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Neuroscience |
1 Department of Neurology, Harvard Medical School and Center for Neurologic Diseases, Brigham and Women's Hospital, Boston, MA 02115, USA
2 Laboratory for Neurodegenerative Research, Conway Institute for Biomedical and Biomolecular Research, University College Dublin, Dublin 4, Ireland
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Abstract |
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(Received 16 December 2005;
accepted after revision 7 February 2006;
first published online 9 February 2006)
Corresponding author D. J. Selkoe: Center for Neurologic Diseases, Harvard Institutes of Medicine, 77 Ave Louis Pasteur, Room 730, Boston, MA 02115-5716, USA. Email: dselkoe{at}rics.bwh.harvard.edu
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Introduction |
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Transgenic mice have provided important insights into the chronology of events leading to the neuritic plaques, gliosis and neurofibrillary tangles that characterize AD. For instance, triple transgenic mice (PS1M146V, APPSWE, tauP301L) develop Aß pathology prior to tau alterations, despite using the same promoter to drive expression of mutant human APP and tau (Oddo et al. 2003). Those same authors also found that the appearance of Aß aggregation is correlated with impairments in long-term potentiation (LTP). Moreover, immunizing the triple transgenic mice with anti-Aß antibodies reduces Aß accumulation and slows the emergence of tau-containing tangles, suggesting that the build-up of Aß precedes tau aggregation (Oddo et al. 2004; Billings et al. 2005). It has also been shown that Aß accelerates tangle-like cytopathology in tau transgenic mice (Gotz et al. 2001; Lewis et al. 2001). In humans, familial forms of AD caused by missense mutations in APP or presenilin (the active site component of 
-secretase (Wolfe et al. 1999) lead to severe tau cytopathology (Sudo et al. 2005). Thus, excessive accumulation of Aß may be one of the earliest pathogenic events in AD.
We have chosen to study natural oligomers of human Aß that are secreted by cultured cells expressing APPV717F, a mutant form of APP known to cause an aggressive form of familial AD. The Aß assemblies secreted by this ‘7PA2’ CHO cell line have been extensively characterized biochemically (Podlisny et al. 1995, 1998; Walsh et al. 2000). Cell-derived Aß is distinct from the widely used synthetic Aß preparations in at least four ways that make it attractive for understanding the neurophysiological properties of Aß. First, the Aß produced by the 7PA2 cells is naturally generated from human APP and has heterogeneous N- and C-termini similar to those that occur in brain, in contrast to synthetic Aß peptides of a single defined length. Second, it has biological effects at low nanomolar to high picomolar concentrations, similar to those in human brain and cerebrospinal fluid (Motter et al. 1995; Mehta et al. 2000; Walsh et al. 2002), whereas synthetic Aß typically needs to be applied to neurons at 2–4 orders of magnitude higher concentrations to achieve similar biological effects. Third, the 7PA2 cells naturally generate stable and soluble oligomers of Aß, in addition to abundant monomers (Podlisny et al. 1995). We have previously reported that these low-n oligomers inhibit synaptic function, suggesting that cell-derived Aß oligomers are in a biologically active conformation that may resemble the physical state of some Aß species in the hippocampus of AD patients (Walsh et al. 2000; Kokubo et al. 2005). Fourth, the cell-derived oligomers interrupt LTP rapidly, robustly and consistently (Walsh et al. 2002, 2005; Klyubin et al. 2005), indicating that the electrophysiological action of Aß can be readily assayed before significant compensatory effects, inflammatory reaction, neuritic degeneration or apoptosis have occurred. Importantly, the same Aß oligomers microinjected intraventricularly into healthy behaving rats impair their ability to recall a complex learned behaviour (Cleary et al. 2005).
Here, we report a detailed electrophysiological characterization of the effects of secreted human Aß on hippocampal synaptic plasticity in wild-type Swiss Webster mice. With improved separation of Aß oligomers by size-exclusion chromatography (SEC), we show that a trimer species is a particularly potent inhibitor of LTP. Aß oligomers have the most pronounced effect on induction rather than expression of LTP, yet have little effect on presynaptic release. Moreover, to the best of our knowledge, we show for the first time that not all forms of LTP are affected by Aß oligomers. Thus, the effects of natural Aß oligomers are selective for certain forms of synaptic plasticity.
