± 13.6%; p = 0.04) and 4 h (38.6 ± 2.9%; p = 0.004 n = 4) post-training times but not at 6 h (73.0 ± 2.4%) or 12 h (data not shown) as compared with naive control. By contrast, no learning-induced change in sAPP containing the Kunitz protease inhibitor-like domain was observed (F4,13 = 1.18, p = 0.37; Fig. 3c). Remarkably, these results discriminated two learning-dependent mechanisms of APP degradation in the hippocampal dentate gyrus.

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Figure 3. Soluble amyloid precursor protein (APP)695 expression in the CSF transiently decreases following passive avoidance training. (a) Representative immunoblots of soluble APP695 (sAPP); (b) APP containing a Kunitz protease inhibitor-like domain (APP-KPI) (sKPI) in the CSF, as single bands of 97 kDa (APP695) and approximately 200 kDa (APP-KPI). Equal protein loading is illustrated by unchanged albumen (AL) immunoreactivity. (c) Quantitative densitometric analysis of sAPP695 (▪) and sAPP-KPI (□) expression in the CSF in naive (N; n = 3) and trained animals killed at increasing post-training times (2 h, n = 3; 4 h, n = 4; 6 h, n = 3). Significant changes in protein expression were determined using a one-way anova followed by Bonferroni post-hoc analysis. *Values significantly different from naive animal (p < 0.05).

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Endosomal trafficking of dentate gyrus amyloid precursor protein during memory consolidation

Regulation of APP expression following avoidance conditioning was further investigated by evaluating the involvement of the endosomal pathway in the cellular events that immediately follow training. Initially, the association of APs AP1 and AP2 with APP and their interaction with AP180 was examined, as these proteins can modulate clathrin-coated pit formation at synaptic sites (Hao et al. 1999; Yao et al. 2002). Using a monoclonal antibody that recognizes a sequence common to the AP1 and AP2 β-subunit, two bands of approximately 110 kDa were detected in the rat dentate gyrus (Fig. 4a). The upper band corresponded to the expected weight of the AP1 and the lower band corresponded to that of AP2 (Ahle et al. 1988). Following passive avoidance training no change was observed in rat dentate gyrus AP1 or AP2 expression in the 0–12 h post-training period (data not shown).

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Figure 4. Amyloid precursor protein (APP) associates with adaptin proteins (APs) following passive avoidance training. (a) Anti-adaptin monoclonal antibody detected both AP1 and AP2 in the rat dentate gyrus. Co-immunoprecipitation using this antibody detected AP2 and not AP1 and the AP2 adaptin was found to coprecipitate dentate gyrus APP695 but not dentate gyrus APP containing a Kunitz protease inhibitor-like domain (APP-KPI). (b) Association of AP2 with AP180 in immunoprecipitates isolated with anti-AP180 from the dentate gyrus of naive (N), passive and trained animals killed at 2 h following training. (c) Quantitative densitometric analysis of dentate gyrus AP2 immunoisolated with anti-AP180 in naive, trained and passive animals killed at 2 h following training. The quantitative data are expressed as percent of protein expression in the naive animal and are the mean ± SEM (n = 3). Significant changes in protein expression were determined using a Student's t-test. *Values significantly different from naive animal (p < 0.05). DG, homogenized whole dentate gyrus; IP, immunoprecipitating antibody; IB, immunoblotting antibody; NC, negative control (primary precipitating antibody omitted); 2P, 2 h passive.

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As the adaptin antibody employed had previously been used to demonstrate an association of adaptins with GABAA receptors in neuronal membranes (Kittler et al. 2000), we also determined whether a similar association occurred with the clathrin endocytosis machinery in the rat dentate gyrus during memory consolidation. We found APP695 to coprecipitate with the β-subunit of AP2 but, importantly, we failed to observe any association of APP-KPI with either the AP1 or AP2 APs (Fig. 4a). The expression of the AP180 AP, which associates with AP2 to enhance clathrin-coated pit formation in vitro (Hao et al. 1999), remained unchanged following avoidance training (data not shown). However, AP180 was found to coprecipitate with AP2 and this association was significantly increased at the 2 h post-training time point (126.7 ± 17.0) (Figs 4b and c). This modulation appeared to be learning specific as a similar modulation in the association of AP180 and AP2 was not detected in 2 h passive controls (106.6 ± 10%).

