Deficits in Synaptic Transmission and Learning in Amyloid Precursor Protein (APP) Transgenic Mice Require C-Terminal Cleavage of APP

Discussion

The data presented herein describe evidence implicating cleavage of APP at D664 as an important participant in the mechanism of synaptic dysfunction and behavioral disturbance in a model of amyloid-associated pathology in mice. We have shown that, despite the presence of high amounts of Aβ₄₀, Aβ₄₂, and corresponding amyloid deposits, a single amino acid replacement, that prohibits cleavage at D664, leads to a protection from the predicted dysfunction in synaptic transmission and learning behavior. Specifically, D664A(B21) mice express APP and Aβ₄₂ at levels between the PDAPP(J9) and PDAPP(J20) lines and yet are protected from the synaptic dysfunction and learning deficits present in both of those lines. Furthermore, the even higher expressing line, D664A(B254), also shows preservation of synaptic function, even at an age when plaque accumulation has begun. Whether the deficits in the PDAPP(J20) mice are attributable to APP or Aβ overexpression cannot be ascertained from the experiments in this study; however, several groups have demonstrated that Aβ immunotherapy reverses cognitive deficits in APP transgenic mice (Dodart et al., 2002; Kotilinek et al., 2002; Wilcock et al., 2004), suggesting that Aβ likely plays an important role in the deficits observed in APP-overexpressing transgenic mice.

The ability of Aβ to induce dysfunction is well established at the cellular, synaptic, and behavioral level (Walsh and Selkoe, 2004). Although much research has focused on the β-secretase and γ-secretase cleavage of the APP protein and the creation and role of Aβ, several groups have suggested that the C terminus of APP may also play a role in AD (Cao and Sudhof, 2001; Galvan et al., 2002; Kim et al., 2003). Critical to the present study is recent evidence showing that the D664A mutation indeed prevents cleavage at D664 and thus the formation of the C31 fragment in vivo (Galvan et al., 2006), confirming previous in vitro findings (Lu et al., 2003b). This study was also in agreement with our data (Fig. 1) that the D664A mutation does not affect formation or accumulation of Aβ. Finally, the study provided preliminary evidence suggesting that cleavage at D664 may be playing a role in synaptic health, because the loss in synaptophysin staining in PDAPP(J20) transgenic mice was prevented in D664A(B21) mice.

Reduction in synaptophysin puncta, commonly used for measuring synaptic loss, has been reported in human AD patients (Terry et al., 1991; Masliah et al., 2001), as well as in multiple APP transgenic lines (Hsia et al., 1999; Mucke et al., 2000; Spires et al., 2005), although not in all studies (for example, see King and Arendash, 2002). However, in addition to inherent difficulties in using immunofluorescence as a quantitative technique, this method also requires the assumption that every puncta is a mature synapse with a corresponding postsynaptic specialization and functionality. A more rigorous histological study combining both synaptophysin staining and electron microscopy of synapses recently confirmed synaptic loss in the hippocampus of APP transgenic mice compared with age-matched controls (Buttini et al., 2005).

Because of the difficulty in interpreting the functional significance of histological synaptic measurements, however, synaptic function has also been directly measured electrophysiologically in at least five independent APP transgenic mouse lines, including PDAPP (Indiana) (Hsia et al., 1999; Larson et al., 1999; Giacchino et al., 2000; Hartman et al., 2005), PDAPP(J9) (Hsia et al., 1999), Tg2576 (Chapman et al., 1999; Fitzjohn et al., 2001), APP London (Moechars et al., 1999; Dewachter et al., 2002), and the 3xTg-AD line (Oddo et al., 2003). Although the interpretation of these results can be complicated by the use of different physiological techniques, expression levels of transgenes and ages and background of animals, a clear consensus has emerged that synaptic function is disrupted in APP-overexpressing transgenic mice. Moreover, this dysfunction often occurs before histological detection of plaques. Two assays for measuring synaptic function are most commonly used: first, measurement of the magnitude of the fEPSPs after evoked stimulation, which is often referred to as “basal synaptic transmission,” and, second, measurement in activity-dependent changes in synaptic strength or LTP.