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Methods |
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Aß was collected and prepared from 7PA2-cell conditioned (CM) as previously described (Walsh et al. 2005). 7PA2 cells are a CHO line that stably expresses human APP751 containing the V717F mutation. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, penicillin/streptomycin, L-glutamine, and G418 for selection. Once the cells reached approximately 95% confluency, they were washed and cultured overnight (
15 h) in serum-free medium. CM was collected, spun at 1000 g to remove dead cells and debris, supplemented with a protease inhibitor cocktail (Sigma P1860 at 1:1000) and stored at –80°C. When 300 ml of medium had been collected, it was centrifuged (3000 g) at 4°C in YM-3 Centricon tubes to concentrate proteins larger than 3 kDa. This procedure concentrated the medium 15-fold, with some loss (30–50%) of the 4 kDa monomer species through the Centricon filter. The concentrated CM was pooled and aliquoted to produce a large number of identical medium samples for experiments. These aliquots were stored at –80°C until use. For experiments with 2x concentrated CM from 7WD4 CHO cells (stably expressing wild-type human APP751), the medium was spun to 30-fold concentration in YM-3 Centricon tubes, and then diluted 1:15 in artificial cerebrospinal fluid (ACSF).
Immunoprecipitation
Immunoprecipitation of Aß from 8 ml of CM prepared as above was performed as described (Walsh et al. 2005). A cocktail of protease inhibitors (mg ml–1: leupeptin 1, pepstatin 1, aprotinin 0.1, EDTA 40, and 1,10-phenanthroline 0.4) was added. Samples were precleared with protein A sepharose for 30 min. The CM was then immunoprecipitated overnight with a 1:75 dilution of our polyclonal antibody R1282. The beads were washed with STEN buffers (mM: NaCl 150, Tris 50, EDTA 2, NP-40 0.2%, pH 7.4). Our standard wash protocol is 20 min 0.5 STEN, 20 min STEN + 0.1% SDS, 20 min STEN. The samples were then resuspended in 2x Tricine sample buffer, boiled, and the supernatant was frozen at –80°C or loaded directly onto Tricine SDS-PAGE gels.
Size-exclusion chromatography
To physically separate natural Aß oligomers, 7PA2 CM was run on two Superdex 75 prep grade 20 x 500 mm columns (100 ml volume) arranged in series. Five millilitres of 15x concentrated CM was injected onto the columns and eluted with 50 mM ammonium acetate pH 8.5. An Amersham AKTA fast protein liquid chromatograph (FPLC) (Amersham Biosciences, Piscataway, NJ, USA) was used to collect 1 ml fractions, which were stored at –20°C until lyophilized. Blots of CHO-control CM (not shown) were similar to our previously reported results (Walsh et al. 2005). Individual SEC column fractions were resuspended in 15 ml of ACSF for electrophysiological experiments. Alternatively, the fractions were resuspended in 1x Tricine sample buffer, and half-fractions run on SDS-PAGE. The SEC columns were cleaned with H2O, 44% formic acid, 1 M NaOH and 1 M Tris-base.
Western blots
Samples were electrophoresed on 10–20% Tricine gels (Invitrogen or Bio-Rad), and the proteins transferred to 0.2 µm Optitran nitrocellulose. The membranes were boiled in water or phosphate-buffered saline (to enhance the exposure of Aß epitopes) and blocked for 1 h in 50% Odyssey blocking buffer diluted in PBS. Blots were probed with the monoclonal antibody 2G3 (Elan), which is specific for Aß peptides ending at residue 40, or with 6E10 (Signet), which recognizes amino acids 4–8 near the N-terminus of Aß. Immunoreactive bands were detected and quantified using a Licor Odyssey imaging system.
Electrophysiology
Field potential recordings were made from coronal sections of postnatal day 16–28 male and female Swiss Webster mice. Mice were deeply anaesthetized with isoflurane before decapitation, in compliance with Harvard University's Animal Resources and Comparative Medicine policies for use of laboratory animals. The brain was rapidly removed from the skull and submerged in oxygenated (95% O2, 5% CO2) 4°C ACSF (mM: sucrose 206, KCl 2.8, CaCl2 1, MgCl2 1 MgSO4 2, NaH2PO4 1.25, NaHCO3 26, D-glucose 10, sodium ascorbate 0.4, pH 7.4; osmolarity 297) (Moyer & Brown, 1998). After 2 min, the cerebellum was removed and the remaining brain was bisected at the midline. The two hemispheres were glued to the sectioning chamber and re-immersed in 4°C ACSF. Coronal sections (350 µm) were prepared on a Vibratome 1000 Plus using stainless steel razor blades (Electron Microscopy Science). The sections were placed in oxygenated ACSF at 27°C (mM: NaCl 124, KCl 2.8, CaCl2 3.6, MgSO4 2, NaH2PO4 1.25, NaHCO3 26, D-glucose 10, sodium ascorbate 0.4, pH 7.4; osmolarity 306) in a custom slice recovery chamber designed to provide a circulating perfusion of aerated ACSF. The slices were allowed to recover for at least 75 min. The order of treatments was randomized for slices prepared from a given animal.