Role of low-density lipoprotein receptor-related protein-mediated endocytosis during memory consolidation

As a lack of APP-KPI coprecipitation with AP2 suggested its mode of internalization to be dissimilar to APP695, we were prompted to determine whether interactions of the Kunitz protease inhibitor domain of APP with LRP (Knauer et al. 1996) contributed to its endocytosis following passive avoidance training. Using a monoclonal antibody raised against human LRP1 (Nykjaer et al. 1992), a significant temporal modulation in post-training LRP protein expression was found (F5,42 = 10.72; p < 0.0001). Post-hoc analysis demonstrated LRP expression to significantly increase at the 2 h (137.8 ± 7.14%; p < 0.001) and 4 h (155.8 ± 12.2%; p < 0.001) post-training times (Figs 5a and b). The increase in LRP expression at the 2 h post-training time was paradigm specific as no similar change was evident in the control animals (2 h passive; 107.81 ± 4.59%).

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Figure 5. Low-density lipoprotein receptor-related protein (LRP) expression and association with apolipoprotein E (ApoE) transiently increases in the rat dentate gyrus following passive avoidance training. (a and c) Immunoblots of dentate gyrus LRP and ApoE immunoisolated with anti-LRP, respectively. (b and d) Quantitative densitometric analysis of the LRP [N, n = 15; 2 h, n = 9; 2 h passive (2P), n = 3; 4 h, n = 6; 6 h, n = 6] and ApoE (N, n = 9; 2 h, n = 6; 2P, n = 3; 4 h, n = 5; 6 h, n = 3) immunoblots, respectively. The quantitative data are expressed as percent of protein expression in the naive animal and are the mean ± SEM. Significant changes in protein expression were determined using a one-way anova followed by Bonferroni post-hoc analysis. *Values significantly different from naive animal (p < 0.05). IP, immunoprecipitating antibody.

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Given the significant learning-associated increase in LRP expression, we further investigated the expression of its ApoE ligand, the ε4 allele of which is a known risk factor in the development of AD (Strimmatter et al. 1993). Following training, no modulation in ApoE expression was observed (data not shown). However, coimmunoprecipitation studies demonstrated an increase in ApoE–LRP association at the 2 h post-training time and this was significant in respect of the naive but not passive control animals (100.8 ± 3.5 vs. 132.70 ± 7.6%; p = 0.03, two-tailed Student's t-test; Figs 5c and d). ApoE/LRP binding returned to basal expression by 6 h post-training and remained at this level up to the 12 h post-training time.

The availability of the RAP, a chaperone protein that inhibits LRP–ligand interactions, allowed us to demonstrate a direct requirement for LRP-mediated endocytosis of APP during the early period of memory consolidation. The LRP antagonist was administered as a GST fusion protein at 3 h following training and task recall was assessed 48 h post-training. GST alone served as a control. Injection of RAP-GST significantly decreased the 48 h escape latency compared with a 3 h post-training injection of GST control (RAP, 275.3 ± 76.5 s vs. GST, 489.7 ± 47.9 s; p < 0.05) (Fig. 6a). The involvement of LRP in consolidation events associated with the 2–4 h post-training period was further supported by the lack of amnesia when RAP-GST was administered at 1 h following training (data not shown). Intraventricular administration of RAP also prevented APP-KPI internalization and subsequent degradation following training. In comparison to sham-operated and GST-treated controls, RAP administration at the 3 h post-training time prevented the learning-associated decrease in APP-KPI expression (Figs 6b and c). The significant increase in APP-KPI expression following RAP administration further demonstrated impaired LRP-dependent endocytosis of APP-KPI. Moreover, RAP had no effect on the post-training increase in LRP protein expression when compared with the naive control (RAP, 141.6 ± 7.3%, p < 0.001; Fig. 7). Moreover, RAP prevented the increased association of LRP and ApoE at the 4 h post-training time.