Reductions in basal synaptic transmission in PDAPP (Indiana) mice have been shown clearly in vitro (Hsia et al., 1999; Larson et al., 1999) as well as in vivo (Giacchino et al., 2000). Deficits also have been observed in APP London mice (Moechars et al., 1999), as well as Tg2576 (Swedish) APP transgenic mice (Fitzjohn et al., 2001). An analysis of these data show that the level of dysfunction in basal synaptic transmission often increases with the animals' age and concurrently with Aβ levels and amyloid deposits. It is therefore not surprising to see studies reporting smaller changes or no changes in basal synaptic transmission when experimenting on younger animals (Larson et al., 1999; Giacchino et al., 2000). Although there is consensus that basal synaptic transmission is reduced in APP transgenic mice, one study showed no change (Chapman et al., 1999). One possible explanation for this discrepancy is the method used in the measurement and analysis of basal synaptic transmission. As explained above, we feel normalization of fEPSP using the fiber volley amplitude, correcting for alterations in electrode/stimulator location, is critical for accurate interpretation of basal synaptic transmission data.

Deficits in the ability to induce long-term synaptic changes have also been observed in several different APP transgenic lines. LTP deficits in PDAPP (Indiana) mice (Larson et al., 1999; Giacchino et al., 2000; Hartman et al., 2005), APP London (Moechars et al., 1999; Dewachter et al., 2002), Tg2576 (Chapman et al., 1999), and the 3xTg-AD line (Oddo et al., 2003) all have been reported. Exceptions include two studies in which no deficits were uncovered; these include studies of PDAPP (Indiana) mice (Hsia et al., 1999) and Tg2576 mice (Fitzjohn et al., 2001), both lines in which LTP deficits have been noted by other groups. Discrepancies in the field regarding LTP in APP transgenic mice may be attributable to differences in LTP induction protocols or the age of the mice when the experiments were conducted (as with basal synaptic transmission, LTP deficits often increase with age). Additionally, because basal synaptic transmission is affected in APP transgenic mice (in most of these cases), it is unclear how reduced synaptic drive might influence LTP or whether the experimenter can compensate for this fact via normalization of baseline drive.

PDAPP(J9) mice were reported previously to have deficits in basal synaptic transmission, even at younger (2–4 months) ages (Hsia et al., 1999). However, LTP was not investigated in the J9 line [only PDAPP (Indiana) mice were studied]. PDAPP(J20) mice, which were examined in this manuscript, were manufactured by the introduction of the same transgene as the J9 line; however, APP and Aβ expression levels are much higher (presumably attributable to increased copy number). Therefore, it was not surprising that these animals showed deficits in basal synaptic transmission (Fig. 2). Moreover, we also demonstrated a significant reduction in LTP in these mice, a novel finding (Fig. 4).

Many APP transgenic mouse lines have been shown to have behavioral learning deficits, including both the PDAPP(J9) and the PDAPP(J20) lines (Raber et al., 2000; Palop et al., 2003). In general agreement with a recent study, despite a different protocol and analysis (Galvan et al., 2006), we found that PDAPP(J20) mice showed learning deficits, whereas D664A(B21) mice learned a Morris Water Maze task normally, despite their elevated APP and Aβ levels. Memory retention, in the form of a probe trial, did not reach significance between groups. In examining the data, it appeared that the PDAPP(J20) mice performed at “normal” levels, perhaps reflecting the short interval between the last training session and the probe trial session. Nevertheless, our data reveal an important deficit in learning, which is prevented in the D664A(B21) line.

Considering the strong evidence for disruption of synaptic function in APP- and Aβ-overexpressing mice, it is important to note that a single amino acid substitution, D664A, which prohibits cleavage at D664, was able to normalize phenotypes that have been shown to be and would be predicted to be abnormal (Figs. 2, 4, 5). In addition, to avoid issues of transgene expression level, we chose to characterize two separate lines of D664A mice: the D664A(B21), which had similar levels of APP expression as the PDAPP(J20) mice with which they were compared, and a second D664A(B254) line, which expressed much higher levels of the APP transgene, presumably making prevention of any AD phenotypes much more difficult.