Glass electrodes (G8510T-3, Warner Instruments) were pulled on a P-97 Sutter Instruments pipette puller to resistances of 3–6 M
. Electrodes were filled with ACSF for field recordings. Electrical stimulation to the Schaeffer collaterals of the hippocampus was delivered through a World Precision Instruments bipolar (TM33A05) or unipolar (TM33CCNON) electrode. Field potential recordings were made at room temperature (27°C) using an Axon Instruments 200B amplifier and digitized with a Digidata 1322 A. Electrodes were specifically placed just below the surface of the slice to maximize the exposure to circulating Aß. (It has not yet been determined how quickly or deeply the Aß can penetrate the slice). Data were stored and analysed on an IBM PC running pClamp 9.1 software. Recordings were sampled at 10 kHz and low-pass filtered at 5 kHz. The slope of the EPSP was estimated from 10 to 60% of the evoked response. The intensity of the stimulus was set to 20–30% of the maximum evoked EPSP or until a population spike was elicited. A typical stimulation intensity was approximately 7.5 µA. Slices were perfused for 20 min in ACSF to establish a steady baseline. During this interval, 1 ml of 15x concentrated CM (with Sigma protease inhibitors) was thawed to 37°C and diluted to 1x in 15 ml ACSF. This 1x CM/ACSF solution was recirculated over the slice using a P720 Instech peristaltic pump at 2.5–3 ml min–1 while being continuously aerated with 95% oxygen. LTP was induced 20 min later by delivering four 100 Hz stimuli every 5 min. The slope of the EPSP was monitored for 1 h after the last high-frequency stimulation.
For chemical-LTP experiments, brain slices were bathed in ACSF containing 0 mM Mg2+, 10 µM picrotoxin, 200 µM glycine (Lu et al. 2001). LTP was prevented by 100 µMD,L-2-amino-5-phosphonovaleric acid (AP5).
To demonstrate retention of Aß on brain slices, three brain sections were continuously perfused for 2 h with recirculating 7PA2 CM or its FPLC fractions. The brain sections were homogenized in 1 ml of Tris-EDTA buffer (50 mM Tris, pH 7.4, 5 mM EDTA and protease inhibitor cocktail as described above) containing 1% Triton X-100. Samples were sonicated for 5 s, and the volume adjusted to 10 ml in Tris-EDTA buffer (without Triton). Aß was immunoprecipitated overnight with the polyclonal antibody R1282. Immunoprecipitation of the perfusate showed no detectable Aß remaining in the solution (data not shown), indicating near complete transfer to the tissue and tubing. Recovery of Aß from slices was considerably less than that found in the starting 7PA2 CM, suggesting some loss to the perfusion tubing or lower efficiency of immunoprecipitation in the presence of brain homogenate.
Chemicals
Reagents for electrophysiological solutions were from Sigma. AP5 was from Tocris, and brain derived neurotrophic factor (BDNF) was acquired from Peprotech and Cell Sciences. BDNF was used on the day of dilution, since we found that the bioactivity of BDNF fell rapidly after solubilizing (Kang & Schuman, 1995).
Statistics
Typical comparisons were done using Student's t test. For comparing the slope of the t values for regression lines, t values were calculated as t= (C1–C2)/
[(S.D. 1 xS.D. 2/n1) + (S.D. 2 xS.D. 2/n2)]. P values were calculated from the calculated t value and the degrees of freedom. Error bars indicate the S.E.M.
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Results |
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The cell-derived human Aß used throughout these experiments was obtained from the CM of a CHO cell line that stably expresses human APP751V717F. These cells (7PA2) secrete biochemically well-characterized monomeric and oligomeric Aß species whose identities have been confirmed by both radiosequencing and selective immunoprecipitation with numerous N- and C-terminal-specific Aß antibodies (Podlisny et al. 1995; Walsh et al. 2000). We recently reported a method for the fractionation of the 7PA2 CM that employs SEC to separate oligomers from monomers (Walsh et al. 2005). Only fractions containing the oligomers have been shown to impair LTP in vitro and in vivo. To achieve better separation of the oligomeric species, we used two Sephadex 75 SEC columns run in series. The resulting fractions were lyophilized and analysed by Western blotting. The use of the tandem SEC columns allowed for separation of six principal Aß-immunoreactive species that could be detected by monoclonal antibodies specific for amino acids 4–8 (6E10) and the C-terminus (2G3) (Fig. 1A). The fractionation produced a clear laddering by size, such that early eluting fractions contained larger oligomers and later eluting fractions contained mostly monomer, with the exception of a range of Aß species observed in a very early eluting fraction (no. 21). The appearance of a tetrameric species as well as several low-molecular-weight Aß bands in this early eluting fraction may indicate a larger but labile multimer that depolymerizes during SDS-PAGE. The improved separation of different-sized Aß oligomers achieved with this modified SEC method enabled us to assess for the first time the effects of individual oligomeric assemblies on synaptic plasticity.
