Deficits in synaptic transmission and learning in amyloid precursor protein (APP) transgenic mice require C-terminal cleavage of APP

Abstract

Synaptic dysfunction has been shown to be one of the earliest correlates of disease progression in animal models of Alzheimer's disease. Amyloid-beta protein (Abeta) is thought to play an important role in disease-related synaptic dysfunction, but the mechanism by which Abeta leads to synaptic dysfunction is not understood. Here we describe evidence that cleavage of APP in the C terminus may be necessary for the deficits present in APP transgenic mice. In APP transgenic mice with a mutated cleavage site at amino acid 664, normal synaptic transmission, synaptic plasticity, and learning were maintained despite the presence of elevated levels of APP, Abeta42, and even plaque accumulation. These results indicate that cleavage of APP may play a critical role in the development of synaptic and behavioral dysfunction in APP transgenic mice.

Figure 1.

Transgenic lines, expression of APP transgene, Aβ₄₂ levels, and plaque deposition in transgenic mouse lines. A, Diagram of PDAPP (J9 and J20) and D664A(B21 and B254) transgenes, including familial Alzheimer's mutations (FAD) and location of D664A mutation. TMD, Transmembrane domain. B, Cortical brain extracts from 3- to 6-month-old mice were used for Western blot analysis of relative expression levels of APP protein using the antibody CT-15. NTg, Nontransgenic. C, ELISA detection of Aβ₄₀ levels in individual 3- to 6-month-old mice are shown for all lines of mice examined (weight per 100 mg of wet weight brain tissue). D, ELISA detection of Aβ₄₂ in individual 3- to 6-month-old transgenic mice (averages noted with horizontal lines; weight per 100 mg of wet weight tissue). Note the large increase in average Aβ₄₂ levels in transgenic line D664A(B254) (**p < 0.01). This is particularly apparent between 3- and 6-month-old animals (6 month animals are noted by } symbol). E, Thioflavin-S staining of amyloid deposits in 12-month-old nontransgenic control (top) PDAPP(J20) (middle) and D664A(B21) (bottom) hippocampal sections. Magnification, 4×. Scale bar, 800 μm.

Figure 2.

C-terminal cleavage of PDAPP transgene is necessary for deficits in basal synaptic transmission in APP transgenic mice. Basal synaptic transmission was assayed by measuring extracellular field potentials (fEPSPs) within the stratum radiatum of the CA1 region of the hippocampus evoked by a bipolar electrode placed at the CA3–CA1 border. For each level of stimulation, the maximum amplitude of the presynaptic fiber volley (FV; arrow) and the initial slope of the fEPSP were measured (see also supplemental Fig. 1, available at www.jneurosci.org as supplemental material). A, Basal synaptic transmission is impaired in PDAPP(J20) animals (left). Representative responses from nontransgenic (NTg) control and PDAPP(J20) animals show a large reduction in fEPSP in PDAPP(J20) animals. Note the large input (see inset with expanded timescale) needed to elicit a relatively small EPSP in transgenic PDAPP(J20) mice. Average fEPSP slopes of nontransgenic (filled squares) and PDAPP(J20) (open circles) plotted as a function of the average fiber volley amplitude (right). B, D664A(B21) animals (open circles) show no impairments in basal synaptic transmission compared with littermate controls (filled squares). C, Even D664A(B254) animals, which express the highest levels of the PDAPP transgene, show no gross impairment in fEPSP. D, Average basal synaptic transmission levels of transgenic PDAPP(J20), B21, and B254 lines (open circles) normalized to nontransgenic littermate controls (filled squares) demonstrating prevention of deficits by the D664A mutation in the PDAPP transgene. **p < 0.001.

Figure 3.

Histological detection of plaques in 6-month-old D664A(B254) mice. After physiological recordings, slices from D664A(B254) (n = 5) and nontransgenic (NTg) control (n = 5) animals were fixed, resectioned, and mounted on glass slides for thioflavin-S processing. Higher APP and Aβ₄₂ expression in D664A(B254) transgenic mice resulted in the earlier development of thioflavin-S-sensitive amyloid deposits in the hippocampus. Plaques were most notable in the dentate gyrus (DG, top) and the CA1 subregion (Sub, bottom) of the hippocampus. No staining was observed in nontransgenic control slices. Scale bar, 300 μm.

Figure 4.

D664A mutation prevents LTP deficits observed in PDAPP(J20) animals at the Schaffer collateral to CA1 synapse. After a 20 min baseline, LTP was induced by four tetani delivered 10 s apart, each at 100 Hz for 1 s. fEPSPs were monitored for 60 min after tetanus. Representative traces are superimposed from the last 10 min of the baseline period and 55–60 min after LTP induction. A–C, Average time course of LTP in PDAPP(J20), D664A(B21), and D664A(B254) transgenic mice (open circles) compared with nontransgenic control animals (filled squares). LTP in PDAPP(J20) animals was significantly lower compared with control (A). In contrast to PDAPP(J20) animals, LTP is normal in both D664A(B21) (B) and the higher expressing D664A(B254) lines (C). D, Cumulative probability distribution of LTP measured as the percentage of potentiation 55–60 min after tetanus compared with baseline period. LTP in PDAPP(J20) animals was significantly lower than D664A(B21), D664A(B254), or nontransgenic (NTg) controls. Calibration: 0.25 mV, 10 ms.

Figure 5.

D664A mutation prevents learning deficits observed in PDAPP(J20) animals. Animals were trained for 4 d (2 sessions per day) in a hidden platform Morris water maze. Distance to platform (in centimeters) and latency to platform were measured; however, only distance is shown. A, At 3–4 months, PDAPP(J20) mice appeared to show longer distance-to-platform measurements in the last four sessions, but the data did not reach statistical significance. B, D664A(B21) animals learn normally. C, At 8–12 months, PDAPP(J20) mice show significant learning impairments (**p < 0.0001; n = 19). Probe trials were not significantly different between groups at either age (data not shown). D, At 8–12 months, D664A(B21) mice do not show any learning impairment. NTg, Nontransgenic.

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.