Effects of secreted oligomers of amyloid beta-protein on hippocampal synaptic plasticity: a potent role for trimers

Abstract

The accumulation of amyloid beta-protein (Abeta) in brain regions serving memory and cognition is a central pathogenic feature of Alzheimer's disease (AD). We have shown that small soluble oligomers of human Abeta that are naturally secreted by cultured cells inhibit hippocampal long-term potentiation (LTP) in vitro and in vivo and transiently impair the recall of a complex learned behaviour in rats. These results support the hypothesis that diffusible oligomers of Abeta initiate a synaptic dysfunction that may be an early event in AD. We now report detailed electrophysiological analyses that define conditions under which acute application of soluble Abeta inhibits hippocampal synaptic plasticity in wild-type mice. To ascertain which Abeta assemblies contribute to the impairment of LTP, we fractionated oligomers by size-exclusion chromatography and found that Abeta trimers fully inhibit LTP, whereas dimers and tetramers have an intermediate potency. Natural Abeta oligomers are sensitive to heat denaturation, primarily inhibit the induction phase of LTP, and cause a sustained impairment of LTP even after extensive washout. We observed no effects of Abeta oligomers on presynaptic vesicle release. LTP in juvenile mice is resistant to the effects of Abeta oligomers, as is brain-derived-neurotrophic-factor-induced LTP in adult hippocampus. We conclude that specific assemblies, particularly timers, of naturally secreted Abeta oligomers are potent and selective inhibitors of certain forms of hippocampal LTP.

Figure 1. Amyloid β-protein trimers potently inhibit long-term potentiation in the CA1 region of mouse hippocampal slices

A, secreted human amyloid β-protein (Aβ) oligomers from 7PA2 (APP₇₅₁V717F) conditioned medium (CM) were size-separated by fast protein liquid chromatography (FPLC). Column fractions were lyophilized and examined by tricine SDS-PAGE. Blotting with the Aβ₄₀-specific antibody 2G3 revealed the fractionation of a ladder of oligomers from tetramers down to monomers. B, each lyophilized fraction was resuspended in artificial cerebrospinal fluid (ACSF) and perfused over mouse hippocampal slices for 20 min before four high-frequency stimulations (HFS; 100 Hz, 1 s) were given. Field potential recordings were made in the CA1 region. Size-exclusion chromatography (SEC) fractions 50–55, enriched for Aβ trimers, strongly inhibited long-term potentiation (LTP) at 60 min post-HFS (118 ± 8.8% s.e.m., whereas monomer fractions had no effect (202 ± 19.1% Student's t test, P < 0.01, n = 12 and 13, respectively). Fractions 60–64, which were enriched for dimer, showed an intermediate effect that was significantly different from monomer but not trimer (149 ± 6.7%). C, oligomeric assemblies do not change in size after perfusion over hippocampal slices, indicating high stability. Lyophilized SEC fractions were run directly on a Western blot (left panel) or else used for electrophysiology followed by immunoprecipitation (with R1282) of Aβ species from the homogenized slices. Blots were probed with 6E10. Note that 6E10 showed better detection of the Aβ monomer relative to oligomers than did 2G3. D, summary histogram depicting the potentiation of the EPSP slope 60 min post-HFS for many of the SEC fractions (n = 3 independent SEC fractionation runs). The mean potentiation obtained using CHO-control CM (lacking human Aβ) is shown as a blue bar just above 200%. The black horizontal bands above the histogram depict the relative abundance of each oligomeric band across the fractions. Arrows point to the regions of the histogram representing the greatest LTP inhibition (fractions 18–23 and 50–58). Immunodepletion of the inhibitory fractions with Aβ antibody R1282 restored normal LTP (far right bars). Black vertical histogram bars indicate fractions of primary interest in this study, while the grey bars show results from intervening fractions.

Figure 2. Timing and temperature influence the effect of 7PA2 CM on LTP

A, 7PA2 CM did not significantly affect LTP (measured 60 min post-HFS) when it was applied immediately after the HFS (180 ± 9.4% Student's t test, P > 0.1, n = 7). B, boiling 7PA2 CM eliminated its inhibitory effect on LTP (182 ± 10.5% P > 0.1, n = 8). C, a 2 h washout of slices pretreated with 7PA2 CM did not prevent the inhibitory effect of the CM on LTP (124 ± 9.4% P < 0.01, n = 7) and it remained similar to the effect of acute application of 7PA2 CM, which we have previously reported as 131 ± 8.8%. The inset shows that a considerable amount of Aβ, including oligomers, was retained in the slice even after a 2 h washout period. D, CM from 7WD4 cells (expressing wild-type human APP751) contained Aβ oligomers (inset, compared to control CHO- CM; probed with 6E10 + 2G3) and inhibited LTP when adjusted to have a similar Aβ concentration as 7PA2 CM (132 ± 14.6% P < 0.05, n = 5 for 2× concentrated).

Figure 3. Comparison of 7PA2 CM, 7WD4 CM and synthetic Aβ shows differences in quality and quantity

A, a titration curve of synthetic Aβ demonstrated the high sensitivity of the immunoprecipitation/Western blot assay. Apparent dimers of synthetic Aβ_1–40 were detected at higher concentrations, although they migrated distinctively from the cell-derived Aβ oligomers on Tricine gels. Similar types of Aβ species (monomers, dimers and trimers) were found in 7PA2 and 7WD4 CM, but in different ratios. B, quantifying these ratios demonstrated that compared to 7WD4 CM, 7PA2 cells expressed a higher ratio of trimers relative to monomers (Student's t test, P < 0.05, n = 16 for 7PA2, n = 7 for 7WD4). C, 7PA2 cells also stably expressed β-amyloid precursor protein (APP) at higher levels relative to 7WD4 cells. D, a titration standard of synthetic Aβ was used to estimate the amount of Aβ momomer in SEC fractions (as in Fig. 1). Fractions 89 and 92 (not shown) contained approximately 750 fmol (∼3 ng) of Aβ monomer.