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Our prior studies have shown that cell-derived Aß oligomers are highly stable and resistant to SDS, sample boiling, urea and formic acid treatment (Walsh et al. 2002). However, it is possible that Aß oligomers become altered after they have been perfused over brain tissue. To address this, Aß was immunoprecipitated with polyclonal antibody R1282 from homogenized slices that had been treated with the SEC fractions for 2 h. Compared with adjacent starting fractions that had never been applied to slices, there was no significant change in the size pattern of the oligomeric species after being in contact with the hippocampal slices for 2 h (Fig. 1C). These results demonstrate that the cell-secreted Aß oligomers are highly stable and can be recovered intact after incubation with brain tissue.
Trimers of Aß inhibit LTP more potently than other low-n oligomers
Based on these results, we tested which oligomeric species were most potent at inhibiting hippocampal LTP. Three independent SEC runs were carried out, and the resulting fractions were all individually tested in LTP experiments. The three replicate results for a given fraction number (always measured at 60 min post-HFS) were averaged and are depicted in the histogram in Fig. 1D. The average potentiation achieved in the presence of the parental CHO-control CM (no human APP expression) is shown as a horizontal line just above 200%, while 100% represents no change from the pre-HFS baseline EPSP slope. Gaps in the histogram reflect SEC fractions that were used for other purposes (e.g. Western blotting or immunodepletion (below); small regions of the gel pattern that were of minimal interest or showed overlapping oligomers; or experiments that failed for technical reasons, such as bubbles in the perfusion which perturbed the slice). Although there is some variability that is intrinsic to the technique, the results clearly demonstrate that fractions 50–58 (which correspond principally to a 11–12 kDa trimeric band) are extremely potent inhibitors of LTP (r2= 0.516 anticorrelated with LTP) (Fig. 1B and D), despite being a fainter band by Western blot than are the tetramers (Fig. 1A, fractions 36–41) or the dimers (Fig. 1A, fractions 61–66). While both the 5 kDa Aß species (which we have previously identified by mass spectroscopy as an SDS-stable conformer of the 4 kDa monomer; Walsh et al. 2000) and the 4 kDa monomer itself showed little effect (r2= 0.076 and 0.257, respectively), the fractions enriched principally in tetramers or dimers demonstrated an intermediate inhibition of LTP (Fig. 1B) (t test compared with monomer, P < 0.05). Fractions immediately surrounding no. 21 that contain several Aß species (Fig. 1A), also caused pronounced inhibition of LTP (Fig. 1D). To confirm that the LTP inhibition was specifically due to Aß, some of the inhibitory SEC fractions were immunodepleted of Aß with the polyclonal antibody R1282 and then tested for effects on LTP. Immunodepletion of Aß from these fractions fully restored LTP (Fig. 1D, far right panel). Therefore, the inhibition of LTP by these SEC fractions is attributable specifically to Aß rather than another coeluting protein. Using polydextran standards, we have previously shown that the trimers elute from the column at a molecular weight of 12 kDa (Walsh et al. 2005), providing further evidence that it is the trimers per se, not a larger Aß assembly, that is responsible for the observed inhibition of LTP. We conclude that soluble trimeric assemblies of human Aß are of particular pathogenic interest because of their potent, complete inhibition of hippocampal LTP. Nevertheless, these data also show that all oligomer-containing fractions tested cause some impairment of LTP.
Cell-derived Aß oligomers prevent induction but not expression of LTP and are sensitive to heat denaturation
The electrophysiological mechanisms by which soluble Aß oligomers impair LTP are not well understood. Because multiple Aß oligomer species inhibited LTP, we proceeded to use whole (unfractionated) CM to characterize the detailed effects of oligomers on synaptic function. We began by asking whether the oligomer-rich 7PA2 CM interferes principally with the induction and/or the expression of LTP. 7PA2 CM was applied to hippocampal slices immediately after the HFS used to induce LTP (Fig. 2A). 7PA2 CM was invariably ineffective at inhibiting LTP (measured at 60 min) when applied after the HFS. These data indicate that soluble, low-n Aß oligomers primarily interfere with the induction of LTP, but not its expression, once the signal transduction cascades which mediate LTP have commenced.
