The prion protein as a receptor for amyloid-β

Increased levels of brain amyloid-β, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer’s disease¹. Increased amyloid-β can cause synaptic depression^2,3, reduce the number of spine protrusions (that is, sites of synaptic contacts)^4,5 and block long-term synaptic potentiation (LTP)^6,7, a form of synaptic plasticity; however, the receptor through which amyloid-β produces these synaptic perturbations has remained elusive. Laurén et al.⁸ suggested that binding between oligomeric amyloid-β (a form of amyloid-β thought to be most active^5,6,9-11) and the cellular prion protein (PrP^C)⁸ is necessary for synaptic perturbations. Here we show that PrP^C is not required for amyloid-β-induced synaptic depression, reduction in spine density, or blockade of LTP; our results indicate that amyloid-β-mediated synaptic defects do not require PrP^c.

To test whether PrP^c is required for amyloid-β-induced synaptic depression we infected organotypic hippocampal slice neurons with a Sindbis virus driving expression of APPct100, a precursor of amyloid-β, which leads to increased neuronal production and secretion of amyloid-β that does not perturb the health of the neuron, as assessed electrophysiologically³. Synaptic transmission was depressed in neurons expressing APPct100, in both wild-type and Prnp^−/− mouse slices (where Prnp is the gene that encodes PrP^C) 24 h after infection (Fig. 1a). As previously shown for wild-type tissue³, this depression was blocked if Prnp^−/− slices were maintained during the APPct100 expression period with an inhibitor of NMDA (N-methyl-d-aspartate) receptors (Fig. 1a). Thus, synaptic depression after expression of APPct100, an effect of elevated amyloid-β³, is intact in animals lacking PrP^C.

Figure 1. PrP^C is not required for amyloid-β-induced synaptic deficits.

a, Depression of whole-cell recorded synaptic AMPA receptor currents in a CA1 hippocampal neuron infected with Sindbis virus expressing APPct100 (grey bars) compared to a simultaneously recorded non-infected neuron (white bars) in wild-type (WT; 73 ± 8%, n = 18, P = 0.02) and Prnp^−/− slices (59 ± 9%, n = 25, P = 0.002). Incubation of slices with 100 μM d-amino-phosphono-valeric acid (d-APV) during APPct100 expression abolished synaptic depression in WT (98 ± 11%, n = 12, P = 0.6) and Prnp^−/− slices (105 ± 13%, n = 13, P = 0.6). EPSC, excitatory postsynaptic current. b, Decreased spine density by APP expression in WT (n = 14 APP expressing, n = 9 control dendrites, P = 0.03) and Prnp^−/− slices (n = 13 APP expressing, n = 9 control dendrites, P = 0.01). Representative images of a dendrite in conditions as indicated. c, Spine loss in PrP^C-deficient hippocampal slices is specific to incubation with oligomeric amyloid-β. Aβ42, but not Aβ40, preparation produced high molecular mass oligomers, as indicated by western blot analysis. WT and Prnp^−/− slices were incubated with no peptide (ctrl: WT, n = 22; Prnp^−/−, n = 22), with Aβ40 (WT, n = 22; Prnp^−/−, n = 20), or with Aβ42 (WT, n = 22; Prnp^−/−, n = 22) at 1 μM peptide concentration for 24 h, and spine densities were analysed blind to the experimenter. Spine densities were unaffected by monomeric amyloid-β incubation (WT, P = 0.4; Prnp^−/−, P = 0.9), but significantly reduced by incubation with oligomeric amyloid-β (WT, P = 0.02; Prnp^−/−, P = 0.03). Representative images of a dendrite in conditions as indicated. d, Oligomeric Aβ42 blocks LTP independent of the PrP^C. Hippocampal slices were isolated from 2–3-month-old WT or Prnp^−/− mice. Field excitatory postsynaptic potentials (fEPSPs) were measured on two independent pathways in the presence of either 500 nM Aβ40 or 500 nM Aβ42. A control pathway (open circles) was recorded while theta burst stimulation (ten 100-Hz four shock bursts with 200-ms interburst intervals, arrow) was delivered to the second pathway (filled circles) after recording a 15 min baseline. LTP was significantly depressed by Aβ42 compared with Aβ40 in both WT (P < 0.001) and Prnp^−/− slices (P < 0.001). When compared to their corresponding control pathway, LTP was successfully induced in WT (P < 0.05) and Prnp^−/− slices (P < 0.05) treated with monomers. Block of LTP was found in both WT (P > 0.05) and Prnp^−/− (P > 0.05) oligomer-treated slices when compared to their corresponding control pathway. Statistical comparisons (P) were performed using paired (a) or non-paired (b–d) two-tailed Student’s t-test of log-transformed data. All error bars, s.e.m.; asterisk indicates P < 0.05.

We next examined the effects of amyloid-β on dendritic spines, sites of excitatory synapses. Overexpression of APP⁴ (or exposure to oligomeric amyloid-β⁵) leads to loss of dendritic spines in organotypic slices prepared from wild-type animals, which can be visualized by co-expression of a cytoplasmic marker. The same APP-induced decrease of spines was seen in slices made from mice lacking PrP^C (Fig. 1b). We next determined which species of amyloid-β is responsible for the observed effects. Synthetic amyloid-β(1–40) (Aβ40) peptides remain predominantly in monomeric form, and did not affect spine density (Fig. 1c). Amyloid-β(1–42) peptides (Aβ42) form oligomers, and when exogenously applied they produced a similar loss of dendritic spines in slices prepared from either wild-type or PrP^C-lacking mice (Fig. 1c). Thus, the loss of dendritic spines produced by oligomeric Aβ42 exposure does not depend on PrP^C.

We repeated the experiments as described in Laurén et al.⁸ that studied PrP^C’s role in the Aβ-mediated blockade of LTP. Acutely prepared hippocampal slices from mature wild-type and Prnp^−/− mice were exposed to either species of amyloid-β and LTP was induced by theta burst stimulation. We found that Aβ42 (but not Aβ40) blocked LTP irrespective of whether PrP^C is present or absent (Fig. 1d).

We show that amyloid-β-induced synaptic depression, loss of dendritic spines and blockade of LTP are present in hippocampal slices prepared from PrP^C-deficient animals. In line with our results, amyloid-β oligomers were shown to impair long-term memory equally in PrP^C-lacking and PrP^C-expressing mice¹². Thus, although oligomeric amyloid-β can bind PrP^C, it does not seem to be the receptor responsible for synaptic perturbations caused by oligomeric amyloid-β. Elucidation of the molecular mechanisms by which amyloid-β produces synaptic perturbations remains as a major goal in finding therapeutic treatments of Alzheimer’s disease.

METHODS

C57/BL10 and C57/BL10 Prnp^−/− mice were provided by M. Oldstone (Scripps Research Institute, grant AG04342). Organotypic slice cultures were infected at 7–12 days in vitro with Sindbis virus expressing APPct100³. After 24h simultaneous whole-cell paired recordings were obtained as described previously¹³. At 12–17 days in vitro slices were infected with Sindbis virus expressing tdTomato or APP plus tdTomato for 48 h, or infected with eGFP and incubated with 1 μM monomeric Aβ40 or oligomerized Aβ42¹⁴ for 24 h. Two-photon laser scanning images were taken of dendrites at the site of primary apical dendrite bifurcation. Spine densities were counted as described previously⁴. The protocol for the LTP experiments was as described in Laurén et al.⁸.