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Figure 6. The low-density lipoprotein receptor-related protein (LRP) receptor antagonist receptor-associated protein (RAP) induces amnesia and post-training expression of amyloid precursor protein (APP) containing a Kunitz protease inhibitor-like domain (APP-KPI). (a) Amnesia induced by the LRP RAP antagonist, administered at the 3 h post-training time, on task recall at 48 h following training. The trained animals received the glutathione S-transferase (GST)-RAP fusion protein (▪n = 10) and the controls (□n = 13) the GST protein alone. The avoidance latency data are expressed as the median and interquartile range. *Significance of the recall deficit was determined using the Mann–Whitney U-test (p < 0.05). (b) Representative immunoblots of APP695 and APP-KPI expression 1 h following administration of RAP-GST (n = 12) or GST alone (n = 9) as compared with protein expression in naive controls (n = 8). Equal protein loading is illustrated by unchanged actin immunoreactivity. (c) Quantitative densitometric analysis of dentate gyrus APP695 and APP-KPI at 1 h following administration of RAP-GST or GST alone as compared with protein expression naive controls. The quantitative data are expressed as percent of protein expression in the naive animal and are the mean ± SEM. Significant changes in protein expression were determined using a one-way anova followed by Bonferroni post-hoc analysis. *Values significantly different from naive animal (p < 0.05).

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Figure 7. The low-density lipoprotein receptor-related protein (LRP) receptor antagonist receptor-associated protein (RAP) fails to reduce LRP expression but inhibits its association with apolipoprotein E (ApoE). (a) Representative immunoblots of LRP expression 4 h following passive avoidance training (n = 8). (b) LRP immunoreactivity in anti-ApoE immunoprecipitates at 4 h following passive avoidance training (n = 3). Quantitative analysis of the data is shown in (c). Results are expressed as percent of protein expression in the naive animal and are the mean ± SEM. Significant changes in protein expression were determined using a one-way anova followed by Bonferroni post-hoc analysis. *Values significantly different from naive animal (p < 0.05). DG, homogenized whole dentate gyrus; GST, glutathione S-transferase; NC, negative control (primary precipitating antibody omitted).

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Several in vivo studies have demonstrated APP to be necessary for learning and memory but none have related this functional significance to the neuroplastic events that accompany memory consolidation. Our observations of a significant down-regulation of the major APP695 neuronal isoform in the hippocampal dentate gyrus at 2–4 h following avoidance conditioning reveal a role for APP in synaptic remodelling similar to that observed for other synaptic proteins, such as the neural cell adhesion molecule (Foley et al. 2000). The concomitant decrease of CSF sAPP indicates that the learning-associated loss of APP does not arise from increased surface α-secretase activity but through its internalization and degradation within an intracellular compartment. The observed association of APP695 with AP2 and, in turn, the enhanced association of the latter with AP180 during the same 2–4 h post-training period further indicates internalization of APP695 to be dependent on clathrin-coated pit formation. Moreover, the localization of AP180 to the pre-synapse (Yao et al. 2005) and internalization and degradation of APP695 restricted to the dentate gyrus suggests that the most intensive processing occurs in the terminals of the perforant path and in a period just prior to the extensive synaptic remodelling that accompanies acquisition of spatial learning and avoidance conditioning paradigms (O'Malley et al. 1998, 2000; Eyre et al. 2003).