Figure 4. Effects of 7PA2 CM on LTP are not attributable to alterations in post-tetanic potentiation, paired-pulse facilitation or HFS

A, slices were pretreated with CHO- CM or 7PA2 CM for 20 min, and the NMDA receptor antagonist d,l-2-amino-5-phosphonovaleric acid (AP5) for 10 min. A single HFS (100 Hz, 1 s) was delivered (arrow) to facilitate presynaptic release. The post-tetanic potentiation was indistinguishable for slices treated with either CHO- CM or 7PA2 CM (n = 7 CHO- CM; n = 6 7PA2). B, paired-pulse ratios were measured in slices treated with CHO- CM or 7PA2 CM by delivering two pulses, 50 ms apart. A ratio of the amplitude of second pulse to first pulse was determined (left bars). There was no significant difference between the two treatments (1.57 ± 0.08 for CHO- CM and 1.56 ± 0.15 for 7PA2 CM; Student's t test, P > 0.1, n = 4). A paired-pulse ratio was also measured 1 min after a single HFS (right bars), and no significant difference was observed (1.04 ± 0.03 for CHO- CM and 1.18 ± 0.10 for 7PA2 CM; P > 0.1, n = 4). C, EPSP responses to the first of four HFS (100 Hz, 1 s) were aligned from slices treated with CHO- CM or 7PA2 CM and overlayed. There was no difference in the average peak amplitudes, number of pulses to peak amplitude, or decay kinetics between the two treatments (n = 23 for CHO- CM and for 7PA2 CM). D, an input/output ratio between the amplitude of the fibre volley (presynaptic) and the slope of the EPSP (postsynaptic) over a range of stimulus intensities was performed. A small but significant shift in the slope of the regression curve was detected (P < 0.05). Dotted lines indicate the respective 95% confidence intervals. E, average traces from short-term potentiation (STP) experiments demonstrate a divergence in EPSP slope between slices treated with CHO- CM control and 7PA2 CM. Slices were perfused with CM for 20 min prior to delivering one HFS (100 Hz, 1 s) (arrow). F, histogram depicting the effect of CHO (diamonds) and 7PA2 (circles) CM on STP. Measured as the percentage change in EPSP slope for the given time intervals, we observed that by 5 min, there was a significant difference between CHO- CM- and 7PA2-CM-treated slices (151 ± 8.5% CHO- CM and 121 ± 10.4% 7PA2 CM; t test, P < 0.05, n = 9 and 7, respectively). The difference between CHO- CM and 7PA2 CM persisted for 10 min (144 ± 6.6% CHO- CM and 122 ± 4.9% 7PA2 CM) and 30 min (136 ± 6.1% CHO- CM and 108 ± 5.6% 7PA2 CM).

Figure 5. 7PA2 CM inhibits chemical LTP in older but not younger mice

A, hippocampal slices from postnatal day (P)16–28 mice were treated with CHO- CM or 7PA2 CM for 20 min. The usual ACSF in the perfusate was replaced with ACSF containing 0 mm Mg²⁺. After 5 min, picrotoxin and glycine were added to the perfusion for an additional 15 min to increase spontaneous synaptic activity. This yielded a large increase in the EPSP, which subsided upon restoration with normal ACSF. Slices treated with CHO- CM showed a persistent enhancement of the EPSP at 60 min after wash-in of normal ACSF, whereas 7PA2 CM caused a significant inhibition of this effect (166 ± 8.2% CHO- CM and 112 ± 8.3% 7PA2 CM; Student's t test, P < 0.01, n = 5 for CHO- CM and n = 8 for 7PA2 CM). B, mice of ages P8–9 also responded to the chem-LTP protocol with a persistent enhancement of the EPSP. However, there was no significant difference between CHO- CM- and 7PA2-CM-treated slices at 60 min after restoring normal ACSF (175 ± 10.9% CHO- CM and 155 ± 22.8% 7PA2 CM; P > 0.1, n = 7 for CHO- CM and n = 7 for 7PA2 CM). C, the enhancement of synaptic function was a form of LTP, as it was blocked by the NMDA receptor antagonist AP5 (P < 0.01, n = 5). Also, the effect could not be attributed to residual picrotoxin or glycine in the slice, since chem-LTP could not be induced without perfusing the slice with 0 mm Mg²⁺ (P < 0.01, n = 4). D, as with the slices from older mice, the LTP was blocked by AP5 (P < 0.05, n = 5), and picrotoxin/glycine treatment alone did not augment EPSPs (P < 0.01, n = 5).

Figure 6. BDNF-induced LTP is resistant to the effects of 7PA2 CM

Slices were pretreated with CHO- CM or 7PA2 CM for 20 min. Brain-derived neurotrophic factor (BDNF; 50 ng ml⁻¹) (see Methods) was added to the perfusate, and EPSPs were followed for 30 min. Four HFS (100 Hz, 1 s) were then delivered, and the potentiation followed for an additional 30 min. The average slopes of the EPSPs at 30 min post-BDNF were not significantly different between CHO- CM- and 7PA2-CM-treated samples (147 ± 8.1% CHO- CM and 138 ± 3.4% 7PA2 CM; Student's t test, P > 0.1, n = 9 for CHO- CM and n = 8 for 7PA2 CM). However, 7PA2 CM caused significant inhibition after HFS (compared to CHO- CM) at 30 min post-HFS (P < 0.05).