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We next sought to establish whether the effects of 7PA2 CM on LTP could be reversed by extensive washout of the slices. Hippocampal slices were treated with 7PA2 CM for 20 min and then washed by returning the slices to the slice recovery chamber for 2 h. Thereafter, HFS stimulation was performed as previously described. The prolonged washing failed to prevent the inhibition of LTP caused by the 7PA2 CM (Fig. 2C). After this LTP assay was completed, the slices were homogenized and subjected to immunoprecipitation/Western blot to determine how much of the Aß remained in the slice after the washout procedure. As shown in Fig. 2C (inset), some Aß (oligomers and monomer) was recovered from the slice even after a 2 h washout period, although the amount retained appeared to be less than in a slice that was homogenized immediately after perfusion with 7PA2 CM. Thus, a 2 h washout period did not reverse the inhibition of LTP, nor did it efficiently clear the tissue of Aß species.
Although the 7PA2 cells have been a consistent and reliable source of bioactive Aß oligomers, we wished to perform LTP experiments with CM from a distinct cell line to rule out the unlikely possibility that the LTP inhibition is cell-line specific. 7WD4 cells stably overexpress wild-type human APP751 (Xia et al. 1997) and produce soluble Aß oligomers similar to those of the 7PA2 line (Fig. 2D, inset). When prepared identically to 7PA2 CM (i.e. at 1x concentration), the 7WD4 CM did not significantly inhibit LTP, whereas at a 2x concentration, it did cause a significant reduction in hippocampal LTP at 60 min post-HFS (Fig. 2D). Therefore, soluble low-n oligomers of human Aß produced by both 7PA2 and 7WD4 cell lines are capable of inhibiting hippocampal LTP.
The V717F APP mutation expressed in the 7PA2 cells has been shown to increase the Aß42/Aß40 ratio in the CM and enhance oligomerization (Xia et al. 1997). The observed difference in potency of 7PA2 versus 7WD4 CM (Fig. 2D) could be due to the type of oligomer species and/or to the levels of APP expression in the two cell lines. To address this issue, immunoprecipitation /Western blots were performed on CM and cell lysates of the two lines. Both 7PA2 CM and 7WD4 CM contained a similar dimer doublet and a trimer band that migrated distinctly from the dimers of synthetic Aß on Tricine SDS-PAGE gels (Fig. 3A). However, the 7WD4 cells consistently showed a lower abundance of total Aß and altered ratios of the Aß species. A Licor Odyssey gel analysis system was used to quantify the optical density of each Aß species from the two cell lines. Importantly, the oligomer ratios were found to be different between the two cell lines, with 7PA2 cells showing a significantly higher trimer/monomer ratio than the 7WD4 cells (Fig. 3B). The lower relative abundance Aß trimers in the 7WD4 CM, which has a reduced potency to inhibit LTP, is consistent with our finding above that trimers are particularly potent at inhibiting LTP.
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Minute quantities of natural Aß oligomers are sufficient to impede synaptic function
Next, we wished to establish as accurately as possible the concentration of natural Aß to which the hippocampal neurons were being subjected during a typical LTP experiment. To this end, a titration standard of synthetic human Aß was compared to the SEC fractions containing natural human Aß monomer, using quantification on a Licor Odyssey gel analysis system (Fig. 3D). Although there are limitations to this approach (see Discussion), we estimate that the total amount of secreted Aß monomer in our SEC fractions is 750 fmol (3 ng/15 ml or 50 pM). Based on this estimate of monomer, we calculated the approximate concentration of tetramer, trimer and dimer species based on their relative optical density measurements. By this accounting, tetramer is predicted to be 150 pM, trimer 100 pM and dimer 300 pM, levels that approximate our previous estimates for total oligomer content (Podlisny et al. 1995). Thus, very low concentrations of naturally secreted soluble Aß oligomers are sufficient to robustly and consistently impair hippocampal LTP.
Several aspects of synaptic function are unaffected by the oligomer-rich CM
Our previous work has shown that 7PA2 CM has no effect on baseline field potential recordings in the absence of a HFS (Fig. 2) (Walsh et al. 2005), demonstrating that basal synaptic transmission is unaffected by Aß. We tested the effects of 7PA2 CM on short-term plasticity to determine if, like LTP, it was similarly inhibited by Aß oligomers. Post-tetanic potentiation (PTP) is a short-term enhancement of presynaptic release thought to be caused by residual Ca2+ in the presynaptic terminals following a HFS (Zucker & Regehr, 2002). Slices were treated with AP5 to block NMDA receptors, thereby eliminating postsynaptic potentiation. Slices treated with either control CHO- CM or 7PA2 CM showed an indistinguishable PTP (Fig. 4A), suggesting that this aspect of presynaptic function is not affected by soluble Aß oligomers.
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During the HFS used to elicit LTP, the EPSPs show a characteristic bimodal response, initially facilitating and then depressing. We asked whether Aß oligomers can reduce the response to the HFS, thereby causing suboptimal stimulation. This scenario seemed unlikely, because 4 HFS is considered to be a saturating LTP stimulus. Indeed, when the HFS profiles were compared, there was no detectable difference between slices treated with CHO- CM or 7PA2 CM (number of stimuli to peak amplitude, peak amplitude, decay). Therefore, although 7PA2 CM inhibits LTP during its induction phase, we found no evidence that Aß oligomers alter the pattern of EPSPs during the HFS stimulation.