These observations may have a direct relevance to understanding the pathophysiology of AD as β/γ-secretase-derived β-amyloid is formed following internalization of cell surface APP into the endosomal compartment (Haass et al. 1992; Koo and Squazzo 1994; Perez et al. 1999). Also, the learning-related APP turnover was restricted to the dentate gyrus, a region that receives extensive sensory input from the perforant path. Moreover, transection of this pathway in the APPswe/PS1-E9 transgenic mouse results in a significant reduction in age-related plaque formation (Lazarov et al. 2002; Sheng et al. 2002).

The lack of any demonstrable interaction of APP-KPI with the AP2 adapter protein indicated that alternative processing mechanisms exist for APP isoforms located to the glial compartment. Previous studies have indicated LRP to modulate APP-KPI expression in vitro (Knauer et al. 1996; Ulery et al. 2000; Rebeck et al. 2001) and we have now provided evidence that a similar glia-associated mechanism may operate during the early stages of memory consolidation. Clearly, internalization of APP-KPI involved mechanisms distinct from those employed by APP695 as loss of APP-KPI protein expression was more enduring and did not require association with APs. Also, the significant increase in LRP protein expression and its increased association with ApoE during the 2–4 h post-training period suggested its possible involvement in memory-related internalization of APP-KPI. This was confirmed by use of the LRP RAP antagonist which, when administered at the 3 h post-training time, resulted in task amnesia, inhibition of ApoE association with LRP and prevented processing of APP-KPI. The lack of effect of RAP on APP695 internalization suggests that its consolidation-related degradation is not sufficient for memory formation. This is consistent with previous studies in which intraventricular infusion of anti-APP induced amnesia only if administered within 2.5 h following task acquisition (Doyle et al. 1990). This would suggest that signalling by cell surface proteins, such as APP695 and neural cell adhesion molecule 180, may be of greater importance to the process of memory consolidation than their subsequent targeted degradation.

An exception to the above rule may relate to the post-training internalization of APP-KPI/LRP/ApoE complexes by glial cells. Perturbation of this function by the RAP antagonist results in amnesia, possibly mediated by one or more mechanisms that would interfere with the synaptic restructuring that occurs at 6 h post-training (O'Connell et al. 1997; O'Malley et al. 1998; Eyre et al. 2003). By way of example, RAP antagonism blocks temporal post-training modulation of LRP binding to the ApoE cholesterol transport protein. ApoE is required for the regulation of cholesterol content in synaptic plasma membranes (Igbavboa et al. 1997, 2005; Mauch et al. 2001) and its uptake, via lipoprotein receptors, is increased during hippocampal synaptic remodelling in the adult rodent (Poirier et al. 1993; Petit-Turcotte et al. 2005). Disruption of LRP/ApoE-dependent cholesterol trafficking causes deficits in long-term potentiation and thus could, at least in part, underlie the learning deficits observed in these studies (Trommer et al. 2004). Synaptic restructuring in vitro is positively regulated by glial cells and is, in part, dependent on their release of ApoE-conjugated cholesterol (Nagler et al. 2001; Ullian et al. 2001; Goritz et al. 2005). Moreover, simultaneous, learning-dependent APP processing in both the neuronal and glial compartments points to a role in the glial unsheathing of synaptic connections, a process observed during neurohypophyseal plasticity associated with cycles of dehydration or lactation (Nothias et al. 1997; Hoyk et al. 2001). This unsheathing process increases axon–synaptic contact, an event required for synaptic growth and elimination in vitro (Zhu et al. 1995). Co-ordinated down-regulation of APP695 and APP-KPI would influence interactions between neurones and glial cell populations during the early post-training time frame, in preparation for dentate gyrus-specific synapse restructuring. Consistent with these views, the internalization of both neural cell adhesion molecule and APP in the 2–6 h post-training period immediately precedes the learning-dependent synaptic growth at 6–8 h following training (O'Malley et al. 1998, 2000; Eyre et al. 2003).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Financial support from the Health Research Broad (HRB) Ireland and the contribution of Dr Helen Gallagher in preparing the grant application are gratefully acknowledged. KJM and CMR are Science Foundation Ireland Investigators.

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  6. Acknowledgements
  7. References
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