On the surface, the present data might appear to challenge the role of Aβ in the synaptic toxicity present in AD mouse models. On the contrary, our data strengthen a previously proposed hypothesis implicating both Aβ and APP in a mechanistic pathway underlying cellular dysfunction (Lu et al., 2003). First, it has been shown that loss of APP attenuates Aβ toxicity (Lorenzo et al., 2000). Second, we have previously proposed a model (Fig. 6) based on cell culture experiments that Aβ can initiate a series of events by binding to and causing the APP transmembrane protein to form a complex, an event that may be necessary for one component of Aβ-induced cell death (Lorenzo et al., 2000; Lu et al., 2003; Shaked et al., 2006). In these studies, the dimerization of APP has been shown to lead to the recruitment of caspase 8 and the cleavage of APP at D664 (Lu et al., 2003). Furthermore, the dimerization of APP appears to be a critical step, given that artificial crosslinking of APP results in cytotoxicity even in the absence of soluble Aβ. Dimerization alone, however, is not sufficient for cytotoxicity because the D664A mutation (abrogating cleavage at D664 and C31 formation) attenuates cell death caused by either Aβ or artificial dimerization of APP. The inference from all of these studies is that the D664A mutation leads to a loss of caspase cleavage at that position, reducing generation of C31 and, in so doing, lowering cell death. However, the precise mechanism by which the D664A mutation attenuates cytotoxicity in our studies remains to be defined.

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Figure 6.

Schematic of proposed role of Aβ and D664 cleavage in the development of cellular dysfunction. Aβ may function, in part, by binding to APP and causing APP to dimerize, an event that leads to the recruitment of caspases and cleavage of APP at D664. This model demonstrates a possible mechanistic pathway leading from Aβ to caspase cleavage at D664 to synaptic dysfunction.

While in a cell culture environment, Aβ treatment leads to D664 cleavage and cell death, our data suggest that, in vivo, this cleavage event leads to an initial loss or toxicity to functional synaptic connections. This dysfunction, as opposed to losses in overall cell numbers, is consistent with previous observations in AD patients and APP transgenic mice that synaptotoxicity occurs well before other neuropathological events (Hsia et al., 1999; Giacchino et al., 2000). Our data suggest that preventing cleavage of the PDAPP transgene at position D664 renders the APP protein and its cleavage products ineffective at disrupting synaptic transmission. We propose, therefore, that Aβ may serve as an upstream factor, causing the dimerization of APP, which leads to cleavage of APP at the D664 site. The fact that D664 cleavage is known to occur in AD patients as well as in APP transgenic mice (Zhao et al., 2003; Galvan et al., 2006) lends additional support to the hypothesis that this cleavage may be an important downstream event in Aβ-induced synaptic dysfunction.

With cleavage of APP at D664 now well established in vitro and in vivo, one logical hurdle remains to be elucidated: if prevention of cleavage at D664 is protective, one has to assume that cleavage at D664 (and creation of the C31 fragment) has a functional role in synaptic and/or cellular dysfunction. A number of groups have speculated on potential mechanistic roles for C31 and other APP C-terminal cleavage products (Cao and Sudhof, 2001; Galvan et al., 2002; Kim et al., 2003; Shaked et al., 2006), with much focus on the potential for a role as a transcription factor. Another possibility is that the new domain in the remaining N-terminal fragment of APP after D664 cleavage may have a toxic function, although this is unlikely because APP deleted of the C-terminal 31 amino acids has no effect in cell culture (Lu et al., 2003).

Although our current data fit into this model pathway, the present experiments focused on the long-term effects of APP and Aβ overexpression. Other pathways also are likely involved in Aβ-induced toxicity. For example, acute effects of Aβ, which were not examined in the present study, have been shown to be dependent on excitatory postsynaptic receptor activity (Kamenetz et al., 2003). Even with this in mind, if prevention of D664 cleavage is indeed protective for synaptic function in the presence of Aβ, then uncovering the mechanism by which this cleavage contributes to dysfunction has implications both for understanding synaptic dysfunction as well as developing drugs to treat Alzheimer's disease.