The input/output ratio compares the size of the fibre volley (representing the evoked action potentials in the Schaeffer collaterals) with the size of the evoked EPSP in the CA1 pyramidal neurons. This measure provides an estimate of the amount of presynaptic stimulation required to elicit a given postsynaptic response and imparts information regarding the effectiveness of the synaptic connections. For these experiments, slices were treated with CHO- CM or 7PA2 CM, and the stimulation intensity adjusted between a minimal and maximal evoked EPSP. As shown in Fig. 4D, 7PA2-CM-treated slices showed a slight but significant increase in slope in the regression curve, compared to those in CHO- CM (P < 0.05). This result indicates that slices treated with 7PA2 CM show a larger EPSP for a given input stimulation. Preliminary follow-up studies suggest that this is probably due to the secreted APP ectodomain (APPs) that is also found in the 7PA2 CM. However, this small shift is not likely to account for the effects of the oligomer-rich 7PA2 CM on LTP.
Finally, we compared the short-term potentiation after stimulation of hippocampal slices with a single HFS (100 Hz, 1 s). At 5, 10 and 30 min post-HFS, there was a significant difference between slices treated with control CHO- CM and 7PA2 CM (Fig. 4E and F). These data demonstrate that Aß oligomers inhibit even the earliest stages of synaptic plasticity following a HFS.
Not all forms of LTP are vulnerable to Aß oligomers
APP transgenic mice typically do not show memory deficits for several months after birth, presumably because sufficient levels of bioactive Aß species must accumulate to induce neuronal dysfunction. However, an additional explanation could be that very young mice differ in their susceptibility to Aß, particularly because the mechanisms involved in LTP differ from those in older animals (Yasuda et al. 2003). Because postnatal day (P)8–9 mice have immature synapses that are not capable of following HFS, we modified our LTP protocol to induce a chemical LTP by using picrotoxin, glycine and low Mg2+ to elevate spontaneous synaptic activity in brain slices (Neuhoff et al. 1999; Lu et al. 2001; Ehlers, 2003). In the presence of CHO- CM, this protocol induced a long-lasting potentiation of the EPSP in both P8–9 and P16–28 mice 60 min after induction (Fig. 5A and B). While 7PA2 CM inhibited this chemical LTP in older animals (Fig. 5A), there was no significant inhibition in the P8–9 mice (Fig. 5B). The chemical LTP was blocked by AP5, demonstrating its dependence on NMDA receptors (Fig. 5C and D), and it was not due to residual picrotoxin or glycine in the slices, as there was no effect on the EPSP slope unless the Mg2+ concentration was lowered during the induction protocol. We conclude that soluble Aß oligomers do not exert a significant effect on hippocampal LTP in P8–9 mice.
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Discussion |
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One obstacle to understanding how Aß inhibits LTP is the ambiguity over which species are responsible. We previously reported that soluble low-n Aß oligomers (but not monomers) can inhibit LTP (Walsh et al. 2002, 2005). Using an improved SEC method to separate naturally secreted Aß oligomers by size, we now demonstrate that a 12 kDa trimeric species is particularly potent at inhibiting LTP. While it is unknown whether a similar Aß trimer is found in the AD brain, we have previously reported that low-n oligomers are detectable in human cerebral spinal fluid (Walsh et al. 2000). Other low-n oligomers, including dimers and tetramers, produced a partial inhibition, but consistently less than did the trimer. Both the 4 kDa classical monomeric band and the band migrating just above it at 5 kDa in 7PA2 medium have been confirmed by mass spectrometry to be conformers of the Aß monomer (Walsh et al. 2000), and intensive efforts are underway to obtain mass spectrometry data on the other oligomer species. We also noted that a few early eluting fractions (21) contained several low-n Aß species. It is unknown at this time whether this represents a larger oligomer that breaks down during SDS PAGE, or whether this fraction may contain an unknown Aß binding protein. Intriguingly, these early fractions did show a fairly strong inhibition of LTP. Additional studies will be needed to characterize these fractions.
Several groups have made important advances in the preparation of synthetic Aß to generate low-n oligomers (Lambert et al. 1998; Barghorn et al. 2005). It has now been shown that nanomolar concentrations of these synthetic Aß42 assemblies can inhibit LTP. Moreover, Ashe and colleagues have proposed that the appearance of a 12-mer Aß species in APP transgenic mice correlates with disease progression (K. Ashe, personal communication). It will therefore, be important to identify commonalities among these different sources, different concentrations, and different sizes of Aß assemblies, to improve our understanding of Aß pathology. Nevertheless, it also important to pursue the study of Aß with various distinct preparations, and to resist the temptation to identify a single amyloidgenic Aß species, as it is currently unclear how any of the Aß multimers mediates the inhibition of LTP.
We show here that very low concentrations of cell-derived human Aß are sufficient to inhibit hippocampal LTP. Using a standard curve of synthetic Aß, we estimate that the concentration of Aß oligomers in the SEC fractions that were applied to the hippocampus is likely to be only 100–300 pM in the 15 ml of circulating medium. Although there is some error inherent in this method, for example, the efficiency of resuspending a lyophilized sample in a small volume for electrophoresis and the potentially different affinities of the antibody for synthetic and cell-derived Aß, other methods of measuring secreted Aß such as ELISA have similar limitations. The 3 ng of Aß monomer in any one SEC fraction will underestimate the amount of monomer in the whole CM, because (a) the monomer is distributed over several SEC fractions (b) some monomer is lost during the Centricon concentration and SEC steps, and (c) inhibitors of the Aß-degrading enzymes could not be included in the CM used for electrophysiology. However, Aß oligomers are resistant to degradation by Insuline Degrading Enzyme (IDE) (Wang et al. 2002) and certain other proteases, and thus we can approximate the oligomer concentrations in whole medium. Our previous estimate of total oligomer concentration of <1 nM is consistent with the findings shown here. Moreover, we can now approximate the relative abundance of each oligomer and demonstrate their relative effects on LTP. Interestingly, Aß trimers showed the strongest inhibition of LTP and yet were the weakest band on the gel, as detected by both a C-terminal (2G3) and amino acids 4–8 directed antibody (6E10) (data not shown). In summary, hippocampal synapses are exquisitely sensitive to very small quantities of naturally secreted, diffusible oligomers of human Aß.
Because LTP requires the activation of multiple, sequential signal transduction cascades (reviewed in Lynch, 2004), we tested whether the oligomer-rich 7PA2 CM disrupts early and/or late events. Our results indicate that the Aß oligomers inhibit the induction but not the expression of LTP. Application of 7PA2 CM shortly after the HFS had no effect on early LTP (1 h post-HFS). This result suggests that Aß oligomers disrupt the primary steps in the signal transduction cascades that initiate LTP. Two studies have reported that application of micromolar quantities of synthetic Aß to hippocampal slices immediately after a HFS can interfere with late LTP, although just as we report, early LTP was not substantially affected (Kim et al. 2001; Chen et al. 2002). Our use of cell-derived Aß at far lower concentrations may account for this difference. Nevertheless, all three studies suggest that the most pronounced effect of Aß is on the initial events during LTP induction.
The inhibitory effect of 7PA2 CM was also found to be sensitive to heat denaturation. It has been hypothesized that the ß-sheet conformation of Aß may contribute to its cytotoxicity (Glenner & Wong, 1984; Kirschner et al. 1986), and high temperatures may destroy this conformation. By SDS-PAGE, we have observed no difference in the migration of trimer and dimer bands in samples that have been boiled or allowed to equilibrate to room temperature before loading onto a gel (data not shown). Therefore, it may not be possible to detect changes in the conformation of these oligomers as a shift in size on an SDS-PAGE gel. The continued development of conformation-specific antibodies will be an important tool (Chang et al. 2003; Kayed et al. 2003), since the relative abundance of oligomers alone may not be sufficient to conclude whether the Aß species are in an active conformation. In this regard, we have tested the conformation-specific antibody A11, reported by Kayed et al. (2003), but it does not recognize our naturally secreted low-n oligomers, consistent with the authors' report that it recognizes intermediate assemblies of >40 kDa.
We also demonstrate here that despite a 2 h washout period, 7PA2 CM continues to inhibit LTP in brain slices. By assaying the retention of Aß on those slices, we show that a substantial amount of Aß remains in the tissue even after it was washed extensively. With longer wash periods, additional Aß clearance may be possible. Moreover, the slice preparations may not clear Aß as effectively as the intact brain, where both microglial uptake and Aß transport across the blood–brain barrier may actively decrease Aß levels (Deane et al. 2003; Streit, 2004). Nevertheless, our data demonstrate that Aß oligomers strongly adhere to brain tissue and continue to impair synaptic function.
Like the 7PA2 cell line, 7WD4 cells, which stably express wild-type APP751, secrete an array of soluble Aß oligomers. We found that the 7WD4 CM also inhibited LTP, although higher concentrations were required to obtain similar quantities of Aß oligomers and thus similar effects on LTP. The reduced potency of 7WD4 CM may be explained by two factors: the lower expression of APP (and lower total Aß levels) in the 7WD4 cells, and their decreased trimer/monomer ratio versus the 7PA2 cells. The difference in oligomer ratios is presumably caused by the reported Aß42-elevating effect of the V717F mutation in the 7PA2 cells (Xia et al. 1997). This mutation is known to cause an aggressive form of AD in humans, and it may similarly result in more potent Aß species in the AD family carrying it. While additional studies will be necessary to answer these question, our results make the important observation that Aß oligomers from two different cell lines cause an inhibition of LTP in a dose-dependent fashion.
Because LTP is so profoundly affected by soluble Aß oligomers, we examined other electrophysiological features of synaptic function and plasticity. Importantly, post-tetanic potentiation, paired-pulse ratios and the profile of the synaptic potential evoked during HFS were all found to be normal in the presence of the oligomer-rich 7PA2 medium. Therefore, there was no indication that presynaptic vesicle release was affected, nor the ability of the pyramidal cells to follow the presynaptic stimulation. Based on the tests that we performed, our data is agreement with previous work using Aß-derived diffusible ligands (ADDLs) or APP transgenic mice (Wang et al. 2002; Zhang et al. 2005). Nevertheless, we cannot rule out that a more extensive study of presynaptic function might detect an effect of Aß on presynaptic release. We did observe a small but significant difference between the CHO- CM (control) and 7PA2 CM-treated slices in the slope of the regression line of the input/output curve. However, the shift was in the opposite direction than that expected if 7PA2 CM were inhibiting synaptic transmission. Preliminary studies with a cell line that only expresses the soluble APP ectodomain APPs-
indicate that this product of APP processing in the 7PA2 CM may account for this finding. Our experiments did not detect any significant effect of 7PA2 CM on presynaptic vesicle release. However, short-term potentiation measured 5 min after HFS was significantly reduced in the presence of 7PA2 CM. The latter results are consistent with other work in hippocampal slices (Wang et al. 2004) but differ somewhat from the effects of the same CM in vivo (Klyubin et al. 2005), where short-term potentiation (STP) was not significantly affected. There are many potential explanations for this difference including the exact quantity of Aß oligomers applied in vivo and in vitro. Nevertheless, our results suggest that as with LTP, Aß oligomers interfere with the early stages of synaptic plasticity following a HFS.
To further address the mechanisms by which Aß inhibits LTP, we examined whether early postnatal mice were similarly susceptible to 7PA2 CM. Juvenile mice generally express a different complement of cell-surface receptors than older mice and employ different signal transduction cascades for LTP (Yasuda et al. 2003), both factors that could affect Aß-mediated synaptic inhibition. Unlike the P16–28 mice used throughout most of this study, P8–9 mice showed no significant difference in synaptic potentiation between CHO- CM and 7PA2 CM. While the reason for this difference remains unknown, these results do reveal that 7PA2 CM does not invariably inhibit LTP, suggesting that there is molecular specificity to the inhibition. It cannot be concluded that Aß has no effect on young neurons, but rather that LTP is unaffected by short applications of Aß in young hippocampal neurons. These results may have important implications for dissociated neuronal culture studies, in which it is common to use immature neurons.
BDNF can induce a stable potentiation of synapses that is independent of stimulus-induced LTP (Kang & Schuman, 1995). We therefore examined whether this form of LTP is similarly affected by 7PA2 CM. Intriguingly, this pathway was resistant to inhibition by Aß oligomers in the same slices that then showed impaired HFS-induced LTP. Although the BDNF signal transduction pathway has not been fully elucidated, it is known that the autophosphorylation of TrkB activates the Erk/MAPK and PI3K pathways, ultimately leading to CREB phosphorylation (Lu & Chow, 1999). It remains to be determined whether BDNF activates the LTP downstream of an Aß block or whether BDNF activates a parallel pathway.
In conclusion, we report that the acute application of cell-derived soluble oligomers of human Aß can rapidly inhibit LTP in normal mouse hippocampus. Fractionation of the medium suggests that the Aß trimer is particularly potent, even at very low concentrations (100 pM). We have also determined that several measures of synaptic function remain normal in the presence of 7PA2 CM, despite the highly reproducible inhibition of LTP. Finally, young mice appear to be resilient to the immediate effects of Aß on LTP, and BDNF-induced LTP is also spared. These results suggest that small diffusible Aß oligomers specifically target certain molecular components that mediate synaptic plasticity. The chronic failure of certain synapses to function normally in the ongoing presence of natural Aß oligomers might be expected to lead to a loss of synaptic spines (Papa & Segal, 1996; Collin et al. 1997; Kossel et al. 1997; Neuhoff et al. 1999), and thus is likely to contribute to the downstream neuropathology of AD.
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